TWI865391B - A method for expanding circulating tumor cells and applications thereof - Google Patents

Abstract

The present invention provides a method for expanding circulating tumor cells and application thereof. The expansion method involves providing tumor cells, centrifuging the tumor cells in an ultra-low attachment multiple well plate to aggregate the cells into a 3D sphere, administering Matrigel to coat the 3D sphere, and suspension culturing the cells with a serum-free expansion medium, wherein the serum-free expansion medium contains fibroblast growth factor 3 and glutamine.

Description

Methods for expanding circulating tumor cells and their applications

The present invention relates to a method for expanding tumor cells, and more particularly to a tumor cell expansion technique for detecting circulating tumor cells.

Circulating tumor cells (CTCs) refer to tumor cells that break away from the primary tumor and enter the peripheral blood circulation or lymphatic system. They are potential sources of cancer metastasis. The detection and analysis of circulating tumor cells is of great value in early cancer assessment, metastasis risk assessment, and postoperative effect assessment. The detection only requires blood samples, which is a non-invasive detection method. It can also be used for real-time monitoring of cancer patients undergoing treatment. It can serve as a good indicator of treatment efficacy and whether the tumor has metastasized, especially in some patients after surgery. The tumor tissue sections cannot be obtained, so the detection of circulating tumor cells in the blood has great clinical applicability.

However, the proportion of circulating tumor cells in the blood is extremely small, only one in about 10 ⁸ to 10 ⁹ blood cells, so the detection of circulating tumor cells usually requires the use of high-sensitivity and high-precision technology to identify and separate extremely small amounts of circulating tumor cells. The current analysis and screening of circulating tumor cells is through microfluidic systems, flow cytometers, and magnetic bead sorting systems, which are expensive and difficult to be widely used. Therefore, in order to solve the above clinical pain points, a simple, low-cost technology that can effectively expand circulating tumor cells is an important issue that needs to be solved in this field.

The content of the invention is intended to provide a simplified summary of the invention so that readers can have a basic understanding of the disclosure. This content of the invention is not a complete overview of the invention, and its intention is not to point out the important/key elements of the specific embodiments of the invention or to define the scope of the invention. The sole purpose of the content of the invention is to provide a simplified description of certain concepts disclosed by the invention to facilitate the understanding of the implementation methods described later.

In one aspect, the present invention provides a method for expanding tumor cells, comprising: Providing a tumor cell; Placing the tumor cell in an ultra-low attachment multi-well dish for centrifugation to aggregate the cells into a 3D spherical structure; Applying a matrix gel (Matrigel) to coat the 3D spherical structure; and Performing suspension culture with a serum-free expansion medium, wherein the serum-free expansion medium contains fibroblast growth factor 3 (Fibroblast Growth Factor 3, FGF-3) and glutamine (glutamine, Gln).

In some embodiments, the culturing is carried out in a hypoxic environment with an oxygen concentration of 1 to 3% (v/v).

In some specific embodiments, the centrifugation speed is 700 to 900 x g , and the centrifugation time is 6 to 10 minutes.

In some embodiments, the concentration of fibroblast growth factor 3 is 8 to 12 ng/ml.

In some embodiments, the concentration of glutamine is 1 to 3 mM.

In some specific embodiments, the tumor cell is a lung cancer tumor cell, a breast cancer tumor cell, and/or a colorectal cancer tumor cell.

On the other hand, the present invention provides a method for detecting circulating tumor cells, which comprises: Providing peripheral blood; Performing density gradient centrifugation on the peripheral blood to separate a peripheral blood mononuclear cell sample; Performing migration ability screening on the peripheral blood mononuclear cell sample to obtain a target cell sample; Placing the target cell sample in an ultra-low attachment multi-well plate for centrifugation to aggregate the cells into a 3D spherical structure; Applying a matrix gel to coat the 3D spherical structure; Performing suspension culture with a serum-free expansion medium, wherein the serum-free expansion medium contains fibroblast growth factor 3 and glutamine; and Analyze the cell proliferation of circulating tumor cells to determine whether the peripheral blood contains circulating tumor cells.

The method for detecting circulating tumor cells of the present invention is to apply the tumor cell expansion method of the present invention to the culture of the target cell sample, and then observe the expansion situation as a basis for judging whether the peripheral blood contains circulating tumor cells.

In some embodiments, the blood sample is from a subject.

In some specific embodiments, the screening is performed using 8 μm transwells and Matrigel to simulate extracellular matrix (ECM) to screen out circulating tumor cells with migration ability.

In some embodiments, the culturing is carried out in a hypoxic environment with an oxygen concentration of 1 to 3% (v/v).

The circulating tumor cell detection method provided by the present invention screens circulating tumor cells with migration ability in blood cell samples through the design of through-hole chambers and matrix gel. Since the proportion of circulating tumor cells in the blood is extremely small, the number of circulating tumor cells screened out is very rare. The tumor cell expansion method of the present invention can effectively expand rare tumor cells. The cells are aggregated into 3D spheres by centrifugation, and the extracellular matrix environment is simulated by matrix gel. Suspension culture is performed in a specially designed serum-free expansion medium under hypoxia and 3D tumor-like microenvironment, and finally the number of circulating tumor cells is effectively expanded. Subsequently, the expansion of circulating tumor cells is detected as a basis for cancer detection, and further circulating tumor cell analysis can be performed to achieve the purpose of precision medicine.

The expansion method of the present invention does not require a microfluidic system or a magnetic bead sorting system, which not only greatly reduces the difficulty and cost of circulating tumor cell detection, but also allows circulating tumor cells to be effectively expanded to a purity of more than 90% without a sorting system, which is of great value for the development of cancer detection, clinical medicine and precision medicine.

After reading the following implementation methods, a person with ordinary knowledge in the technical field to which the present invention belongs can easily understand the basic spirit and other invention purposes of the present invention, as well as the technical means and implementation modes adopted by the present invention.

The content of circulating tumor cells in the blood is very rare, with only one in about 10 ⁸ to 10 ⁹ blood cells, so the detection threshold is high. The current analysis and detection of circulating tumor cells requires microfluidic systems, flow cytometers, and magnetic bead sorting systems, which are costly and complicated to operate, and are therefore difficult to be widely used.

In view of this, the present invention first provides a method for expanding tumor cells. Please refer to FIG. 1 . The method for expanding tumor cells of the present invention comprises steps 11 to 14.

Step 11: providing a tumor cell. The tumor cell includes but is not limited to lung cancer tumor cells, breast cancer tumor cells, colorectal cancer tumor cells, epithelial cell cancer tumor cells, lung adenocarcinoma tumor cells, peritoneal cancer tumor cells, liver cancer tumor cells, gastrointestinal cancer tumor cells, pancreatic cancer tumor cells, cervical cancer tumor cells, ovarian cancer tumor cells, bladder cancer tumor cells, kidney cancer tumor cells, prostate cancer tumor cells or thyroid cancer tumor cells.

Step 12: placing the tumor cells in an ultra-low attachment multi-well dish for centrifugation to aggregate the cells into a 3D spherical structure, wherein the centrifugation speed is 700 to 900 x g , such as 700 x g , 710 x g , 720 x g , 730 x g , 740 x g , 750 x g , 760 x g , 770 x g , 780 x g , 790 x g , 800 x g , 810 x g , 820 x g , 830 x g , 840 x g , 850 x g , 860 x g , 870 x g , 880 x g , 890 x g or 900 x g . The centrifugation time is 6 to 10 minutes, such as 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes.

Step 13: applying a matrix gel to coat the 3D spherical structure, wherein the matrix gel is used to simulate the environment of the extracellular matrix coating the cells.

Step 14: Perform suspension culture in a serum-free expansion medium, wherein the serum-free expansion medium contains fibroblast growth factor 3 (FGF-3) and glutamine, wherein the concentration of FGF-3 is 8 to 12 ng/ml, such as 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml. The concentration of glutamine is 1 to 3 mM, for example, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM or 3 mM.

In some embodiments, the culturing is carried out in a hypoxic environment with an oxygen concentration of 1 to 3% (v/v), such as 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% or 3%.

In certain specific embodiments, in the tumor cell expansion method of the present invention, during expansion culture, the serum-free expansion medium is replaced every 7 days, and after 18 days of suspension culture, it can be changed to attachment culture.

In another aspect, the present invention provides a method for detecting circulating tumor cells. Please refer to FIG. 2 . The method for detecting circulating tumor cells of the present invention comprises steps 21 to 27.

Step 21: Providing peripheral blood. The peripheral blood comes from a subject, wherein the subject is an individual whose circulating tumor cells in the blood are to be detected. In some specific embodiments, the peripheral blood can be other blood samples.

Step 22: Perform density gradient centrifugation on the peripheral blood to separate the peripheral blood mononuclear cell sample. In some specific embodiments, the density gradient centrifugation is performed by centrifugation using a Ficoll-Paque density gradient separation reagent to separate the peripheral blood mononuclear cells, wherein the peripheral blood mononuclear cells may contain a relatively rare amount of circulating tumor cells.

Step 23: Screen the peripheral blood mononuclear cell sample for migration ability to obtain a target cell sample. In some specific embodiments, the screening is performed using an 8 μm through-hole chamber (transwell) and matrigel to simulate extracellular matrix (ECM) to screen circulating tumor cells with migration ability that can penetrate the simulated extracellular matrix. This step will also screen out immune cells in the sample. In some specific embodiments, the target cell sample may contain circulating tumor cells.

Step 24: placing the target cell sample in an ultra-low attachment multi-well dish for centrifugation to aggregate the cells into a 3D spherical structure, wherein the centrifugation speed is 700 to 900 x g , such as 700 x g , 710 x g , 720 x g , 730 x g , 740 x g , 750 x g , 760 x g , 770 x g , 780 x g , 790 x g , 800 x g , 810 x g , 820 x g , 830 x g , 840 x g , 850 x g , 860 x g , 870 x g , 880 x g , 890 x g or 900 x g . The centrifugation time is 6 to 10 minutes, such as 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes.

Step 25: applying a matrix gel to coat the 3D spherical structure, wherein the matrix gel is used to simulate the environment of extracellular matrix coating cells.

Step 26: Perform suspension culture in a serum-free expansion medium, wherein the serum-free expansion medium contains fibroblast growth factor 3 (FGF-3) and glutamine, wherein the concentration of FGF-3 is 8 to 12 ng/ml, such as 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml or 12 ng/ml. The concentration of glutamine is 1 to 3 mM, such as 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM or 3 mM. The culture is effective to expand circulating tumor cells in the target sample. In some embodiments, the culture period is 7 to 21 days, such as 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the culture is carried out in a hypoxic environment with an oxygen concentration of 1 to 3% (v/v), such as 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3%.

Step 27: Analyze the number of circulating tumor cells to determine whether the peripheral blood contains circulating tumor cells. If the peripheral blood to be tested contains circulating tumor cells, a significant increase in the number of circulating tumor cells can be observed. On the contrary, if the peripheral blood to be tested does not contain circulating tumor cells, no circulating tumor cells can be observed. In certain specific embodiments, the cell proliferation of circulating tumor cells can reflect the circulating tumor cell content in the subject's blood, and can be used as a basis for cancer assessment, metastasis risk assessment, and postoperative effect assessment. The expanded circulating tumor cells can also be further analyzed for cancer types and treatment methods, etc., to achieve the effect of precision medicine.

Among them, step 24 to step 26 is to apply the tumor cell expansion method described in the present invention to the circulating tumor cell detection method described in the present invention. Specifically, the target cell sample after screening is expanded for tumor cells, and then the expansion situation is observed as a basis for judging whether the peripheral blood contains circulating tumor cells, and the risk of cancer metastasis can also be evaluated.

It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative, but not intended to limit the rights claimed by the present invention. Certain details of one or more embodiments of the present invention are described in the following description. Other features or advantages of the present invention will be apparent from the following non-exhaustive list of representative embodiments and from the attached claims.

Unless otherwise defined below, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. References to the techniques used herein are intended to indicate techniques commonly understood in the art, including variants or equivalents of those techniques or alternative techniques developed later, which are obvious to those skilled in the art.

It should be noted that, as used herein, the singular terms "a", "an", and "the" include plural referents unless expressly limited to one referent. Unless the context clearly indicates otherwise, the term "or" is used interchangeably with the term "and/or".

The terms "about", "approximately" or "nearly" as used herein substantially represent that the numerical value or range described is within 5%, preferably within 3%, and more preferably within 1%. The numerical quantities provided herein are approximate values, which means that they can be inferred even if the terms "about", "approximately" or "nearly" are not used.

As used herein, the term "comprising" is open-ended, indicating that such embodiments may include additional elements. Conversely, the term "consisting of" is closed-ended, indicating that such embodiments do not include additional elements (except for trace impurities). The term "consisting essentially of" is partially closed-ended, indicating that such embodiments may also include elements that do not substantially alter the basic characteristics of such embodiments.

Unless otherwise defined herein, scientific and technical terms used in conjunction with this document shall have the meanings commonly understood by persons of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. In general, the nomenclature described herein to link the following technologies, as well as the techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, immunology, genetics and protein and nucleic acid chemistry and hybrid reactions are all known and frequently used in the art. Unless otherwise indicated, the methods and techniques of the present invention can generally be performed according to conventional methods known in the art and are described in various general and more specific references cited and discussed in this specification.

As used herein, the term "expansion" refers to any process that increases the number or purity of cells. As used herein, the term "expansion medium" refers generally to cell culture media used in the expansion process of the present invention, including but not limited to basal medium, non-essential amino acids, galactosylceramide, hydroxyethylpiperazineethanesulfonic acid, sodium pyruvate, 2-hydroxyethanol, fetal bovine serum, penicillin/streptomycin, or nutrients. As used herein, the term "serum-free expansion medium" refers generally to expansion medium that does not contain animal serum.

As used herein, the term "migration ability" refers to the ability of cells to penetrate the extracellular matrix or cell layer. Cells with migration ability include but are not limited to tumor cells. As used herein, the term "migration ability screening" generally refers to screening based on whether cells have the ability to penetrate the extracellular matrix or cell layer to screen out cells with migration ability.

As used herein, the term "peripheral blood mononuclear cell" (PBMC) refers to any cell with a round nucleus in the blood excluding the bone marrow, including but not limited to lymphocytes (such as T cells, B cells and NK cells), monocytes, eosinophils, neutrophils, basophils, and dendritic cells, excluding red blood cells and platelets. Among them, if the in situ tumor cells of a cancer patient pass through the extracellular matrix into the blood circulation and become circulating tumor cells, the peripheral blood mononuclear cells of the above patient include circulating tumor cells.

As used herein, the term "subject" refers to an animal, more specifically a non-human mammal and a human organism. Non-human animal subjects may also include pre-natal forms of animals, such as embryos or fetuses. Non-limiting examples of non-human animals include: horses, cows, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, pigs. In certain specific embodiments, the subject is a human. Human subjects may also include fetuses.

As used herein, the term "magnetic bead separation system" refers to a separation system that uses nano-scale superparamagnetic beads to bind to highly specific antibodies against specific cell surface antigens to separate desired specific cells.

The present invention is further illustrated by the following embodiments, which should not be interpreted as further limiting in any way. The entire contents of all references cited in this application (including references, approved patents, published patent applications, and patent applications in the same application) are hereby expressly incorporated into this application by reference.

Embodiment 1. Proliferation of lung cancer tumor cells

In this example, lung cancer tumor cells (PC9 lung cancer cells, RRID number: CVCL_B260) were expanded and cultured using the tumor cell expansion method of the present invention to verify the expansion effect of the tumor cell expansion method of the present invention on lung cancer tumor cells.

Materials and methods

In this example, tumor cells were divided into three groups with different culture conditions, namely a control group, a hyperoxic environment group, and a hypoxic environment group. The control group was cultured in a 21% (v/v) oxygen concentration environment using a conventional serum-free culture medium (without the addition of fibroblast growth factor 3 and glutamine); the hyperoxic environment group was cultured in a 21% (v/v) oxygen concentration environment using a serum-free expansion culture medium (with the addition of fibroblast growth factor 3 and glutamine); the hypoxic environment group was cultured in a 2% (v/v) oxygen concentration environment using a serum-free expansion culture medium (with the addition of fibroblast growth factor 3 and glutamine); The cells were cultured in serum-free expansion medium (supplemented with fibroblast growth factor-3 and glutamine) at 37°C (v/v) oxygen concentration. The serum-free expansion medium described in this embodiment is RPMI 1640 medium (Gibco, product number: 11875093, USA) supplemented with 10 ng/ml fibroblast growth factor 3 (FGF-3), 2 mM glutamine and 1% penicillin/streptomycin (Gibco, product number: 15140122, USA), and the conventional serum-free medium described in this embodiment is RPMI 1640 medium supplemented with 1% penicillin/streptomycin. Each group was repeated three times (n=3), and the Student's t - test was used to statistically analyze the differences between the experimental data. The symbols of statistical significance * and # represent p < 0.05 compared with the control group, the hyperoxic environment group and the hypoxic environment group, respectively, while ** and ## represent p < 0.01 compared with the control group, the hyperoxic environment group and the hypoxic environment group, respectively.

Tumor cells were placed in an ultra-low attachment round bottom 96-well plate at a density of 50 cells per well and centrifuged at 800 x g for 8 minutes to aggregate the cells into 3D spherical structures. uL of Matrigel coated 3D spherical cell clusters were cultured in suspension for 18 days in serum-free expansion medium or regular serum-free medium according to the group design. The cells were then transferred from the ultra-low attachment round-bottom 96-well plate to the flat-bottom 96-well cell culture plate for attachment culture until the 21st day. The culture medium was changed every 7 days, and the number of lung cancer tumor cells was calculated on the 1st, 7th, 14th and 21st days of expansion. Among them, the proliferation of tumor cells in the hypoxic environment group was observed under a microscope on days 0, 11th, 18th and 20th.

result

Please refer to Figure 3A. Under the microscope, it can be observed that the number of lung cancer tumor cells in the hypoxic environment group has a significant and gradual increase after culture. Please refer to Figure 3B. Compared with the control group, the number of cells in the hypoxic environment group and the hyperoxic environment group increased significantly on the 7th and 14th days of expansion culture. Please refer to Figure 3C. After 21 days of culture, the number of lung cancer tumor cells in the hypoxic environment group increased from 50 to about 3 x ¹⁰⁷ lung cancer tumor cells, which has a significant expansion effect compared with the hyperoxic environment group or the control group. The experimental results show that the serum-free expansion culture medium of the present invention can effectively expand lung cancer tumor cells, and the expansion effect is more significant when cultured in a hypoxic environment, which proves that the tumor cell expansion method of the present invention has the effect of expanding tumor cells.

Embodiment 2. Proliferation of breast cancer tumor cells

In this example, breast cancer tumor cells (MDA-MB-231 breast cancer cells, ATCC number: CRM-HTB-26) were expanded and cultured using the tumor cell expansion method of the present invention to verify the expansion effect of the tumor cell expansion method of the present invention on breast cancer tumor cells.

In this embodiment, the experimental design and material methods are the same as those described in Example 1.

result

Please refer to Figure 4A. Compared with the control group, the number of cells in the hypoxic environment group and the hyperoxic environment group increased significantly on the 14th and 21st days of expansion culture. Please refer to Figure 4B. After 21 days of culture, the number of breast cancer tumor cells in the hypoxic environment group expanded from 50 to about 3.5 x ¹⁰⁷ breast cancer tumor cells, which has a significant expansion effect compared with the hyperoxic environment group or the control group. In addition, although the hyperoxic environment group also has a significant expansion effect compared with the control group, the expansion effect is far less than that of the hypoxic environment group. The experimental results show that the serum-free expansion culture medium of the present invention can effectively expand breast cancer tumor cells, and culturing in a hypoxic environment can produce a more significant expansion effect, which proves that the tumor cell expansion method of the present invention has the effect of expanding tumor cells.

Embodiment 3. Proliferation of colorectal cancer tumor cells

In this example, the tumor cell expansion method of the present invention is used to expand and culture colorectal cancer tumor cells (DLD-1 colorectal cancer cells, ATCC number: CCL-221) to verify the expansion effect of the tumor cell expansion method of the present invention on colorectal cancer tumor cells.

In this embodiment, the experimental design and material methods are the same as those described in Example 1.

result

Please refer to Figure 5A. Compared with the control group, the number of cells in the hypoxic environment group and the hyperoxic environment group increased significantly on the 14th and 21st days of expansion culture. Please refer to Figure 5B. After 21 days of culture, the hypoxic environment group expanded from 50 colorectal cancer tumor cells to about 6 x ¹⁰⁷ colorectal cancer tumor cells, which has a significant expansion effect compared with the hyperoxic environment group or the control group. In addition, although the hyperoxic environment group also has a significant expansion effect compared with the control group, the expansion effect is far less than that of the hypoxic environment group. The experimental results show that the serum-free expansion culture medium of the present invention can effectively expand colorectal cancer tumor cells, and culturing in a hypoxic environment can produce a more significant expansion effect, which proves that the tumor cell expansion method of the present invention has the effect of expanding tumor cells.

Embodiment 4. Expansion and detection of circulating tumor cells

This embodiment uses the circulating tumor cell detection method of the present invention to expand and culture peripheral blood samples containing circulating tumor cells to verify that the circulating tumor cell detection method of the present invention can effectively expand the circulating tumor cells in the peripheral blood and serve as a basis for detection.

Materials and methods

This embodiment uses peripheral blood samples from patients with non-small cell lung cancer for testing, wherein the sample contains about 10 circulating tumor cells per 8 mL of peripheral blood. First, 8 mL of the patient's peripheral blood is centrifuged through a Ficoll-Paque density gradient separation reagent to separate a peripheral blood mononuclear cell sample, wherein the peripheral blood mononuclear cell sample contains about 10 circulating tumor cells. 50 uL of Matrigel was added to an 8 μm transwell to simulate the extracellular matrix (ECM), and then the peripheral blood mononuclear cell sample was added to the upper chamber. The cells with migration ability would penetrate the simulated extracellular matrix to reach the lower chamber, thereby screening out the cells with migration ability. In addition, since immune cells cannot penetrate the simulated extracellular matrix to reach the lower chamber, the screening can also effectively remove immune cells and prevent them from affecting the subsequent growth of circulating tumor cells. After screening, the cell samples in the lower chamber were collected and transferred to an ultra-low attachment round bottom 96-well plate. The cells were centrifuged at 800 x g for 8 minutes to aggregate into 3D spherical structures. 50 uL of Matrigel was added to coat the cell clusters in the 3D spherical structure to simulate the environment of extracellular matrix coating cells.

The cell samples were divided into a control group and an experimental group. The control group was cultured in a serum-free medium under a hypoxic environment of 2% (v/v) oxygen concentration for 21 days, while the experimental group was cultured in a serum-free expansion medium (containing FGF-3 and glutamine) under a hypoxic environment of 2% (v/v) oxygen concentration for 21 days. The medium was changed every 7 days in each group. The proportion of circulating tumor cells was analyzed by flow cytometry on the 1st, 7th, 14th and 21st days of expansion, and the number of circulating tumor cells was calculated. The symbol of statistical significance * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

In the flow cytometric analysis of this embodiment, a cell fluid sample was added with a flow cytometry staining buffer. For the analysis of circulating tumor cells, Alexa Fluor ^® 488-labeled anti-CD326 antibody (Anti-Hu CD326 Alexa Fluor ^® 488, purchased from EXBIO, product number: A4-582-T100) and phycoerythrin (PE)-labeled anti-cytokeratins antibody (Anti-Cytokeratins PE, purchased from EXBIO, product number: 1P-108-C100) were used for identification and analysis. After incubating the fluorescent markers for 30 minutes in the dark, the flow was collected by flow cytometry (BD FACS AriaIII, BD Biosciences, USA). The fluorescent signals were acquired and analyzed by drawing areas and box selection in the analysis software to calculate the number of cells expressing in the cell fluid, and then the proportion of the cells in the total cell sample and other data results were calculated.

result

Please refer to Figure 6. On the first day of expansion culture, the number of circulating tumor cells in the cell sample accounted for about 0.63% of the total cell sample. After culture, the proportion of circulating tumor cells in the experimental group increased to 14.78%, 56.99% and 93.27% of the total cell sample on the 7th, 14th and 21st days, respectively. In contrast, the proportion of circulating tumor cells in the control group on the 7th, 14th and 21st days was 2.34%, 2.62% and 3.90%, respectively. The proportions of circulating tumor cells in the cell samples were 0.58%, 1.51% and 1.84% respectively. Please refer to Figure 7A. The proportion of circulating tumor cells in the experimental group on days 7, 14 and 21 showed a significant upward trend and was significantly higher than that in the control group. Please refer to Figure 7B. The number of circulating tumor cells in the experimental group on days 7, 14 and 21 showed a significant increase, especially on day 21, the number of circulating tumor cells expanded to about 1.4 x 10 ⁷ , which was significantly higher than that in the control group. The experimental results show that the expansion technology described in the present invention can effectively expand a very small number of circulating tumor cells in peripheral blood mononuclear cell samples. The initial approximately 10 circulating tumor cells can be expanded to approximately 10 ⁷ after 21 days of culture, and the expansion effect is a million times. Moreover, the purity of circulating tumor cells can reach more than 90% without the need for a sorting system. After expansion, it is easy to identify whether the original sample contains circulating tumor cells, which becomes the basis for the detection of circulating tumor cells. The expanded circulating tumor cells can also be further analyzed.

In the method for detecting circulating tumor cells of the present invention, if the subject's peripheral blood does not contain circulating tumor cells, the proliferation of circulating tumor cells will not be observed after the expansion culture of the present invention. On the contrary, if the subject's peripheral blood contains circulating tumor cells, even if the number is very rare, circulating tumor cells will still be observed after the expansion culture of the present invention. The large-scale expansion of circulating tumor cells can be used as a basis for detecting whether there are circulating tumor cells in peripheral blood. The results are very intuitive and sensitive, and there is no need to go through a microfluidic system or a magnetic bead sorting system, which greatly reduces the difficulty and cost of circulating tumor cell detection. The expanded circulating tumor cells can be further analyzed later, which is of great value to the fields of clinical detection and precision medicine.

The present invention is disclosed as above through the above-mentioned embodiments, which are only some of the preferred embodiments of the present invention, but they are not used to limit the present invention. Any person familiar with this technical field with common knowledge, after understanding the above-mentioned technical features and embodiments of the present invention, and making equivalent changes or modifications without departing from the spirit and scope of the present invention, still falls within the scope of the present invention, and the scope of patent protection of the present invention shall be defined by the claim items attached to this specification.

11-14: Steps 21-27: Steps

FIG1 is a flow chart of the tumor cell expansion method of the present invention.

FIG. 2 is a flow chart of the method for detecting circulating tumor cells of the present invention.

FIG3A is a microscopic image of the hypoxic environment group of the lung cancer tumor cell expansion experiment in Example 1 of the present invention on days 0, 11, 18 and 20. FIG3B is a line graph of the cell number on days 1, 7, 14 and 21 of the lung cancer tumor cell expansion experiment in Example 1 of the present invention. FIG3C is a graph of the cell number results of the lung cancer tumor cells in Example 1 of the present invention on day 21 of expansion culture.

FIG4A is a line graph of the cell counts on days 1, 7, 14 and 21 of the breast cancer cell expansion experiment in Example 2 of the present invention. FIG4B is a graph of the cell counts on day 21 of the breast cancer cell expansion culture in Example 2 of the present invention.

FIG5A is a line graph of the cell counts on days 1, 7, 14 and 21 of the colorectal cancer tumor cell expansion experiment in Example 3 of the present invention. FIG5B is a graph of the cell counts on day 21 of the colorectal cancer tumor cell expansion culture in Example 3 of the present invention.

FIG6 is a flow cytometer analysis result diagram of Example 4 of the present invention on days 1, 7, 14 and 21 of expansion culture.

FIG7A is a line graph showing the percentage of circulating tumor cells on days 1, 7, 14 and 21 of the expansion culture of Example 4 of the present invention. FIG7B is a line graph showing the number of circulating tumor cells on days 1, 7, 14 and 21 of the expansion culture of Example 4 of the present invention.

Claims (8)

A method for expanding tumor cells, comprising: providing a tumor cell; placing the tumor cell in an ultra-low attachment multi-well plate for centrifugation to aggregate the cells into a 3D spherical structure; applying a matrix gel to coat the 3D spherical structure; and performing suspension culture in a serum-free expansion medium, wherein the serum-free expansion medium comprises RPMI 1640 culture medium, 8 to 12 ng/ml fibroblast growth factor 3, and 1 to 3 mM glutamine. A method for expanding tumor cells as described in claim 1, wherein the culture is carried out in a low oxygen environment with an oxygen concentration of 1 to 3% (v/v). The method for expanding tumor cells as described in claim 1, wherein the centrifugation speed is 700 to 900 x g and the centrifugation time is 6 to 10 minutes. A method for expanding tumor cells as described in claim 1, wherein the tumor cells are lung cancer tumor cells, breast cancer tumor cells or colorectal cancer tumor cells. A method for detecting circulating tumor cells, comprising: providing peripheral blood; subjecting the peripheral blood to density gradient centrifugation to separate peripheral blood mononuclear cell samples; screening the peripheral blood mononuclear cell samples for migration ability to obtain a target cell sample; placing the target cell sample in an ultra-low attachment multi-well plate for centrifugation to aggregate the cells into a 3D spherical structure; applying a matrix gel to coat the 3D spherical structure; Performing suspension culture with a serum-free expansion culture medium, wherein the serum-free expansion culture medium comprises RPMI 1640 culture medium, 8 to 12 ng/ml fibroblast growth factor 3 and 1 to 3 mM glutamine; and analyze the number of circulating tumor cells to determine whether the peripheral blood contains circulating tumor cells. A method for detecting circulating tumor cells as described in claim 5, wherein the peripheral blood is from a subject. A method for detecting circulating tumor cells as described in claim 5, wherein the screening is performed using an 8μm transwell and matrigel to simulate the extracellular matrix to screen out circulating tumor cells with migration ability. A method for detecting circulating tumor cells as described in claim 5, wherein the culture is carried out in a hypoxic environment with an oxygen concentration of 1 to 3% (v/v).