CN117405908A - Method for preparing and analyzing biopsies and biological samples - Google Patents

Abstract

Matrix-assisted methods and compositions for solidifying and preparing liquid biopsies and other liquid samples for three-dimensional high-resolution imaging and diagnosis by microscopy.

Description

Methods for preparing and analyzing biopsies and biological specimens

Technical Field

The present invention relates to matrix-assisted methods and compositions for analyzing biological specimens, in particular, liquid biopsies, and other liquid samples by microscopy, including fluorescence microscopy.

Background Art

Liquid biopsies are typically obtained from bodily fluids such as peripheral blood, bone marrow, cerebrospinal fluid, urine, saliva, sputum, tears, semen, or other tissue sources. Biomarkers or components in liquid samples can be evaluated or measured for various diagnostic applications such as disease screening, detection, staging, and monitoring.

Biomarkers in liquid biopsies may include cells and extracellular components, the selection of which may depend on a variety of factors, such as underlying medical conditions or treatment status. For example, biomarkers may correspond to antigens or other properties that distinguish rare circulating cells, such as circulating tumor cells (CTC) and CTC clusters derived from solid tumors or metastases, and circulating endothelial cells (CEC) associated with cardiovascular and other pathologies. See, for example, Lim et al. 2019, NPJ Prec. Oncol. 3, 23; Rostami et al. 2019, J. Sci: Adv. Mat. Dev. 4, 1-18; Schmidt et al. 2015, Trends Cardiovasc. Med. 25, 578-587. These cells can be further processed for genetic abnormalities and other molecular characteristics. Biomarkers can also identify extracellular components, such as circulating factors, secreted proteins, released vesicles and exosomes and cell-free nucleic acids derived from tumors. Cell-free nucleic acids include cell-free tumor DNA (ctDNA), which is used in cancer monitoring, and cell-free fetal DNA (cffDNA), which is found in maternal blood and is used in non-invasive prenatal testing. Campos et al. 2018, Cancer J. 24, 93-103; Sifakis et al. 2014, Mol. Med. Rep. 11, 2367-2372. Subsequent genomic and protein processing can allow further analysis of extracellular biomarkers.

Typical biopsy methods can include a variety of methods. See, for example, Harouaka et al., 2014, Pharmacol. Ther. 141, 209-221. A common method is based on immunoaffinity, such as microfluidics and microchip-based methods, which allow the detection of antibodies bound to cells and extracellular targets. These methods rely on antibody-antigen binding between floating cells and antibody-coated surfaces, and are therefore limited to antigens present on the surface of target cells, such as membrane proteins. In addition, the efficacy of these methods depends on sufficient levels of cell surface antigens to allow effective and specific recognition by antibodies (for example, lower EpCAM expression on the cell surface can cause poor cell binding to the chip surface). These methods generally have lower cell flow rates, thereby hindering the effective analysis of complex samples. Therefore, these methods, as well as other immunoaffinity methods (such as those based on magnetic beads), are limited in scope, sensitivity, specificity, and processing volume.

Another common method is cytology: examining cells directly under a microscope. However, due to the light scattering properties of cells, this method is limited to examining only a small number of cells at a time (e.g., as a monolayer on a slide); therefore, using this method to detect rare targets that may be present in millions of cells in even 2 c.c. of blood is impractical and economically unfeasible.

In another method, flow cytometry, thousands of cells per second pass through one or more laser beams one by one, which can produce different light scattering patterns (depending on, for example, cell size and granularity) and fluorescence emissions (depending on which fluorescent probes are bound to the cells). See, for example, Flow cytometry: retrospective, fundamentals and recent instrumentation, Cytotechnology, 2012 Mar; 64 (2): 109-130. However, when the cells of interest are rare, flow cytometry methods do not provide high resolution and confidence. For example, it does not provide direct visual inspection of cells to confirm their potential morphology or functional properties. In addition, there may be problems with the gating of pixels captured based on the intensity of the fluorescent signal and by the detector (which does not provide information about the labeling quality or morphological details of the analyzed sample). For example, only small gating changes or interferences can lead to the exclusion of CTC cells and other rare cells (e.g., CECs) that are small in size or have weak fluorescent signals.

In view of these and other limitations, there is still a need to further improve liquid biopsy analysis. The present invention addresses these and other needs in the art by providing methods and compositions for labeling, dispersing and capturing biopsy components in 3-dimensional gels or other non-liquid forms. When maintained in this form, discontinuous and rare biomarkers can be detected with high resolution and sensitivity by rapid imaging methods such as light sheet fluorescence microscopy and other methods. More generally, these methods are applicable to components in any sample, biological or non-biological, whose resolution can be improved by dispersing in a liquid and then capturing and imaging in a non-liquid state.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic overview of a matrix-assisted protocol for preparing and analyzing liquid biological samples according to certain embodiments of the present disclosure. The stages in the protocol may include (1) cell preparation; (2) sample gelation and clarification; (3) sample mounting and imaging; and (4) sample observation and analysis.

2A-2C depict images obtained from a light sheet microscopy imaging chamber as described in Example 2. Scale bar in Fig. 2C (20 μm).

3A-3C depict images obtained from a light sheet microscopy imaging chamber as described in Example 3. Scale bars: FIG. 3A (50 μm); FIG. 3B (300 μm); FIG. 3C (50 μm).

Figures 4A-4D depict images obtained from a light sheet microscopy imaging chamber as described in Example 4. Scale bar (30 μm) in Figure 4B.

Figures 5A and 5B depict 3D gel data for patient A, as described in Example 5. Scale bar in Figure 5A (150 μm).

Figures 6A and 6B depict 3D gel data for patient B, as described in Example 5. Scale bars: Figure 6A (200 μm); Figure 6B (2 μm).

Summary of the invention

The present disclosure provides the matrix-assisted method of preparing and analyzing the components in the biological and non-biological samples including liquid biological specimens as further described in this article, such as the method based on gel formation.Liquid specimens can be derived from any source, including humans and animals.In an embodiment, it can be obtained from liquid biopsy, and this biopsy is obtained from the peripheral blood, bone marrow, cerebrospinal fluid and other tissue sources that can be further processed.In an embodiment, it can be obtained from the liquid dispersion of material, and the material is obtained from other sources, including solid sources, such as from solid tissue biopsy.

In an embodiment, the matrix-assisted method includes adding a solidifying agent (e.g., a gelling agent) to a biological specimen containing a biological material; producing a solidified sample (e.g., a gelled sample) containing dispersed biological material; and imaging the solidified sample to identify one or more components in the biological material. The biological material may include any biomolecule, including nucleic acids, proteins, and small molecules, which, in an embodiment, may serve as a biomarker for a medical condition or disease state. For example, the biomolecule may serve as a biomarker for rare circulating cells in the blood, such as circulating tumor cells, circulating endothelial cells, and other cells and cell clusters that may be present in a biological specimen. In an embodiment, the biological material specimen may be enriched, for example, by concentrating cells from a large amount of blood or other sample sources. However, advantageously, the method disclosed herein does not require any pre-selection or pre-screening of cells; alternatively, the method allows for unbiased analysis of samples of unselected or unscreened cells by detecting biomarker markers, such as antibodies or nucleic acid probes, bound to the biological material in the specimen, for example, by detecting labeled antibodies that identify specific cell surface markers in the specimen.

In embodiments, the step of adding a solidifying agent in any method may include adding a liquid gel solution, such as low melting point agarose or a hydrogel precursor, to the sample; adding the agent directly to the sample under conditions that allow the formation of a solid such as a gel; forming a hydrogel, or other methods as disclosed herein. Prior to adding the solidifying agent, the method may include subjecting the components of the biological specimen to a fixation procedure as described herein, and it may also include labeling one or more biomolecules, such as proteins or nucleic acids, with molecular probes. Molecular probes include antibodies, dyes, and nucleic acid probes known in the art, including those described herein. In embodiments, probes can be used to identify circulating tumor cells and clusters, as well as cell-free tumors or fetal-derived DNA, and genetic and structural changes in the cell nucleus, such as DNA and chromosomal abnormalities, amplifications, deletions, and translocations.

In an embodiment, the step of adding a solidifying agent to the liquid specimen comprises mixing a sample containing biological material (e.g., a clump containing biological material) with a liquid (molten) gel solution having a temperature raised above its gel point. Thus, in an embodiment, a cell clump, such as labeled and washed in PBST, is resuspended in the gel solution and allowed to cool into a gel. The resuspension step also allows any trace amounts of liquid associated with the clump after treatment (such as PBST) to be diluted and mixed in the gel solution, thereby ensuring a uniform sample for imaging.

In an alternative embodiment, the step of adding a curing agent to the liquid specimen includes mixing a sample containing a biological material (e.g., a mass containing a biological material) with a mixture or solution containing one or more hydrogel precursors, and changing the conditions of the mixture to induce curing (e.g., gelation). The formation of hydrogels is known in the art and can be accomplished by various methods according to the present disclosure. For example, according to methods known in the art, a second agent (e.g., Ca+ ions or a cross-linking agent) can be added in an amount sufficient to induce gelation of a hydrogel precursor.

For example, the specimen can be processed as described herein, including the steps of centrifuging the sample to obtain a pellet, and resuspending in an alginate-hydrogel precursor solution and mixing with a sufficient amount of _CaCl2 solution (e.g., 0.2 M) to initiate gelation. In this exemplary method, the gel is formed in a short period of time (e.g., about 15 minutes) and can then be mounted for refractive index matching (if necessary) and imaging.

The step of producing a solidified sample containing dispersed biological material in any method may include, after adding a solidifying agent, transferring the sample to a sample holder and allowing solidification to occur. In other embodiments, the sample may be prepared directly in a sample holder, to which a solidifying agent is added, thereby merging the pre-gelling and solidification steps in a single tube. Before solidification, the sample may be stirred, oscillated, vibrated or otherwise agitated in the sample holder to ensure the dispersion of the material. In an embodiment, the solidified sample has a shape suitable for imaging, such as a square, a cylindrical shape, or any other form compatible with the desired imaging system. In an embodiment, the solidified sample containing dispersed biological material is transferred to a clear (or balanced) solution to obtain a refractive index match. In other embodiments, refractive index matching is not necessary due to, for example, the suitable optical properties of the specific solidifying agent used. For example, if a sample of dispersed cells is prepared in a transparent matrix, the labeled molecules in or on the surface of the individual dispersed cells can be appropriately imaged without pre-cultivation in a refractive index matching material (e.g., a refractive index matching solution). In certain embodiments, solidification includes mixing the biological specimen with a refractive index matching material (eg, a refractive index matching solution), thereby preventing the need for a separate treatment with the refractive index matching material.

In embodiments, the step of imaging the solidified sample may include imaging a suitably shaped solid sample such as a block by various microscopic techniques including fluorescence microscopy, and more specifically, fluorescence light sheet microscopy. In embodiments, imaging allows identification of single cells in the solidified sample, and more specifically, for example, may include detection of one or more cancer cells, such as circulating tumor cells, or cancer markers. The size of the solidified sample may vary depending on the application, sensitivity, and biomolecule being assayed. In embodiments, imaging may be used to analyze cell-cell interactions, as well as morphological and structural characteristics of cells, such as size, shape, and nuclear to cytoplasmic ratio and characteristics of organelles.

The methods are suitable for many applications, including but not limited to, for example, assessing, diagnosing or monitoring disease by microscopic analysis of fluids and/or tissue biopsies; screening candidate therapeutic agents for their effects on samples (e.g., blood or tissue samples) under disease conditions; and evaluating the expression of a panel of biomarkers in a sample.

DETAILED DESCRIPTION

The present disclosure provides for the presence of one or more biomarkers to analyze liquid samples, such as the matrix-assisted method and composition of liquid biopsy. In an embodiment, the method and composition are used to solidify the dispersed material in the sample so that it is captured and fixed in a three-dimensional state. Subsequently, the biomarker of interest, such as rare disease markers, that may be present in the material can be detected and resolved with high sensitivity and specificity. The matrix-assisted method includes using a solidifying agent such as a molten gel solution or a hydrogel precursor to convert the liquid sample into a solid sample with dispersed components.

In embodiments, the present disclosure provides three-dimensional imaging methods for detecting biomarkers including rare molecules such as cancer cell markers using matrix-assisted methods and compositions for analyzing liquid biopsies and other liquid samples by microscopy including fluorescence microscopy.

In an embodiment, the biomarker indicates a cellular component, such as a component of rare circulating cells, such as circulating tumor cells and circulating endothelial cells. For example, the biomarker may include a cell surface protein, morphological marker, or nucleic acid sequence that can identify these tumor cells. In an embodiment, the biomarker indicates an extracellular component, such as extracellular DNA, protein, or vesicle, which indicates a specific disease or health state.

Biomarkers can be labeled by known techniques as described herein, and even in the case of complex samples, they can be detected by a wide range of imaging methods, and more specifically, by fluorescence microscopy including light sheet fluorescence microscopy and other microscopy methods.

Terms and Definitions

Unless otherwise defined, technical and scientific terms used herein all have the same meaning as commonly understood by those of ordinary skill in the art. Any methods, devices and materials similar or equivalent to those described herein can be used to practice the present invention. The following definitions are provided to facilitate the understanding of certain terms frequently used in this article and are not intended to limit the scope of this disclosure.

As used herein, the term "about" or "approximately" means a range of values that one skilled in the art would consider to be reasonably similar to a specified value, including the specified value. In the embodiments, "about" means within the standard deviation using measurements generally accepted in the art. In the embodiments, "about" means a range extending to +/- 10% of the specified value. In the embodiments, "approximately" means the specified value.

It should be understood that, regardless of whether the term "about" is explicitly used, each quantity given herein is intended to refer to both the actual given value and the approximate value of this given value that is reasonably inferred based on ordinary skills in the art, including equivalent values and approximate values due to the experimental and/or measurement conditions of this given value. Therefore, for any embodiment of the present disclosure in which a numerical value begins with "about" or "approximately", the present disclosure includes embodiments in which the exact value is listed. Conversely, for any embodiment of the present disclosure in which a numerical value does not begin with "about" or "approximately", the present disclosure includes embodiments in which a numerical value begins with "about" or "approximately".

Unless otherwise indicated, concentrations provided as percentages or weight (wt) percentages refer to weight/volume (w/v) concentrations. For example, 2% or 2 wt% of a component in 100 ml of solution corresponds to 2 grams of that component.

Unless expressly stated otherwise, the terms "a", "an" and "the" as used herein are to be understood to refer to both the singular and the plural. Thus, "a", "an" and "the" (and, where appropriate, grammatical variations thereof) refer to one or more.

Furthermore, although items, elements, or components of the embodiments may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated.

The terms "comprising" and "including" are used herein in their open, non-restrictive sense. Unless otherwise expressly stated, other terms and phrases used in this document and their variations should be understood to be open, not restrictive. As an example of the foregoing: the term "example" is used to provide an exemplary case of the items discussed, rather than an exhaustive or limiting list thereof. Adjectives such as "conventional", "ordinary", "known" and terms of similar meaning should not be understood to limit the described items to a given period of time or to items available as of a given time, but should instead be interpreted as including conventional or common technologies that may be available or known at any time now or in the future. Similarly, when this document refers to technologies that are obvious or known to a person of ordinary skill, these technologies cover those that are obvious or known to a person skilled in the art at any time now or in the future.

As used herein, the term "solidified sample" refers to a sample in a non-liquid form, for example, a solid or gel form, wherein the solid or gel material provides a support matrix to capture the biological material in a dispersed state, that is, fixed in a 3-dimensional sample. The dispersed material in the sample can then be effectively identified and imaged in 3 dimensions. As used herein, the term "solidifying agent" includes gelling agents capable of forming a gel, as well as, for example, epoxy resins and other agents that may be desirable when supporting dispersed biological materials at a higher density.

As used herein, the terms "biological sample" and "biological specimen" (and, depending on the context, "sample" or "specimen") refer to any biological material that contains or is believed to contain biological molecules such as nucleic acids or proteins. Samples that can be conditioned by the compositions and methods provided herein can be obtained from in vivo or in vitro sources, and thus include specimens excised from subjects such as rodent models, such as cells, tissues, viruses, and organs, as well as specimens grown in vitro, such as cells, tissues, and micro-organs. Exemplary biological specimens include solid tissues and organs, including but not limited to liver, spleen, kidney, lung, intestine, thymus, colon, tonsils, testicles, skin, brain, heart, muscle, and pancreatic tissues and organs. In an embodiment, the sample is a whole organ obtained from an animal including a mouse, a rat, and other animals. In an embodiment, the biological specimen is a brain tissue or whole brain such as from a rodent, and more specifically, from a mouse. Other biological specimens include cells, viruses, and other microorganisms. In an embodiment, the biological specimen is from a human, an animal, or a plant. In embodiments, the sample is from a human, a companion animal such as a dog or cat, an agricultural animal such as a cow, sheep, and pig, a rodent such as a rat or mouse, a zoo animal, a primate such as a monkey, and the like.

Exemplary biological samples include, but are not limited to, materials from biopsy, bone marrow samples, organ samples, skin fragments, organisms, and materials obtained from clinical or forensic environments. In an embodiment, the biological sample is a tissue sample, preferably an organ sample. The sample can be obtained from an animal or human subject that is affected by a disease or other condition or suspected of being affected (normal or sick), or is considered normal or healthy. Specimens such as organ and tissue samples can be collected and processed using the methods described herein and subjected to microscopic analysis immediately after processing, or can be preserved and, for example, after being stored for a long time, subjected to microscopic analysis at a future time. In an embodiment, the methods described herein can be used to analyze living cells, and in other embodiments, the methods described herein can be used to analyze fixed cells.

In certain embodiments, the biological sample is a liquid biopsy obtained from a body fluid such as peripheral blood, bone marrow, cerebrospinal fluid, urine, saliva, sputum, tears, semen, or other tissue source.

As used herein, the term "biomolecule" is interchangeable with a molecule and refers to a molecule present in a biological sample or specimen. In one aspect, a biomolecule is an endogenous biomolecule. In another aspect, a biomolecule is an exogenous biomolecule. Non-limiting examples of exogenous biomolecules include artificially implanted biomolecules, for example, biomolecules transferred or expressed by viruses or plasmids. Biomolecules include, but are not limited to, proteins, nucleic acids, lipids, carbohydrates, steroids, metabolites, and other subcellular structures or components within cells, tissues, or organs. Non-limiting examples of proteins include enzymes, membrane proteins, transcription factors, synaptic proteins, and neuronal markers. In some non-limiting embodiments, biomolecules are selected from subunits, receptors, receptor subunits, membrane proteins, intermediate filament proteins, membrane pumps, transcription factors, and combinations of the above items of macromolecules. In other non-limiting embodiments, biomolecules are Olig2 (oligodendritic cell transcription factor), NeuN (neuronal nuclear antigen), NKCC2 (Na+K+Cl-cotransporter 2). In other non-limiting embodiments, biomolecules include RNA. In other non-limiting embodiments, biomolecules include DNA molecules. In an embodiment, the biomolecule is located on a structure, examples of which include flagella, cilia, synapses, synaptic spines, extracellular matrix (ECM), cell wall, outer cell envelope, membrane, cytoplasm, Golgi network, mitochondria, endoplasmic reticulum (ER) (e.g., rough ER or smooth ER), nucleus, centriole, ribosome, polysome, lysosome, liposome, cytoskeletal component, vesicle, granule, peroxisome, vacuole, protoplast, vacuole membrane, plasmodesmata, chloroplast, pseudopodia, blood vessel-associated structures of the brain, dense stellate cell network of the brain, or a combination of the above. In an embodiment, the biomolecule is a cellular marker, such as a protein expressed on the surface of a cancer cell.

In an embodiment, a "biomolecule" is evaluated or measured in a liquid biopsy sample and for diagnostic applications such as screening, detecting, staging or monitoring (monitoring) a disease condition such as cancer or a medical condition such as a metabolic disorder. In an embodiment, the biomolecule is evaluated or measured in a non-invasive prenatal screening or diagnostic test.

As used herein, the term "label" refers to any technique and reagent now known or discovered in the future that can provide a signal-based indication of the presence or absence of a specific target moiety within a sample of the present disclosure. Non-limiting examples of labeling agents include small molecules, dyes, antibodies, enzymes, nanoparticles, nucleic acid probes, or a combination of the above. In some non-limiting embodiments, the labeling agent includes a label, for example, a chromogenic label, a fluorescent label, a radionuclide-conjugated label, or a combination of the above.

Aspects of the present invention provide a method for analyzing a liquid sample containing biological material for one or more target components. In an exemplary embodiment, the method includes adding a solidifying agent to a specimen obtained from a liquid sample containing biological material, producing a solidified sample containing dispersed biological material, and imaging the solidified sample to identify one or more target components in the dispersed biological material.

In an embodiment, the method of analyzing a liquid sample according to the present disclosure further includes labeling a specimen obtained from the liquid sample with one or more probes for one or more target components before adding a curing agent; and/or labeling the cured sample with one or more probes for one or more target components. In an exemplary embodiment, the method includes labeling a specimen obtained from the liquid sample with one or more probes for one or more target components before adding a curing agent. In an embodiment, the method further includes introducing a refractive index matching material into the cured sample.

In an embodiment, the liquid sample is a liquid blood sample. For example, a specimen obtained from a liquid blood sample may be processed to remove red blood cells and platelets from the liquid blood sample, and/or may be a specimen comprising peripheral blood mononuclear cells (PBMCs) separated from a liquid blood sample. Alternatively, in other applications, red blood cells or other components of the liquid sample may be separated. Alternatively, the specimen may be obtained from other biological fluids and fluids other than blood obtained from a mammal (e.g., a human or a rat).

In certain embodiments, one or more target components include nucleic acids, proteins, viruses, or vesicles. In certain embodiments, one or more target components include extracellular targets. In certain embodiments, one or more target components include cells or intracellular targets.

In an embodiment, labeling as described in any of the above embodiments comprises contacting a specimen obtained from a liquid sample with a molecular probe; and/or contacting the solidified sample from step (b) with a molecular probe. The molecular probe may be, for example, an antibody, a fluorescent dye or a nucleic acid probe.

In an embodiment, the method of analyzing a liquid sample according to the present disclosure further comprises transferring the specimen to a sample holder. In an exemplary embodiment, the method may further comprise oscillating or vibrating the sample in the sample holder. In an exemplary embodiment, the solidified sample is a solid or gel block suitable for imaging.

In an embodiment, the method of analyzing a liquid sample according to the present disclosure further comprises performing a fixation procedure on the specimen, such as by incubating the specimen (if performed before curing), or the cured sample in a fixative solution. In an exemplary embodiment, the fixative solution comprises glutaraldehyde, formaldehyde, epoxy resin, or a mixture of any two or more of the foregoing.

Imaging as described in any of the above embodiments can be achieved, for example, using microscopy and photography techniques known to those of ordinary skill in the art. For example, in an embodiment, imaging is performed by fluorescence microscopy such as light sheet fluorescence microscopy. In an exemplary embodiment, imaging identifies the presence or absence of a specific cell type in a solidified sample.

An exemplary embodiment of the present disclosure provides a method for analyzing a liquid blood sample for the presence of rare circulating cells such as, but not limited to, circulating tumor cells. In one embodiment, the method includes labeling a specimen containing isolated peripheral blood mononuclear cells (PBMCs) obtained from a liquid blood sample with one or more probes for rare circulating cells; adding a solidifying agent to the labeled specimen containing peripheral blood mononuclear cells (PBMCs); producing a solidified sample containing dispersed peripheral blood mononuclear cells (PBMCs); optionally, introducing a refractive index matching material into the solidified sample to provide an optically transparent solidified sample having a refractive index suitable for imaging; and imaging the solidified sample or the optically transparent solidified sample to determine the presence of one or more probes, thereby determining the presence of rare circulating cells in the liquid blood sample. Labeling may further include adding a probe for white blood cells that can serve as, for example, a control.

In an embodiment, one or more probes recognize cancer-specific antigens or tumor-specific DNA or RNA sequences. For example, one or more probes can be selected from antibodies or nucleic acid probes. In an embodiment, one or more probes confer detection of one or more of EpCAM, HER2, CDX2, CK20, CK19, PD/PDL-1, and EGFR antigens or corresponding nucleic acid sequences.

In an embodiment, the method of analyzing a liquid sample according to any of the above embodiments further comprises introducing a refractive index matching material into the solidified sample.In certain embodiments, an optically transparent gelled sample immersed in a solution comprising a refractive index matching material is introduced into a sample holder.

In one embodiment, the curing agent comprises a component selected from low melting point agarose, agarose and a hydrogel precursor. In an exemplary embodiment, the step of producing a solidified sample comprises adding a curing agent to the sample above room temperature and allowing the sample to reach a low temperature to become a solid gel. In other embodiments, the step of producing a solidified sample comprises adding an agent to a hydrogel precursor to induce gelation.

In one embodiment, the present disclosure provides a solidified sample suitable for imaging, the sample comprising dispersed biological material obtained from a liquid biopsy (e.g., a blood sample) and fixed within the solidified sample. The solidified sample may further comprise probes for one or more target components in the liquid biopsy. The biological material includes peripheral blood mononuclear cells (PBMCs) and rare circulating cells, such as circulating tumor cells, circulating epithelial cells, and circulating endothelial cells.

Low melting (LM) agarose is commercially available and is the result of a chemical derivatization process known in the art, such as hydroxyethylation, which reduces the number of intrachain hydrogen bonds present in standard agarose, thereby resulting in relatively low melting and gelling temperatures. LM agarose generally has a number of properties, including: (i) relatively low melting and gelling temperatures when compared to standard agarose; and (ii) higher clarity (gel transparency) compared to standard agarose gels.

In certain exemplary embodiments, the LM agarose is a molecular biology grade LM agarose having a gelling temperature of ≤30°C (e.g., 26°C-30°C) and/or a melting temperature of ≤65°C (both at 1.5 wt% concentration) and/or a gel strength of ≥200 g/ ^cm2 (at 1 wt% concentration), thereby producing a gel having greater sieving properties and higher clarity than normal melting point agarose. Examples of commercially available LM agarose suitable for use according to the present disclosure include, but are not limited to, SeaPrep ^™ agarose (Lonza Catalog #50302), SeaPlaque ^™ agarose (Lonza Catalog #50104), and UltraPure ^™ low melting point agarose (ThermoFisher Scientific Catalog #16500500).

In an embodiment, the labeling agent comprises a small molecule capable of binding to a specific target portion within the tissue. Examples of small molecule dyes include DAPI, propidium iodide, lectin, phalloidin, and any other small molecule capable of binding to a target portion within the tissue. In an embodiment, the small molecule intrinsically generates a signal, such as a fluorescent signal generated by DAPI, propidium iodide, or acridine orange. In an embodiment, the small molecule is coupled to an indicator that generates a signal, such as, for example, in the case of a lectin dye, an indicator that generates a fluorescent signal, or an indicator that generates a non-fluorescent signal, for example, a colorimetric indicator (e.g., horseradish peroxidase (horseradish peroxidase; HRP) or 3,3'-diaminobenzidinetetrahydrochloride (3,3'-diaminobenzidinetetrahydrochloride; DAB)). In an embodiment, the stain comprises an antibody as further described herein. In an embodiment, staining includes detection activity targeting a modified nucleic acid chain. In other embodiments, staining includes in situ hybridization so that the stain comprises a nucleotide-based probe capable of hybridizing to a predetermined nucleic acid sequence within the tissue. In an embodiment, the nucleotide-based probe comprises a label (e.g., one or more of the labels provided above) that enables signal generation and detection of the nucleotide-based probe. In other embodiments, the nucleotide-based probe comprises a fluorescent label, such as in fluorescent in situ hybridization (FISH).

In an embodiment, a biological sample such as a cell, tissue, organ, organism, or organ substructure provides an endogenous signal, for example, an endogenous fluorescent molecule. Examples of endogenous fluorescent molecules include fluorescent protein reporters (e.g., green fluorescent protein (GFP) or red fluorescent protein (RFP)). In other embodiments, the sample is derived from a transgenic model and the fluorescent molecule is expressed by a constitutive or inducible promoter. In other aspects, the organism is infected with a recombinant virus or transfected with a plasmid encoding a fluorescent protein. Exemplary fluorescent protein reporters include: green fluorescent protein (GFP), EGFP (enhanced GFP), BFP (Blue fluorescent protein), CFP (indigo), red fluorescent protein (RFP), wtGFP (White GFP), YFP (yellowfluorescent protein), dsRed, mCherry, mVenus, mCitrine, tdTomato, luciferase, mTurquoise2, etc.

As mentioned above, the liquid specimen can be labeled with a molecular probe such as an antibody. In an embodiment, the antibody is a primary antibody, which contains a marker that directly or indirectly generates a signal, such as a biotin label, a fluorescent label (fluorophore), an enzyme label (e.g., HRP or DAB), a coenzyme label, a chemiluminescent label, or a radioisotope label. In other aspects, the primary antibody is used as a single stain (e.g., with or without additional reagents, such as labeled streptavidin or an enzyme/coenzyme substrate to provide a signal). In an embodiment, the primary antibody does not contain a label and is detected by a secondary antibody coupled to a label instead.

Examples of fluorophores that can be attached to primary or secondary antibodies include: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, or Alexa Fluor 750. Other exemplary fluorophores include BODIPY FL, coumarin, Cy3, Cy5, Fluorescein (FITC), Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, Tetramethylrhodamine (TRITC), Texas Red, APC-eFluor 780, eFluor 450, eFluor 506, eFluor 660, PE-eFluor 610, PerCP-eFluor 710, Super Bright 436, Super Bright 645, Super Bright 702, Super Bright 780, Super Bright 600, Qdot 525, Qdot 565, Qdot 605, Qdot 655, Qdot 705, Qdot 800. R-phycoerythrin (R-PE) and allophycocyanin (APC).

The solidified sample can be imaged by any microscopy-based application, and therefore in certain embodiments, the disclosed subject matter is not limited to the specific imaging technique used. Examples of microscopy-based applications include, but are not limited to, immunofluorescence, confocal microscopy, two-photon microscopy, super-resolution microscopy, light sheet microscopy, and x-ray microscopy, etc. The term "detectable agent" or "detectable marker" refers to a molecule that can be used to directly or indirectly detect a biomarker. A variety of detectable agents are known in the art and can be easily identified and used by those skilled in the art. Suitable detectable agents include, but are not limited to, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (fluorescein isothiocyanate; FITC), OregonGreen ^TM , rhodamine, Texas Red, tetrarhodamine isothiocynate (tetrarhodamine isothiocynate; TRITC), Cy3, Cy5, Alexa Fluor, ... 647、Alexa 555. Alexa 488), fluorescent protein markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, metals and their analogs.

The term "immunofluorescent marker" refers to a detectable agent that targets the delivery of a fluorescent dye to an antibody or a functional fragment thereof to a specific molecule in or on a cell. Immunofluorescent markers can be used in methods for immunostaining of a desired sample using a fluorescent microscope. Immunofluorescent markers can also be used in immunocytochemistry (ICC) or immunohistochemistry (IHC) methods described herein. For example, in the context of the present disclosure, immunofluorescent markers can be used to detect rare circulating cells (e.g., CTCs or CTC mimetics) as described herein.

The term "antibody" refers to any immunoglobulin or derivative thereof, whether natural or completely or partially synthetically produced. All antibody derivatives that retain specific binding capabilities can also be used in the disclosed methods. The antibodies of the present disclosure may specifically bind to a biomarker. For example, an antibody may specifically bind to a single biomarker (e.g., chondroitin sulfate proteoglycan 4 (CSPG4)). In addition, the antibody may be pan-specific. For example, the pan-specific antibodies of the present disclosure may specifically bind to one or more members of a biomarker family (e.g., one or more members of a chondroitin sulfate proteoglycan family, including chondroitin sulfate proteoglycans 1, 2, 3, 4, 5, 6, 7, and 8). The antibody may have a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain and may be derived from a natural source, or may be partially or completely synthetically produced. The antibody may be a single or multiple clone antibody. In some embodiments, the antibody is a single-chain antibody. In some embodiments, the antibody comprises a single-chain antibody fragment. In certain embodiments, the antibody may be an antibody fragment, including but not limited to Fab, Fab, F(ab)2, scFv, Fv, dsFv bifunctional antibodies and Fd fragments. Due to its smaller size, antibody fragments can provide advantages over complete antibodies in certain applications. Alternatively or in addition, the antibody may include multiple chains, such as those linked together by disulfide bonds, and any functional fragments obtained from these molecules, wherein these fragments maintain the specific binding properties of the parent antibody molecule. Those skilled in the art recognize that antibodies may include any of the various forms of, for example, humanization, partial humanization, chimeric, chimeric humanization, etc. to provide. Antibodies can be prepared using any suitable method known in the art. For example, antibodies can be produced enzymatically or chemically by the segmentation of complete antibodies or they can be recombinantly produced by genes encoding partial antibody sequences.

The term "biomarker" refers to a biomolecule or a fragment of a biomolecule, the change and/or detection of which can be associated with a specific physical condition or state of a rare circulating cell (e.g., CTC, CTC mimetic, or CEC) or other target component. The terms "marker" and "biomarker" can be used interchangeably throughout this disclosure. These biomarkers include, but are not limited to, biomolecules comprising nucleotides, nucleic acids, nucleosides, amino acids, sugars, fatty acids, steroids, metabolites, peptides, polypeptides, proteins, carbohydrates, lipids, hormones, antibodies, regions of interest that serve as surrogates for biomacromolecules and combinations of the above (e.g., glycoproteins, ribonucleoproteins, lipoproteins). The term also encompasses parts or fragments of biomolecules, for example, peptide fragments of proteins or polypeptides. In the context of the present disclosure, for example, exemplary biomarkers of CTCs such as circulating melanoma cells (CMCs) include chondroitin sulfate proteoglycan 4 (CSPG4), premelanosome protein (Pmel17), and S100 calcium-binding protein A1 (S100A1).

method

In an embodiment, the present disclosure provides for analyzing materials in liquid samples that may contain biological or non-biological components. Thus, the methods can be used to analyze liquid biopsies, as well as other samples having biological components. Biological components can include cellular material, as well as extracellular material such as vesicles and cell-free DNA, secreted proteins, and other cell-free biomolecules.

In an embodiment, the method includes a step of solidifying the liquid sample, thereby capturing (fixing) the dispersed material in a form that can be subsequently imaged by microscopic applications (or other imaging methods). The resulting scanned image allows detection of components that are fixed and spatially separated in the solid sample in 3 dimensions, allowing for higher resolution and sensitivity. Selectively labeled components, such as those labeled with fluorescent tracer antibodies, can then be sensitively and rapidly identified, such as by fluorescence microscopy, to detect the labeled target of interest.

Thus, the described compositions and methods effectively provide a 3D scan or image of a liquid biopsy. Rare biomarkers such as, but not limited to, rare circulating cells such as circulating tumor cells (and even complete circulating tumor cell clusters) or cell-free nucleic acids can be present as discrete signals in the resulting scanned sample. This is in contrast to other methods such as microfluidics-based applications that detect these biomarkers indirectly, or conventional cytology methods based on examining smaller samples of overlapping cells on standard slides.

In an embodiment of the present disclosure, the present method provides the additional advantage of not imposing any morphological or cell size cutoffs on the analyzed components. For example, existing CTC detection techniques based on specific enrichment methods may inadvertently miss rare biomarkers. In addition, the potential multiple processing steps of existing CTC detection methods can induce changes in cell biomarkers, thereby further reducing the sensitivity and accuracy of the detection method. In contrast, the embodiments of the methods described in the present disclosure do not require any specific selection criteria for the analyzed biological materials. Alternatively, it allows the capture of a range of complex cells and extracellular materials, regardless of the shape and morphology captured, preserved and spatially separated in a three-dimensional solidified sample.

In an exemplary embodiment, the currently disclosed method allows for individual analysis and identification of individual cells in a solidified sample, which is attributed to their spatial dispersion (separation) in the matrix of the solidified sample. Further, the currently disclosed method allows for analysis of specific details of the identified cells themselves (e.g., morphological details of the cells) and the spatial relationship of the identified cells to other cells within the sample. For example, in an exemplary embodiment, the currently disclosed method allows for analysis of cell clusters, where one cell in the cluster is a specific cell of interest. The morphology of the specific cell of interest can also be compared in its unbound state to the morphology of the cell in the cluster in order to determine, for example, the clinical progression of a disease state in a subject. In other exemplary embodiments, cell fragments (e.g., cell fragments in white blood cells), or biomolecules secreted by cells can be analyzed according to the currently disclosed method.

Matrix-assisted methods

The present disclosure provides, in part, matrix-assisted methods for processing and analyzing liquid samples, such as biopsy samples, that may contain rare biomarkers such as circulating tumor cells or cell-free tumor DNA.

In an exemplary embodiment, steps in a method are used to generate an image of dispersed components of a sample in a 3D spatial configuration. In an embodiment, the method includes solidifying a liquid sample containing dispersed biological material in a form that allows rapid imaging, such as by light sheet microscopy or other microscopy techniques. In an embodiment, the method includes: (a) adding a solidifying agent to a liquid specimen containing biological material; (b) generating a solidified sample containing biological material; and (c) imaging the solidified sample to identify one or more components in the dispersed biological material.

In an embodiment, the liquid sample contains biological material and undergoes a number of processing steps, which may include but are not limited to the steps shown in the stages depicted in FIG. 1 as described in detail below.

Phase 1 - Cell preparation

In embodiments, the method comprises preparing or procuring a liquid sample having biological material that can be dispersed and subsequently captured in solid form, thereby allowing high resolution detection.

Although depicted as a blood sample in Figure 1, the liquid sample can be derived from a variety of sources, such as bone marrow, cerebrospinal fluid, urine, saliva, sputum, tears, semen or other fluid sources. It should also be noted that although the exemplary disclosure relates to CTC cells, it can also be applied to other biomarkers that may be present in liquid biopsy samples.

In an embodiment, the material in the liquid sample is obtained from a processed blood sample, such as a sample obtained in a liquid biopsy. The processing may involve one or more steps. For example, the processing may include red blood cell removal and separation (collection) of PBMC cells. More specifically, the blood sample may be subjected to centrifugation to remove red blood cells (erythrocytes) and platelets. The processing may also involve fractionating the blood sample into different components by well-known separation techniques such as density gradient centrifugation. For example, these separation techniques may include red blood cell (RBC) removal and peripheral blood mononuclear (PBMC) collection or separation.

As shown in Figure 1, in an exemplary embodiment, a blood sample can be provided in a test tube or container comprising an anticoagulant, such as in an evacuated blood collection tube with EDTA or heparin. The blood sample can be processed with a cell separation medium such as Lymphoprep ^TM or Ficoll-Paque ^TM , and centrifuged, and the supernatant is removed. Alternatively, a PBMC separation tube (e.g., a SepMate ^TM tube available from Stemcell ^TM Technologies) can be used. If necessary, a blood sample can also be subjected to RBC lysis according to known techniques, such as based on a known scheme to a person skilled in the art, using an available or synthetic ammonium chloride solution (e.g., an ammonium chloride solution available from Stemcell ^TM Technologies), which can be performed before or after centrifugation. In other embodiments, RBC lysis is not used because trace RBC does not affect the imaging and analysis of the sample.

In an exemplary embodiment, a pellet (e.g., a pellet containing isolated PBMCs) is obtained, for example, by centrifugation and further processed as described below. In certain exemplary embodiments, the volume of the pellet may be at least 5 μl, or at least 10 μl, or at least 15 μl. In other exemplary embodiments, a lower volume of pellet is processed. In any case, the entire pellet is encapsulated in the gel and has a size several orders of magnitude larger than the typical working volume encountered in microfluidic processes known in the art.

In an embodiment, the dispersed material in the liquid sample may be derived from other tissue sources. For example, it may be derived from a biopsy obtained from a body fluid other than blood, such as bone marrow, cerebrospinal fluid, urine, saliva, sputum, tears, semen or other fluid sources. Alternatively, the dispersed material in the liquid sample may reflect a liquid dispersion of material from a solid biopsy, such as a tissue sample obtained from a tumor or other solid samples derived from a structure or organ of interest. In an embodiment, the dispersed material may be derived from a non-tissue source. In an embodiment, the biological material in the specimen may be enriched by concentrating a large amount of samples, for example, by collecting and agglomerating cells from a larger volume of blood or other sample sources such as 2 ml, 4 ml, 8 ml or more than 8 ml.

In embodiments, biological material in a specimen can be enriched by concentrating it from a relatively large volume of sample, for example, by centrifuging and collecting a large volume of blood (eg, 0.5 cc or 1 cc or more) or other tissue-derived cell pellet.

Before being dispersed in the liquid sample, the material may undergo additional processing steps. In an embodiment, before being dispersed in the liquid sample, the biological material may undergo a fixation step, as further discussed herein. Alternatively, fixation may occur after the biological material is collected in the liquid sample. Fixation may also occur after the cells are labeled. Specific fixatives applicable according to the present disclosure are not limited and include those known to those of ordinary skill in the art. In an exemplary embodiment, the fixative is a solution comprising one or more of the following: glutaraldehyde, formaldehyde, epoxy resin, or a cross-linked product of one or more of the foregoing.

In the case of rare targets such as CTCs, a large number of liquid biopsy samples can be collected serially and collected into a single tube. For example, 2 ml or more of blood can be collected and processed and cell pellets pooled and resuspended in a liquid sample buffer such as PBS.

In an embodiment, the biological material includes circulating tumor cells (CTCs), circulating tumor cell fragments, circulating tumor cell mimics, circulating epithelial cells (CECs), and similar rare circulating cells.

In an embodiment, the method further comprises labeling one or more targets of interest and adding a solidifying agent that allows the dispersed labeling material to be captured in a solid form comprising a three-dimensional cross-linked network. In other words, the solidifying agent provides a solid matrix that supports 3D observation of sample components.

Labeling can involve any method known in the art for identifying biomolecules, including immunological and molecular means. For example, a protein target of interest, whether on the cell surface, intracellular or extracellular, can be labeled with an antibody (or related immunological reagent) that is detected directly, such as with a fluorescently coupled antibody, or indirectly, such as with immunohistochemistry or a primary antibody and a coupled secondary antibody. Labeling (e.g., chemical and immunological labeling) can occur in one step, or in alternative embodiments, labeling is a multi-step process.

For example, multiple antibodies from different host species can be introduced at the same time or at about the same time, or at different times. In an exemplary embodiment, the primary antibody can be coupled (or pre-labeled) with a label such as a fluorescent dye or an enzyme, for example, a fluorophore, or the corresponding secondary antibody can be introduced as a mixture in one step. In certain embodiments, one-step labeling is preferred over multi-step labeling because the labeling step usually requires subsequent washing, and therefore additional centrifugation and supernatant removal steps can result in cell loss or cell damage and potentially reduce signal sensitivity.

Likewise, biomolecular targets such as DNA or RNA can be labeled with nucleic acid probes that are detected directly or indirectly, such as for fluorescence in situ hybridization (FISH). If desired, the signal can be further amplified by available techniques such as biotin-streptavidin binding-based systems and polymerase chain reaction. In embodiments, probes can identify other disease biomarkers, such as cell-free tumors or fetal-derived DNA, or can observe genetic and structural changes in the cell nucleus, such as DNA and chromosomal abnormalities, amplifications, deletions, and translocations.

Samples may also be labeled by other methods known in the art, such as using various dyes for cellular components, including fluorescent dyes such as DAPI and PI (binding to nuclear components) and or DiD or DiL (binding to membrane components).

Labeling may also involve other steps as appropriate and known in the art, such as cell permeabilization or fixation, to allow efficient and specific binding of immune or molecular reagents to the target (biomolecule) of interest. In certain embodiments, labeling is applied prior to sample gelation and clarification. In certain embodiments, labeling is preferably applied prior to obtaining a pellet, or otherwise separating components from a sample, such as while dispersing the biological material in a liquid (e.g., blood) sample.

Labeling can also be performed after cells are fixed in a gel state. For example, cells collected from liquid samples (e.g., PBMC obtained from blood) can be introduced into PFA solution and gel can be formed directly before labeling. Then, gel samples can be labeled with probes passively or by other active immune labeling approaches, such as those methods involving and electrophoresis or approaches based on pressure. Therefore, in an embodiment, after solidification, the processed gel sample can be immunolabeled (i.e., labeled for the first time or further labeled). Although this labeling method after gelation can consume longer processing time, it can also enhance the retention of target cell number. In the embodiment of labeling after solidification, the fixation procedure can be performed for the solidified sample after gel formation and after labeling.

In other embodiments, when it is necessary or desirable to enhance sample transparency for imaging, delipidation may be performed on a solidified (e.g., gelled) cell sample. In embodiments, the solidified sample may be further cross-linked with a hydrogel precursor or epoxy resin and delipidated following the CLARITY method. See, e.g., “Advances in CLARITY-based tissue clearing and imaging,” Exp Ther. Med. 2008; 16(3): 1567-1576. However, it should be noted that, due to the smaller cell packing in the sample dispersed in the solidified sample, a delipidation step is generally not necessary according to most embodiments. As discussed below, in some embodiments, even a refractive index matching step is not necessary.

Stage 2 - Sample solidification and clarification

In certain embodiments, solidifying the sample includes introducing a solidifying agent (e.g., a gelling agent) that allows the biomaterial to be captured in a solid form such as a gel that is compatible with subsequent imaging and detection of the labeled biomolecules of interest. Solidifying agents may include, but are not limited to, agents known in the art, such as agarose (including low melting point agarose) solutions, polyacrylamide precursors, natural gums, starches, pectins, agar, and gelatin. In embodiments, these agents are based on polysaccharides or proteins. See, for example, Kar et al. 2019, Current developments in excipient science: in Fundamentals of Drug Delivery, 29-83. Alternatively, in certain embodiments, the solidifying agent may consist of, or consist essentially of, a refractive index matching solution itself, which is modified, if necessary, to provide an appropriate viscosity, thereby providing a rigid solid gel.

In an embodiment, the curing agent is or comprises a viscosity modifier, such as made from a natural or synthetic polymer (eg, Triplex, Pemulen ^™ , Carbopol ^™ , Velvesil ^™ plus or other polyacrylic acid derivatives).

In embodiments, the solidifying agent comprises a mixture of polysaccharides (eg, agar/agarose, gellan gum) that produces a solid gel having a solid three-dimensional matrix or network.

In an embodiment, the curing agent comprises a mixture of chemical monomers and a cross-linking agent in order to produce a synthetic chemical gel, ie, having a chemically cross-linked polymer network.

In an exemplary embodiment, solidification (eg, gelling) may include dispersing or resuspending the biological material in a gelling material in liquid form, such as a low melting point agarose solution added at a temperature above its gelling point, eg, 37°C.

During the pre-gelling stage, according to this exemplary embodiment, the sample is maintained in a fluid or molten state and the material can be maintained in a dispersed state before being transferred to the imaging holder in stage 3. For example, a curing agent can be introduced into a labeled specimen (e.g., a labeled PBMC mass as indicated in FIG. 1 ) and the mixture mixed, such as using a pipette or via a vortex mixer. Thus, the gelling agent is a reversible gelling agent in certain embodiments, because the gelling agent (and the sample) can be heated to achieve a fluid or molten state if desired.

In certain embodiments, pre-gelling comprises dispersing or resuspending the biological material in a solution comprising low melting point agarose. In embodiments, the final concentration of the low melting point agarose is greater than 0.3% or greater than 0.5%. In embodiments, the final concentration of the low melting point agarose is less than 1.0% or less than 1.6% or less than 2% or less than 10%. In embodiments, the final concentration of the low melting point agarose is between 0.1% and 10% or between 0.3% and 1.6% or between 0.5% and 1.5% (e.g., about 1%).

Thus, in certain embodiments, pre-gelling comprises dispersing or resuspending the biomaterial in a solution comprising agarose (i.e., conventional melting point agarose). In embodiments, the final concentration of agarose is greater than 0.3% or greater than 0.5%. In embodiments, the final concentration of agarose is less than 1.0% or less than 1.6% or less than 2% or less than 10%. In embodiments, the final concentration of agarose is between 0.1% and 10% or between 0.3% and 1.6% or between 0.5% and 1.5% (e.g., about 1%).

In certain exemplary embodiments, pre-gelling includes dispersing or resuspending the biological material in a solution comprising a mixture of low melting point agarose and regular agarose. In certain exemplary embodiments, a gelling agent solution (such as, but not limited to, agarose or a low melting point agarose solution) is combined with a sufficient amount of an RI matching material discussed below to provide the desired refractive index of the final, cured gel. In certain exemplary embodiments, the pre-gelling material is directly dissolved in the RI matching solution to constitute the gelling solution.

In an embodiment, dispersing or resuspending the biological sample in the mixture and forming a gel generally allows the components in the sample to be stabilized with a spatial distribution having sufficient properties such as transparency, thereby allowing subsequent imaging. In certain embodiments, a refractive index matching material having a certain refractive index (e.g., having a refractive index between 1.3-1.6 or 1.33-1.5, or an RI approximately equivalent to water) may be introduced into the gelled or pre-gelled mixture, while in other embodiments, RI matching materials having other refractive indices may be applicable. As is known in the art, refractive index matching materials (also referred to as RI matching or RIM) are able to penetrate tissue/cells to achieve tissue/cell transparency and include but are not limited to CUBIC-R+, RapidClear, RIMS or ScaleView. See Neuropathol Appl Neurobiol, 2016 Oct; 42(6): 573-87.

In some embodiments, index matching materials are not necessary. For example, for smaller samples, the laser light can still penetrate the solidified sample and excite labeled cells or other target components. That is, when the laser does not need to travel too deep to cause light refraction, the refractive index difference of the solidified sample is unimportant. Using, for example, a two-photon laser with greater power, the laser can penetrate even deeper without bending. This system is only limited by possible photodamage of the fluorophores, and this problem does not necessarily require the addition of index matching materials, such as index matching solutions, to avoid such damage.

In an embodiment, pre-gelling comprises dispersing or resuspending the biomaterial in a gelling material comprising one or more hydrogel precursors such as polymerizable materials, monomers or oligomers, including monomers selected from the group consisting of water-soluble groups containing polymerizable ethylenically unsaturated groups. The monomer or oligomer may comprise one or more substituted or unsubstituted methacrylates, acrylates, acrylamides, methacrylamides, vinyl alcohols, vinylamines, allylamines, allyl alcohols. The precursor may also include polymerization initiators, crosslinkers and other components as known in the art, such as described in WO2019023214 and WO/2020/013833.

In embodiments, the method includes transferring the pre-gelled mixture in a liquid or molten state to a sample well in a holder. In embodiments, the well in the holder allows for the formation of a solid sample suitable for imaging. For example, the solid sample may be in a block or other form customized for imaging by fluorescence microscopy. Alternatively, in certain embodiments, the pre-gelling step may be performed in a combined test tube-sample holder, thereby allowing solidification (e.g., gelling) to occur in the same test tube, thereby eliminating the need to subsequently transfer the liquid solution to a separate sample holder.

In the case of an agarose mixture, solidification or gelling can be achieved by transferring the pre-gelling mixture to a temperature below the gelling point, for example 4° C. In other embodiments, such as for hydrogel formation, the transferred sample is subjected to further processing, such as the addition of a polymerizing agent.

After curing, the sample can be processed through additional steps prior to imaging. For example, the sample can be equilibrated in a refractive index material so that it is properly matched (e.g., clarified) for imaging in step 3 (RI matching). Alternatively, in certain embodiments, the RI matching material is added simultaneously with the curing agent. In further embodiments, the RI matching material is added with the curing agent, and then a second round of RI matching material is added to the cured sample in order to provide the final, desired transparency of the sample.

RI matching materials are known in the art and can be used in an appropriate amount to provide the desired transparency of the cured gel sample. In certain embodiments, the RI matching material has an RI of about 1.39 to about 1.65 or about 1.49 to about 1.55 (e.g., about 1.52). For example, and without limitation, the RI matching material can be the RI matching material obtained from Table 3 of "Bringing CLARITY to the human brain: Visualization of Lewy pathology in three dimensions" by Liu et al., available at <https://www.researchgate.net/figure/Comparison-of-different-refractive-index-matching-solutions-Abbreviations-BABB_tbl3_283493188>.

Step 3 - Sample Mounting and Imaging

In an exemplary embodiment, this step entails mounting the sample on an imaging holder and imaging the solid sample by a suitable imaging means such as fluorescence microscopy.

In an embodiment, the method may include, prior to imaging, mounting the sample on an imaging holder such as a 3D printed sample holder as shown in FIG. 1 and imaging the sample, for example, in a water chamber or RI matching solution. The sample holder may be customized according to a specific sample preparation. In an embodiment, it may include a base and two side walls. In embodiments such as using a sample prepared in a viscous medium, the holder may have four walls that surround and maintain the spatial configuration of dispersed components in the sample. In an embodiment, imaging may be performed in a sample cuvette using a standard epifluorescence microscope. 3D gel samples may be mounted on a sample holder with the aid of a mounting gel (e.g., agarose, poly-L-lysine, super glue). See, for example, Asano et al., Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissue, Current Protocols in cell biology (2018), especially pages 34-36 - "Sample mounting".

Fluorescence microscopy methods include, but are not limited to, conventional confocal microscopy, resonant scanning confocal microscopy, spinning disk microscopy, and light sheet microscopy as described in FIG. 1 . In a particular embodiment, the gel sample is rapidly imaged by light sheet microscopy, such as light sheet fluorescence microscopy (LSFM) using a detection lens and an illumination lens as shown in FIG. 1 . In certain embodiments, a Gaussian beam may be used, or alternatively a dedicated beam profile such as a non-diffracting Behssel beam may be applicable, as described in FIG. 1 . This imaging allows the material in the 3D sample block to be scanned and the individual presence of labeled biomarkers to be assessed. As is known in the art, a processor may communicate with a microscope to receive output therefrom and combine (i.e., “stitch”) adjacent target areas imaged sequentially. See, for example, U.S. Pat. Nos. 10,746,981, 10,876,870, U.S. Published Patent Application No. 2016/0041099, and International Published Patent Application No. WO 2017/031249, which techniques and apparatuses may be applicable in light of the presently disclosed subject matter.

Other detection light sources and corresponding microscopy applications may also be used. For example, light microscopy can be used to examine visible dye-stained cells, such as those stained with H&E (hematoxylin and eosin) or by immunohistochemistry (IHC). In addition, X-ray microscopy can be used to detect liquid components labeled with appropriate metal labels.

Other imaging techniques may be applicable, including those included in commercially available smartphones (e.g., based on or images captured by a high-resolution camera in a smartphone or related light detection system.

Step 4 - Observation and Analysis

The present method can also be used to assess, diagnose or monitor disease. For example, a liquid biopsy (e.g., whole blood processed as described above) can be microscopically analyzed to detect the presence of rare cells therein. For example, the currently disclosed method can detect, for example, colorectal adenocarcinoma cells, leukemia cells, the type of cancer, the extent of cancer development, whether the cancer responds to therapeutic intervention, etc.

In embodiments, the disclosed methods can be used to detect cellular biomarkers associated with other diseases and conditions such as inflammatory, metabolic, gastrointestinal, endocrine, immune, musculoskeletal, cardiovascular, cardiopulmonary, genitourinary, hepatology, respiratory, viral, and neurological diseases and conditions according to embodiments of the present disclosure.

In addition to detecting the presence of biomarkers such as certain cellular or extracellular components (e.g., circulating tumor-derived factors, secreted proteins, released vesicles and exosomes and cell-free nucleic acids and other biomarkers), the presently disclosed methods can be used to determine cell-cell interactions, cell volume, nucleus to cytoplasm ratio, and other phenomena. In an embodiment, imaging can be used to analyze cell morphology and structure, including analyzing changes in cell morphology and structure as compared to its native or healthy state. For example, imaging methods can be used to analyze the appearance of a cell, such as its size, shape, or other external characteristics. In addition, imaging methods can be used to analyze the form and structure of internal cellular components such as the nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria, or other organelles.

As another example, a biopsy may be prepared by liquid dispersion of a sample of diseased tissue, such as from the kidney, heart, stomach, liver, pancreas, intestine, brain, etc., in order to determine the pathology of the tissue, the extent of disease progression, the likelihood of success of the tissue, etc. The methods herein may be used herein to assess morphological changes in a cell population that may be indicative of a disease state, such as through the use of dyes that target membrane or cellular components.

The methods can also be used in other applications. In one application, liquid biological samples can be used to screen candidate therapeutic agents for their effects on tissues or diseases. For example, liquid samples obtained from subjects such as mice, rats, dogs, primates, humans, etc., that have been contacted with a candidate agent can be prepared by the methods disclosed herein and microscopically analyzed for one or more cell or tissue parameters, i.e., properties or characteristics of measurable subcellular components.

In another application, the method can also be used to observe the distribution of genetically encoded markers in a liquid sample prepared by dispersing material from tissue. These markers may include, for example, chromosomal abnormalities (inversions, duplications, translocations), loss of genetic heterozygosity, the presence of genetic markers indicating a tendency toward a disease state or a healthy state. This detection may be useful, for example, for diagnosing and monitoring diseases, such as in personalized medicine, studying parent-child relationships, or other applications.

CTC Detection

In certain exemplary embodiments, the methods disclosed herein are used to detect CTCs and address their clinical significance by immune or molecular means for diagnostic purposes. CTCs are rare cells that circulate in blood or other fluids along with millions of other circulating cells. The present method captures these rare cells in a solidified 3D form of a composite liquid sample or liquid biopsy. When the sample is subsequently imaged, for example, by light sheet fluorescence microscopy, any CTC can be detected as a discrete signal in the sample block. Advantageously, the method allows this CTC detection without reducing its biological heterogeneity. The method may also include less intervention than the intervention required by microfluidics or traditional cytology that can destroy morphology or hinder sensitivity.

Example

The present disclosure will be further illustrated by the following non-limiting examples. It should be understood that these examples are exemplary only and should not be construed as limiting the scope of one or more embodiments as defined by the appended claims.

Example 1 - Matrix-Assisted Detection of CTCs in Patient Blood Samples

background

CTCs are rare cells that circulate in the blood or other fluids with millions of other circulating cells that belong to, for example, the hematopoietic compartment and do not adhere spontaneously. These poor adhesion properties hinder existing methods for detecting CTCs in the art, such as using solid supports to separate and fix CTCs. The currently disclosed method allows these rare cells (if present) to be detected in a complex 3D presentation of a liquid sample without limiting its biological heterogeneity and reducing interventions that can destroy the morphology. In addition, methods based on modified supports and matrices coated with anti-adhesion molecules (or other binding proteins) can cause biological reactions that change the morphology of CTCs, resulting in inaccurate analysis. Immunofixation relies on the strong affinity between the coated antibodies and cell membrane proteins. The lower expression of target surface proteins or lower antibody affinity can lead to a lower capture rate of target cells. Similarly, cells are centrifuged until they are on a support such as a microscope slide, followed by fixation and subsequent labeling, which can destroy cells or interfere with their morphology, thereby hindering diagnostic assessment. In contrast, microscope slide methods require cells to be plated in a monolayer to allow imaging, greatly reducing the throughput, or number of cells imaged and analyzed.

Detection methods that rely on microfluidics, nanostructures, and channels may suffer from similar limitations. See, e.g., WO2012016136; WO2013049636. These methods may induce flow stresses on cellular components in a liquid sample, thereby impairing their morphology and other properties. More generally, these methods typically select homogenous cell populations based on the size or expression of surface membrane proteins, thereby limiting the biological heterogeneity that would otherwise be suitable for diagnostic analysis of clinical or biological samples.

Therefore, there is a need to identify biopsy methods that can minimize lengthy procedures, interacting external stimuli, and prolonged processing. These improvements can help improve both the natural state and heterogeneity of cells and other components in liquid biopsies, thereby providing more accurate tools for diagnostic applications, such as early diagnosis of possible metastatic processes.

CTCs in peripheral blood can be considered as an extension of the tumor. Tumors are usually heterogeneous, meaning that through mutations, the tumor can develop multiple different cell types therein. Each cell type can have its own characteristics, which can vary from mild to invasive. CTCs are rare cancer cells released from tumors into the bloodstream and are believed to play a key role in cancer metastasis. See, for example, Harouaka et al., 2014, Pharmacol. Ther. 141, 209-221.

In U.S. Published Patent Application No. 2019/0113423, which is incorporated herein by reference, it is described how molecular characteristics such as membrane proteins or DNA/RNA information in tumors can indicate treatment outcomes. This reference involves fixing or embedding a sample such as a solid tissue or solid cell mass in a solution of hydrogel monomers, crosslinking the monomers, clarifying the crosslinked sample, staining the clarified sample with one or more detectable markers, and imaging the stained sample using COLM or a similar imaging process.

In contrast, in certain embodiments, the present disclosure provides methods for analyzing individual cells dispersed and captured in a three-dimensional solidified sample (e.g., from a liquid biological sample). No enrichment or selection of cells is required, and all cells can be labeled and visually screened by imaging the sample.

Again, in certain embodiments, delipidation (e.g., SDS-based delipidation) or other sample clarification methods are generally not required, and certain embodiments of the currently disclosed methods are defined by not including such delipidation or other sample clarification steps. See, for example, Jensen and Berg, 2017, J. Chem. Neuroanat. 86, 19-34. Therefore, as compared with, for example, U.S. Published Patent Application No. 2019/0113423 and methods involving CLARITY or other clarification schemes, more cell information is retained. According to the present disclosure, individual cells or cell clusters can be observed and analyzed separately from each other in a three-dimensional array, thereby providing comprehensive cell information such as cell size, morphology, biomarker distribution, nucleus-cytoplasm ratio and more.

According to the present disclosure, a variety of tumor types can be identified, such as lung, liver and colon cancer, and other cancers that can be detected as CTCs in liquid biopsy. For example, after using EpCAM markers, detecting CTCs from liquid biopsy (e.g., blood samples), the information obtained can be provided by providing information such as the number (or type) of cancer cells in, for example, 1cc, 2cc, 3cc, 4cc or more blood to indicate whether the patient has cancer (or other characteristics about risk, prediction and treatment response). EpCAM is an epithelial marker for invasive cancer cells that indicate epithelial mesenchymal transition (epithelial mesenchymal transition; EMT), which is the main cause of cancer invasion. Cancer invasion usually begins with EMT from a smaller group of tumor cells, which stimulates blood vessel growth, providing cells as CTCs to invade the bloodstream.

A variety of different cancer cells from different tissues may be present in the bloodstream, and different biomarkers may be used to identify it. For example, according to the present disclosure, a variety of cancer markers may be used together to identify CTC, such as but not limited to HER2 (breast), CDX2 (colon), CK20 (colorectal, transitional cell carcinoma and Merkel cell carcinoma), CK19 (breast), PD/PDL-1 (multiple cancers, including NSCLC, melanoma and renal cells) and EGFR (lung). The identification of different CTC cell subtypes in a blood sample may provide information about the source of cancer. In turn, this information may guide more detailed follow-up studies, such as high-resolution analysis by MRI of the location of the lesion and tissue biopsy for pathological examination.

Cell heterogeneity in blood can also be applied to healthy cells such as white blood cells. According to the present disclosure, by applying parameters such as cell morphology (e.g., the nucleus of CTC can be larger than that of white blood cells) and molecular markers (e.g., EpCAM-CTC+/WBC-&CD45 CTC-/WBC+), the differences between CTCs and white blood cells can be distinguished.

In addition, because CTC escapes from the primary tumor into the bloodstream, it is usually highly invasive. These invasive cells can make cancer difficult to treat and cure, due to its ability to metastasize, and its greater mutation possibility, thereby increasing the chance of resistance to chemotherapy and other therapeutic interventions. This emphasizes the importance and value of identifying the molecular characteristics of these invasive cells in order to determine the correct treatment plan. For example, if the CTC present in the blood shows a high level of PDL-1, immunotherapy may be a more effective method. The PDL-1 level can be determined, for example, using a PDL-1 antibody probe that quantifies the number of PDL-1+CTCs relative to all CTCs in the sample. Because tumor development is almost always heterogeneous, it means that one tumor site does not represent all tumor populations, and because CTC can be derived from all tumor sites, this measurement result can help predict the effectiveness of, for example, PD-1/PDL-1 inhibitory therapeutic agents. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627043/) Since CTCs are rare in the blood, these predictive measurements are most accurate if CTCs are efficiently captured and analyzed as provided by the currently disclosed methods.

Another example according to the present disclosure involves determining the number and cell type of CTCs and administering therapy based on this determination (e.g., determining whether to continue the current course of treatment or establish a different or additional course of treatment). For cancer patients under chemotherapy or cell therapy, the number of CTCs can indicate the effectiveness of the treatment. For stage III colon cancer patients, the number of CTCs in 1cc of blood can vary from tens of thousands for advanced cases to hundreds for less advanced cases. In some advanced stage III cases, after 3 months of chemotherapy or other therapeutic interventions, the number of CTCs may drop to 5,000 or even approach zero after 12 months. However, in some cases, the number of CTCs may only decrease to about 2,000 after 6 months of chemotherapy and rise back to tens of thousands after 6 months. This indicates the resistance of cancer cells to therapeutic interventions. Chemotherapy, for example, can kill all drug-sensitive CTCs, but other subtypes that are resistant are not affected.

According to the present disclosure, by performing blood tests for CTC detection and identification on a regular basis (e.g., weekly or monthly), doctors and medical professionals can quickly confirm the resistance of a tumor to chemotherapy and modify the treatment plan accordingly. Therefore, the research herein addresses the above limitations by describing formulations and methods for observing biopsy samples in three dimensions by capturing dispersed components in the sample in a solidified state. This approach has many advantages, such as increasing the sensitivity of the analysis by separating individual components, increasing the speed of the analysis by allowing the use of rapid imaging techniques such as light sheet fluorescence microscopy, and enhancing the specificity of the analysis by reducing the number of processing steps that may otherwise destroy the morphology and integrity of sample components such as cell markers.

Materials and methods

Sample collection and fixation:

Blood samples (e.g., 2cc, 8cc, or 10cc) are collected from the subject and red blood cells are removed by density gradient centrifugation using standard methods, such as, for example, centrifugation at 1000 rpm for five minutes at room temperature. See, e.g., Farahina et al. 2020, Circulating Tumor Cell Separation of Blood Cells and Sorting in novel Microfluidic approaches: a review. 10.20944/preprints202010.0622.v1; and Lowes et al. 2014, Circulating tumor cells as a real-time liquid biopsy: isolation and detection systems, molecular characterization, and clinical applications; in Pathobiology of human disease: a dynamic encyclopedia of disease mechanisms (McManus and Mitchell, eds.).

The supernatant is removed, transferred to a separate tube, and re-centrifuged to collect the remaining cells and other biological components including any CTCs. The pellet is optionally resuspended in a fixative solution such as a 4% paraformaldehyde solution, shaken for a few minutes, centrifuged, washed in phosphate buffered saline (PBS), and centrifuged again. The pellet is resuspended in blocking buffer and shaken for two minutes, and centrifuged again.

Biomarker markers:

The fixed and washed pellets are resuspended in blocking and permeabilization buffer, placed on a shaker for a few minutes, centrifuged, and resuspended in the premixed labeling solution for 30 to 60 minutes. Depending on the binding affinity of the antibody, which can directly affect the final imaging quality, the pellets can be resuspended and incubated in the labeling mixture for longer periods of time (e.g., up to 10 or 20 hours) as needed.

The protein biomarker of interest can be labeled with an antibody (or related fragment or derivative), which is detected directly, for example, with a fluorescently coupled antibody, or indirectly, for example, with immunohistochemistry or a primary antibody and a coupled secondary antibody. Similarly, the nucleic acid of interest can be labeled with a molecular probe that detects directly or indirectly. If necessary, the signal can be further amplified by available techniques such as a biotin-streptavidin-based system and polymerase chain reaction. Optionally, the sample can be centrifuged and the pellet washed once or multiple times.

In this example, using Fab from Jackson ImmunoResearch Laboratories, Inc. (https://www.jacksonimmuno.com/catalog/31), 200 μL of labeling solution in PBST blocking buffer was mixed with propidium iodide (PI) for nuclear staining (1:2000), EpCAM antibody for cancer cell detection (1:250 for antibody concentrations greater than 0.1 mg/ml), CD45 antibody for immune cell detection (1:250 antibody concentration), and a secondary antibody matching the primary antibody host (2x the primary antibody by weight).

Alternatively, sequential labeling can be performed starting with incubation of primary antibodies for 60 minutes or more and washing, then secondary antibodies and washing, then staining with PI in PBS solution for 5 minutes, followed by washing. For other targets, surface proteins can be labeled with antibodies or DNA/RNA targets can be labeled with probes. Sequential labeling with direct conjugates of secondary antibodies or fluorescent molecules can also be used. Biotinylated antibodies can also be used with complementary signals based on binding affinity with, for example, streptavidin, as known in the art. After adding the labeled antibody or any probe or chemical dye, the pellet can be resuspended in 4% PFA at room temperature for 10 minutes as appropriate to fix all labeled materials on the cells so that they are not washed off during the curing step.

Curing:

After the labeling reaction, the sample is washed one or more times as appropriate, and then resuspended in a solution containing a solidifying agent (e.g., a gelling agent), such as a low melting point agarose (LMP) solution that can be solidified (e.g., gelled) or a solution containing a hydrogel precursor that can be polymerized after adding a sufficient amount of a cross-linking agent and an ion-containing component (e.g., a component containing Ca ²⁺ ions) to induce gelation. For example, the amount of cross-linking agent introduced can be adjusted depending on whether a gel-type solid is desired, or increased to provide a higher density solidified sample.

In an embodiment, the solution containing the solidifying agent contains, for example, 1.2% or 2.4% or 10% liquid agarose in an RI matching solution to accelerate subsequent imaging. Alternatively, the solidifying agent (e.g., gelling agent) is in a solution with a solvent that crosslinks and/or becomes solid (e.g., gel-like) at lower temperatures and/or higher ionic concentrations, and melts at higher temperatures and/or lower ionic concentrations.

In this example, the gel solution containing the dispersed material is transferred to a desired imaging holder, such as a holder containing sample wells having a block shape (or other desired shape), at 25-37° C., and the holder may be shaken or vibrated to ensure dispersion of the material in each well. The holder containing the sample solution is transferred to a lower temperature, such as by incubating the sample dispersed in LMP agarose at 4° C. for about 15-30 minutes, allowing gelation in the wells to occur.

It should be noted that the original liquid blood sample has millions of non-cancerous cells that obscure CTCs that are rare and present at very low levels. Gelation allows cells to be dispersed and separated in 3D space to facilitate imaging (described below). A key point is that millions of cells are condensed from a higher volume (e.g., 10cc of blood) to a 20μl gel block ( ^2.71mm3 ), which then allows imaging within an acceptable imaging time. In alternative embodiments, the volume can be reduced even more to allow fast high-resolution imaging (e.g., up to 20x or 40x). In certain embodiments, the cell mass is reduced to give the smallest possible volume. In these situations where relatively small volumes are used, the close distance between cells may require concern that gel clarification should be performed due to the higher density of the lipid bilayer (cell membrane), but this can be solved, for example, using an optimized RI matching solution or a higher power laser such as a two-photon microscope.

Imaging:

Once the gel block (with dispersed inclusions) is formed, it is clarified by immersion in a refractive index (RI) matching solution for about 30 minutes to make it transparent for imaging. The RI matching gel sample can then be imaged to detect the presence of more biomarkers that are not discretely isolated in the gel block.

This imaging can occur by microscopy, including by effective fluorescence imaging methods such as light sheet microscopy. In the case of fluorescently labeled biomarkers, for example, samples can be imaged quickly using non-diffracted Behssel beams by light sheet fluorescence microscopy (LSFM). For example, using light sheet microscopy allows hundreds or thousands of cells to be imaged from a single field of view (FOV). For example, by setting the imaging z step to 1 micron, each cell with an average cell diameter of 15 microns is imaged from top to bottom about 10 to 15 times. Different from indirect gating on the integrated traces by flow cytometry, the obtained LFSM data from the imaged sample discloses individual cells that can be visually inspected for antibody labeling effectiveness, intensity, distribution, etc.

Application of the methods herein can allow for rapid detection of rare biomarkers such as CTCs in solid arrays, thereby allowing spatial discrimination of labeled biomarkers. The methods also provide a more sensitive and accurate diagnostic method by reducing the need for processing steps that can reduce sample heterogeneity or alter its properties.

In addition, the methods described herein allow for the fixation of PBMCs immediately after RBC removal, allowing for the capture of their distribution and association in their initial state. Advantageously, this allows for direct observation of sample characteristics such as cell-cell interactions, cell clusters (e.g., circulating tumor microemboli), and other properties that may indicate disease state or provide other prognostic or diagnostic applications.

Example 2 - 3D matrix-based imaging of blood samples

Cell preparation

Transfer anticoagulated blood collected from humans in EDTA-coated tubes to 15 ml tubes and add cell separation medium (e.g., ), and the mixture was subjected to density gradient centrifugation (1200RPM) for five minutes. After centrifugation, the buffy coat (the portion containing white blood cells and platelets) was transferred to a 2 ml test tube without collecting any separation medium. The resulting mixture was centrifuged again at 500g for 5 minutes to allow cell clumps to form at the bottom of the test tube. The supernatant was carefully removed without disturbing the cell clumps.

mark

The pellet was then resuspended in 200 μl of labeling solution (1:1000 PI in 4% PFA in PBS) to label the nuclei. The labeling solution was added for 20 minutes at 4°C. The mixture was then centrifuged at 500 g for 5 min, the supernatant was carefully collected and discarded. 1 ml of 4% PFA in PBS was then added to fix the chemical dye at 4°C for 20 minutes, and the mixture was centrifuged again at 500 g for 5 min, and the supernatant was discarded again.

Curing

At the same time, prepare a solidification solution mixture containing about 0.5wt% LM agarose. The solidification solution is heated in a microwave to above its gel point until it melts, then allowed to cool at room temperature, where it is provided in a liquid state until it reaches its gel point again. Using a pipette, 20 μl of the cooling solidification solution is slowly added to the mass so that the mass is resuspended, and the solidification solution is slowly added to avoid bubble formation. Make the mixture reach 4°C and keep a period of time (less than about 30 minutes) that allows gel formation.

Installation and RI Matching

Once the sample gelled, the gel was carefully removed from the tube with a pipette by poking the side of the gel, providing a 20 μl volume of gel. The gel was then mounted on a sample holder, in this case a 2 mm diameter 3D gel rod (same composition as the solidification solution prefabricated into a holding shape mounted on a 3D printed plastic surface) at room temperature and cooled at 4°C for 10 minutes to allow it to gel. The sample gel on the holder was immersed in RI matching solution for 5-30 min to prepare it for imaging.

Imaging

The sample was transferred to the light sheet imaging chamber and imaged (10X objective). For a 20 μl gel sample, imaging a single channel took approximately 3 min at 10X resolution with a z-step of 4 μm, where imaging time can be reduced by reducing gel volume (increasing cell density), reducing imaging resolution (lower magnification objective or higher z-step), or by optimizing microscope settings such as z-step, as well as other means known to those of ordinary skill in the art.

2A-2C depict images obtained from a light sheet microscopy imaging chamber.

The nuclei of stained white blood cells are shown in cyan. In FIG2A, the image represents the entire 20 μl gel sample from the top down view. FIG2A is a 100 μm overlay and reduced image of the 25x4 μm image. In FIG2B, the image represents the entire 20 μl gel sample from the side view. In FIG2C, the image is magnified to capture individual white blood cells in greater detail, with the smaller horizontal line in the lower left corner equal to 20 μm in scale.

Example 3 - Cell Compatibility

Cells from the CACO2 cell line obtained from ATCC; and cells from the HL60 cell line also obtained from ATCC were harvested and processed as briefly described below. CACO2 cells were cultured and collected via trypsinization. HL60 is a floating cell line that does not require trypsinization. CACO2 and HL60 cells were each collected, washed with PBS, and centrifuged to collect cell clumps.

Immunolabeling - Cancer cell line CACO2

CACO2 cells were stained with trypan blue and counted using a cell counter. An appropriate number of cells were resuspended in 100 μl PBS. EpCAM (Dako) antibody was added to the cell solution at a 1:100 ratio and secondary antibody was added at a 1:2 primary to secondary antibody molar ratio.

The mixture was shaken at room temperature for 30 minutes. The mixture was centrifuged at 500g for 5min, and the supernatant was carefully removed. The cell mass was washed by resuspension in PBS, and centrifuged again at 500g for 5min, and the supernatant was discarded. The cells were then resuspended and cultivated at 4°C in 1ml PI solution in 4% PFA (1:1000) for 20 minutes so that the binding antibody was fixed and the nucleus was labeled, the mixture was centrifuged again at 500g for 5min, and the supernatant was discarded.

Immunolabeling - Leukocyte cell line HL60

HL60 cells were stained with trypan blue and counted using a cell counter. An appropriate number of cells were collected in 100 μl PBS. CD45 (Dako) antibody was added to the cell solution at a 1:100 ratio and secondary antibody was added at a 1:2 primary to secondary antibody molar ratio.

The mixture was shaken at room temperature for 30 minutes. The mixture was centrifuged at 500g for 5min, the supernatant was carefully removed, and 1ml of PBS was added to wash, and the mixture was centrifuged at 500g for 5min again, and the supernatant was carefully collected and discarded again. The cells were then resuspended and cultivated at 4°C in 1ml PI solution in 4% PFA (1:1000) for 20 minutes to fix the binding antibody and at 4°C, the cell nucleus was labeled for 20 minutes, and the mixture was centrifuged at 500g for 5min again, and the supernatant was carefully collected and discarded again.

Curing

At the same time, prepare a solidification solution containing about 1wt% LM agarose. The solidification solution is heated to melting in a microwave, then allowed to cool to room temperature, where it is ultimately provided in a gel state. Using a pipette, 20 μl of the cooling solidification solution is slowly added to the mass so that the mass is resuspended, slowly adding the solidification solution to avoid bubble formation. Make the mixture reach 4°C and keep a period of time (less than about 20 minutes) that allows gel formation.

Imaging

Gels with immunolabeled CACO2 and HL60 cells were imaged using light sheet microscopy with a 10X objective as previously described.

FIG. 3A depicts CD45/PI stained HL60 cells as captured by light sheet microscopy. FIG. 3A is digitally magnified as indicated by the scale bar in the lower left corner, where the white bar corresponds to 50 μm. Yellow indicates PI staining of the labeled nuclei and green indicates CD45 immunolabeling. FIG. 3B-3C depicts EpCAM/PI stained CACO2 cells as captured by light sheet microscopy. Images were obtained with a 10x objective. FIG. 3B is a presentation of the entire gel volume. FIG. 3C is digitally magnified as indicated by the scale bar. That is, in FIG. 3B , the scale bar represents 300 μm and in FIG. 3C , the scale bar represents 50 μm.

Example 4 - Multiplex labeling of CACO2 cells in blood samples

Cancer cell spikes in blood samples

Cancer cell count

A study simulating CTC detection in cancer patients was performed. The study was designed to evaluate the rare cell detection efficiency of the 3D liquid biopsy method and the percentage of cell loss from the cell preparation process.

Cell preparation

The sample of CACO2 cells was trypsinized, counted and inoculated in a freshly collected peripheral blood sample in an appropriate volume to prepare a blood sample spiked with cancer cells. Peripheral blood mononuclear cells (PBMC) were collected via an erythrocyte lysis method. Anticoagulated blood collected from humans in an EDTA-coated tube was transferred to a 15 ml tube. For 1 c.c. of blood, 8 c.c. of ammonium chloride solution was added to lyse red blood cells. The lysis process was performed on ice for 15 minutes, centrifuged, the supernatant was removed, and the collected PBMC cell pellet containing the spiked cancer cells was resuspended in 100 μL PBS for further processing.

EpCAM (Dako) and CD45 (Dako) antibodies were added to the cell solution at 1:100 and matching secondary antibodies were added at a 1:2 primary to secondary antibody molar ratio to achieve a one-step labeling process. The cell samples were incubated at room temperature for 1 hour to allow antibody binding. Subsequently, the samples were centrifuged at 500g for 5 minutes to collect the pellets. The pellets were washed with 1 ml PBS and centrifuged again and the supernatant was removed. Subsequently, 4% PFA with 1:1000 PI was added to the pellets to fix the labeled antibodies and stain the cell nuclei for 30 minutes. Subsequently, the cells were centrifuged again to collect the pellets.

As previously described, cell mass is mixed in solidification solution. Then, mixed PBMC and CACO2 cells are identified in the sample to confirm the ability to detect multiple cell types by multiple antibody-labeled targets in the sample. Fig. 4A depicts the whole 3D gel data of the multiple labels of CACO2 in PBMC, wherein EpCAM marker is shown in magenta and PI is shown in blue. Fig. 4B depicts the amplification, superposition images of CACO2-EpCAM (magenta), leukocyte-CD45 (green) and PI (blue). Fig. 4C and 4D depict images of CTCs identified with and without EpCAM (magenta signal) for confirmation.

In these superimposed or separated figures, EpCAM labeling for cancer cell detection is confirmed as a viable approach in 3D presentation. CACO2 shows higher EpCAM expression without CD45 expression and PBMC (white blood cells) show variable CD45 expression but no EpCAM expression.

Cell counting

The trypsinized CACO2 counts were inoculated into 2 cc of blood, stained, and labeled as illustrated in the following table to prepare three sets of gels (a, b, and c):

Health Spiked cell lines Cell number Probe 2c.c. CACO2 2000 PI, EpCAM, CD45

As described in Examples 1, 2, and 3, 3-dimensional gels were formed and imaged via light sheet microscopy. Each image was denoised and feature enhanced for all immune marker signals used for cell detection. Cells with positive EpCAM and PI signals, and negative CD45 were counted as positive; cells with negative EpCAM signals and positive PI signals were counted as negative; and signals with negative PI signals were counted as invalid. Other parameters such as cell or nuclear roundness, cell/nuclear volume, and nucleus-cytoplasm ratio (N:C ratio) of positive cancer cells can be quantified using MATLAB-based or Python-based cell detection algorithms.

FIG. 4A depicts the output of 3d imaging from three replicates of a gel sample with 2000 spiked CACO2 cells.

In order to better observe the test results, 3D imaging data is shown in the maximum projection mode of all 2D images superimposed on each other to generate single plane imaging data. EpCAM (coloring) labeled CACO2 cells can be easily visually identified by digitally amplifying the data set. EpCAM positive cells are screened for negative CD45 signals and positive PI signals. The cell detection algorithm screens all cells with those parameters and outputs the CTC images with separation and combination signals, as shown in Figure 4B. These images are stored in a single file for each sample, and all detected cell images are further confirmed by human eyes or signal detection algorithms.

The table below depicts the final cell counts from three sets of gels with a, b, and c. The assay results were within a reasonable approximation of the seed cell counts.

Sample ID Number of spiked cells Detected cells a 2000 1987 b 2000 2058 c 2000 2011

The total number of cells in the entire gel can be quantified by the total PI positive signal. The total number of positive cancer cells divided by the total number of PI signals will output a ratio. Multiplying this ratio by one million gives the number of cancer cells in one million PBMCs.

Example 5 - CTC Detection in Cancer Patients

An IRB study was performed to detect the presence of CTCs in the blood of patients with colon cancer. The study was designed to evaluate the efficiency of rare cell detection of the 3D liquid biopsy method. The enrolled patients included healthy and disease subjects. In the following example, patients with stage II to stage IV colon cancer that metastasized to the liver or lungs are shown.

Blood sample preparation

Anticoagulated blood collected from humans in EDTA coated tubes was transferred to 15 ml tubes. Peripheral blood mononuclear cells (PBMC) were collected via an erythrocyte lysis method. For 2 cc of blood, 16 cc of ammonium chloride solution was added to lyse erythrocytes. The lysis process was performed on ice for 15 minutes, centrifuged, the supernatant was removed, and the collected PBMC cell pellet containing the spiked cancer cells was resuspended in 100 μL PBS for further processing.

Patient A

EpCAM (Dako) and CD45 (Dako) antibodies were added to the cell solution at 1:100 and matching secondary antibodies were added at a 1:2 primary to secondary antibody molar ratio to achieve a one-step labeling process. The cell samples were incubated at room temperature for 1 hour to allow antibody binding. Subsequently, the samples were centrifuged at 500g for 5 minutes to collect the pellets. The pellets were washed with 1 ml PBS and centrifuged again and the supernatant was removed. Subsequently, 4% PFA with 1:1000 PI was added to the pellets to fix the labeled antibodies and stain the cell nuclei for 30 minutes. Subsequently, the cells were centrifuged again to collect the pellets.

The cell clumps were mixed in a solidifying solution (1% LM agarose) and imaged as described above. FIG. 5A depicts 3D gel data (single stack block) with a resolution of 0.42 μm x 0.42 μm x 1 μm, where the EpCAM marker is shown in magenta, the CD45 marker is shown in green, and PI is shown in blue. FIG. 5B depicts a magnified image of EpCAM positive, CD45 negative, and PI positive cell targets. Using the above parameters for screening positive CTCs, we were able to detect 195 CTCs in approximately 160,000 PBMCs, equivalent to 1,218 CTCs/million PBMCs.

Patient B

CK20 (Ventana) and EpCAM (Dako) antibodies were added to the cell solution at 1:100 and matching secondary antibodies were added at a 1:2 primary to secondary antibody molar ratio to achieve a one-step labeling process. Labeled cell mass collection, gelation and imaging were performed as described above for patient A. FIG6A depicts 2 consecutive stacks (600 μm stacks placed on 1200 μm stacks) of 3D gel data with a resolution of 0.42 μm x 0.42 μm x 1 μm, where the CK20 marker is shown in yellow, the EpCAM marker is shown in magenta, and PI is shown in blue. FIG6B depicts magnified single cell images of CK20-positive, EpCAM-positive, and PI-positive cell targets. Using the above parameters for screening positive CTCs, we were able to detect 12 CTCs in approximately 46,200 PBMCs, equivalent to 259 CTCs/million PBMCs.

Compared with other methods for viewing cells in complex samples such as tissue samples, the method herein does not require a clarification step to remove lipids or destroy other cell information in order to achieve tissue transparency for 3D imaging. Instead, the method herein combines gelation of dispersed cell samples in three-dimensional space with refractive index matching to achieve complete cell sorting and 3D observation of complete cell information within liquid samples. It is a novel method for screening rare cells or any cells in liquid biopsy samples by simple antibody labeling.

Those skilled in the art will appreciate that the examples and embodiments described herein do not limit the scope of the invention. The description including the examples is intended to be exemplary only, and it will be apparent to those skilled in the art that various modifications and changes may be made in the present invention without departing from the scope or spirit of the invention as defined in the appended claims.

Furthermore, although certain details in this disclosure are provided in order to convey a comprehensive understanding of the invention as defined by the appended claims, it will be apparent to those skilled in the art that certain embodiments may be implemented without these details. Additionally, in certain cases, well-known methods, procedures, or other specific details are not described to avoid unnecessarily obscuring aspects of the invention as defined by the appended claims.

Numbered Examples

The present disclosure is further directed to the following examples.

A method for analyzing a biological sample, comprising:

adding a solidifying agent to a liquid specimen containing a biological material;

Producing solidified samples containing dispersed biological material

The solidified sample is imaged to identify one or more components in the dispersed biological material.

The method of Example 1, wherein the specimen is obtained from a liquid blood sample.

The method of Example 2, wherein the specimen is obtained from the liquid blood sample by a process comprising removing red blood cells and platelets from the liquid blood sample.

The method of any of the foregoing embodiments further comprises labeling the biomolecule target in the sample before adding the solidifying agent.

The method of Example 4, wherein the biomolecule target is a nucleic acid, a protein or a vesicle.

The method of embodiment 4 or 5, wherein labeling comprises contacting the specimen with a molecular probe.

The method of Example 6, wherein the molecular probe is an antibody.

The method of Example 6, wherein the molecular probe is a fluorescent dye.

The method of Example 6, wherein the molecular probe is a nucleic acid probe.

The method of any one of embodiments 1 to 9, wherein the biomolecule target is an extracellular target.

The method of any one of embodiments 1 to 9, wherein the biomolecule target is a cellular target.

The method of any of the preceding embodiments further comprises transferring the specimen to a sample holder after adding the curing agent.

The method of Example 12 further comprises shaking the sample in the sample holder.

The method of Example 12 further comprises vibrating the sample in the sample holder.

The method of any of the preceding embodiments, wherein the cured sample is a solid block suitable for imaging.

The method of any of the preceding embodiments, wherein prior to imaging, the solidified sample comprising dispersed biological material is transferred to a solution in order to ensure a desired transparency of the sample for imaging.

The method of Example 16, wherein the solution is a refractive index matching solution.

The method of any of the preceding embodiments, wherein step (c) further comprises detecting one or more cancer cells or cancer markers in the solidified sample.

The method of any one of the preceding embodiments, wherein imaging is performed by fluorescence microscopy.

The method of Example 19, wherein imaging is performed by light sheet fluorescence microscopy.

The method of any of the preceding embodiments, wherein the curing agent is a gelling agent and the cured sample is a gel sample.

The method of any of the preceding embodiments, wherein prior to step (a), the biological specimen is subjected to a fixation procedure.

The method of Example 22, wherein the fixation procedure comprises incubating the biological specimen in a fixative solution.

The method of Example 23, wherein the fixative solution comprises glutaraldehyde or formaldehyde.

The method of any preceding embodiment, wherein step (c) allows for single cell identification in the solidified sample.

Claims (40)

1. A method of analyzing a liquid sample containing biological material for one or more target components, comprising: (a) adding a curing agent to a specimen obtained from said liquid sample comprising said biological material; (b) produce a solidified sample containing dispersed biological material; and (c) Imaging the solidified sample to identify the one or more target components in the dispersed biological material. 2. The method of claim 1, further comprising: (i) labeling step (a) with one or more probes for the one or more target components prior to adding the curing agent. the specimen obtained from the liquid sample in; and/or (ii) labeling the solidified sample from step (b) with one or more probes directed to the one or more target components . 3. The method of claim 1, comprising labeling the material obtained in step (a) with one or more probes for the one or more target components before adding the curing agent. said specimen of said liquid sample. 4. The method of claim 1, further comprising introducing a refractive index matching material into the cured sample. 5. The method of claim 1, wherein the liquid sample is a liquid blood sample. 6. The method of claim 5, wherein the specimen is obtained from the liquid blood sample by a process including removing red blood cells and platelets from the liquid blood sample. 7. The method of claim 6, wherein the specimen comprises peripheral blood mononuclear cells (PBMC) isolated from the liquid blood sample. 8. The method of claim 1, wherein the one or more target components comprise nucleic acids, proteins, viruses, or vesicles. 9. The method of claim 2, wherein labeling comprises: contacting the specimen obtained from the liquid sample in step (a) with a molecular probe; and/or contacting the specimen from step (b) The solidified sample is contacted with a molecular probe. 10. The method of claim 9, wherein the molecular probe is an antibody. 11. The method of claim 9, wherein the molecular probe is a fluorescent dye. 12. The method of claim 9, wherein the molecular probe is a nucleic acid probe. 13. The method of claim 1, wherein the one or more target components comprise extracellular targets. 14. The method of claim 1, wherein the one or more target components comprise cellular or intracellular targets. 15. The method of claim 1, further comprising transferring the specimen to a sample holder. 16. The method of claim 15, further comprising oscillating or vibrating the sample in the sample holder. 17. The method of claim 1, wherein the solidified sample is a solid or gel mass suitable for imaging. 18. The method of claim 1, wherein imaging is performed by fluorescence microscopy. 19. The method of claim 1, wherein imaging is performed by light sheet fluorescence microscopy. 20. The method of claim 1, further comprising a fixation procedure for the specimen. 21. The method of claim 20, wherein the fixation procedure includes the step of incubating the specimen or the solidified sample in a fixative solution. 22. The method of claim 21, wherein the fixative solution contains glutaraldehyde, formaldehyde, epoxy resin, or a mixture of any two or more of the foregoing. 23. The method of claim 1, wherein the imaging identifies the presence or absence of a specific cell type in the solidified sample. 24. A method of analyzing a liquid blood sample for the presence of rare circulating cells, comprising: (a) labeling a specimen comprising isolated peripheral blood mononuclear cells (PBMCs) obtained from the liquid blood sample with one or more probes directed to the rare circulating cells; (b) adding a curing agent to a labeled specimen containing peripheral blood mononuclear cells (PBMC); (c) producing a solidified sample containing dispersed peripheral blood mononuclear cells (PBMC); (d) optionally, introducing a refractive index matching material into the cured sample to provide an optically clear cured sample having a refractive index suitable for imaging; and (e) imaging the solidified sample from step (c) or the optically clear solidified sample from step (d) to determine the presence of the one or more probes, thereby determining rare circulating cells in the liquid blood sample The presence. 25. The method of claim 24, wherein labeling further comprises adding a probe for white blood cells. 26. The method of claim 24, wherein the one or more probes recognize cancer-specific antigens or tumor-specific DNA or RNA sequences. 27. The method of claim 26, wherein the one or more probes are selected from antibodies or nucleic acid probes. 28. The method of claim 27, wherein the one or more probes confer an affinity for one or more of EpCAM, HER2, CDX2, CK20, CK19, PD/PDL-1 and EGFR antigens or corresponding nucleic acid sequences. detection. 29. The method of claim 24, wherein the solidifying agent comprises a component selected from the group consisting of low melting point agarose, agarose, and hydrogel precursors. 30. The method of claim 24, further comprising introducing an index matching material into the cured sample. 31. The method of claim 24, wherein producing the solidified sample includes adding a curing agent to the sample above room temperature and allowing the sample to reach a reduced temperature to become a solid gel. 32. The method of claim 24, wherein producing the solidified sample includes adding a reagent to a hydrogel precursor to induce gelation. 33. The method of claim 24, wherein an optically clear gelled sample immersed in a solution containing a refractive index matching material is introduced into the sample holder. 34. The method of claim 24, wherein imaging is performed using light sheet microscopy. 35. The method of claim 24, wherein the rare circulating cells are circulating tumor cells. 36. A solidified sample suitable for imaging, said specimen comprising dispersed biological material obtained from a liquid biopsy and immobilized within said solidified specimen. 37. The solidified sample of claim 36, further comprising a probe for one or more target components in the liquid biopsy. 38. The solidified sample of claim 36, wherein the liquid biopsy is a blood sample and the biological material includes peripheral blood mononuclear cells (PBMC). 39. The solidified sample of claim 38, further comprising rare circulating cells. 40. The solidified sample of claim 39, wherein the rare circulating cells are selected from the group consisting of circulating tumor cells, circulating epithelial cells and circulating endothelial cells.