A method for constructing tumor-like tissue based on circulating tumor cells and drug sensitivity test

Abstract

The present application relates to the field of tumor organoids and personalized medicine screening, and particularly relates to a tumor-like tissue construction and drug sensitivity testing method based on circulating tumor cells. The present application collects various cells through gradient centrifugation and annular microfluidic sorting, and then establishes a standardized method for separating, screening and culturing multi-cell subtype tumor-like tissues through self-made materials and culture solution. This method is universal, and the obtained tumor-like tissue contains at least circulating tumor cells, tumor stem cells, macrophages and other multi-cell subtypes. The personalized drug sensitivity test can be performed within two weeks of obtaining the patient's peripheral blood. After drug screening and clinical data comparison, the consistency of the present application and the actual drug use of the patient reaches more than 80%. Compared with the prior art, the present application has the advantages of rapidity, cheapness and accuracy, and can save the time and risk of the patient's drug trial, and has great clinical significance in personalized tumor drug guidance, especially for patients with advanced cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tumor organoids and personalized medicine screening, and in particular to a method for constructing tumor-like tissue based on circulating tumor cells and drug sensitivity testing. BACKGROUND

[0002] Circulating tumor cells (CTCs) and cancer stem cells (CSCs) are small cell populations found in cancer patients. CTCs have been recognized as the tumor avatar for real-time monitoring of cancer, while CSCs are the most malignant tumor cells that play a dominant role in drug resistance and metastasis. These two types of cells share the same surface markers, such as epithelial cell adhesion molecule (EpCAM), CD44 and CD133 (Table 1 and Table 2). CTCs are generally identified as nucleated cells expressing epithelial marker cytokeratin (CK) / EpCAM but not CD45. However, the drawback of current CTC capture techniques by recognizing epithelial-specific markers is that they cannot detect CTCs that have undergone epithelial-mesenchymal transition (EMT) and no longer overexpress EpCAM.

[0003] In traditional medicine, physicians must continuously try various anticancer drug regimens based on existing guidelines and their own experience to determine the final effective treatment regimen, resulting in limitations in tumor treatment. Therefore, capturing CTCs from blood samples is crucial. Although these rare cells can be captured, their expansion remains challenging due to their limited number. Generally, CTC capture methods from patients can be divided into chemical and physical methods. Common enrichment methods for CTCs include filter membrane, immunomagnetic beads (positive, negative), and microfluidic chips. The most reliable method is currently the use of cell surface markers, such as EpCAM. EpCAM is a glycoprotein that is expressed on most tumor cells, including those of the lung, pancreas, prostate, breast, esophagus, and liver. Other surface markers can also be used to capture CTCs from specific cancer cells, such as prostate-specific membrane antigen (PSMA) for prostate cancer. However, CTCs captured from these systems are difficult to grow in vitro using traditional cell culture protocols. Immunomagnetic bead enrichment of CTCs is the most commonly used technique for capturing CTCs. It relies on magnetic bead separation technology, which uses antibodies coupled to magnetic beads to enrich CTCs from blood samples. Antibodies bind to CTCs by targeting epithelial or tumor-specific antigens or exclude CTCs by targeting blood cell antigens (e.g., CD45). With technological advancements, material-based methods, including cell size-based filtration, cell density-based separation, hydrodynamic separation, DEP-based cell sorting, acoustics, and immunomagnetic separation, have been proposed over the past decade. Nanomaterials have great potential in improving capture efficiency due to their high surface-to-volume ratio and enhanced cell-surface interactions. Although many nanostructured surfaces have been proposed for cell culture, only a few capture systems, such as Cytolumina's CytoTrapNano (also known as NanoVelcro chips), are available on the market. CTCs have also been published using cell size separation. Filter membrane is one of the most representative ways to separate CTCs using cell size separation, which separates CTCs by taking advantage of the size difference between tumor cells and normal cells. CTCs have a diameter of about 10-20 μm, while blood cells are 7-12 μm in size. By filtering, larger volume CTCs are left behind. However, there are two drawbacks. First, the separation effect is poor, and the difference in diameter between CTCs and white blood cells is not sufficient to support effective separation. Second, the recovery rate is low, and CTCs can deform through small pores, resulting in CTCs being missed, but these cells may be more tumor stem-like, making the prognosis worse in clinical practice. Based on the characteristic that CTCs are larger in volume than blood cells, CTCs are captured.Newer research is using spiral particle sorting to separate cells of different sizes and masses. By taking advantage of the size difference between tumor cells and normal cells, tumor cell populations can be successfully separated.

[0004] Currently, CTCs are difficult to grow in vitro using existing screening methods. One possible reason is that capturing CTCs using cell surface markers (such as EPCAM) hinders cell adhesion and proliferation in subsequent cell culture. Therefore, we hypothesized that capturing CTCs without blocking cell surface markers may be a reliable method for expanding cells in vitro while maintaining the phenotype of captured CTCs. CTC detection technology has continued to develop in recent years. However, due to different enrichment and detection methods, CTC detection methods have not yet been standardized in clinical practice, and their main applications are still limited to simple counting and determination of tumor metastasis potential.

[0005] CTCs in peripheral blood contain a certain number of CSCs. Furthermore, immune cells play a crucial role in the tumor microenvironment, possessing dual functions of promoting tumor growth and killing tumors. Therefore, collecting immune cells from peripheral blood and combining them with tumor-associated cells to construct tumor-like tissues can better reconstruct the tumor microenvironment and is crucial for in vitro anticancer drug sensitivity testing.

[0006]

[0007] Table 1: Surface markers of CTCs and CSCs

[0008]

[0009] Table 2: CSCs surface markers Summary of the Invention

[0010] In response to the problems in the background technology, the present invention proposes a method for constructing tumor-like tissue and drug sensitivity testing based on circulating tumor cells. The present invention increases the number of circulating tumor cells collected and successfully conducts multi-cellular three-dimensional culture on the surface of the material. This method is both rapid and relatively inexpensive. By comparing the drug sensitivity test of the tumor-like tissue with the clinical situation, the drugs screened by this patent are consistent with the drugs used by patients who have received effective treatment. Therefore, this method can provide effective personalized drug selection in the future, eliminating the pain, risk and economic loss of patients testing drugs on themselves.

[0011] The present invention proposes a tumor-like tissue construction based on circulating tumor cells, comprising the following steps:

[0012] S1. Draw peripheral blood from the vein of patients with liver cancer, brain cancer, and other tumors. Isolate mononuclear cells by gradient centrifugation using Ficoll-Paque solution at 400-500g for 10-15 minutes.

[0013] S2, cell subpopulation sorting by ring microfluidic separation technology; the flow channel has 8-12 turns, the flow channel inlet width is 300-600 pm, the flow channel width is 400-700 pm, the flow channel gap is 200-500 pm, and the five flow channel outlet widths are 70-100 pm; the sorting types include, but are not limited to, circulating macrophages, circulating tumor cells and cancer stem cells;

[0014] S3, after sorting, the cells are inoculated on the surface of the self-made Binary Colloidal Crystals (BCCs) material in a cell co-culture manner;

[0015] S4, multi-cell subtypes are cultured in a three-dimensional tumor-like tissue culture by using a self-made culture solution;

[0016] S5, after 7-14 days of culture, based on the tumor-like tissue formation of circulating tumor cells, drug sensitivity test of anticancer drugs can be directly or transferred to a new plate.

[0017] Preferably, in S2, the ring microfluidic channel is sorted according to the cell size, and only the larger size macrophages and tumor cells are collected for co-culture.

[0018] Preferably, in S3, the cells are inoculated on the surface of the BCCs material pre-coated with gelatin (0.05-0.5%), collagen (0.01-2%) or Matrigel solution (0.05-2%) at a cell concentration of 2*10 4 -5*10 6 cells / ml.

[0019] Preferably, in S4, the self-made culture solution formula includes, but is not limited to, DMEM / F12, platelet growth factor, B-27 supplement, N2 supplement, Recombinant Human FGF basic Protein (bFGF), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Forskolin, A8301 and Y27632.

[0020] Preferably, in S5, the culture solution is replaced with 30-80% volume of new culture solution every day.

[0021] The application further provides a tumor tissue-like drug sensitivity test method based on circulating tumor cells, which comprises the tumor tissue-like structure, and the drug sensitivity test is performed on the tumor tissue-like structure in situ or on the surface of new BCCs; the prepared tumor tissue-like structure is used to test the cell activity of different anti-cancer drug types, drug concentrations and drug mixing methods, and the optimal drug scheme is sorted out.

[0022] Compared with the prior art, the application has the following obvious beneficial technical effects:

[0023] The application provides a tumor tissue-like structure construction and drug sensitivity test method based on circulating tumor cells. The method combines an efficient cell separation method, uses a special component culture medium and a new type of cell culture material. Compared with the existing tumor cell sorting and culture technology, the application can improve the number of collected circulating tumor cells, has higher efficiency, and successfully performs multi-cell three-dimensional culture on the surface of the material. The method is fast and relatively inexpensive, is not affected by immunofluorescence dyes, can be successfully cultured in vitro, forms a multi-cell tumor tissue-like structure, makes circulating tumor cells, tumor stem cells and tumor macrophages grow together in the form of cell balls, and makes the circulating tumor cells have higher activity. Through comparison between the drug sensitivity experiment of the tumor tissue-like structure and the clinical situation, the drug screened by the application is consistent with the drug used by the patient who is effectively treated. Therefore, the method can provide effective personalized drug selection in the future, and can save the patient from the pain, risk and economic loss caused by drug testing. For tumor patients, only 10-20 milliliters of blood is needed, and after in vitro culture, the cells can be used for further detection of anti-cancer drug screening. Compared with expensive and time-consuming analysis such as gene sequencing, the method is fast and relatively inexpensive, and can inform the patient and the doctor of the effective drug selection in a short time (only about 7-10 days), so that the treatment time of the patient is saved, and the tumor treatment is greatly helped, and the method can be used for personalized precision medicine with great individual differences. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 A method flowchart of an embodiment of the application;

[0025] Figure 2 A schematic diagram of a ring-shaped microfluidic channel and cell enrichment in an embodiment of the application;

[0026] Figure 3 A schematic diagram of coating Matrigel on the surface of a Binary Colloidal Crystals (BCCs) material in an embodiment of the application;

[0027] Figure 4Figure 1 is a schematic diagram of the morphology of cell spheres in one embodiment of the present application, wherein (A)-(B) are tumor-like tissue cultures of blood of liver cancer patients (A) LC-43 and (B) LC-45, and (C)-(D) are tumor-like tissue cultures of blood of brain metastatic cancer patients (C) BC-08 and (D) BC-07;

[0028] Figure 5 Figure 2 is a schematic diagram of the results of identifying CTC and CSC surface markers in tumor-like tissue of a brain metastatic cancer patient (patient code BC-08) by immunofluorescence in one embodiment of the present application (10x10 group of images);

[0029] Figure 6 Figure 3 is a schematic diagram of the results of identifying CSC and macrophage surface markers in tumor-like tissue of a brain metastatic cancer patient (patient code BC-08) by immunofluorescence in one embodiment of the present application (10x10 group of images);

[0030] Figure 7 Figure 4 is a schematic diagram of the results of identifying CTC and macrophage surface markers in tumor-like tissue of a brain metastatic cancer patient (patient code BC-08) by immunofluorescence in one embodiment of the present application (10x10 group of images);

[0031] Figure 8 Figure 5 is a schematic diagram of the results of identifying CSC surface markers in tumor-like tissue of a brain metastatic cancer patient (patient code BC-08) by immunofluorescence in one embodiment of the present application (10x10 group of images);

[0032] Figure 9 Figure 6 is a schematic diagram of the results of identifying CSC and macrophage surface markers in tumor-like tissue of a brain metastatic cancer patient (patient code BC-09) by immunofluorescence in one embodiment of the present application (10x10 group of images);

[0033] Figure 10 Figure 7 is a schematic diagram of the results of identifying the amount of cell death after drug treatment of a patient with intrahepatic cholangiocarcinoma (patient code LC-43) by lactate dehydrogenase (LDH) analysis in one embodiment of the present application;

[0034] Figure 11 Figure 8 is a schematic diagram of the results of identifying the amount of cell death after drug treatment of a patient with intrahepatic cholangiocarcinoma (patient code LC-14) by lactate dehydrogenase analysis in one embodiment of the present application;

[0035] Figure 12 Figure 9 is a schematic diagram of the prognosis results of a patient with primary liver cancer (patient code LC-14) 2.5 months after treatment in one embodiment of the present application, wherein the CT image of the patient's intrahepatic lesion is shown, and the arrow indicates the tumor tissue;

[0036] Figure 13For an embodiment of the present application, a liver cancer patient (patient code LC-14) has bone metastasis, and the CT image of the patient's liver lesion after 2.5 months of treatment shows that the tumor tissue is in the circle.

[0037] Figure 14 A 6-month survival rate chart for an embodiment of the present application includes 49 patients with advanced liver, gallbladder and pancreatic cancer. DETAILED DESCRIPTION

[0038] Example 1 (liver cancer)

[0039] As shown in Figure 1 , the present application proposes a tumor tissue-like structure based on circulating tumor cells, which includes the following steps:

[0040] S1, extract peripheral blood (10-20 mL) from a liver cancer patient, store at room temperature (<3 h) or low temperature (<8 h), and separate mononuclear cells from whole blood cells by gradient centrifugation with Ficoll-Paque solution;

[0041] S2, use ring microfluidic separation technology to sort cell subpopulations, and collect cells at the outlet 1 to outlet 3 of the flow channel. Figure 2 ).

[0042] Further, in S2, the collected cell types should include, but are not limited to, circulating macrophages, circulating tumor cells and cancer stem cells;

[0043] S3, seed the sorted cells on the surface of self-made Binary Colloidal Crystals (BCCs) material in a cell co-culture manner. Figure 3 ).

[0044] Further, in S3, seed the sorted cells on the surface of BCCs material pre-coated with diluted Matrigel solution (0.05-2%) at a cell concentration of 2*10 4 -5*10 6 cells / ml.

[0045] S4. Using self-made culture medium, multi-cell subtypes of three-dimensional tumor-like tissue culture were carried out; the culture medium formula comprises DMEM / F12 culture medium, human platelet lysate (hPL), B-27 additive, N2 additive, recombinant human FGF basic protein (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), forskolin, A8301 inhibitor and ROCK inhibitor (Y27632).

[0046] S5. Cell culture for 7-10 days, various cells aggregated into liver tumor-like tissue Figure 4 (A)-4(C).

[0047] Example 2 (brain cancer)

[0048] As Figure 1 shown, the application proposes a tumor-like tissue construction based on circulating tumor cells, the steps comprising:

[0049] S6. Peripheral blood (10-20 mL) of brain cancer tumor patients was extracted, and the whole blood cells were gradient centrifuged with Ficoll-Paque solution to separate out mononuclear cells;

[0050] S7. Cell subpopulation sorting was carried out using ring microfluidic separation technology, and cells at the outlet 1 to outlet 3 of the flow channel were collected Figure 2 ).

[0051] Further, in S7, the collected cell types should include, but are not limited to, circulating macrophages, circulating tumor cells and cancer stem cells;

[0052] S8. The sorted cells were inoculated on the surface of self-made Binary Colloidal Crystals (BCCs) material in a cell co-culture manner Figure 3 ).

[0053] Further, in S8, the sorted cells were inoculated on the surface of BCCs material coated with diluted gelatin solution (0.05-2%) at a cell concentration of 2*10 4 -5*10 6 cells / ml.

[0054] S9, using self-made culture solution, three-dimensional tumor-like tissue culture of multiple cell subtypes is carried out; the culture medium formula comprises DMEM / F12 culture medium, human platelet lysate (hPL), B-27 additive, N2 additive, recombinant human FGF basic protein (bFGF), epidermal growth factor (EGF), forskolin, A8301 inhibitor and ROCK inhibitor (Y27632).

[0055] S10, cell culture for 7-10 days, various cells will gather into brain tumor-like tissue Figure 4 (D)-4(F))).

[0056] S11, one of the brain tumor-like tissues is taken out for further immunofluorescence identification, and the CTCs, CSCs and circulating macrophage-related markers such as Pan-CK, EPCAM, CD44, CD133 and CD68 all have obvious performance Figures 5-9 )。

[0057] Example 3 (drug screening)

[0058] The application further provides a drug sensitivity test method based on tumor-like tissue of circulating tumor cells, which comprises the above-mentioned tumor-like tissue, and the above-mentioned liver tumor-like tissue is taken out to the surface of new BCCs for drug sensitivity test; the prepared liver tumor-like tissue is tested for cell activity of different anti-cancer drug types (such as A, B and C), drug concentrations (90nM-100μM) and drug mixing methods (such as A+B, A+B+C), and the best drug regimen is sorted out.

[0059] The application identifies the death value of cells after drug treatment by lactate dehydrogenase analysis (LDH assay), and compares it with the group of cells without drug treatment, so as to judge the effective degree of anti-cancer drugs Figures 10-11 )。

[0060] The following is a tracking record of one case, Cai, a 64-year-old male, diagnosed as intrahepatic cholangiocarcinoma with multiple bone metastases, underwent quadruple therapy of calea libi 200mg q3w+ lenvatinib 8mg qd+ gemcitabine 1900mg d1 / d8+ oxaliplatin 190mg d1 from October 30, 2010 to December 20, 2020. The patient still has tumor progression and severe diarrhea, high fever and other intolerable side effects, and stops using anti-tumor drugs.

[0061] On December 25, 2020, the patient entered the Department of Hepatobiliary Surgery of Shenzhen University General Hospital, and on December 28, 2020, blood was drawn and drug sensitivity screening of tumor-like tissue was performed. The CTC drug screening results showed that the tumor cells in the patient's body were sensitive to Sorafenib Figure 11 ), which is consistent with the PD-1 monoclonal antibody commonly used for liver cancer, Sorafenib 0.4g bid combined with Camrelizumab 200mg q3w. The patient was in good condition after taking the drug, with no special side effects, and the tumor did not progress Figure 12 ), and even some areas of the tumor in the body had a shrinking trend Figure 13 ).

[0062] According to the doctor's advice, blood was drawn from 49 patients with tumors that had reached the middle and late stages and were difficult to treat, and tumor-like liver tissues were constructed and tested for drug sensitivity. Among them, 30 patient samples successfully constructed tumor-like liver tissues and screened the best drugs, and 19 patient samples could not perform the final drug screening. In the 6-month prognosis tracking process, 24 patients were still alive, with a survival rate of 80% Figure 14 ). After comparison, the drugs used by these 24 patients were effective, which was consistent with the results of this patent screening.

[0063] This patent collected a total of 49 liver cancer and 11 brain cancer patients (Tables 3-4), obtained 10-20 milliliters of patient peripheral blood, and cultured tumor-like tissues, and finally performed drug sensitivity testing. The entire process only takes 2 weeks. Table 3 is a detailed information statistical table of 49 hepatobiliary and pancreatic cancer patients. Table 4 is a detailed information statistical table of 11 brain cancer patients.

[0064] The present application proposes a kind of tumor tissue construction and drug sensitivity test method based on circulating tumor cell.Combined with efficient cell separation method, special ingredient culture medium and new cell culture material are used.Compared with existing sorting and culturing tumor cell technology, the number of circulating tumor cells collected can be improved using the technical solution of the present application, and the efficiency is higher, and multi-cell three-dimensional culture on the surface of material is successfully carried out, this method is fast and relatively cheap, and is not affected by immunofluorescence dye, can be successfully cultured in vitro, and form multi-cell tumor tissue, make circulating tumor cells, tumor stem cells and tumor macrophages grow together in cell ball form, make circulating tumor cells have higher activity.Through the drug sensitivity test of tumor tissue and the comparison with clinical situation, the drugs screened out by the present application are consistent with the drugs used by the patients who get effective treatment.Therefore, the present method can provide effective personalized drug selection in the future, and eliminate the pain, risk and economic loss of patients trying drugs.For tumor patients, only 10-20 milliliters of blood is needed, after in vitro culture, these cells can be used for further detection of anticancer drug screening, compared with expensive and time-consuming analysis such as gene sequencing, this method is fast and relatively cheap, which can tell patients and doctors effective drug selection in a short time, and strive for the treatment time of patients, which is of great help to tumor treatment, and can be used for personalized precision medicine with great individual differences.

[0065]

[0066] Table 3

[0067]

[0068] Table 4

[0069] The embodiments of the present application are described in detail above in combination with the drawings, but the present application is not limited thereto, and various changes can be made within the knowledge range of those skilled in the art without departing from the purpose of the present application.

Claims

1. A method for constructing a tumor-like tissue based on circulating tumor cells, characterized by the steps of The method comprises the following steps: S1, taking blood from a tumor patient, and performing gradient centrifugation for 10-15 minutes at 400-500 g with a Ficoll-Paque solution to separate mononuclear cells; S2, using a ring-shaped microfluidic separation technology to sort cell subgroups; the flow channel has 8-12 turns, the flow channel inlet width is 300-600 μm, the flow channel width is 400-700 μm, the flow channel gap is 200-500 μm, and the five flow channel outlet widths are 70-100 μm; the sorting types include circulating macrophages, circulating tumor cells and cancer stem cells; S3, after sorting, the cells are inoculated on the surface of a self-made binary colloidal crystal (BCCs) material in a cell co-culture manner; S4, using a self-made culture solution to perform three-dimensional tumor-like tissue culture of multiple cell subtypes; in S4, the self-made culture solution formula comprises DMEM / F12 culture medium, human platelet lysate (hPL), B-27 additive, N2 additive, basic fibroblast growth factor recombinant protein, epidermal growth factor, hepatocyte growth factor, forskolin, A8301 inhibitor and Y27632 inhibitor; S5, after 7-14 days of culture, based on the formation of tumor-like tissues of circulating tumor cells, direct or transfer to a new plate to perform drug sensitivity test of anticancer drugs.

2. The method according to claim 1, wherein the method is characterized by, In S2, the ring-shaped microfluidic channel has 5-8 outlets, and according to the size of the cells, only the circulating macrophages, circulating tumor cells and circulating cancer stem cells with large size are collected for co-culture.

3. The method according to claim 1, wherein the method is characterized by, In S3, the cells are seeded at a cell concentration of 2*10 4 -5*10 6 cells / ml on the surface of the BCCs material previously coated with a gelatin, collagen or Matrigel solution; The gelatin solution has a concentration of 0.05-0.5%; the collagen solution has a concentration of 0.01-2%; and the Matrigel solution has a concentration of 0.05-2%. In S5, the culture solution is replaced with 30-80% of new culture solution every day.

4. The method according to claim 1, wherein the method is characterized by, The above tumor-like tissues are tested for drug sensitivity in situ or after being taken out to the surface of new BCCs; the prepared tumor-like tissues are tested for cell activity of different anticancer drug types, drug concentrations and drug mixing methods, and the best drug regimen is sorted out.

5. A method for tumor tissue-like pharmaresistance test based on circulating tumor cells, the method comprising using the tumor tissue-like pharmaresistance test based on circulating tumor cells according to any one of claims 1-4, characterized in that, ​

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