TWI703215B - A method of in vitro diagnostic for prediction of drug efficacy - Google Patents

Abstract

A 3D organoid is used for diagnosis or assay, wherein the 3D organoid is constructed from a tumor cell. The tumor cell in the 3D organoid is from a cell line, from circulating tumor cells isolated from a patient, or from a tumor tissue. The 3D organoid is constructed using an aqueous gel material and an adhesion molecule. The aqueous gel material is hydrogel. The adhesion molecule is ICAM-1 or galectin-3. The 3D organoid is constructed by forming a scaffold with an aqueous gel material, followed by adding a mixture of the tumor cell and an adhesion molecule.

Description

In vitro diagnostic methods for predicting drug efficacy

The present invention generally relates to diagnostic or analytical methods for predicting the efficacy or effects of drugs.

Traditional 2D cell cultures in preclinical research can provide many important predictions of drug efficacy. However, in the clinic, the patient's response to drugs or gene performance is very different from them in the preclinical environment. Therefore, the new 3D organoid system can provide an improved drug testing environment and a cell model that simulates the clinical environment to illustrate the effects of drugs.

3D organoids are mimics of miniaturized organs produced in vitro in 3D. 3D organoids are usually derived from one or several cells from tissues, embryonic stem cells or induced pluripotent stem cells, which can propagate and self-organize in 3D culture due to their self-renewal and differentiation capabilities.

3D organoid models can be used to establish the structure and function of specific tissues. Since its structure and model are very similar to real tissues, 3D organoid models can provide appropriate cell-cell and cell-cell matrix interactions, thereby making up for the shortcomings of the conventional 2D in vitro cell culture systems, which lack Tumor heterogeneity and molecular diversity, cell heterogeneity, tumor microenvironment and tissue characteristics can be seen in clinical patients. In vitro models based on 3D organoids can be used for anti-cancer drug development/testing, and can also provide timely analysis data for screening therapeutic agents for recurrent tumors.

The embodiment of the present invention relates to a 3D organoid system for diagnosing or analyzing the efficacy of drugs. One aspect of the present invention relates to 3D organoids for diagnosis or analysis. The 3D organoid according to an embodiment of the present invention can be constructed by tumor cells. The tumor cells in 3D organoids can be derived from cell lines, circulating tumor cells isolated from patients, or tumor tissues. 3D organoids can be constructed using water-based gel materials and adhesion molecules. The aqueous gel material may be a hydrogel. The adhesion molecule can be ICAM-1 or galectin-3. 3D organoids can be constructed by forming a scaffold with a hydrogel material, and then adding a mixture of tumor cells and adhesion molecules.

One aspect of the present invention relates to a method for analyzing the efficacy or response of drugs using the 3D organoids of the present invention. The method according to an embodiment of the present invention includes adding a drug to the 3D organoid and observing the effect of the drug on the cells in the 3D organoid. The effects of drugs are analyzed using imaging systems to analyze the expression levels of genes or proteins. The effect of the drug is analyzed by fluorescent labeling, luminescence or bright light.

Other aspects of the present invention will become apparent from the following embodiments and drawings.

The embodiment of the present invention relates to a 3D organoid system for diagnosing or analyzing the efficacy of drugs. The embodiments of the present invention also relate to a method for establishing a 3D organoid system and a method for using 3D organoids to analyze the therapeutic effects of drugs.

3D organoid tumor culture can be used to establish the structure and function of tissues. The structure and function of 3D organoids are highly similar to actual tissues. It can provide cell-cell interaction as well as cell-matrix interaction. Therefore, 3D organoid models can overcome the shortcomings of in vitro 2D cell cultures. 2D cell cultures usually cannot present the clinically observed tumor heterogeneity and molecular diversity, cell heterogeneity, tumor environment and tissue characteristics. The use of 3D organoid models can provide timely information about anti-tumor drug testing and drug screening information for the treatment of tumor recurrence.

The 3D organoid according to an embodiment of the present invention can be constructed by tumor cells. The tumor cells in 3D organoids can be derived from cell lines, circulating tumor cells isolated from patients, or tumor tissues. Water-based gel materials and adhesion molecules can be used to construct 3D organoids. The aqueous gel material may be a hydrogel. The 3D organoid of the present invention can be constructed by forming a scaffold with a hydrogel material, and then adding a mixture of tumor cells and adhesion molecules.

The inventors of the present invention found that adding adhesion molecules to the culture can greatly improve 3D organoid formation. The adhesion molecule can be any suitable adhesion molecule known in the art, such as ICAM-1 and/or galectin-3.

The 3D organoids of the present invention can be used to analyze the efficacy of drugs. The drug analysis method according to an embodiment of the present invention includes adding a drug to a 3D organoid and observing the effect of the drug on the cells in the 3D organoid. Any suitable method can be used, such as using an imaging system to analyze the effects of drugs to analyze the expression levels of genes or proteins. For example, using imaging systems, microscopes and fluorescent markers, luminescence or white light can be used to analyze the effects of drugs.

To test drug efficacy, tumor cells from patients, such as tumor tissues or circulating tumor cells (CTC) isolated from liquid biopsies, can be used to establish 3D organoid models. Alternatively, an organoid system can be established using cells with the same genetic background as the tumor of interest in the patient. Several 3D organoid systems or tumor models have been established.

For drug efficacy analysis, 3D organoids can be created in 24-well or 96-well plates. Then, various concentrations of drugs can be added to each well. Next, the effect of the drug on the cells will be monitored, for example by monitoring enzymes or markers. For example, ATPase activity can be monitored as an indicator of cell activity.

In addition, optical methods can be used to image 3D organoid cells to understand the effects of drugs. For example, single-molecule RNA FISH can be used to monitor the performance of specific genes known to be affected by drugs. Bioimaging systems can be used to monitor and quantify gene expression. The quantitative information from this type of analysis can be used to obtain IC50 values or to obtain z-scores from various drugs.

According to embodiments of the present invention, 3D organoids can be used to establish drug dosage relationships. For example, the cancer cells are seeded in 24-well or 96-well plates. Once a 3D organoid model is established from cancer cells. The drug to be tested can be added to the 3D organoid system at different concentrations (for example, 10 nM-1,000 nM). After incubation with drugs, the total RNA of the cells can be extracted to produce cDNA (for example, using PCR). Then, cDNA can be used in qPCR to monitor the performance of specific markers under the action of drugs.

For example, 3 genes are known to be associated with lung cancer. Based on these three genes, corresponding cell lines can be found to produce 3D organoids. After the establishment of 3D organoids, these 3D organoids can be used to evaluate the effect of targeted therapy on the expression of these three genes.

The following specific examples will be used to further illustrate the embodiments of the present invention. Those familiar with the art will understand that these examples are only for illustration and not for limiting the scope of the present invention. Those skilled in the art will understand that these examples can be modified and changed without departing from the scope of the present invention. Example 1: Galectin-3 improves 3D tumor organoid formation

Two different lung cancer cell lines (HCC-827 and NCI-H727) derived from in vitro culture were used to establish 2D cell culture and 3D organoid cell culture, respectively. In short, 1×10 ⁵ cells/ml lung cancer cells were added to each well of a 96-well low-attachment plate, where each well also contained a specific concentration of adhesion molecules (such as galectin-3). The cells are cultured to form 3D organoids. The growth and size changes of 3D organoids can be monitored using a microscope.

As shown in Figure 1, on the 4th day, 3D organoids contained adhesion molecules (galectin-3, Gal-Galectin-3, Gal- 3) The hole is established and mature. In the presence of adhesion molecules, the size of 3D organoids is about 50% larger. In addition, NCI-H727 cells will form a ribbon structure in the presence of adhesion molecules. These results indicate that adhesion molecules can support the formation of larger 3D organoids. Example 2: Hydrogel and Galectin-3 can stimulate the formation of vascular 3D organoids

Hydrogels can be prepared with different ratios of collagen and PEG, such as 1:2, 1:4, 1:6, and 1:8 (collagen:PEG). For example, 3 mg/ml type I collagen and 300 mg/ml PEG (for example, 7500 MW) are added to the cell culture medium (containing 10% bovine serum or 5% human serum). This solution is thoroughly mixed with a reconstituted solution composed of acetic acid and 10X MEM cell culture medium, and then allowed to stand in a cell culture incubator for 20 minutes or more to form a hydrogel.

A homogeneous mixture of 1×10 ⁵ cells/ml tumor cells, 2×10 ⁴ cells/ml vascular endothelial cells (tumor cells: vascular endothelial cells = 5:1) and 1 μg/mL galectin-3 was prepared. This homogeneous mixture is injected into the hydrogel prepared above. The hydrogel was placed in a cell incubator (37°C, 5% CO ₂ ) for incubation. An automatic multifunctional optical imaging system (Cytation 5, Bio-Tek, USA) was used to monitor the growth process. Figure 2 shows the progression of 3D organoid formation over time with and without Gal-3 and with or without EC (endothelial cells).

As shown in Figure 2, hydrogels prepared with collagen:PEG ratios of 1:4, 1:6, and 1:8 can better support the formation of cell masses (organoids). With the addition of Gal-3 (adhesion molecule), the formation of 3D organoids can be further enhanced. By adding endothelial cells (EC), the formation of blood vessels can be observed. Example 3-1: Compared with 3D organoid system, genes behave differently in 2D cell culture system

In this example, 4 different lung cancer cell lines (HCC-827, NCI-H727, A549 and H1975) derived from in vitro cultures were used to establish 2D cell and 3D organoid culture systems. The procedure is as follows.

A 96-well low-attachment plate was seeded with 1×10 ⁵ cells/ml lung cancer cells and a specific concentration of adhesion molecules to cultivate 3D organoids. Observe and record the size of the 3D organoids under a microscope (see Figure 1).

In order to establish a 2D cell culture model, the procedure is as follows: Inoculate 4 human lung cancer cell lines (1×10 ⁵ cells/ml) in a 24-well plate. The plates were incubated overnight at 5% CO ₂ and 37°C. Confirm under the microscope that the cells completely cover the hole, and then collect the cells.

In order to establish a 3D organoid model, the procedure is as follows: 4 human lung cancer cell lines (6×10 ⁵ cells/ml) were respectively inoculated in a low-attachment 24-well plate. Incubate the plates under the conditions of 5% CO ₂ and 37°C until the 7th day. On the 7th day, a 3D organoid cell population was collected.

After collecting cells from 2D cell culture and 3D organoids, total RNA was extracted from these cells respectively. Next, cDNA is synthesized from total RNA to construct a cDNA form.

Real-time quantitative polymerase chain reaction (qPCR) was used to analyze the gene expression levels of PIK3CA, EGFR and KRAS in cDNA from 2D cell cultures and 3D organoids.

The expression levels of PIK3CA, EGFR and KRAS from these analyses are shown in Figure 3. As shown in Figure 3, in most lung cancer cell lines, the expression levels of EGFR, PIK3CA and KRAS in 3D organoid cultures are relatively low compared with those in the 2D culture system. Example 3-2: Therapeutic effect of PLX3397 (CSF1R inhibitor)

testing method:

2D cell culture: 4 human lung cancer cell lines (1×10 ⁵ cells/ml) were respectively inoculated in a 24-well plate. The plates were incubated overnight under the conditions of 5% CO ₂ and 37°C.

It was confirmed under the microscope that the cells completely covered the wells, and then 10, 100, and 1,000 nM PLX3397 preformed solutions were added to the wells. Then, incubate the plate for 24 hours under the conditions of 5% CO ₂ and 37°C.

3D organoid culture: 4 human lung cancer cell lines (6×10 ⁵ cells/ml) were respectively inoculated in a low-attachment 24-well plate. Incubate the plates under the conditions of 5% CO ₂ and 37°C until the 7th day. On the 7th day, 10, 100, and 1,000 nM preformed solutions of PLX3397 were added to the wells, respectively. Then, incubate the plate for 24 hours under the conditions of 5% CO ₂ and 37°C.

After collecting cells from 2D cell culture and 3D organoids, total RNA was extracted from these cells respectively. Next, cDNA is synthesized from total RNA to construct a cDNA form.

Real-time quantitative polymerase chain reaction (qPCR) was used to analyze the gene expression levels of PIK3CA, EGFR and KRAS in cDNA from 2D cell cultures and 3D organoids. The expression levels of PIK3CA, EGFR and KRAS after drug treatment are shown in Figure 4.

As shown in Figure 4, using the 2D cell culture system, in response to the treatment of Pexidartinib (Pexidartinib, PLX3397; CSF1R inhibitor) in HCC-827 and NCI-H727 cells, the gene expression levels of PIK3CA, EGFR and KRAS will increase. But it did not increase in A549 and H1975 cells. These results were found in both wild-type and mutant KAS. However, with 3D organoid system, only PIK3CA will increase after treatment with PLX3397, but EGFR and KRAS will not increase.

These results clearly indicate that the 2D cell culture system and the 3D organoid system produce inconsistent results. As discussed in the following article section, the results of these 3D organoid systems can be verified by in vivo animal xenograft tests, but the results from the 2D cell culture system cannot be supported by in vivo animal models. Example 4: Using 3D organoid tumor culture system to evaluate the therapeutic effect of drugs

To perform these tests, several lung cancer cell lines cultured in vitro (for example, HCC-827, NCI-H460, NCI-H727, and NCI-H1975) and colorectal cancer cells (for example, HCT-116 and HT-29) are used. ) Establish 2D cell culture and 3D organoid tumor system. In short, 1×10 ⁴ cells/ml tumor cells were placed in each well of a 96-well low attachment plate to cultivate 2D cell cultures and 3D organoid tumor systems.

As an example of drug efficacy testing, 11 different chemotherapeutics, targeted therapeutics and immunomodulators (0, 0.01, 0.1, 1, and 10 nM) and caspase 3/7 fluorescence detection kit Group reagents were individually tested in 2D cell cultures and 3-day-old 3D organoid tumor systems. The reaction was allowed to proceed for 24 hours.

After 24 hours, the caspase 3/7 fluorescence yield in 2D cell cultures and 3D organoids was quantified using an automatic multifunctional optical imaging system (Cytation 5, Bio-Tek, USA). The result is shown in Figure 5.

As shown in Figure 5, different therapeutic agents have different effects on different tumor cells. This department is well known. It is worth noting that there is a discrepancy between the 2D cell culture system and the 3D organoid system. For example, Paclitaxel, Gefitinib, and Erlotinib have been shown to be effective against HCC-827 lung cancer cells in a 2D cell culture system, and these same drugs are effective in 3D organoids. Invalid in the system. In contrast, the 3D organoid system shows that only Afatinib is effective against HCC-827. This inconsistency is due to the inaccurate results of the 2D cell culture system, because the results of the 3D organoid system were verified in the in vivo animal xenograft model, but the results of the 2D cell culture system were not verified (see the following section and figure 6). Example 5: Using animal xenotransplantation models to verify 2D cell culture system and 3D organoid system

HCC-827 lung cancer cells were injected subcutaneously to establish a xenograft model, which was used to verify the results of the drug efficacy test from the 2D cell culture system and the 3D organoid system. Briefly, 0.1 ml of HCC-827 lung cancer cells (2×10 ⁶ cells/ml) were injected subcutaneously into the back of mice. After 1 week, the tumor size was measured with a digital caliper. When the tumor grows to a size of 100-150 mm ³ , drug testing can begin.

Afatinib was administered via an oral feeding tube 5 times a week, and paclitaxel was administered once a week for 4 weeks. The tumor size and animal body weight were measured twice a week for 4 weeks. The animal test results are shown in Figure 6.

As shown in Figure 6, paclitaxel at 5 mpk or 20 mpk is not effective in inhibiting tumor growth. In contrast, 5 mpk or 20 mpk (mg/kg) afatinib is very effective in inhibiting tumor growth. These results are consistent with the results observed using the 3D organoids of the present invention, thereby verifying that the 3D organoids of the present invention are used for drug efficacy testing.

Referring to Figure 5, the 2D cell culture system also shows that paclitaxel is effective. This is not verified using in vivo xenograft models. This result proves that the conventional 2D cell culture system is not as reliable as the 3D organoid system of the present invention. Example 6: Patient-derived tumor reconstruction in 3D organoid system

The patient's colorectal cancer cells were inoculated into mice to establish xenografts. A 300 mm ³ colorectal cancer tissue was taken out from the xenograft, and the tissue was digested with collagenase NB4G (0.5 PZU/mL) for 40 minutes to separate the cells.

Wash the cells with HBSS buffer. Centrifuge and take cell suspension for experiment. Approximately 5×10 ⁴ cells were added to the cell culture medium of the hydrogel in each well of 96 wells, and galectin-3 (Gal-3, 1 μg/mL) was added to each well. An automated multifunctional optical imaging system (Cytation 5, Bio-Tek, USA) was used to analyze the size of the tumor mass in the hole in the next few days. The result is shown in Figure 7.

As shown in Figure 7, with or without the addition of adhesion molecules (Gal-3), tumor masses basically grew over 4 days. However, the holes with Gal-3 produced better organized tumor masses than the holes without added adhesion molecules. Example 7: 3D organoid model containing vascular cells

The hydrogel prepared from collagen and PEG (MW=7500; ratio 1:2) was placed in the wells of a 96-well plate. In short, the cell culture medium (containing 10% bovine serum or 5% human serum), 3 mg/ml type I collagen, 300 mg/ml PEG (7500 MW) and a reconstitution solution composed of acetic acid and 10X MEM cell culture medium Mix, and then allow the mixture to stand for 20 minutes or more in a cell incubator to form a hydrogel.

Mix 1×10 ⁵ cells/ml tumor cells, 2×10 ⁴ cells/ml vascular endothelial cells (tumor cells: vascular endothelial cells = 5:1) and 1 μg/ml Gal-3. The mixture was poured into the hydrogel and the plate was incubated in a cell incubator (37°C, 5% CO ₂ ) for 2 days to allow the formation of tumor masses containing blood vessels.

Add 2 μl of anti-CD31 antibody [JC/70A] (Alexa Flour ^® 647) ab215912 to each well from the above-incubated plate to stain vascular endothelial cell markers, and allow the reaction to proceed in an incubator for 60 minutes (ensure Block the lights).

Add 0.05% Triton X100 to permeabilize the cells and allow the reaction to proceed on ice for 15 minutes. Next, 450 nM DAPI (4',6-diamidino-2-phenylindole dihydrochloride, D1306, Invitrogen™) stain was added to stain the cells. The staining was allowed to proceed for 1 hour in the dark, and then the container was placed in a cell incubator for 12-16 hours.

An automatic multifunctional optical imaging system (Cytation 5, Bio-Tek, USA) was used to measure the fluorescence intensity of anti-CD31 and DAPI staining. The result is shown in Figure 8.

As shown in Figure 8, the procedure for 3D organoids has been repeated as Test 1, Test 2 and Test 3. The image shows the formation of blood vessels in organoids. All three experiments produced consistent results.

The above examples clearly show that 3D organoids can be effectively constructed with the aid of adhesion molecules (for example, galectin-3, ICAM-1 or similar adhesion molecules). In terms of representing the microenvironment in vivo, 3D organoids are more accurate than 2D cell cultures, and therefore, 3D organoids can be used to accurately assess the therapeutic effects of drugs. In addition, 3D organoids including blood vessel formation can be established to more accurately represent the tumor microenvironment in vivo.

The embodiments of the present invention have been described with a limited number of examples. However, those familiar with the art will understand that these specific procedures are only for illustration, and other changes and modifications can be made without departing from the scope of the present invention. Therefore, the scope of the present invention should be determined by the scope of the attached patent application.

Figure 1 shows the improvement of 3D organoid cultures by adding adhesion molecules. The concentrations of adhesion molecules (galectin-3, Gal-3) were 0.3 μg/mL, 0.6 μg/mL, 1.25 μg/mL, and 2.5 μg/mL. In the presence of adhesion molecules, the size of 3D organoids is about 50% larger. In addition, NCI-H727 cells form a ribbon structure in the presence of adhesion molecules. These results indicate that adhesion molecules can support the formation of larger 3D organoids.

Figure 2 shows the effect of different hydrogels prepared with different collagen/PEG ratios on organoid formation. Collagen:PEG ratios of 1:4, 1:6 and 1:8 can better support the formation of cell masses (organoids). With the addition of Gal-3 (adhesion molecule), the formation of 3D organoids can be further enhanced. By adding endothelial cells (EC), the formation of blood vessels can be observed.

Figure 3 shows the differential gene expression in 2D cell cultures compared to 3D organoids. For example, in most lung cancer cell lines, the expression levels of EGFR, PIK3CA, and KRAS in 3D organoid cultures are relatively low compared with those in 2D culture systems.

Figure 4 shows the different effects of drug treatment in 2D cell cultures compared to 3D organoids. In response to the treatment of pexidanib (PLX3397; CSF1R inhibitor) in HCC827 and NCI-H727 cells, the gene expression levels of PIK3CA, EGFR and KRAS will increase. Compared to those in 2D cultures, lower expression levels were observed in 3D organoid cultures. PLX3397 treatment showed no dose-dependent effect in A549 and H1975 cells.

Figure 5 shows the effects of various therapeutic agents in 2D cell cultures or 3D organoids. Different therapeutic agents have different effects on different tumor cells. However, the 2D cell culture system and the 3D organoid system showed different results. For example, paclitaxel, gefitinib, and erlotinib have been shown to be effective on HCC-827 lung cancer cells in a 2D cell culture system, while these same drugs are ineffective in a 3D organoid system. In contrast, the 3D organoid system shows that only afatinib is effective against HCC-827. It is shown that this inconsistency is due to the inaccurate results of the 2D cell culture system.

Figure 6 shows the results of verifying the 3D organoid system using in vivo animal models. Paclitaxel at 5 mpk or 20 mpk is not effective in inhibiting tumor growth. In contrast, 5 mpk or 20 mpk (mg/kg) afatinib is very effective in inhibiting tumor growth. These results are consistent with the results observed using the 3D organoids of the present invention, thereby verifying that the 3D organoids of the present invention are used for drug efficacy testing.

Figure 7 shows the results of 3D organoids established with patient-derived xenografts. Compared with those without the addition of adhesion molecules, the addition of Gal-3 produced better organized tumor mass.

Figure 8 shows a 3D organoid model containing vascular cells.

Claims (12)

A 3D organoid used for diagnosis or analysis, wherein the 3D organoid is constructed by tumor cells and adhesion molecules, wherein the tumor cells are lung cancer or colorectal cancer cells, and wherein the adhesion molecule is galectin-3. The 3D organoid of claim 1, wherein the tumor cells in the 3D organoid are derived from a cell line, a circulating tumor cell isolated from a patient, or a tumor tissue. Such as the 3D organoid of claim 2, wherein the 3D organoid is constructed using hydrogel materials and adhesion molecules. The 3D organoid of claim 3, wherein the hydrogel is prepared with PEG and collagen at a ratio of PEG:collagen=1 to 25:1. Such as the 3D organoid of claim 1, wherein the concentration of the adhesion molecule is 0.3 μg/mL to 2.5 μg/mL. The 3D organoid of claim 1, wherein the 3D organoid is constructed by forming a scaffold with a hydrogel material, and then adding the mixture of tumor cells and adhesion molecules. The 3D organoid of claim 6, wherein the aqueous gel material comprises PEG and collagen, wherein the ratio of PEG: collagen is 1 to 25:1. Such as the 3D organoid of claim 6, wherein the concentration of the adhesion molecule is 0.3 μg/mL to 2.5 μg/mL. The 3D organoid of claim 1, which further comprises blood vessels formed from endothelial cells in the 3D organoid. A method for analyzing the efficacy or response of a drug using a 3D organoid as claimed in Claim 1, which comprises adding a drug to the 3D organoid and observing the effect of the drug on the cells in the 3D organoid. The method of claim 10, wherein the effect of the drug is analyzed using an imaging system to analyze the biochemical activity and/or expression level of the gene or protein. The method of claim 11, wherein the effect of the drug is to use fluorescent labeling, luminescence or white light analysis.

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