Kit for predicting the efficacy of chemotherapy drugs in patients with colorectal cancer and its application

Abstract

The present invention discloses a kit for predicting the efficacy of chemotherapy drugs for patients with colorectal cancer and its application. The kit includes a washing buffer, a digestion buffer, a rectal cancer chemotherapy drug, a stem cell culture fluid, a first cell filter, and a second cell filter; the washing buffer is a phosphate buffer containing a penicillin-streptomycin solution; the digestion buffer is a DMEM culture fluid containing type IV collagenase and a penicillin-streptomycin solution; the stem cell culture fluid is a DMEM / F12 culture fluid containing glutamine, non-essential amino acids, bFGF, 2-mercaptoethanol, serum substitutes, and penicillin-streptomycin solution; the pore size of the first cell filter is 100-500μm; the pore size of the second cell filter is 20-70μm. The present invention can accurately predict the clinical chemotherapy effect of patients, provide doctors with reliable simulation results, and then guide and judge clinical treatment plans to achieve individualized treatment.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and relates to a kit for predicting the efficacy of chemotherapy drugs in patients with colorectal cancer and its application, which is embodied in the application of constructing intestinal cancer organoids, etc. Background Art

[0002] At present, the classic treatments for malignant tumors include chemotherapy, radiation and surgical resection. When patients cannot undergo radical surgery, drug therapy including chemotherapy will directly determine the prognosis and outcome of cancer patients. However, there is a certain heterogeneity in the response of individuals to drugs, which will directly lead to the differences in the same standardized treatment and the complexity of individualized treatment. Most previous studies have studied cancer patients as a whole, while ignoring the differences between individuals. Currently, more and more researchers have begun to pay attention to individualized cancer research and personalized treatment. In this research field, preclinical tumor models are very important. They are similar to real malignant tumors and can predict drug responses in vivo.

[0003] Colorectal cancer is the third most common cancer. In clinical treatment, only a small part of colorectal cancer responds to chemotherapy drugs 5-fluorouracil (5-FU), irinotecan or oxaliplatin. However, the side effects of chemotherapy, delays in treatment timing and the cost of money are all issues that clinicians need to consider when choosing treatment options. Therefore, if the effect of chemotherapy on the patient can be predicted before the patient receives chemotherapy, and then a personalized treatment plan can be formulated, it will provide great convenience and benefits to both clinicians and patients.

[0004] Drug sensitivity testing refers to the use of tumor models in vitro to detect the inhibitory effect of a drug on tumor growth in a laboratory environment. It has the advantages of being independent of the in vivo environment and having high repeatability and operability. In this detection method, the tumor model plays a core role. Currently, commonly used tumor models include animal models, tumor cell lines, primary patient-derived tumor xenografts (PDX) and primary tumor organoids (PDO). A variety of drug sensitivity testing schemes have been developed based on these models, but most of them are limited by low success rates, high operating difficulties, long testing time and high costs, weak consistency in predicting clinical efficacy, lack of unified indicators, etc., and are rarely used in clinical or commercial purposes.

[0005] Current research suggests that cell culture models with three-dimensional structures have higher biomimetic activity and can better simulate the growth environment in vivo than traditional two-dimensional culture, so they are particularly suitable for drug sensitivity testing. Organoids are micro-organs with three-dimensional tissue structures formed by stem cells in the body under special culture conditions in vitro. They reproduce the structure and function of the corresponding organs in vivo in many aspects. Commonly used three-dimensional cell culture methods include matrix gel method, hydrogel method, suspension method, hanging drop culture method and non-adhesive matrix method. Based on this technology, tumor organoids have been established from various cancers, including gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, and ovarian cancer. In addition, in order to solve the problem that clinical samples of some tumor tissues are difficult to obtain and need to be repeatedly sampled, researchers are committed to establishing organoid models from small samples or even puncture biopsy and endoscopic biopsy tissues by optimizing culture conditions and simplifying operation steps.

[0006] The mainstream method for constructing organoids in vitro is the Matrigel method. However, since the organoids constructed by the Matrigel method are completely derived from tumor stem cells, drug resistance will inevitably occur during in vitro drug sensitivity assays, and it is impossible to accurately predict the sensitivity of cancer patients to chemotherapy drugs. Matrigel is a commercial matrix gel that is a widely recognized and applied extracellular matrix component for culturing organoids.

[0007] To predict drug efficacy, more mature primary cell extraction technology and more stable organoid culture systems are still needed, while finding predictive indicators that are more in line with clinical practice. In this way, clinicians can quickly determine whether a drug is effective for a specific individual, thereby guiding clinical treatment plans, reducing patients' adverse reactions and treatment costs, finding the best treatment plan for patients, and improving the effectiveness of clinical guidance and disease prognosis. Summary of the invention

[0008] In order to accurately predict the clinical chemotherapy effect of colorectal cancer patients and solve at least one of the above technical problems, the present invention discloses a kit for predicting the efficacy of chemotherapy drugs for colorectal cancer patients, including a washing buffer, a digestion buffer, a rectal cancer chemotherapy drug, a stem cell culture medium, a first cell filter, and a second cell filter;

[0009] The washing buffer is a phosphate buffer containing a penicillin-streptomycin solution; the digestion buffer is a DMEM culture medium containing type IV collagenase and a penicillin-streptomycin solution; the stem cell culture medium is a DMEM / F12 culture medium containing glutamine, non-essential amino acids, bFGF, 2-mercaptoethanol, serum substitutes, and a penicillin-streptomycin solution; the pore size of the first cell filter is 100-500 μm; and the pore size of the second cell filter is 20-70 μm.

[0010] Furthermore, the pore size of the first cell filter is 100 μm; and the pore size of the second cell filter is 40 μm.

[0011] In some embodiments, the rectal cancer chemotherapy drug is selected from one or more of oxaliplatin, 5-fluorouracil, folinic acid, irinotecan and capecitabine.

[0012] In some embodiments, the washing buffer is a phosphate buffer containing a 1X penicillin-streptomycin solution; the 1X penicillin-streptomycin solution is prepared by diluting a 2-100X penicillin-streptomycin solution; wherein the content of penicillin in the 100X penicillin-streptomycin solution is 5-20 kU / ml, the content of streptomycin is 5-20 mg / ml, and it is prepared with 0.9% sodium chloride. Preferably, the content of penicillin in the 100X penicillin-streptomycin solution is 10 kU / ml, the content of streptomycin is 10 mg / ml, and it is prepared with 0.9% sodium chloride.

[0013] In some embodiments, the digestion buffer is a DMEM culture medium containing 0.5-5 mg / ml type IV collagenase and 1X penicillin-streptomycin solution; the stem cell culture medium is a DMEM / F12 culture medium containing 2 mM glutamine, 0.1 mM non-essential amino acids, 1-50 ng / mL bFGF, 0.1 mM 2-mercaptoethanol, 1-10% serum replacement, and 1X penicillin-streptomycin solution.

[0014] Furthermore, the digestion buffer is a DMEM culture medium containing 0.5 mg / ml type IV collagenase and 1X penicillin-streptomycin solution; the stem cell culture medium is a DMEM / F12 culture medium containing 2 mM glutamine, 0.1 mM non-essential amino acids, 8 ng / mL bFGF, 0.1 mM 2-mercaptoethanol, 5% serum replacement, and 1X penicillin-streptomycin solution.

[0015] In some embodiments, the efficacy standard of the XELOX regimen is also included; the efficacy standard of the XELOX regimen is: using the kit for predicting the efficacy of chemotherapy drugs in colorectal cancer patients to construct colorectal cancer organoids by the suspension method, and according to the drug dose-response curve of the colorectal cancer organoids under the XELOX regimen, the growth half-inhibitory concentration (IC50) of the colorectal cancer organoids from different colorectal cancer patients is calculated; if the IC50 value is <4μM, the colorectal cancer organoid belongs to the sensitive group, if the IC50 value is >10μM, the colorectal cancer organoid belongs to the resistant group; if 4≤IC50≤10μM, the colorectal cancer organoid belongs to the intermediate group.

[0016] On the other hand, the present invention also provides the use of the above-mentioned kit for predicting the efficacy of chemotherapy drugs in patients with colorectal cancer in constructing intestinal cancer organoids for predicting the efficacy of chemotherapy drugs in patients with colorectal cancer.

[0017] In a third aspect, the present invention provides a method for constructing a colorectal cancer organoid, comprising the following steps:

[0018] S1. Lysing colorectal cancer tissue into single cells or cell clusters using the kit for predicting the efficacy of chemotherapy drugs in colorectal cancer patients as described above;

[0019] S2. Using the kit for predicting the efficacy of chemotherapy drugs in colorectal cancer patients as described above, the single cells or cell clusters obtained in step S1 are subjected to stemness induction and organoid formation of colorectal cancer cells.

[0020] In some embodiments, step S1 comprises the following steps:

[0021] S1.1, taking colorectal cancer tissue from a patient, and washing it with the washing buffer;

[0022] S1.2, cutting the tissue cleaned in step S1.1 into minced meat;

[0023] S1.3, transferring the chopped tissue in step S1.2 to a centrifuge tube, adding the digestion buffer, and shaking digesting at 37°C to obtain a lysed cell / tissue suspension;

[0024] S1.4, filtering the cell / tissue suspension after lysis in step S1.3 through the first cell filter to remove residual tissue and retain the filtrate;

[0025] S1.5, centrifuging the filtrate obtained in step S1.4, discarding the supernatant, and obtaining the precipitate, which is the single cell or cell cluster.

[0026] In some embodiments, the source of the colorectal cancer tissue in step S1.1 includes:

[0027] 1) Obtained through endoscopic (colonoscopy) biopsy before the implementation of neoadjuvant chemotherapy to predict the effect of neoadjuvant chemotherapy in patients.

[0028] 2) After the patient undergoes surgical treatment, tissue is obtained by collecting surgical specimens to predict the effect of postoperative adjuvant chemotherapy.

[0029] 3) For patients with colorectal cancer liver metastasis, biopsy tissue of liver metastasis lesions is obtained by puncture to predict the chemotherapy effect of patients and assist in the selection of chemotherapy regimens.

[0030] 4) For patients with colorectal cancer liver metastasis, after surgical treatment, the tissue of liver metastasis lesions is obtained by collecting surgical specimens to predict the effect of postoperative adjuvant chemotherapy.

[0031] 5) For patients with recurrent / recurrent and metastatic colorectal cancer, biopsy tissue is obtained through colonoscopy / puncture to predict the chemotherapy effect of the patient and assist in the selection of chemotherapy regimen.

[0032] 6) For patients with colorectal cancer with dual / multiple lesions, tumor lesions in different locations can be collected simultaneously to predict the chemotherapy effects separately.

[0033] In some embodiments, step S2 comprises the following steps:

[0034] S2.1, resuspending the precipitate obtained in step S1.5 with the washing buffer, then centrifuging, discarding the supernatant, and retaining the precipitate;

[0035] S2.2, resuspending the precipitate obtained in step S2.1 with the washing buffer, filtering the obtained cell suspension with the second cell filter, retaining the cell clusters intercepted on the second cell filter, and filtering out the single cells, and discarding the filtrate;

[0036] S2.3, washing the second cell filter and the intercepted cell clusters from top to bottom with the washing buffer to remove the remaining single cells;

[0037] S2.4, inverting the second cell filter treated in step 2.3, and washing the cell clusters from the second cell filter with the stem cell culture medium;

[0038] S2.5. Cultivate the cell clusters eluted in step 2.4 with the stem cell culture medium to form a distinct spherical structure with a smooth surface; continue culturing to form the intestinal cancer organoids.

[0039] In a fourth aspect, the present invention further provides an application of a colorectal cancer organoid in an in vitro chemotherapy drug sensitivity test, wherein the colorectal cancer organoid is obtained by the construction method described above; the application of the colorectal cancer organoid in an in vitro chemotherapy drug sensitivity test comprises the following steps:

[0040] S3.1, selecting the colorectal cancer organoids with a diameter greater than 200 um obtained in step S2.5 for in vitro chemotherapy drug sensitivity test; establishing a concentration gradient of the rectal cancer chemotherapy drug;

[0041] S3.2, adding the colorectal cancer chemotherapy drug of the corresponding concentration in step S3.1 to the colorectal cancer organoid selected in step S3.1;

[0042] S3.3. The intestinal cancer organoids treated by the step S3.2 are cultured in 5% CO2 at 37°C, during which images are captured, and the area of ​​the intestinal cancer organoids during the culture process is measured. The growth / inhibition efficiency is calculated by comparing the area of ​​the intestinal cancer organoids during the culture process with their initial size. A drug dose-response curve is plotted with a drug dose of 0 μM as the baseline, and the data is expressed as the growth percentage starting from 0 μM, thereby obtaining the associated area under the curve (AUC) of the intestinal cancer organoids. According to the AUC of the intestinal cancer organoids and compared with the efficacy standard, it is concluded that the intestinal cancer organoids belong to the sensitive group, intermediate group or resistant group of the colorectal cancer chemotherapy drug.

[0043] There are different efficacy standards for different medication regimens. For example, the efficacy standard of the XELOX regimen is: the colorectal cancer organoids are constructed by the suspension method using the kit for predicting the efficacy of chemotherapy drugs in colorectal cancer patients, and the IC50 of the colorectal cancer organoids from different colorectal cancer patients is calculated based on the drug dose-response curve of the colorectal cancer organoids under the XELOX regimen; if the IC50 value is <4μM, the colorectal cancer organoids belong to the sensitive group, if the IC50 value is >10μM, the colorectal cancer organoids belong to the drug-resistant group; if 4≤IC50≤10μM, the colorectal cancer organoids belong to the intermediate group.

[0044] Those skilled in the art can use the kit and the method for constructing intestinal cancer organoids provided by the present invention to establish the efficacy standards of other medication regimens through small sample studies. The basic principle is: calculate the corresponding area under the curve (AUC) of the drug dose-response curve, and divide it into 3 groups according to the numerical value after standardization, with the highest value being 1 and the lowest value being 0. The values ​​in the range of 67-100% are the drug-resistant group, the values ​​in the range of 34-66% are the medium group, and the values ​​in the range of 0-33% are the sensitive group; compare with the clinical data, if it is basically consistent, adopt it, if there is a large discrepancy, adjust it adaptively; then correspond to the IC50 of each group, and obtain the IC50 corresponding to the drug-resistant group, the medium group and the sensitive group.

[0045] The kit, construction method and application of the present invention can collect specimens multiple times in multiple time periods of clinical treatment and predict the effect of chemotherapy on patients.

[0046] The chemotherapeutic drugs that can be evaluated in the present invention include:

[0047] (1) The kit includes oxaliplatin and 5-fluorouracil, which can be used to detect XELOX, a first-line treatment for colorectal cancer.

[0048] (2) By increasing the types of drugs tested, other treatment options for colorectal cancer can be personalized, including FOLFOX (oxaliplatin, fluorouracil, folinic acid), FOLFIRI (irinotecan, fluorouracil, folinic acid), etc.

[0049] The drug 5-fluorouracil provided by the kit of the present invention is an effective component of the clinical colorectal cancer treatment drug capecitabine after metabolism in the body, so 5-fluorouracil is selected for in vitro drug evaluation.

[0050] The kits of the present invention are not suitable for predicting the effect of radiotherapy on patients.

[0051] The kit of the present invention is not suitable for predicting the effect of drugs whose targets are located in non-tumor cells, including:

[0052] (1) Immunotherapy drugs: PD-1 monoclonal antibody, CTLA4 monoclonal antibody.

[0053] (2) Anti-angiogenic drugs: bevacizumab.

[0054] The kit of the present invention is not suitable for predicting the drugs included in the medication regimen clearly specified in the colorectal cancer treatment guidelines:

[0055] (1) Cetuximab: The guidelines recommend it for patients with KRAS mutations.

[0056] The kit of the present invention provides a standard for predicting the efficacy of the XELOX regimen (oxaliplatin + 5-fluorouracil): We previously drew a drug dose-response curve for 12 colorectal cancer patients ( Figure 1 ), calculate the AUC values ​​(area under the curve) of different patients, and divide the colorectal cancer organoids from different patients into three groups: resistant, moderate, and sensitive according to the AUC values ​​( Figure 2 ). The efficacy criteria for predicting other colorectal cancer chemotherapy drugs can be obtained by referring to the efficacy criteria for predicting the XELOX regimen (oxaliplatin + 5-fluorouracil).

[0057] The advantages of the present invention are: the kit involved in the present invention uses an in vitro tumor organoid construction method to construct an organoid model from clinically derived colorectal cancer tissue, and in vitro drug sensitivity tests can accurately predict the patient's clinical chemotherapy effect, provide doctors with reliable simulation results, and then guide and judge clinical treatment plans to achieve individualized treatment. The kit involved in the present invention uses a suspension method to construct colorectal cancer organoids, which is simple to operate and has a high success rate. At the same time, the colorectal cancer organoid construction time is greatly shortened compared to the three-dimensional (3D) culture method, and the construction of colorectal cancer organoids and in vitro drug sensitivity experiments can be completed within one week, and the predicted results of in vitro drug efficacy can be reported at the same time. In the initial 12 test cases, the consistency rate between the predicted results of the kit and the patient's clinical chemotherapy results reached 91.66% (Table 1).

[0058] The kit involved in the present invention is combined with the method of suspended tumor sphere culture to construct an organoid model for in vitro drug screening (i.e., the method for constructing colorectal cancer organoids involved in the present invention). Its advantages also lie in that a large number of high-purity colorectal cancer cells can be extracted in a short period of time, and the constructed colorectal cancer organoids are highly consistent with the tumor cells in the primary tumor, and are therefore very suitable for in vitro drug efficacy prediction.

[0059] Through comparative experiments, it was found that the intestinal cancer organoids obtained by the method for constructing intestinal cancer organoids involved in the present invention contain cancer stem cells (CSC) and non-CSC components, while the intestinal cancer organoids constructed by the Matrigel method are completely derived from CSC. Since the primary tumor also contains CSC and non-CSC components, the organoids constructed by the method of the present invention are highly consistent with the tumor cells in the primary tumor ( Fig.11 ).

[0060] As the culture time increases, the difference between the organoids obtained by the method for constructing intestinal cancer organoids of the present invention and the organoids obtained by the Matrigel method gradually decreases, and there is no significant difference between them in terms of growth state and number of consecutive reproductions ( Fig.10 ). In terms of culture time, the time of the organoids constructed by the present invention is significantly shortened ( Fig.12 ).

[0061] The method for constructing intestinal cancer organoids involved in the present invention is greatly improved from the previously disclosed method for establishing tumor organoids in vitro by adding two components: a second cell filter (especially a cell filter with a pore size of 40 μm) and 2-mercaptoethanol. The use of a 40 μm cell filter can effectively capture tumor spheres with a diameter greater than 40 μm. These tumor spheres can retain the characteristics of the original tumor in vitro and form tumor organoids under the stimulation of 2-mercaptoethanol.

[0062] Compared with CN112080474A (a method for establishing tumor organoids in vitro), the method of CN112080474A is to construct tumor organoids using the Matrigel method, and then digest them into single cell suspensions for chemotherapeutic drug sensitivity determination, with the purpose of using a suspension culture method in the later stage to reduce the drug resistance problem caused by the Matrigel method. The present invention adopts a suspension method to construct organoids, and the suspension method is used for drug sensitivity determination, so as to avoid the drug resistance problem caused by the Matrigel method to the greatest extent, and can greatly shorten the experimental cycle.

[0063] Compared with CN113201479A (a method for isolating and culturing motile mouse small intestinal organoids in vitro), the method of CN113201479A is to construct mouse small intestinal organoids in vitro, which is different from the intestinal cancer organoids constructed by the present invention in terms of methodology and research objects. Wnt3A is the main ligand in the Wnt signaling pathway, a representative signaling protein of the Wnt family, and a key inducer of signal transduction. For the cultivation of intestinal organoids, the supplementation of Wnt3A is necessary, so the present invention does not additionally supplement Wnt3A, so that normal intestinal organoids cannot be propagated in the culture system of the present invention, thereby selectively culturing tumor organoids.

[0064] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The drug dose-response curves for 12 patients with colorectal cancer.

[0066] Figure 2 It is the standard AUC value and grouping for predicting the efficacy of the XELOX regimen.

[0067] Figure 3 It is the overall technical route of the kit involved in the present invention and its application.

[0068] Figure 4 This is the growth process of the organoid in Example 1.

[0069] Figure 5 is the drug dose-response curve of Example 1.

[0070] Figure 6 These are imaging pictures of the patient in Example 1 before and after chemotherapy.

[0071] Figure 7 This is the growth process of the organoid in Example 2.

[0072] Figure 8is the drug dose-response curve of Example 2.

[0073] Fig. 9 These are the imaging pictures of the patient in Example 2 before and after chemotherapy.

[0074] Fig.10 It is the morphology of intestinal cancer organoids constructed by the Matrigel method and the method for constructing intestinal cancer organoids involved in the present invention (referred to as the present invention in the figure).

[0075] Fig.11 It is to detect the proportion of tumor stem cells in the original tumor, the Matrigel method and the colorectal cancer organoid construction method involved in the present invention (referred to as the present invention in the figure).

[0076] Fig.12 It is to detect the growth rate of colorectal cancer organoids constructed by the Matrigel method and the colorectal cancer organoid construction method involved in the present invention (referred to as the present invention in the figure). DETAILED DESCRIPTION

[0077] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific diagrams. However, the invention is not limited to the following implementation cases.

[0078] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0079] Figure 3 The overall technical route of the kit and its application involved in the present invention is shown. A biopsy tissue of the tumor is taken from a patient clinically diagnosed with colorectal cancer or rectal cancer; the patient is given chemotherapy, and images of the lesion are taken before and after chemotherapy to obtain the actual clinical chemotherapy results; at the same time, the biopsy tissue taken out is cultured into intestinal cancer organoids using the kit involved in the present invention; the cultured intestinal cancer organoids are subjected to in vitro chemotherapy drug sensitivity tests; a drug dose-response curve is drawn, and the predicted chemotherapy results are obtained according to the size of the AUC; the predicted chemotherapy results are compared with the actual clinical chemotherapy results, and it is found that the consistency can reach 91.66%, indicating that the kit involved in the present invention has high accuracy in predicting the efficacy of chemotherapy drugs in patients with colorectal cancer.

[0080] On this basis, clinicians can use the kit involved in the present invention to predict the efficacy of corresponding chemotherapy drugs before chemotherapy, and consider using this drug for patients in the sensitive group (which may also include the intermediate group); and switch to other drug treatments for patients in the resistant group (which may also include the intermediate group).

[0081] Example 1

[0082] 1. A 62-year-old male was clinically diagnosed with colorectal cancer. Rectal magnetic resonance imaging (MR) diagnosed colorectal cancer (T4N2); CRM (+); EMVI (+). Before neoadjuvant therapy, a biopsy tissue was obtained by endoscopic means.

[0083] 2. Composition of the kit:

[0084] (1) Washing buffer: phosphate-buffered saline (PBS) containing 1X penicillin-streptomycin solution.

[0085] The content of penicillin in the penicillin-streptomycin solution (100X) is 10 kU / ml and the content of streptomycin is 10 mg / ml. The solution is prepared with 0.9% sodium chloride.

[0086] Phosphate buffered saline (PBS), containing 135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, pH 7.3.

[0087] (2) Digestion buffer: DMEM culture medium containing 0.5 mg / ml type IV collagenase and 1X penicillin-streptomycin solution.

[0088] (3) 100μm cell strainer, 40μm cell strainer.

[0089] (4) Stem cell culture medium: DMEM / F12 culture medium, 2 mM glutamine, 0.1 mM non-essential amino acids, 8 ng / mL bFGF, 0.1 mM 2-mercaptoethanol, 5% serum replacement, 1X penicillin-streptomycin solution.

[0090] (5) Chemotherapy drugs: oxaliplatin, 5-fluorouracil.

[0091] 3. Testing steps:

[0092] (1) Lyse the biopsied tumor tissue into single cells or cell clusters:

[0093] a) Take several rice-sized pieces of biopsy tumor tissue from the patient and wash them several times with washing buffer until they are clean to the naked eye.

[0094] b) Use disposable surgical blades to mince the tissue in a 6 cm cell culture dish.

[0095] c) Transfer the minced tissue to a 15 ml centrifuge tube, add an appropriate amount of digestion buffer, and digest with shaking at 37°C for 1 hour.

[0096] d) Filter the fully lysed cell / tissue suspension through a 100 μm cell strainer to remove residual tissue.

[0097] e) Centrifuge the filtrate at 1500 rpm for 5 minutes, discard the supernatant, and the resulting precipitate is single cells or cell clusters, which can be transferred to the next step.

[0098] (2) Stemness induction and organoid formation of colorectal cancer cells:

[0099] a) Resuspend the cell pellet in the washing buffer, then centrifuge at 1500 rpm for 5 minutes and discard the supernatant.

[0100] b) Resuspend the pellet with 2 ml of washing buffer and filter the resulting cell suspension with a 40 μm cell strainer, retaining the cell clusters intercepted on the upper layer of the strainer. At this time, single cells are filtered out and the filtrate is discarded.

[0101] c) Wash the filter and the intercepted cell clusters from top to bottom with washing buffer to remove the remaining single cells;

[0102] d) Invert the cell strainer and use stem cell culture medium to wash the cell pellet off the strainer.

[0103] e) The washed cell clusters were cultured in a stem cell culture medium for 24 hours to form a clear spherical structure with a smooth surface.

[0104] f) Growth process of intestinal cancer organoids derived from Example 1 ( Figure 4 ).

[0105] g) Select colorectal cancer organoids with a diameter greater than 200 μm and proceed to the next step.

[0106] (3) In vitro chemotherapy drug sensitivity test of colorectal cancer organoids:

[0107] a) The colorectal cancer organoids prepared in the previous step were used for in vitro drug sensitivity testing. The drug combination selected was oxaliplatin combined with 5-fluorouracil. The concentration gradient was established as follows: 0, 0.25, 0.5, 1, 3, 6, 13, 25, 50, and 100 μM; both oxaliplatin and 5-fluorouracil followed this concentration gradient.

[0108] b) Add the corresponding dose of the drug to the colorectal cancer organoid culture system and culture it in a 96-well plate with 10 to 20 colorectal cancer organoids per well.

[0109] c) Cultured at 37°C in 5% CO2 for 7 days, captured images on days 1, 3, 5, and 7, measured the area of ​​colorectal cancer organoids, calculated the growth / inhibition efficiency by comparing the area of ​​colorectal cancer organoids with their starting size, plotted drug dose-response curves with a drug dose of 0 μM as the baseline, and the data were expressed as percentage of growth starting from 0 μM.

[0110] d) According to the criteria provided by the kit to predict the efficacy of the XELOX regimen (oxaliplatin + 5-fluorouracil), this case was evaluated as the moderate group ( Figure 5 ).

[0111] e) Clinical evaluation results of this case: imaging results after chemotherapy showed that the patient's tumor had not shrunk significantly. Rectal MRI diagnosis: changes in rectal cancer after chemotherapy, the solid part of the mass is basically the same as before, CRM (+); EMVI (+) ( Figure 6 ). The clinical evaluation result of this case was moderate group.

[0112] f) In this case, the results of evaluating the efficacy of chemotherapy drugs using a kit method to predict the efficacy of chemotherapy drugs in patients with colorectal cancer were consistent with the clinical evaluation results (i.e., the actual chemotherapy results).

[0113] Example 2

[0114] 1. A 65-year-old male was clinically diagnosed with rectal cancer. Rectal MRI diagnosis: pseudo-rectal cancer (mrT3N2), MRF (+), EMVI (-). Before neoadjuvant therapy, a biopsy tissue was obtained by endoscopic means.

[0115] 2. Composition of the kit:

[0116] (1) Washing buffer: phosphate-buffered saline (PBS) containing 1X penicillin-streptomycin solution.

[0117] The content of penicillin in the penicillin-streptomycin solution (100X) is 10 kU / ml and the content of streptomycin is 10 mg / ml. The solution is prepared with 0.9% sodium chloride.

[0118] Phosphate buffered saline (PBS), containing 135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, pH 7.3.

[0119] (2) Digestion buffer: DMEM culture medium containing 0.5 mg / ml type IV collagenase and 1X penicillin-streptomycin solution.

[0120] (3) 100μm cell strainer, 40μm cell strainer.

[0121] (4) Stem cell culture medium: DMEM / F12 culture medium, 2 mM glutamine, 0.1 mM non-essential amino acids, 8 ng / mL bFGF, 0.1 mM 2-mercaptoethanol, 5% serum replacement, 1X penicillin-streptomycin solution.

[0122] (5) Chemotherapy drugs: oxaliplatin, 5-fluorouracil.

[0123] 3. Testing steps:

[0124] (1) Lyse the biopsied tumor tissue into single cells or cell clusters:

[0125] a) Take several rice-sized pieces of biopsy tumor tissue from the patient and wash them several times with washing buffer until they are clean to the naked eye.

[0126] b) Use disposable surgical blades to mince the tissue in a 6 cm cell culture dish.

[0127] c) Transfer the minced tissue to a 15 ml centrifuge tube, add an appropriate amount of digestion buffer, and digest with shaking at 37°C for 1 hour.

[0128] d) Filter the fully lysed cell / tissue suspension through a 100 μm cell strainer to remove residual tissue.

[0129] e) Centrifuge the filtrate at 1500 rpm for 5 minutes, discard the supernatant, and the resulting precipitate is single cells or cell clusters, which can be transferred to the next step.

[0130] (2) Stemness induction and organoid formation of colorectal cancer cells:

[0131] a) Resuspend the cell pellet in the washing buffer, then centrifuge at 1500 rpm for 5 minutes and discard the supernatant.

[0132] b) Resuspend the pellet with 2 ml of washing buffer and filter the resulting cell suspension with a 40 μm cell strainer, retaining the cell clusters intercepted on the upper layer of the strainer. At this time, single cells are filtered out and the filtrate is discarded.

[0133] c) Wash the filter and the intercepted cell clusters from top to bottom with washing buffer to wash away the remaining single cells.

[0134] d) Invert the cell strainer and use stem cell culture medium to wash the cell pellet off the strainer.

[0135] e) The washed cell clusters were cultured in a stem cell culture medium for 24 hours to form a clear spherical structure with a smooth surface.

[0136] f) Growth process of intestinal cancer organoids derived from Example 2 ( Figure 7 ).

[0137] g) Select colorectal cancer organoids with a diameter greater than 200 μm and proceed to the next step.

[0138] (3) In vitro chemotherapy drug sensitivity test of colorectal cancer organoids:

[0139] a) The colorectal cancer organoids prepared in the previous step were used for in vitro drug sensitivity testing. The drug combination selected was oxaliplatin combined with 5-fluorouracil, and the concentration gradient was established as follows: 0, 0.25, 0.5, 1, 3, 6, 13, 25, 50, and 100 μM.

[0140] b) Add the corresponding dose of the drug to the colorectal cancer organoid culture system and culture it in a 96-well plate with 10 to 20 colorectal cancer organoids per well.

[0141] c) The cells were cultured at 37°C in 5% CO2 for 7 days, images were captured on days 1, 3, 5, and 7, the area of ​​the colorectal cancer organoids was measured, the growth / inhibition efficiency was calculated by comparing the area of ​​the colorectal cancer organoids with their starting size, drug dose-response curves were plotted with a drug dose of 0 μM as the baseline, and the data were expressed as percentage of growth starting from 0 μM.

[0142] d) According to the criteria provided by the kit for predicting the efficacy of the XELOX regimen (oxaliplatin + 5-fluorouracil), this case was evaluated as the sensitive group ( Figure 8 ).

[0143] e) Clinical evaluation results of this case. Imaging results after chemotherapy showed that the patient's tumor was smaller than before. Rectal MRI diagnosis: Rectal Ca changes after chemotherapy: The primary lesion and some lymph nodes were smaller than before, CRM (+); EMVI (-) ( Fig. 9 ). The clinical evaluation result of this case was sensitive group.

[0144] f) In this case, the results of evaluating the efficacy of chemotherapy drugs using a kit method to predict the efficacy of chemotherapy drugs in patients with colorectal cancer were consistent with the clinical evaluation results (i.e., the actual chemotherapy results).

[0145] Example 3

[0146] The other 10 patients with colorectal cancer or rectal cancer also used the kit and detection steps described in Example 1. After combining the results of the two patients in Examples 1 and 2, the results of the kit method for predicting the efficacy of chemotherapy drugs in colorectal cancer patients (i.e., predicting chemotherapy results) for the 12 patients were as follows: Figure 1As shown. The drug dose-response curve was drawn with a drug dose of 0 μM as the baseline, and the data were expressed as the percentage of growth starting from 0 μM. The corresponding area under the curve was calculated by statistical software, and the standardized corresponding area under the curve was obtained by dividing the obtained value by the maximum area of ​​the concentration range measured for each case. The range of the standardized value is between 0-1 (such as Figure 2 As shown). The corresponding area under the normalized curve was divided into three groups according to the numerical value. The highest value (67-100%) was the drug-resistant group, the medium value (34-66%) was the medium group, and the lowest value (0-33%) was the sensitive group. At this time, the corresponding IC50 (growth half-inhibitory concentration) of each group was: IC50 < 4μM for the sensitive group, IC50 for the medium group was between 4-10μM, and IC50 for the drug-resistant group was > 10μM.

[0147] The results of the kit method for predicting the efficacy of chemotherapy drugs for colorectal cancer patients (i.e., predicted chemotherapy results) and the clinical evaluation results (i.e., actual chemotherapy results) of 12 patients are shown in Table 1. According to the data in Table 1, the consistency rate between the predicted chemotherapy results of the kit involved in the present invention and the actual clinical chemotherapy results of the patients reached 91.66%.

[0148] The groups that predicted chemotherapy outcomes were:

[0149] Predicted chemotherapy sensitivity: IC50 < 4 μM.

[0150] Predicted moderate chemotherapeutic activity: 4 ≤ IC50 ≤ 10 μM.

[0151] Predicted chemotherapy-resistant group: IC50>10μM.

[0152] The actual chemotherapy results were grouped as follows:

[0153] Actual chemotherapy sensitivity: obvious regression or disappearance.

[0154] Actual chemotherapy is moderate: partial regression or no significant change.

[0155] Actual chemoresistance: tumor progression.

[0156] Table 1 Prediction effect of the kit of the present invention on the efficacy of chemotherapy drugs for colorectal cancer patients

[0157]

[0158] Example 4

[0159] 1. A 54-year-old male was clinically diagnosed with rectal cancer and tumor tissue was obtained through surgical specimens.

[0160] 2. Composition of the kit:

[0161] (1) Washing buffer: phosphate-buffered saline (PBS) containing 1X penicillin-streptomycin solution.

[0162] The content of penicillin in the penicillin-streptomycin solution (100X) is 10 kU / ml and the content of streptomycin is 10 mg / ml. The solution is prepared with 0.9% sodium chloride.

[0163] Phosphate buffered saline (PBS), containing 135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, pH 7.3.

[0164] (2) Digestion buffer: DMEM culture medium containing 0.5 mg / ml type IV collagenase and 1X penicillin-streptomycin solution.

[0165] (3) 100μm cell strainer, 40μm cell strainer.

[0166] (4) Stem cell culture medium: DMEM / F12 culture medium, 2 mM glutamine, 0.1 mM non-essential amino acids, 8 ng / mL bFGF, 0.1 mM 2-mercaptoethanol, 5% serum replacement, 1X penicillin-streptomycin solution.

[0167] 3. The contrast reagents used include:

[0168] (1) Washing buffer: phosphate-buffered saline (PBS) containing 1X penicillin-streptomycin solution.

[0169] The content of penicillin in the penicillin-streptomycin solution (100X) is 10 kU / ml and the content of streptomycin is 10 mg / ml. The solution is prepared with 0.9% sodium chloride.

[0170] Phosphate buffered saline (PBS), containing 135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, pH 7.3.

[0171] (2) Digestion buffer: DMEM culture medium containing 0.5 mg / ml type IV collagenase and 1X penicillin-streptomycin solution.

[0172] (3) 100 μm cell strainer.

[0173] (4) Commercial Matrigel (Cat. No. 356235, brand: Corning, New York, USA).

[0174] (5) Commercialization of Human IntestiCult TMOrganoid Growth Medium (Cat. No. 06010, brand STEMCELL, British Columbia, Canada).

[0175] 4. Testing steps:

[0176] (1) Lyse the biopsied tumor tissue into single cells or cell clusters:

[0177] a) Take several rice-sized pieces of biopsy tumor tissue from the patient and wash them several times with washing buffer until they are clean to the naked eye.

[0178] b) Use disposable surgical blades to mince the tissue in a 6 cm cell culture dish.

[0179] c) Transfer the minced tissue to a 15 ml centrifuge tube, add an appropriate amount of digestion buffer, and digest with shaking at 37°C for 1 hour.

[0180] d) Filter the fully lysed cell / tissue suspension through a 100 μm cell strainer to remove residual tissue.

[0181] e) Centrifuge the filtrate at 1500 rpm for 5 minutes, discard the supernatant, and the resulting precipitate is single cells or cell clusters, which can be transferred to the next step.

[0182] (2) Inducing organoid formation using the method for constructing intestinal cancer organoids of the present invention:

[0183] a) Resuspend the cell pellet in the washing buffer, then centrifuge at 1500 rpm for 5 minutes and discard the supernatant.

[0184] b) Resuspend the pellet with 2 ml of washing buffer and filter the resulting cell suspension with a 40 μm cell strainer, retaining the cell clusters intercepted on the upper layer of the strainer. At this time, single cells are filtered out and the filtrate is discarded.

[0185] c) Wash the filter and the intercepted cell clusters from top to bottom with washing buffer to wash away the remaining single cells.

[0186] d) Invert the cell strainer and use stem cell culture medium to wash the cell pellet off the strainer.

[0187] e) The washed cell clusters were cultured in a stem cell culture medium for 24 hours to form a clear spherical structure with a smooth surface.

[0188] f) Growth process of intestinal cancer organoids extracted by the kit method from Example 4 ( Fig.10 ).

[0189] (3) Inducing organoid formation using Matrigel method:

[0190] a) The cell pellet obtained in (1) was resuspended in washing buffer, and then centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded.

[0191] b) Inoculate the cell pellet into Matrigel and cover with human IntestiCult TM Organoid Growth Medium.

[0192] c) Growth process of colorectal cancer organoids extracted by Matrigel method from Example 4 ( Fig.10 ).

[0193] (4) Comparison of the proportion of tumor stem cells in the intestinal cancer organoids constructed by the method of the present invention and those extracted by the Matrigel method:

[0194] a) Organoids extracted by both methods were collected and washed with PBS, and then digested into single-cell suspension using 0.25% trypsin / EDTA.

[0195] b) Collect the cell precipitate obtained in (1) and proceed to the next step.

[0196] c) The cells were washed with PBS and then incubated with fluorescently conjugated primary antibodies in PBS containing 0.5% BSA at 4°C in the dark for 30 minutes. The antibodies used included CD133 (Cat. No. 372805, brand Biolegend, USA) and CD166 (Cat. No. 343903, brand Biolegend, USA). Samples were detected using flow cytometry (BD Biosciences, USA) and analyzed using FlowJo software (Tree Star) according to the manufacturer's instructions.

[0197] d) Percentage of CD133 / CD166 positive cells of original tumor cells and intestinal cancer organoids derived from Example 4 ( Fig.11 ).

[0198] (5) Comparison of the growth rates of the intestinal cancer organoids constructed by the method of the present invention and those obtained by the Matrigel method:

[0199] The organoids obtained by the two methods were cultured at 37°C in 5% CO2 for 13 days. Images were captured on days 1, 3, 5, 7, 9, 11, and 13, and the area of ​​the intestinal cancer organoids was measured. The growth efficiency was calculated by comparing the area of ​​the intestinal cancer organoids with their initial size, and the growth curve was drawn with the first day as the baseline ( Fig.12 ).

[0200] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. Application of a kit for predicting the efficacy of chemotherapy drugs in patients with colorectal cancer in constructing intestinal cancer organoids for predicting the efficacy of chemotherapy drugs in patients with colorectal cancer; The kit for predicting the efficacy of chemotherapy drugs for colorectal cancer patients comprises a washing buffer, a digestion buffer, a colorectal cancer chemotherapy drug, a stem cell culture fluid, a first cell filter, and a second cell filter; The washing buffer is a phosphate buffer containing penicillin-streptomycin solution; the digestion buffer is a DMEM culture medium containing type IV collagenase and penicillin-streptomycin solution; the stem cell culture medium is composed of glutamine, non-essential amino acids, bFGF, 2-mercaptoethanol, serum substitute, penicillin-streptomycin solution and DMEM / F12 culture medium; the pore size of the first cell filter is 100-500 μm; the pore size of the second cell filter is 40 μm; The kit for predicting the efficacy of chemotherapy drugs in colorectal cancer patients uses a suspension method to construct colorectal cancer organoids, and the suspension method does not involve Matrigel or other matrix gels; according to the drug dose-response curve of the colorectal cancer organoids under the XELOX regimen, the IC50 of the colorectal cancer organoids from different colorectal cancer patients is calculated; if the IC50 value is <4μM, the colorectal cancer organoids belong to the sensitive group, if the IC50 value is >10μM, the colorectal cancer organoids belong to the resistant group; if 4≤IC50≤10μM, the colorectal cancer organoids belong to the intermediate group.

2. The use according to claim 1, characterized in that The washing buffer is a phosphate buffer containing 1X penicillin-streptomycin solution; the 1X penicillin-streptomycin solution is prepared by diluting 2-100X penicillin-streptomycin solution; wherein the content of penicillin in the 100X penicillin-streptomycin solution is 5-20 kU / ml, the content of streptomycin is 5-20 mg / ml, and the solution is prepared with 0.9% sodium chloride.

3. The use according to claim 2, characterized in that The digestion buffer is a DMEM culture medium containing 0.5-5 mg / ml type IV collagenase and 1X penicillin-streptomycin solution; the stem cell culture medium is a DMEM / F12 culture medium containing 2 mM glutamine, 0.1 mM non-essential amino acids, 1-50 ng / mL bFGF, 0.1 mM 2-mercaptoethanol, 1-10% serum replacement, and 1X penicillin-streptomycin solution.

4. A method for constructing intestinal cancer organoids, characterized in that: The following steps are involved: S1. Lysing colorectal cancer tissue into single cells or cell clusters using the kit for predicting the efficacy of chemotherapy drugs in colorectal cancer patients as described in any one of claims 1 to 3; S2. Using the kit for predicting the efficacy of chemotherapy drugs for colorectal cancer patients as described in any one of claims 1 to 3, the single cells or cell clusters obtained in step S1 are subjected to stemness induction and organoid formation of colorectal cancer cells; The step S1 comprises the following steps: S1.1, taking colorectal cancer tissue from a patient, and washing it with the washing buffer; S1.2, cutting the tissue cleaned in step S1.1 into minced meat; S1.3, transferring the chopped tissue in step S1.2 to a centrifuge tube, adding the digestion buffer, and shaking digesting at 37°C to obtain a lysed cell / tissue suspension; S1.4, filtering the cell / tissue suspension after lysis in step S1.3 through the first cell filter to remove residual tissue and retain the filtrate; S1.5, centrifuging the filtrate obtained in step S1.4, discarding the supernatant, and obtaining the precipitate, which is the single cell or cell cluster; The step S2 comprises the following steps: S2.1, resuspending the precipitate obtained in step S1.5 with the washing buffer, then centrifuging, discarding the supernatant, and retaining the precipitate; S2.2, resuspending the precipitate obtained in step S2.1 with the washing buffer, filtering the obtained cell suspension with the second cell filter, retaining the cell clusters intercepted on the second cell filter, and filtering out the single cells, and discarding the filtrate; S2.3, washing the second cell filter and the intercepted cell clusters from top to bottom with the washing buffer to remove the remaining single cells; S2.4, inverting the second cell filter treated in step 2.3, and washing the cell clusters from the second cell filter with the stem cell culture medium; S2.

5. Cultivate the cell clusters eluted in step 2.4 with the stem cell culture medium to form a distinct spherical structure with a smooth surface; continue culturing to form the intestinal cancer organoids.

5. Use of intestinal cancer organoids in an in vitro chemotherapeutic drug sensitivity test, characterized in that: The intestinal cancer organoid is obtained by the construction method according to claim 4; the use of the intestinal cancer organoid in an in vitro chemotherapy drug sensitivity test comprises the following steps: S3.1, selecting the colorectal cancer organoids with a diameter greater than 200 um obtained in step S2.5 for in vitro chemotherapy drug sensitivity test; establishing a concentration gradient of the rectal cancer chemotherapy drug; S3.2, adding the colorectal cancer chemotherapy drug of the corresponding concentration in step S3.1 to the colorectal cancer organoid selected in step S3.1; S3.

3. Cultivate the intestinal cancer organoids treated in step S3.2 at 37°C in 5% CO2, capture images during the process, measure the area of ​​the intestinal cancer organoids during the culture process, calculate the growth / inhibition efficiency by comparing the area of ​​the intestinal cancer organoids during the culture process with their initial size, draw a drug dose-response curve with a drug dose of 0 μM as the baseline, and express the data as the growth percentage starting from 0 μM to obtain the AUC of the intestinal cancer organoids; compare the AUC of the intestinal cancer organoids with the efficacy standard , it was concluded that the intestinal cancer organoids belonged to the sensitive group, intermediate group or resistant group of the colorectal cancer chemotherapy drug; the efficacy standard was: according to the drug dose-response curve of the intestinal cancer organoids under the XELOX regimen, the IC50 of the intestinal cancer organoids from different colorectal cancer patients was calculated; if the IC50 value was <4μM, the intestinal cancer organoids belonged to the sensitive group; if the IC50 value was >10μM, the intestinal cancer organoids belonged to the resistant group; if 4≤IC50≤10μM, the intestinal cancer organoids belonged to the intermediate group.

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