Method for screening organoid drugs based on colorectal cancer

Abstract

The invention discloses a medicine screening method based on colorectal cancer organoid, which comprises the following steps: adding a colorectal cancer culture medium into the cultured colorectal cancer organoid, culturing for 48-72 hours, adding a to-be-detected colorectal medicine, continuously culturing for 72-96 hours, detecting the cell activity, and screening the medicine capable of reducing the cell activity. According to the method, the tumor microenvironment of the organoid is utilized, the modeling period is shortened, the cost is reduced, the tumor state can be simulated, high-throughput drug screening can be carried out in vitro, and the method has important significance on accurate treatment of lung cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for screening drugs for colorectal cancer. Background Technology

[0002] Colorectal cancer is the third most common cancer worldwide, accounting for approximately 10% of all cancer cases, and it is the second leading cause of cancer-related deaths globally. In 2020, an estimated 1.9 million new cases of colorectal cancer and over 930,000 deaths were reported globally. Significant geographical differences exist in incidence and mortality rates. Europe, Australia, and New Zealand have the highest incidence rates, while Eastern Europe has the highest mortality rates. In my country, both the incidence and mortality rates of colorectal cancer continue to rise. By 2040, the global burden of colorectal cancer is projected to increase to 3.2 million new cases annually (a 63% increase) and 1.6 million deaths annually (a 73% increase). Chemotherapy is a crucial clinical treatment for malignant tumors, and adjuvant chemotherapy is often required after surgery for patients with advanced colorectal cancer. However, chemotherapy resistance is a common problem in colorectal cancer, and empirical chemotherapy, lacking objective evidence, causes considerable suffering for patients. Furthermore, clinical treatment cycles are typically measured in years, making it impossible to determine the effectiveness of drugs for patients within a short period.

[0003] To improve the targeting and sensitivity of medication, in vitro antitumor drug sensitivity testing is crucial before treatment. Drug sensitivity testing using primary cancer cell cultures can guide targeted treatment, but individual differences exist in the sensitivity of colorectal cancer to tumor drugs. The in vitro growth environment of cell lines differs significantly from the in vivo environment, resulting in low personalization, an inability to accurately simulate the actual tumor's response to drugs, and limited predictive ability. Tumor organoids, on the other hand, are 3D-cultured cells with stem cell potential to form similar tissues to corresponding organs. These organoids can maximally mimic the structure and function of in vivo tissues and can be passaged long-term. Furthermore, the genetic background of tumor organoids is highly consistent with that of their source tumor tissue, making them extremely valuable for personalized colorectal cancer treatment. Therefore, a new method for drug sensitivity testing of colorectal cancer organoids is urgently needed to select the correct and effective drugs for each patient's condition, achieving individualized treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a drug screening method based on colorectal cancer organoids. This method directly utilizes the tumor microenvironment of colorectal cancer organoids, shortens the modeling cycle, reduces costs, and enables high-throughput drug screening in vitro by simulating tumor states.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A drug screening method based on colorectal cancer organoids, characterized by the following steps: digesting colorectal cancer organoids into cell clusters of 3-5 cells, then seeding the digested colorectal cancer organoid cells into a drug screening culture plate, adding 10 μL of matrix gel suspension to each well, and after gelation, adding 25 μL / well of colorectal cancer culture medium, culturing for 48-72 h, adding 5 μL of a certain concentration of the test drug, and continuing to culture for 72-96 h, detecting cell viability, analyzing data, and screening for drugs that reduce cell viability as colorectal test drugs; the components of the colorectal cancer culture medium are as follows: 1.5 mM N-acetyl-L-cysteine, 2 mM GlutaMAX™, 10 mM nicotinamide, 10 µM Y-27632, 10 mM HEPES, 1% Penicillin-Streptomycin, 1% gentamicin and amphotericin B total mass fraction, 1× N21-MAX Supplement, 1× B27, 100 ng / mL Noggin, 500 ng / mL R-Spondin 1, 500 nM A83-01, 5 µMSB202190, 10-100 ng / mL EGF, 20 ng / mL FGF-10, 100 ng / mL Wnt3a, 5-15 nM Gastrin I, 1-50 nM Prostaglandin E2, Advanced DMEM / F12.

[0006] Preferably, the method for preparing the colorectal cancer organoids is as follows: colorectal cancer tissue is digested to obtain a suspension of primary colorectal cancer cells, then the suspension of primary colorectal cancer cells is mixed with matrix gel and added to the bottom center of a culture plate. After being placed at 37°C for 5 minutes, the plate is inverted and placed for another 25 minutes to solidify. Then, colorectal cancer culture medium is added to the solidified matrix gel and cell mixture. The cell culture plate is then placed in a 37°C carbon dioxide incubator for at least 7 days.

[0007] The drugs to be tested were folic acid calcium, raltitrexed, 5-fluorouracil, and oxaliplatin; The method for detecting cell viability is to use CTG reagent. The cell concentration of the primary colorectal cancer cell suspension was 1 × 10⁶ cells per milliliter; The seeding density of colorectal cancer organoid digestive cells is 3 × 10³ cells per well; The volume ratio of the matrix gel to the colorectal cancer cell suspension is 7:3; The software used to analyze the data is GraphPad Prism; The preferred method for preparing primary colorectal cancer cells according to the present invention is as follows: Colorectal cancer tissue samples are washed with DPBS containing 2% Penicillin-Streptomycin, then the tissue is minced and transferred to a digestion solution. The samples are incubated on a shaker at 37°C for 15-30 minutes. Digestion is then terminated by adding DPBS containing 2% Penicillin-Streptomycin. The samples are then filtered and centrifuged to collect the precipitate.

[0008] The beneficial effects of this invention are as follows: This invention provides a method for screening drugs based on colorectal cancer organoids. It uses organoid models as a means of efficacy evaluation, constructing tumor organoids from intraoperative or biopsy specimens, and treating these organoids with antitumor drugs in vitro. This method allows for a simple, rapid, and economical identification of colorectal cancer cell specificity, providing a basis for personalized treatment. Furthermore, it can quickly screen for specific drugs for patients, reducing the significant physical suffering caused by multiple chemotherapy sessions. Attached Figure Description

[0009] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 The state of cells on day 1 of culture; Figure 2 The cell state on day 3 of culture; Figure 3 The cell state on day 7 of culture; Figure 4 The curve showing the change in organoid cell viability with leucovorin calcium concentration; Figure 5 The curve showing the change in organoid cell viability with raltitrexed concentration; Figure 6 The curve shows the change in organoid cell viability with 5-fluorouracil concentration; Figure 7 The curve shows the change in organoid cell viability with oxaliplatin concentration. Detailed Implementation

[0010] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0011] Example 1: Methods for organoid culture and drug screening for colorectal cancer. 1. Colorectal cancer organoid culture (1) Wash the fresh tumor tissue samples obtained by surgery or biopsy with DPBS containing 2% Penicillin-Streptomycin + 2% gentamicin and amphotericin B five times and place them in a sterile 1.5mL EP tube. Cut the tissue into small pieces and then transfer it to a digestion solution (1mg / mL collagenase type IV, 0.15 mg / mL hyaluronidase, 10mg / mL dispase II, 0.2mg / mL DNase). Incubate the digestion solution on a shaker at 37°C for 15min-30min. Then add DPBS containing 2% Penicillin-Streptomycin + 2% gentamicin and amphotericin B to stop the digestion. Filter the solution and centrifuge to collect the precipitate.

[0012] (2) Mix Matrigel with the prepared primary colorectal cancer cell suspension, and add 50 µL of each to the bottom center of a 24-well cell culture plate. Incubate at 37°C for 5 min, then invert the plate and incubate for another 25 min to allow it to solidify. Add 600 µL of colorectal cancer culture medium to the solidified Matrigel and cell mixture. Incubate the cell culture plate in a 37°C CO2 incubator. Observe the cell growth status and take photos daily. The results are as follows: Figures 1-3 As shown in the figure. The results showed that colorectal cancer organoids grew on day 3 of culture.

[0013] 2. Drug screening methods for colorectal cancer (1) The expanded and cultured colorectal cancer organoids were digested into cell clusters of 3-5 cells. The digested cells were then seeded into 384-well culture plates at a volume ratio of 70% between the matrix gel and the colorectal cancer cell suspension. Only the experimental group and the control group were seeded. The volume of the mixed gel per well was 10 μL, and there were 3 × 103 cells per well. After gelation, 25 μL of colorectal cancer culture medium (including the blank group) was added to each well and the plates were placed in an incubator at 37°C and 5% CO2 saturated humidity.

[0014] (2) After 48-72 hours of inoculation, the organoid culture plates were removed. The experimental group was given 5 μL of the test drug (leucovorin calcium, raltitrexed, 5-fluorouracil, oxaliplatin) at a certain concentration, and the control group and blank group were given 5 μL of culture medium. The plates were then cultured at 5% CO2 and 37℃ for 72-96 hours.

[0015] (3) Remove the organoid culture plate and equilibrate the cell culture plate to room temperature for 10 min. Add a certain amount of CellTiter-Glo 3D cell viability assay reagent to each well. Shake at room temperature for 5 min to promote cell lysis and incubate for 25 min to allow the luminescence signal to stabilize.

[0016] (4) Use a multi-functional microplate reader with chemiluminescence detection function to perform chemiluminescence detection.

[0017] (5) Analyze the data, calculate the cell survival rate and drug inhibition rate, and plot the growth inhibition curve.

[0018] Experimental results of drug screening for organoids in colorectal cancer are as follows: Figures 4-7 As shown, the results indicated that this colorectal cancer organoid was sensitive to leucovorin, raltitrexed, 5-fluorouracil, and oxaliplatin. This suggests that the model could be considered a preferred treatment option for this patient, providing a basis for individualized clinical treatment.

[0019] In this embodiment, the composition and concentration of the colorectal cancer culture medium are as follows: N-acetyl-L-cysteine ​​1.5 mM; GlutaMAX™ 2mM, glutamine supplement; Nicotinamide 10mM; HEPES (n-2-hydroxyethylpiperazine-n-2-ethanesulfonic acid) 10mM; The mass fraction of penicillin-streptomycin is 1%; N21-MAX Supplement 1×, cell culture additive; B27 1×, cell culture additive; Noggin 100ng / ml, BMP modifier; R-Spondin 1 500ng / ml, a modulator of the Wnt / β-catenin signaling pathway; A83-01 500nM, an ALK inhibitor; Y-27632 10μM, a ROCK inhibitor; CHIR99021 200nM, GSK-3α / β inhibitor; SB202190 5μM, a TGF-β receptor kinase inhibitor; Activin A 100 ng / mL; EGF 100ng / mL, epidermal growth factor; FGF-10 20ng / mL, heparin-bound growth factor; FGF-7 100ng / mL promotes cell proliferation and differentiation; Wnt3a 100ng / mL, cell culture additive; Gastrin I 15 nM, gastrin; Prostaglandin E2 50nM; Advanced DMEM / F12, a modified DMEM basal medium.

[0020] The above-described embodiments merely illustrate several drug screening methods for colorectal cancer treatment, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of this invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept, and these all fall within the scope of protection of this invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A drug screening method based on colorectal cancer organoids, characterized in that, The procedure includes the following steps: Colorectal cancer organoids are digested into cell clusters of 3-5 cells each. These digested cells are then seeded into drug screening culture plates. 10 μL of matrix gel suspension is added to each well. After gelation, 25 μL / well of colorectal cancer culture medium is added. After 48-72 hours of incubation, 5 μL of a specific concentration of the test drug is added. The culture continues for 72-96 hours. Cell viability is then assessed, data is analyzed, and drugs that reduce cell viability are selected as the colorectal cancer test drugs. The components of the colorectal cancer culture medium are as follows: 1.5 mM N-acetyl-L-cysteine, 2 mM GlutaMAX™, 10 mM nicotinamide, 10 µM Y-27632, 10 mM HEPES, 1% Penicillin-Streptomycin, 1% gentamicin and amphotericin B, 1× N21-MAX Supplement, 1× B27, 100 ng / mL Noggin, 500 ng / mL R-Spondin 1, 500 nM A83-01, 5 µMSB202190, 10-100 ng / mL EGF, 20 ng / mL FGF-10, 100 ng / mL Wnt3a, 5-15 nM Gastrin I, 1-50 nM Prostaglandin E2, Advanced DMEM / F12.

2. The method for preparing the colorectal cancer organoids is as follows: After digesting the colorectal cancer tissue, a suspension of primary colorectal cancer cells is obtained. Then, the suspension of primary colorectal cancer cells is mixed with matrix gel and added to the bottom center of a culture plate. After being placed at 37°C for 5 minutes, the plate is inverted and placed for another 25 minutes to solidify. Then, colorectal cancer culture medium is added to the solidified matrix gel and cell mixture. The cell culture plate is then placed in a 37°C carbon dioxide incubator for at least 7 days.

3. The drugs to be tested are leucovorin calcium, raltitrexed, 5-fluorouracil, and oxaliplatin; The method for detecting cell viability is to use CTG reagent. The cell concentration of the primary colorectal cancer cell suspension was 1 × 10⁶ cells per milliliter; The seeding density of colorectal cancer organoid digestive cells is 3 × 10³ cells per well; The volume ratio of the matrix gel to the colorectal cancer cell suspension is 7:3; The software used to analyze the data is GraphPad Prism; The method for preparing primary colorectal cancer cells is as follows: colorectal cancer tissue samples are washed with DPBS containing 2% Penicillin-Streptomycin, then the tissue is cut into small pieces and transferred to a digestion solution. The tissue is incubated on a shaker at 37°C for 15-30 minutes. Then, DPBS containing 2% Penicillin-Streptomycin is added to terminate the digestion. The sample is filtered, centrifuged, and the precipitate is collected.

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