Renal cell carcinoma circulating tumor cell culture medium, culture method and application

Abstract

The invention discloses a renal cell carcinoma circulating tumor cell culture medium as well as a culture method and application thereof. The invention belongs to the field of biological medicine, and particularly relates to a renal cell carcinoma circulating tumor cell culture medium and a culture method and application. The invention provides a composition for culturing circulating tumor cells, the composition contains a cell culture medium and a biological scaffold, the cell culture medium contains a reagent named as RFGI, and the RFGI contains fetal calf serum, insulin, glucose and R-spondin-1 protein. Enriching the isolated circulating tumor cells through density gradient centrifugation; the composition is used for culturing the enriched circulating tumor cells to obtain the circulating tumor cells, so that the survival rate of the circulating tumor cells is effectively improved, and the composition has huge potential and application prospects for subsequent drug screening.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a culture medium for circulating tumor cells of renal cell carcinoma, a culture method thereon, and its application. Background Technology

[0002] Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system, and it is highly malignant. Approximately one-third of RCC patients already have metastases at initial diagnosis. Although localized RCC can be surgically removed, 20%-30% of patients eventually progress to metastatic RCC after surgery. Once RCC metastasizes, the prognosis is poor, with a median survival of only 17-26 months.

[0003] Metastatic renal cell carcinoma is insensitive to both radiotherapy and chemotherapy. In recent years, the use of molecularly targeted drugs and immune checkpoint inhibitors has improved the prognosis of patients with metastatic renal cell carcinoma to some extent. However, due to the heterogeneity and complexity of the pathogenesis of renal cell carcinoma, the long-term efficacy of these molecularly targeted drugs and immune checkpoint inhibitors is still unsatisfactory. For example, complete remission or long-term benefit from targeted therapy such as sunitinib and sorafenib is rare in clinical practice, with an objective response rate of only 30%-40%. Most renal cell carcinoma patients develop secondary resistance or disease progression after 6-11 months of treatment, requiring reassessment of the patient's tumor status and a change in treatment regimen. Although multiple tumor tissue sampling to monitor the dynamic changes of tumor cells is important, 50%-80% of metastatic tumor patients suffer from cachexia and cannot tolerate multiple surgeries or biopsies, making this approach difficult to implement in clinical practice.

[0004] Liquid biopsy for tumors utilizes various techniques to detect circulating tumor cells, cell-free tumor DNA, and exosomes released from tumors into bodily fluids, particularly peripheral blood, enabling precise tumor diagnosis and treatment. Compared to invasive tissue biopsies, liquid biopsies offer advantages such as being non-invasive, readily available samples, and providing dynamic monitoring of disease progression. Circulating tumor cells (CTCs) are tumor cells that detach from tumor lesions (primary or metastatic) and enter the peripheral blood circulation. They are considered the "seeds" for metastatic lesions and play a crucial role in tumor metastasis. Compared to other liquid biopsy techniques, CTCs are viable, intact tumor cells carrying more comprehensive multi-omics information (genomics, transcriptomics, proteomics, metabolomics, etc.). Viable CTCs allow for in vitro morphological and functional analysis of tumor cells, and can also be cultured in vitro or in vivo to assess drug sensitivity. Furthermore, CTC-derived xenograft (CDX) models can be constructed to replicate the tumor characteristics of patients. Therefore, CTC research is considered one of the most promising "liquid biopsy" technologies.

[0005] Currently, the practical application of renal cell carcinoma (CTC) faces several challenges: ① The concentration of CTCs in peripheral blood is low, typically yielding only <20 CTCs from 10 mL of blood; ② Existing CTC extraction and enrichment methods have limitations, resulting in very low detection rates, which severely restricts the clinical and research applications of CTCs; ③ The optimal culture conditions required for CTCs are not yet clear, and effective culture techniques are lacking, making it extremely difficult to culture and amplify CTCs in vitro, thus hindering in-depth and reproducible research. Through search and analysis, there are currently no research reports on the successful in vitro culture of renal cell carcinoma CTCs. How to achieve efficient culture, amplification, and application of a very small number of target renal cell carcinoma CTCs is an urgent scientific problem to be solved. Summary of the Invention

[0006] The main problem this invention aims to solve is how to optimize the culture method of renal cell carcinoma circulating tumor cells and improve the success rate of renal cell carcinoma circulating tumor cell culture.

[0007] To address the aforementioned problems, the present invention provides a composition for culturing circulating tumor cells of renal cell carcinoma.

[0008] The composition for culturing renal cell carcinoma circulating tumor cells provided by the present invention contains a cell culture medium and a biological scaffold, wherein the cell culture medium contains a reagent named RFGI, and the RFGI contains fetal bovine serum, insulin and glucose.

[0009] The RFGI is a composition.

[0010] The RFGI also contains the R-spondin-1 protein.

[0011] The R-spondin-1 (RSPO1) protein is a secretory activator protein with two cysteine-rich furin-like domains (FU-like CR) and a platelet-reactive protein type 1 domain (TSR). RSPO1 acts as a classic Wnt signaling enhancer.

[0012] In the above composition, the RFGI consists of fetal bovine serum, insulin, glucose and R-spondin-1 protein, and the ratio of fetal bovine serum, insulin, glucose and R-spondin-1 protein in the RFGI is 100 mL fetal bovine serum : 137.6 U insulin : 3.3 mmol glucose : 10 μg R-spondin-1 protein.

[0013] In the above composition, the cell culture medium contains 10 ng / ml of R-spondin-1 protein, 10% fetal bovine serum, 0.1376 units / mL of insulin, and 3.3 mM of glucose.

[0014] In a specific embodiment, the composition of 1L of cell culture medium may be as follows: Advanced DMEM / F128 50ml: HEPES 10ml: GlutaMAX (100X) 10ml: Pen / Strep (100×) 10ml: Primocine 2ml: EGF 50μg: FGF-10 100μg: B27 (50×) 20ml: N-acety-l-cysteine ​​1.25mmol: Y-27632 (10mM) 10μmol: A83-01 (1mM) 5μmol: R-spondin-1 (100ug / ml) 10μg: FBS 100mL: D-(+)-Glucose 3.3mmol: Insulin (40Units / mL) 137.6U.

[0015] The present invention also provides a culture medium for culturing circulating tumor cells of renal cell carcinoma, wherein the culture medium may be the cell culture medium described above.

[0016] The present invention also provides a kit for culturing circulating tumor cells of renal cell carcinoma, the kit containing the RFGI described above.

[0017] In the above text, the bioscaffold is obtained by covalent crosslinking of 2,3-dialdehyde cellulose and polyamine polymer.

[0018] In the above description, the volume ratio of the cell culture medium to the biological scaffold in the composition is 1:1.

[0019] The present invention also provides a method for culturing circulating tumor cells of renal cell carcinoma, comprising the following steps:

[0020] 1) Enrichment of isolated circulating tumor cells;

[0021] 2) The circulating tumor cells from step 1) are cultured using the composition described above to obtain expanded circulating tumor cells.

[0022] In the above method, step 1) describes a method for enriching circulating tumor cells using density gradient centrifugation.

[0023] In this article, the tumor cells are kidney cancer cells.

[0024] The present invention also provides a method for constructing a circulating tumor cell xenograft model, the method comprising implanting circulating tumor cells obtained by the method of culturing circulating tumor cells described above into mice to obtain a circulating tumor cell xenograft model.

[0025] The present invention also provides the use of the composition, culture medium, kit, and method described above in the preparation of circulating tumor cell products, or in the preparation of products for increasing the cell density of renal cell carcinoma circulating tumor cells.

[0026] In the above application, increasing the cell density of renal cell carcinoma circulating tumor cells can be achieved by increasing the cell density of renal cell carcinoma circulating tumor cells cultured in medium No. 4. Medium No. 4 consists of Advanced DMEM / F12, HEPES, GlutaMAX (100×), Pen / Strep (100×), Primocine, EGF, FGF-10, B27, N-acety-l-cysteine, Y-27632:A83-01, and R-spondin-1.

[0027] In the above applications, the No. 4 culture medium can have the following composition (1L system): Advanced DMEM / F129 50ml: HEPES 10ml: GlutaMAX (100×) 10ml: Pen / Strep (100×) 10ml: Primocine 2ml: EGF 50μg: FGF-10 100μg: B27 (50×) 20ml: N-acety-l-cysteine ​​1.25mmol: Y-27632 (10mM) 10μmol: A83-01 (1mM) 5μmol: R-spondin-1 (100ug / ml) 500μg.

[0028] The low success rate of existing CTC culture methods may be due to the excessive exposure of already fragile CTCs to physical damage. Over-selected CTCs lack the protection of the tumor microenvironment, and the CTC expansion process lacks growth factors and three-dimensional spatial support. This study reports a novel CTC culture system (culture medium and method) that achieves a 66.7% success rate in culturing CTCs from peripheral blood of newly diagnosed metastatic renal cell carcinoma patients. Biomarker detection and gene mutation profile analysis confirm that the CTCs originate from renal cell carcinoma cells. The construction of a CDX model effectively demonstrates the tumorigenic potential of CTCs. Furthermore, the application of the mini-CDX model established based on CTC culture in this study indicates that CTCs have promising clinical application prospects for drug screening. This efficient CTC culture method provides an important support platform for basic research and personalized clinical treatment of metastatic renal cell carcinoma. Attached Figure Description

[0029] Figure 1Three methods were used to enrich renal cell carcinoma (CTCs). a. Flowchart comparing the three CTC enrichment methods; b. After culturing CTCs using the schizoaflavins method alone for 2 weeks, gradually apoptotic leukocytes were observed under a light microscope; c. After culturing CTCs using density gradient centrifugation for 2 weeks, large-diameter cells and cell clusters were observed under a light microscope; d. After culturing CTCs using density gradient centrifugation combined with CD45 negative selection for 2 weeks, only a small number of cells were enriched under a light microscope, and no cell proliferation was observed. Scale bar: 50 μm.

[0030] Figure 2 Successful proliferation of CTCs in newly diagnosed metastatic renal cell carcinoma. a. Flowchart of CTC culture; b. 3D scaffold morphology observed under an optical microscope; c. Scaffold morphology observed under an electron microscope; d. Representative phase-contrast microscopic images of CTCs from metastatic renal cell carcinoma (RCC) patients taken on the day of culture, day 14, and day 28. Thick arrows represent circulating tumor endothelial cells, which can self-assemble and proliferate into spheroids. Thin arrows represent CTCs, which attach and proliferate around endothelial cell spheroids. ▲ Gradually apoptotic leukocytes. Scale bar: 50 μm; e. Representative phase-contrast microscopic images of healthy subjects (HD) taken on the day of culture, day 14, and day 28. Under these culture conditions, no cells survived in healthy subjects, and the remaining leukocytes gradually apoptotic within 30 days. ▲ Gradually apoptotic leukocytes. Scale bar: 50 μm.

[0031] Figure 3 To ensure that cultured CTCs highly retain the biological characteristics of renal cell carcinoma cells, the clustered cell spheroids are tumor-associated endothelial cells. HE, IHC, and FISH staining were performed on tumor tissue CTCs and CTECs. PAX-2, PAX-8, CA IX, vimentin, and chromosome 3p deletion are key markers of renal cell carcinoma, showing positivity in tumor tissue and CTCs, but negativity in CTECs. CD31, CD34, and vimentin are key markers of endothelial cells, showing positivity in CTEC spheroids. CD45, as a leukocyte marker, was negative in both CTCs and CTECs. Chromosome 3 probe signal indicated that the cell spheroids were predominantly aneuploid, suggesting that the cell spheroids are aneuploid tumor-associated cells. Scale bar: 50 μm.

[0032] Figure 4 To demonstrate the tumorigenicity of cultured CTCs, cultured CTCs were injected subcutaneously into mice. a. Tumor-bearing mice; b. CDX tumor tissue collected at 6 weeks; c. Tumor volume change curve after implantation, with tumor doubling time ranging from 14 to 21 days. Black dots represent tumor volume; solid lines represent the best-fit growth curve; d. HE, PAX-2, PAX-8, CD31, and CD45 staining images of CDX 1–5 tumor tissues. Scale bar: 50 μm.

[0033] Figure 5 Gene copy numbers and gene mutation profiles of cultured CTCs and matched tumor tissues (CTC-05, 08, 09, 11). a. Venn diagram of common and specific gene mutations in tumor tissues and CTC cultures for each patient; b. Mutations of key renal cell carcinoma genes in tumor tissues and CTC cultures for each patient.

[0034] Figure 6 Copy number variation heatmaps for cultured CTCs and matched tumor tissues (CTC-05, 08, 09, 11). Measured in log2. The bands on the left represent chromosomes 1-22 and X / Y. The data at the bottom shows the correlation between tumor tissues and CTCs, as well as the percentage of overlapping data points.

[0035] Figure 7 MiniPDX models derived from CTCs can be used for drug screening. a. Using a. Flowchart of the Mini-CDX Assay for rapid in vivo drug sensitivity testing; b. Average CTC proliferation rate after treatment with 7 drug regimens tested on the Mini-CDX model; c. Histogram showing the average CTC proliferation rate after treatment with 7 drug regimens tested on the mini-CDX model. Drug sensitivity varies greatly, with the highest average proliferation rate being >100% and the lowest being 30%.

[0036] Figure 8 This section describes the application of the Mini-CDX test in clinical practice. a. Dynamic curve of the maximum tumor diameter measured by chest CT scan. The left arrow indicates the start time of first-line sunitinib treatment, and the right arrow indicates the start time of second-line treatment; b. Chest CT scan image before first-line treatment; c. Chest CT scan image before second-line treatment, showing progression of parietal pleural metastases and the appearance of malignant pleural effusion; d. Chest CT image after two cycles of second-line treatment, showing the patient achieving partial remission. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] The reagent information used in the following examples is shown in Table 1. The basal culture medium preparation information used in the following examples is shown in Table 2. The erythrocyte lysis buffer preparation information used in the following examples is shown in Table 3. The cell culture medium preparation information used in the following examples is shown in Table 4.

[0040] Table 1. Relevant reagent information in the examples

[0041]

[0042]

[0043]

[0044] Table 2. Preparation of basal culture medium

[0045] name Preparation volume (mL) Advanced DMEM / F12 500 HEPES 5 GlutaMAX(100×) 5 Pen / Strep (100×) 5 Primocine 1

[0046] Table 3. Preparation of Red Blood Cell Lysis Buffer

[0047] name Configuration quantity Ammonium chloride lyse (10×) 10ml NH4Cl (ammonium chloride) 8.02mg NaHCO3 (sodium bicarbonate) 0.84mg EDTA (disodium) 0.37mg Deionized water 90ml

[0048] The bioscaffold used in the following examples was a gift from Professor Dazhi Yang and is described in Elizondo DM, Brandy N, Da SR, et al. Pancreatic islets seeded in a novel bioscaffold forms an organoid to rescue insulin production and reverse hyperglycemia in models of type 1 diabetes[J]. Sci Rep, 2020, 10(1):4362. For specific preparation methods, please refer to the experimental methods section of this paper. The detailed steps are as follows:

[0049] A. Synthesis of biological scaffolds

[0050] (1) Selective oxidation of cellulose, covalent crosslinking of 2,3-dialdehyde cellulose with polyamine polymers and reduction of carbon-nitrogen imine double bonds;

[0051] (2) 2,3-dialdehyde cellulose is covalently crosslinked with functional block polymers to form a polyamine-cellulose copolymer with a three-dimensional dense interlocking network;

[0052] (3) The amine groups are protonated in an aqueous environment with a pH value below 9, causing the positively charged copolymer scaffold to form a hydrogel matrix;

[0053] (4) Fourier transform infrared spectroscopy (FTIR) was used to evaluate the functional group characterization of the material.

[0054] To obtain synthetic biological scaffolds.

[0055] B. Pretreatment and use of biological scaffolds

[0056] (1) Transfer of substrate: Use a 10ml pipette to transfer 10ml of synthetic biological scaffold into a 15ml centrifuge tube;

[0057] (2) Collect the substrate: Centrifuge at 250×g for 10 min at room temperature, remove the supernatant, collect the carrier, and transfer it to a 50ml centrifuge tube;

[0058] (3) Replacement of matrix preservation solution: Resuspend the biological scaffold with 4 times the matrix volume of DMEM / F12 basal medium, and invert and mix 20 times repeatedly.

[0059] (4) Natural sedimentation: Afterward, place the centrifuge tubes in a cell culture incubator for at least 12 hours, keeping them upright. Pre-fabricated biological scaffolds can be stored in the cell culture incubator for 3 weeks, while unfabricated biological scaffolds can be stored at room temperature for 2 years.

[0060] (5) Aspirate an appropriate amount of the pre-prepared biological scaffold after centrifugation: Slowly aspirate an appropriate volume of the pre-prepared biological scaffold from the bottom of the compressed 50ml centrifuge tube (containing the pre-prepared biological scaffold) into the 15ml centrifuge tube.

[0061] (6) Add complete tumor culture medium: Add cell culture medium according to the ratio of biological scaffold: cell culture medium = 1:1 to enrich the biological scaffold with nutrients.

[0062] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0063] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicates that there is a significant difference.

[0064] Example 1: Enrichment and Culture of Circulating Tumor Cells (CTCs)

[0065] The peripheral blood samples used in this embodiment were collected from patients with newly diagnosed metastatic renal cell carcinoma. The samples were approved by the ethics committees of Beijing Anzhen Hospital, affiliated with Capital Medical University and Peking University Cancer Hospital, and the patients or their families signed informed consent forms.

[0066] First, peripheral blood samples (15 ml / person) were collected from 5 newly diagnosed metastatic renal cell carcinoma patients. Cytotoxic tumor cells (CTCs) were enriched using three methods: schizoaffective assay, density gradient centrifugation, and density gradient centrifugation combined with CD45 negative selection. The samples were then cultured in the aforementioned culture system for 2 weeks (flowchart shown). Figure 1 (a)

[0067] I. Enrichment of CTC

[0068] 1. Research on three enrichment methods

[0069] 1) Direct Red Crack Method

[0070] ① Dilute the lysis buffer: Add 20ml of 10× concentrate to 180ml of sterile deionized water and store at 4℃ until used;

[0071] ②Aliquoting whole blood: Transfer 1 ml of whole blood into 10 ml sterile conical centrifuge tubes;

[0072] ③ Add lysis buffer: Add 10 ml of 4℃ 1× red blood cell lysis buffer to each tube;

[0073] ④ Lyse red blood cells: Invert or shake for 3 minutes until the solution turns transparent red;

[0074] ⑤ Centrifuge and remove supernatant: Centrifuge at 180×g, 4℃ for 10min, and remove supernatant;

[0075] ⑥ Resuspend cells: Add 1 ml of pre-cooled CTC protection solution to each tube and resuspend the cells;

[0076] ⑦ Collect cells: Transfer the cell suspension from each tube to a new 50ml sterile conical centrifuge tube, and add pre-cooled basal culture medium to bring the volume to 20ml;

[0077] ⑧ Observe the red blood cell cleavage under a microscope: Aspirate 20 μL of cell suspension, observe the degree of red blood cell cleavage under a 40× microscope, and take a picture;

[0078] ⑨ Centrifuge and remove supernatant: Centrifuge at 180×g, 4℃ for 10 min, and remove supernatant;

[0079] ⑩ Re-crack red: Based on the microscopic observation results in step 8, if necessary, re-crack red;

[0080] Collect the cell pellet.

[0081] 2) Density gradient centrifugation method

[0082] ① Rewarming: Samples used for lymphocyte separation medium (Stemcell, catalog number 07801) can be transported at 4℃ and processed on the same day. Upon arrival at the laboratory, rewarm the lymphocyte separation medium and "PBS + 2% FBS" to room temperature (15-25℃). Shake the lymphocyte separation medium well before use;

[0083] ② Add lymphocyte separation medium: Add the lymphocyte separation medium to a 50ml centrifuge tube and set aside for later use;

[0084] ③ Dilute the blood: Transfer the blood to a 50ml centrifuge tube. Add an equal volume of "PBS + 2% FBS" to the blood;

[0085] ④ Blood Preparation: Slowly add the diluted blood onto the lymphocyte separation medium. Tilt the 50ml centrifuge tube and add the blood very slowly along the tube wall. Remember not to disturb the interface between the two.

[0086] ⑤ Centrifugation: Centrifuge at room temperature, 800×g, for 30 minutes, then remove the brake;

[0087] ⑥ Remove the white film layer: Transfer the white film layer (i.e., the PBMC layer) into a new 15mL centrifuge tube;

[0088] ⑦ Wash the white film layer: Add 10 mL of "PBS + 2% FBS", and mix by inverting the test tube. 300×g

[0089] Centrifuge for 10 minutes;

[0090] ⑧ Clean the white film layer again: Same as above;

[0091] ⑨ Collect the cell pellet.

[0092] 3) Density gradient centrifugation combined with CD45 negative selection method

[0093] ① Rewarming: Samples used for lymphocyte separation can be transported at 4℃ and processed on the same day. Upon arrival at the laboratory, rewarm the lymphocyte separation solution and "PBS + 2% FBS" to room temperature (15-25℃). Shake the lymphocyte separation solution well before use;

[0094] ②Incubate CD45 antibody: Add 15 μL of CD45 deletion cocktail liquid per milliliter of peripheral blood and incubate at room temperature for 20 minutes;

[0095] ③ Add lymphocyte separation medium: Add the lymphocyte separation medium to a 50mL centrifuge tube and set aside for later use;

[0096] ④ Dilute the blood: Transfer the blood to a 50mL centrifuge tube. Add an equal volume of "PBS + 2% FBS" to the blood;

[0097] ⑤ Blood Preparation: Slowly add the diluted blood onto the lymphocyte separation medium. Tilt the 50mL centrifuge tube and add the blood very slowly along the tube wall. Remember not to disturb the interface between the two.

[0098] ⑥ Centrifugation: Centrifuge at room temperature, 800×g, for 30 minutes, then remove the brake;

[0099] ⑦ Aspirate the white film layer: Aspirate the white film layer (i.e., the PBMC layer) into a new 15mL centrifuge tube;

[0100] ⑧ Wash the white film layer: Add 10mL of "PBS + 2% FBS", invert the test tube to mix, and centrifuge at 300×g for 10min;

[0101] ⑨ Clean the white film layer again: Same as above;

[0102] ⑩ Collect the cell pellet.

[0103] 2. CTC heavy suspension

[0104] The cell pellet was resuspended in cell culture medium and brought to a final volume of 10 mL.

[0105] 3. Cell counting

[0106] Using an automated cell counter, record the total number of cells, the number of viable cells, and the viability in the cell resuspension in step 2 above. Record the data three times and take the average value.

[0107] 4. Add cell culture medium

[0108] Add cell culture medium at a ratio of 1:1 to enrich the biological scaffold with nutrients.

[0109] 5. Resuspending cell pellets using biological scaffolds

[0110] By repeatedly blowing and aspirating, the cell pellet is resuspended in a biological scaffold (it is essential to ensure that the cells and the biological scaffold are thoroughly and evenly mixed) to obtain a cell-scaffold-culture medium suspension.

[0111] 6. Vaccination

[0112] Seed the cell-scaffold-culture medium suspension from step 5 above into 96-well ultra-low adsorption plates, 200 μL per well. Seed approximately 5-6 wells for every 7.5 mL of blood.

[0113] CTCs were enriched using three methods: the red cleavage assay, density gradient centrifugation, and density gradient centrifugation combined with CD45 negative selection. The cells were then cultured in the aforementioned culture system for 2 weeks (flowchart shown). Figure 1 a) It was found that the simple schizoaffected red blood cell (CTC) assay failed to enrich CTCs, and the culture dish contained only gradually apoptotic leukocytes. Figure 1(b) Density gradient centrifugation, after 2 weeks of culture, revealed large-diameter cells and cell clusters. Figure 1 (c) Density gradient centrifugation combined with CD45 negative selection only enriched a small number of cells, and no cell proliferation was observed within 2 weeks. Figure 1 (d). The results showed that density gradient centrifugation was the optimal method for enriching renal cell carcinoma CTCs compared to the other two CTC enrichment methods. Therefore, subsequent studies in this study will all use CTC enrichment.

[0114] II. Culture of CTCs and Circulating Tumor Endothelial Cells (CTEC)

[0115] This study used Ficoll-Paque density gradient centrifugation to separate erythrocytes, CTC-containing mononuclear cells, and plasma, and then collected the CTC-containing mononuclear cell layer and all cells in the plasma. This method has minimal cell selection bias, causes minimal damage to CTCs, and maximizes the protection of the blood microenvironment.

[0116] This study collected peripheral blood samples from 15 newly diagnosed metastatic renal cell carcinoma (mRCC) patients (see Table 5 for details); in addition, peripheral blood samples from 3 healthy subjects were collected as controls. The samples from healthy subjects were processed and cultured under the same conditions as those from metastatic renal cell carcinoma patients.

[0117] The standardized culture method for CTC is as follows:

[0118] 1. CTC heavy suspension

[0119] The circulating tumor cell pellet was resuspended in cell culture medium and the volume was adjusted to 10 mL.

[0120] 2. Cell counting

[0121] Using an automated cell counter, record the total number of cells, the number of viable cells, and the viability in the cell resuspension obtained in step 1 above. Record the data three times and take the average value.

[0122] 3. Add cell culture medium

[0123] Add cell culture medium at a ratio of 1:1 to enrich the biological scaffold with nutrients.

[0124] 4. Resuspending cell pellets using biological scaffolds

[0125] By repeatedly blowing and aspirating, the cell pellet is resuspended in a biological scaffold (it is essential to ensure that the cells and the biological scaffold are thoroughly and evenly mixed) to obtain a cell-scaffold-culture medium suspension.

[0126] 5. Vaccination

[0127] Cultured CTC cells were seeded into 96-well ultra-low adsorption plates, with 200 μL of cell-scaffold-culture medium suspension per well. Approximately 5-6 wells were seeded with 7.5 mL of blood each.

[0128] Incubate at 37°C in a 5% CO2 cell culture incubator. Figure 2 In section 'a', the process and conditions for CTC culture are described. Figure 2 In the table, b and c are light micrographs and electron micrographs of the biological scaffold, respectively. The culture medium is shown in Table 4.

[0129] The culture media were divided into the following 6 groups:

[0130] Culture medium No. 1: RPMI 1640 medium (Gibco, catalog number 11875093), which is commonly used in commercial renal cell carcinoma cell lines;

[0131] Culture medium No. 2: This is the stem cell culture medium used by Fendler et al. to culture renal cell carcinoma organoids. For specific references, see Fendler A, Bauer D, Busch J, Jung K, Wulf-Goldenberg A, Kunz S, Song K, Myszczyszyn A, Elezkurtaj S, Erguen B, Jung S, Chen W, Birchmeier W. Inhibiting WNT and NOTCH in renal cancer stem cells and the implications for human patients. Nat Commun. 2020 Feb 17; 11(1):929. doi:10.1038 / s41467-020-14700-7;

[0132] Culture medium No. 3: This is the stem cell culture medium used by Grassi et al. to culture renal cell carcinoma organoids. For specific references, see Grassi L, Alfonsi R, Francescangeli F, Signore M, De Angelis ML, Addario A, Costantini M, Flex E, Ciolfi A, Pizzi S, Bruselles A, Pallocca M, Simone G, Haoui M, Falchi M, Milella M, Sentinelli S, Di Matteo P, Stellacci E, Gallucci M, Muto G, Tartaglia M, De Maria R, Bonci D. Organoids as a new model for improving regenerative medicine and cancer personalized therapy in renal diseases. CellDeath Dis. 2019 Feb27;10(3):201. doi:10.1038 / s41419-019-1453-0;

[0133] Culture medium No. 4: This is the stem cell culture medium used by Hans clevers et al. to culture renal cell carcinoma organoids. For details, please refer to Clevers H. Modeling Development and Disease with Organoids. Cell. 2016 Jun 16; 165(7):1586-1597. doi:10.1016 / j.cell.2016.05.082;

[0134] Medium No. 5: Similar in composition to Medium No. 4, but the concentration of R-spondin-1 (Recombinant Human R-Spondin 1 Protein, R&D Systems, Catalog No. 4645-RS-100) is reduced from 500 ng / mL to 10 ng / mL.

[0135] Medium No. 6: Based on Medium No. 5, fetal bovine serum (FBS), insulin (Insulin, Millipore, catalog number 4506) and D-glucose (Glucose, Procell, catalog number PB180418) were added.

[0136] Table 4. Composition of cell culture medium

[0137]

[0138]

[0139] Note: × indicates that the component is not present, and √ indicates that the component is the main solvent. The concentrations in Table 4 are the final concentrations of each component in the corresponding culture medium system.

[0140] WHO / ISUP classification is based on the assessment made at the 2012 Consensus Conference of the International Society of Urological Pathology.

[0141] The IMDC risk score is determined based on the following six indicators: the interval between diagnosis of primary renal cell carcinoma and systemic treatment is <1 year, Karnofsky behavioral status score is <80, hemoglobin is <lower limit of normal, serum calcium is >upper limit of normal, absolute neutrophil count is >upper limit of normal, and absolute platelet count is >upper limit of normal.

[0142] The results are shown in Table 5: CTCs were standardized using medium No. 6, and CTC cultures were established from 10 samples with a success rate of 66.7%.

[0143] Furthermore, during culture, it was found that CTCs and CTECs can coexist. CTECs can self-assemble and proliferate into spheres, while CTCs attach, grow, and expand around the endothelial cell spheres. The remaining leukocytes gradually undergo apoptosis within 30 days. Figure 2 (d).

[0144] Peripheral blood samples from 3 healthy subjects were used as controls. Under culture conditions of medium #6, no cells survived in the healthy subjects. Figure 2 (e). This observation indicates that enrichment methods using minimal selection bias and physical damage are beneficial to the survival of CTCs, and that preserving CTECs, leukocytes, and other blood microenvironment cells can promote the proliferation of CTCs.

[0145] Table 5. Data and culture results of newly diagnosed metastatic renal cell carcinoma patients with circulating tumor cells.

[0146]

[0147]

[0148] Note: ccRCC is an abbreviation for clear cell renal cell carcinoma.

[0149] III. Verification of the Renal Cell Carcinoma Origin of CTC

[0150] This study used HE, IHC, and FISH staining to label renal cell carcinoma-specific biomarkers to identify the renal cell carcinoma origin of 10 successfully cultured CTCs obtained in step two. Among them, four enrolled patients provided paraffin sections of primary tumor tissue matched with their CTCs.

[0151] The specific methods for labeling renal cell carcinoma-specific biomarkers using HE, IHC, and FISH staining are as follows:

[0152] A. HE staining:

[0153] (1) Baking slices: Place the tissue slices in a 60℃ oven and bake for more than 1 hour;

[0154] (2) Dewaxing: Place the slices in xylene I for 10 minutes, xylene II for 10 minutes, and xylene III for 10 minutes;

[0155] (3) Hydration: Soak the slices in a gradient of 100%, 95%, 95%, 85%, 85%, and 75% alcohol for 5 minutes each;

[0156] (4) Staining: Hematoxylin for 5 minutes, rinse twice with water; differentiation solution for 5 seconds, rinse once with water; blue solution for 5 seconds, rinse once with water; eosin solution for 3 minutes, rinse twice with water;

[0157] (5) Dehydration: Starting with 95% alcohol, proceed in reverse order to xylene;

[0158] (6) Sealing: Xylene transparent sealing.

[0159] B. IHC staining:

[0160] (1) Baking slices: Place the tissue slices in a 60℃ oven and bake for more than 1 hour;

[0161] (2) Dewaxing: Place the slices in xylene I for 10 minutes, xylene II for 10 minutes, and xylene III for 10 minutes;

[0162] (3) Hydration: Soak the slices in a gradient of 100%, 95%, 95%, 85%, 85%, and 75% alcohol for 5 minutes each;

[0163] (4) Place the slide in the antigen retrieval box, add PBS solution, and wash 3 times on a shaker for 3 minutes each time;

[0164] (5) Antigen retrieval: Set the microwave oven to high power for 4 minutes. Once the retrieval solution boils, switch to the defrost setting and run for 12 minutes.

[0165] (6) Transfer the slide into the antigen retrieval box and wash with PBS 3 times, 3 minutes each time;

[0166] (7) Blocking peroxidase: Place the slide in a humidified chamber, add 200 μL of 3% H2O2 to the tissue at room temperature, and incubate in the humidified chamber in the dark for 20 min at room temperature;

[0167] (8) Wash with PBS 3 times, 4 min each time;

[0168] (9) Blocking non-specific antigens: Circle the tissue outline with a histochemical pen, add 200ul of sheep serum working solution to each slide to cover the tissue, and incubate at room temperature for 1 hour;

[0169] (10) Incubation of primary antibody: Prepare a suitable concentration of primary antibody, add 200 μL of primary antibody to each glass slide, place in a humidified chamber, and incubate overnight at 4°C;

[0170] (11) Rewarming: Take the wet box out of the 4℃ cold storage and place it at room temperature for 1 hour to rewarm. Wash with PBS 3 times, 3 minutes each time.

[0171] (12) Incubation with secondary antibody: Add secondary antibody A solution to the tissue slide, incubate at room temperature for 20 min, wash with PBS 3 times, 3 min each time; add secondary antibody B solution, incubate at room temperature for 20 min, wash with PBS 3 times, 3 min each time;

[0172] (13) Prepare DAB working solution: Add 50ul of DAB concentrate to 1ml of DAB substrate solution, mix well to prepare DAB working solution. Prepare and use immediately, store away from light and use within 6 hours.

[0173] (14) DAB color development: Prepare DAB color development solution according to the instructions. First, drop it onto the tissue and observe it under a microscope in real time. When the color development depth is appropriate, immediately put the section into distilled water to stop the color development.

[0174] (15) Hematoxylin staining: Based on the location of the target protein molecule in the cell, the section is stained in hematoxylin for 2-5 min, and the staining is stopped with distilled water.

[0175] (16) Differentiation: In 1% hydrochloric acid alcohol, differentiate the slices for 1-3 seconds, and then place them in PBS solution to terminate the differentiation;

[0176] (17) Blueing: Continue to blue in running tap water for 10 minutes;

[0177] (18) Dehydration: Immerse the slices in a gradient of 75%, 85%, 85%, 95%, 95%, 100%, and 100% alcohol for 2 minutes each;

[0178] (19) Clearing: Place the tissue sections in xylene for 10 min to clear them;

[0179] (20) Covering the slide: Add a small amount of neutral resin to the center of the slide and immediately cover it with a coverslip to avoid air bubbles;

[0180] (21) Place the slides in a ventilated place to dry overnight, and then collect and store them in a slide box.

[0181] (22) Taking pictures: Under a microscope, observe and select a suitable field of view to take pictures.

[0182] C. FISH staining:

[0183] (1) Baking slices: Place the tissue slices in a 60℃ oven and bake for more than 1 hour;

[0184] (2) Dewaxing: Place the slices in xylene I for 10 minutes, xylene II for 10 minutes, and xylene III for 10 minutes;

[0185] (3) Hydration: Soak the slices in 100%, 95%, 95%, 85%, 85%, and 75% alcohol for 5 minutes each;

[0186] (4) Antigen retrieval: Incubate the slides in boiling 1× citrate-EDTA buffer for 20 minutes. Cool the slides to 22℃-25℃ in citrate-EDTA buffer. Wash the slides with water for 5 minutes;

[0187] (5) Blocking endogenous peroxidase: Add 100 μL of endogenous peroxidase inhibitor (3% H2O2) to each surrounding section and incubate at 22℃-25℃ for 10 minutes. Gently shake (50 rpm) and wash the slide three times for 5 minutes in 100 mL PBS;

[0188] (6) Blocking: Incubate with 5% BSA in PBS solution for 1 hour while gently shaking (50 rpm) at 22℃-25℃;

[0189] (7) Incubation with fluorescent antibodies: Add Alexa Fluor (AF)488 antibody (green) and AF594 antibody (red), which will mark the 25th gene locus on the centromere of chromosome 3 and the short arm of chromosome 3, respectively. Incubate overnight in a humidified box at 37°C for hybridization.

[0190] (8) Wash with PBS 3 times, 4 min each time;

[0191] (9) Staining cell nuclei: Cell nuclei were stained with 0.2 μmol / L 4',6-diamidinyl-2-phenylindole (DAPI);

[0192] (10) Mounting: Mount the slide with an anti-quenching mounting medium;

[0193] (11) Imaging: Immunofluorescence was imaged using a Zeiss confocal microscope. The major signal count for each FISH probe was assessed and recorded. 3p deletion was defined as the presence of fewer 3p25 probe signals than chromosome 3 probe signals in at least 60% of the tumor cell nuclei. Aneuploidy was defined as having fewer or more than two chromosome 3 probe signals in the cell nucleus. Leukocytes were defined as cells with two 3p25 probe signals and two chromosome 3 probe signals.

[0194] The results are as follows Figure 3 As shown: 1) Clear cell renal cell carcinoma (CTC) exhibits enlarged nuclei and clear cytoplasm in HE staining. HE staining of CTCs obtained in step 2 also revealed enlarged nuclei and clear cytoplasm similar to those in renal cell carcinoma tissue. 2) PAX-2, PAX-8, CA IX, and CK are key biomarkers for pathological diagnosis of renal cell carcinoma. Staining results showed that these biomarkers were positive in CTCs but negative in CTEC spheroids. 3) Vimentin can also be positive in ccRCCs and strongly positive in high-grade WHO / ISUP regions. Staining results showed that vimentin staining was positive in CTCs. 4) FISH detection of 3p deletion is the best molecular pathological method for renal cell carcinoma. Staining results showed that 3p deletion was positive in CTCs, further confirming the renal cell carcinoma origin of CTCs.

[0195] Regarding circulating tumor endothelial cells (CTEC): 1) Vimentin, CD31, and CD34 are biomarkers of endothelial cells. Staining results showed that CTEC spheroids were positive, indicating that the spheroids originated from endothelial cells; 2) Tumor-associated cells often exhibit aneuploid karyotypes due to chromosome instability. The chromosome 3 probe signal showed that the spheroids were mostly non-diploid, suggesting that CTEC is an aneuploid tumor-associated cell; 3) Gradually apoptotic leukocytes were present in the culture system, so it was necessary to rule out false positives that leukocytes might produce. CD45 is considered a biomarker of leukocytes, and staining results showed that CD45 was negative in both CTC and CTEC.

[0196] This study confirmed through HE, IHC, and FISH staining that CTCs can highly retain the biological characteristics of renal cell carcinoma cells. Scattered cells are CTCs derived from renal cell carcinoma, while clusters of cell spheroids are tumor-associated endothelial cells.

[0197] Example 2: Tumorigenicity Study of CTCs in Xenograft Models

[0198] This study used immunocompromised NPSG mice (Beijing imodels Biotechnology Co., Ltd. http: / / www.imodels.tech / ) to construct a xenograft model to demonstrate the tumorigenic capacity of cultured CTCs. The specific experimental steps are as follows:

[0199] The cultured CTCs were injected subcutaneously into the right back near the axilla of NPSG mice. CTCs obtained in Example 1 were implanted (number 125,000 cells / point). Tumor volume was measured within 2 months post-implantation. CTCs from patients (numbers 05, 08, 09, 11, 13, 14) generated tumor tissue in mice (designated CDX 05, CDX08, CDX09, CDX11, CDX13, and CDX14, respectively). Figure 4 (a and b), but CDX-13 died before sampling, causing sampling failure. Tumor doubling time was 14 to 21 days. Figure 4 (c)

[0200] To determine the renal cell carcinoma characteristics of the CDX tumor tissues, the CDX tumor tissues (referred to as CDX05, CDX08, CDX09, CDX11 and CDX14, respectively) were stained with HE and IHC, and the specific operation method was the same as the staining method in step three of Example 1.

[0201] Staining results showed that CTCs could be scattered or nested in tumor tissue, with enlarged nuclei, clear cytoplasm, and positive for renal cell carcinoma markers PAX-2 and PAX-8, retaining biological characteristics similar to renal cell carcinoma tissue. CDX tumor tissue showed CD31-positive luminal structures. Since the CD31 antibody in this study only labeled human endothelial cells, this suggests that these luminal structures are humanized blood vessels formed by CTCs, rather than mouse-derived neovascularization. Figure 4 (d). The formation of humanized blood vessels is beneficial for subsequent screening of anti-angiogenic drugs in mice. CD45 staining was negative in CDX tumor tissue.

[0202] Example 3: Gene mutations in CTC-preserved renal cell carcinoma tumor tissue

[0203] This study performed whole-exome sequencing (WES) on CTC cultures and matched tumor tissues from four renal cell carcinoma patients (patients 05, 08, 09, and 11). The sequencing was commissioned to Beijing Novogene Bioinformatics Technology Co., Ltd. The consistency of gene copy number and key gene mutations between the two was compared.

[0204] This study analyzed somatic mutations in tumor samples and CTCs, with CTCs retaining most of the gene mutations found in the corresponding tumor tissue. Figure 5(a) Analysis of eight key genes in renal cell carcinoma (CTC) (VHL, PBRM1, SETD2, BAP1, KDM5C, MTOR, PIK3CA, TP53) revealed a high degree of consistency in mutation patterns of these eight genes between CTC and tumor tissues. The most common somatic mutation was VHL gene mutation, consistent with the gene mutation spectrum of renal cell carcinoma. Figure 5 (b)

[0205] Copy number variation (CNV) analysis revealed a consistency in DNA copy number variations between CTCs and their corresponding tumors, with CTC-09 containing many novel gene mutations not present in the original tumor. Figure 6 This could be due to genetic evolution within CTCs or heterogeneity of tumor samples.

[0206] Example 4: The mini-CDX model constructed based on CTC culture can be used for drug screening.

[0207] To explore the feasibility of using cultured CTCs as a drug testing platform and a personalized drug sensitivity model, this study constructed a mini-CDX model using tumor tissue from one CDX (CDX-11) patient. The workflow is detailed below. Figure 7 a.

[0208] 1. Mini-CDX Model Construction Method

[0209] (1) BALB / c-nude mice (Beijing Chuangmo Biotechnology Co., Ltd.) were housed subcutaneously, 5-6 week old, female, in an SPF-grade animal facility. The mice were kept in an environment with 12 hours of light / 12 hours of darkness, a temperature of 21°C and 50% relative humidity, and were allowed free access to water and food.

[0210] (2) Immerse the CDX tumor tissue in cell preservation solution, store at 4°C, and transport to the laboratory;

[0211] (3) The tumor tissue was digested to prepare a cell suspension, and the cells were counted. The cells were resuspended, the cell concentration was adjusted, and the suspension was filled into mini-CDX experimental capsules;

[0212] (4) Mini-CDX capsules were injected subcutaneously into BALB / c-nude mice and the administration began on the day of injection.

[0213] (5) After 7 days of continuous administration, mini-CDX in vivo capsules (mini-CDX-11) were obtained. The mini-CDX in vivo capsules (mini-CDX-11) were removed and the cell viability after different administration regimens was detected by testing the ATPase activity in tumor tissue cells.

[0214] (6) Using CellTiter- The Luminescent Cell Viability Assay (G7571, Promega, Madison, WI, USA) measures antitumor activity based on relative fluorescence units (RFU). Proliferation rate was calculated using the following formula: Proliferation rate = (RFU[D7] - RFU[D0]) drug / (RFU[D7] - RFU[D0]) control, where Relative Fluorescence Units (RFU) represent the relative fluorescence units; D7 represents day 7, and D0 represents day 0.

[0215] The Mini-CDX model received seven commonly used first-line or second-line treatment regimens for renal cell carcinoma: ① everolimus combined with lenvatinib; ② everolimus; ③ axitinib combined with bevacizumab; ④ cabozantinib; ⑤ axitinib; ⑥ sunitinib; and ⑦ pazopanib. One mouse was used for each treatment regimen, and each mouse was implanted with three mini-CDX in vivo capsules (mini-CDX-11) containing tumor cells. One mouse received saline as a placebo control. Due to the immunodeficient nature of the mice, drug sensitivity testing for anti-PD-1 / PD-L1 antibodies was not performed. Treatment lasted 7-8 days, after which the implanted capsules were removed, and the mean cell proliferation rate was measured based on relative fluorescence units (RFU).

[0216] The results are as follows: the average proliferation rates of CTCs in the seven treatment regimens were 30%, 38%, 51%, 56%, 72%, 82%, >100%, and 100%, respectively. Figure 7 In (b), drug sensitivity varies greatly, with the highest average proliferation rate being >100% and the lowest being 30%. Figure 7 c) suggests that individualized drug treatment is necessary for this patient.

[0217] 2. Mini-CDX drug sensitivity results and clinical efficacy in patients with renal cell carcinoma

[0218] The patient (Pt-11) who underwent drug screening using the Mini-CDX model was clinically diagnosed with clear cell renal cell carcinoma with left parietal pleural metastasis. Based on clinical guidelines for renal cell carcinoma, this patient received sunitinib as first-line treatment.

[0219] The specific treatment plan is as follows: Sunitinib 50mg, once daily, orally, for 6 weeks per course of treatment. After 4 weeks of treatment, the medication should be discontinued for 2 weeks for observation before resuming treatment.

[0220] During the first 6 treatment cycles, the best response was stable disease (SD). Figure 8 (a and b). Malignant pleural effusion developed at the parietal pleural metastasis site during the 6th cycle, and was diagnosed as sunitinib resistant according to RECIST 1.1 criteria. Figure 8 (c) As mentioned above, in vivo cell viability assays in the mini-CDX model showed that the average proliferation rate of CTCs treated with sunitinib was 82%, and the cell inhibition rate was <20%, suggesting that sunitinib may have a poor therapeutic effect on this patient, which is consistent with the imaging confirmation of rapid resistance to sunitinib in the patient.

[0221] Mini-CDX model drug sensitivity results showed that the average proliferation rate of CTCs treated with axitinib combined with bevacizumab was 51%, suggesting that axitinib combined with bevacizumab may have a good therapeutic effect on this patient.

[0222] In second-line treatment, based on drug sensitivity results and clinical guidelines, patients were treated with axitinib in combination with pembrolizumab and bevacizumab. The specific dosing regimen was as follows: axitinib orally at a dose of 5 mg twice daily; pembrolizumab intravenously at a dose of 200 mg every 3 weeks; and bevacizumab via intrapleural infusion at a dose of 200 mg every 2 weeks. After two cycles of this regimen, the patient achieved partial remission (PR). Figure 8 (d). After a 4-month follow-up, the patient remained in PR status.

[0223] In summary, the patient's drug sensitivity test results are consistent with the actual efficacy of first- and second-line treatments, achieving partial remission. The CTC-based drug sensitivity test benefited the patient, preliminarily demonstrating the potential of this CTC culture system as a drug screening and personalized model, and showing significant clinical application prospects.

[0224] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A composition for culturing circulating tumor cells, characterized in that: The composition contains a cell culture medium and a biological scaffold, wherein the cell culture medium contains a reagent named RFGI, and the RFGI contains fetal bovine serum, insulin, and glucose.

2. The composition according to claim 1, characterized in that: The RFGI also contains the R-spondin-1 protein.

3. The composition according to claim 1 or 2, characterized in that: The RFGI is composed of fetal bovine serum, insulin, glucose and R-spondin-1 protein, and the ratio of fetal bovine serum, insulin, glucose and R-spondin-1 protein in the RFGI is 100 mL fetal bovine serum : 137.6 U : 3.3 mmol glucose : 10 μg R-spondin-1 protein.

4. The composition according to claim 3, characterized in that: The cell culture medium contained 10 ng / mL of R-spondin-1 protein, fetal bovine serum, insulin, and glucose.

5. A culture medium for culturing circulating tumor cells, characterized in that: The culture medium is any of the cell culture media described in claims 1-4.

6. A kit for culturing circulating tumor cells, characterized in that: The kit contains RFGI as described in any one of claims 1-4.

7. A method for culturing circulating tumor cells, characterized in that: Includes the following steps: 1) Enrichment of isolated circulating tumor cells; 2) The circulating tumor cells in step 1) are cultured using the composition according to claims 1-4 to obtain circulating tumor cells, wherein the tumor cells are renal tumor cells.

8. The method according to claim 7, characterized in that: Step 1) describes a method for enriching circulating tumor cells using density gradient centrifugation.

9. A method for constructing a circulating tumor cell xenograft model, characterized in that: The method includes transferring circulating tumor cells obtained by the method of claim 7 or 8 into mice to obtain a circulating tumor cell xenograft model.

10. The use of the composition of any one of claims 1-4, the culture medium of claim 5, the kit of claim 6, or the method of claim 7 or 8 in the preparation of circulating tumor cell products, or in the preparation of products that increase the cell density of renal circulating tumor cells.

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