CN108823169A - A kind of carcinoid tissue, method and purposes prepared from mammal cancerous tissue - Google Patents

Abstract

The invention discloses a method for preparing carcinoid tissue from mammalian cancer tissue, comprising the following steps: A1) digesting the cancer tissue isolated from mammals in a special digestive solution, and separating them into cells; A2) placing the cells in Centrifuge in a centrifuge tube rinsed with BSA, and remove the supernatant; A3) Add a special medium to the centrifuge tube to resuspend the cells; A4) Mix the cell suspension with the thawed Matrigel, inoculate on a culture plate, and wait until the Matrigel The invention also discloses the carcinoid tissue and its application; the invention provides the carcinoid tissue and its function which can be used to evaluate the curative effect of preoperative neoadjuvant radiotherapy and chemotherapy, and the carcinoid tissue which can be efficiently cultivated. Culture medium, digest solution, method.

Description

Carcinoid tissue prepared from mammalian cancer tissue, method and application

technical field

The invention relates to the field of biomedicine, in particular to a carcinoid tissue prepared from a mammalian cancer tissue, a method and an application.

Background technique

Colorectal cancer is one of the three most common malignant tumors in the world. With the change of lifestyle, its incidence is on the rise. Rectal cancer accounts for about one-third of colorectal cancer. Locally advanced rectal cancer refers to the primary tumor found by imaging or pathological examination invading the muscular layer of the intestinal wall until the surrounding well-known structures (c/pT3-4b) or lymph node metastasis in the mesentery and true pelvis (c/pT3-4b) /pN1-2) without distant metastasis (M0) and rectal cancer within 12 cm from the anus (Chinese Expert Consensus on the Diagnosis and Treatment of Locally Advanced Rectal Cancer, 2017). The treatment of locally advanced rectal cancer is one of the key points and difficulties in the field of colorectal cancer treatment. The application of different treatment methods in locally advanced rectal cancer has led to more and more treatment strategies combining different methods. The traditional treatment for locally advanced rectal cancer is surgery combined with radiotherapy and chemotherapy after surgery. However, in the past 10 years, with the release of the results of a number of large-scale phase III clinical studies, preoperative neoadjuvant chemotherapy combined with radical surgery has become the standard treatment mode for locally advanced rectal cancer. Compared with postoperative chemoradiotherapy, preoperative chemoradiotherapy has its clinical and biological advantages. Mainly include: tumor downstaging and shrinkage after radiotherapy can increase the resection rate; for low rectal tumors, the shrinkage of the tumor may increase the chance of retaining the anal sphincter; reduce the probability of intraoperative dissemination; the tumor has fewer hypoxic cells, and the effect of radiotherapy is better than that of postoperative Radiotherapy is sensitive; the peristalsis of the small intestine is larger than that after the operation, and it does not fall into the pelvic cavity, and the adverse reactions of treatment are relatively low.

Although preoperative neoadjuvant chemotherapy for advanced rectal cancer can generally benefit patients, there are still a considerable proportion of patients with poor neoadjuvant treatment effects, which cannot bring benefits such as tumor downstaging and regression. Therefore, if the patients who are really suitable for radiotherapy and chemotherapy are selected, it is helpful to guide their individualized treatment, and it is very important to avoid overtreatment, which is in line with the spirit of precision medicine. In addition, some patients can achieve complete tumor regression after neoadjuvant chemotherapy. Studies have shown that the long-term survival of these patients is no different from neoadjuvant surgery after neoadjuvant therapy. Therefore, if the tumor regresses completely after radiotherapy and chemotherapy, the subsequent treatment may be weakened, but this must be based on an accurate evaluation of the efficacy. The post-neoadjuvant treatment evaluation methods mainly include portal digital examination, imaging and colonoscopy, etc. The prediction accuracy rate is between 30-60%, and the prediction accuracy rate is relatively low. To sum up, it is necessary to find an accurate detection method to identify patients who are resistant to radiotherapy and chemotherapy before neoadjuvant chemoradiotherapy to avoid overtreatment, and to identify patients who are sensitive to neoadjuvant chemoradiotherapy and may obtain complete pathological remission Adopting a watch-and-wait strategy to avoid surgery is currently a research hotspot in the field of advanced rectal cancer.

The evaluation of neoadjuvant chemoradiotherapy for rectal cancer in the prior art includes the following 8 types.

1. Tumor cell lines. Tumor cell lines are relatively easy to cultivate and can be used for large-scale screening of anti-tumor drugs (or therapies). However, tumor cell lines have several major natural defects: 1) A certain tumor cell line is derived from a patient's tumor tissue. As we all know, the tumors of different tumor patients have great heterogeneity, so one or several Tumor cell lines cannot represent every clinical patient; 2) The genetic background and gene expression profile of tumor cell lines are quite different from those of tumor cells in tumor tissues of clinical patients; 3) Tumor cell lines are often passaged many times, Its genetic background and gene expression profile will change, and there is a risk of cross-contamination. Therefore, as of now, tumor cell lines can only be used as basic exploratory research tools before clinical transformation, and do not have direct clinical transformation capabilities. Tumor cell lines cannot be used to predict response to chemoradiotherapy in neoadjuvant patients with rectal cancer.

2. Human-derived tumor xenografts (PDTX, patient-derived tumor xenografts), human-derived tumor xenografts are tumor research tools that separate and process patient tumor tissues and inoculate them in xenogeneic mice. PDTX maintains the degree of differentiation, morphological characteristics, structural characteristics and molecular characteristics of tumor cells. To a certain extent, the blood supply characteristics, matrix characteristics, and necrosis status of transplanted mouse tumors are consistent with the characteristics of human tumors. Compared with tumor cell lines, PDTX can represent the tumor tissue from which it is derived, and its response to drugs or other therapies can more truly represent the response of clinical patients. Therefore, PDTX can be used to predict the clinical treatment effect of drugs or other therapies. PDTX also has many problems in clinical translation research. First of all, the establishment and application of PDTX models take a long time. It often takes several months from obtaining tumor tissue, inoculation, passage, and starting experiments to obtaining data, while clinical patients often only need a few weeks from diagnosis to treatment initiation. Secondly, the PDTX model needs to spend a lot of human and material resources to complete. In addition, PDTX lacks a unified and clear model establishment and efficacy evaluation standard, and the reliability of its experimental results needs to be further improved. Third, the tolerance of experimental animals to drugs or other therapies is quite different from that of humans, which may make it impossible to evaluate treatments that are well tolerated by humans but poorly tolerated by experimental animals. Finally, PDTX inoculates human tissue into experimental animals, and there are certain ethical issues. The above shortcomings greatly limit the application of PDTX in the field of translational medicine. Taking time, cost, reliability, and ethical factors into consideration, it is impossible to use the PDTX model to predict the efficacy of neoadjuvant chemoradiotherapy in patients with rectal cancer.

3. Digital rectal examination, digital rectal examination is the most commonly used method for the diagnosis and curative effect evaluation of rectal cancer. Almost all patients with rectal cancer will have a digital rectal examination at the time of diagnosis, and during and after treatment, clinicians will also use digital rectal examination to roughly judge the extent of tumor regression to evaluate the treatment effect. Digital rectal examination can only roughly evaluate the size of the part of the tumor protruding from the intestinal cavity and the approximate degree of tumor infiltration, which relies heavily on the experience of clinicians and cannot be accurately quantified. It can only be used as a reference in clinical efficacy evaluation. At the same time, digital rectal examination can only obtain physical properties such as the size and hardness of the tumor, and cannot be used to predict the true response of the tumor to drugs or other treatments.

4. Endoscopic ultrasonography, endoscopic ultrasonography is also widely used in the diagnosis and curative effect evaluation of rectal cancer. Endoscopic ultrasonography can accurately determine tumor size, invasion depth, lymph node metastasis and other information. EUS can only obtain the morphological characteristics of the tumor, so it: 1) cannot be used to predict the response of the tumor to drugs or other therapies before treatment; 2) the evaluation of ultrasound morphology after treatment cannot accurately predict the tumor response to drugs or other treatments. real response to therapy.

5. CT, similar to endoscopic ultrasonography, can obtain information such as tumor size, invasion depth, and lymph node metastasis, and is used for the diagnosis and treatment of rectal cancer. Similar to endoscopic ultrasonography, CT can only obtain morphological features of tumors, so it: 1) cannot be used to predict tumor response to drugs or other therapies before treatment; 2) ultrasound morphological evaluation after treatment cannot accurately predict How well a tumor actually responds to drugs or other treatments.

6. Magnetic Resonance Imaging, compared with endoscopic ultrasonography and CT, MRI technology is the most commonly used diagnostic and efficacy evaluation tool for rectal cancer, which can more accurately obtain information such as tumor size, invasion depth, and lymph node metastasis . Especially with the advancement of MRI technology, it has been widely used in the evaluation of the efficacy of neoadjuvant radiotherapy and chemotherapy for rectal cancer, and more and more studies have used MRI-related parameters to predict the efficacy of radiotherapy and chemotherapy. Diffusion weighted imaging (DWI) is an MRI functional imaging technique that can reflect the diffusion ability and movement direction of water molecules in vivo. It can not only be used for the diagnosis of rectal cancer, but its apparent diffusion coefficient (ADC) Dynamic changes during treatment can also be used to predict the efficacy of radiotherapy and chemotherapy. Studies have shown that DWI combined with conventional MRI can improve the accuracy of assessment by 52-64%. In addition, mr tumor regression grade (mrTRG) based on MRI high-resolution sequence T2WI images can predict the long-term survival of patients, and has a high coincidence rate with postoperative pathological tumor regression grade (pTRG). First of all, the information of rectal cancer tumor size, invasion degree, lymph node metastasis, mesorectal fascia and vascular invasion obtained by MRI can only be used to evaluate the curative effect after starting treatment, and cannot be used to predict the response of rectal cancer to drugs before treatment. Secondly, the accuracy of MRI in assessing the curative effect after starting treatment is about 60%, which is still low, and cannot effectively distinguish between pathological complete response (pCR) and residual microscopic lesions under the microscope.

7. PET-CT, PET-CT is also an important tool for tumor diagnosis and monitoring. Studies have shown that the tumor volume of rectal cancer shrinks two weeks after radiotherapy, which can be manifested as a decrease in glucose metabolism uptake, and can predict the efficacy of radiotherapy and chemotherapy. vanStiphout et al. established a model for predicting pCR after radiotherapy and chemotherapy for locally advanced rectal cancer by using the tumor length, the maximum uptake value of 18F-FDG by tumor cells before and after radiotherapy and chemotherapy, and its changes, and achieved good accuracy (AUC= 0.86). First of all, information such as tumor size and metabolic intensity obtained by PET-CT cannot predict the tumor's response to drugs or other therapies before treatment. Second, radiation-induced inflammatory reactions, inflammatory bowel disease, and occasional intestinal perforation may lead to changes in glucose uptake signals, leading to errors in the efficacy evaluation of PET-CT. In addition, the price of PET-CT inspection is relatively expensive (about 8000 RMB), which limits its use. Finally, although the accuracy of PET-CT in evaluating the curative effect of rectal cancer is high, it still needs to be greatly improved.

8. Pathological evaluation, pathological evaluation includes tumor diagnosis before treatment and postoperative pathological examination after neoadjuvant chemotherapy. Postoperative pathological evaluation graded and scored the response to chemotherapy and radiotherapy, namely the TRG score, which was divided into 0 points (complete response, that is, no viable tumor cells were found), and 1 point (moderate response, that is, single or small clusters of cancer cells). Residual), 2 points (mild response, that is, residual cancer focus, interstitial fibrosis), 3 points (poor response, that is, a few or no tumor cells subsided, and a large number of residual cancers). Postoperative pathological evaluation is the gold standard for evaluating the efficacy of neoadjuvant chemoradiotherapy for rectal cancer. The pathological evaluation before treatment is to confirm the diagnosis, but it cannot provide information on the response of the tumor to chemotherapy and radiotherapy, and cannot be used to predict the curative effect. Although postoperative pathological evaluation is the gold standard for evaluating the efficacy of neoadjuvant chemoradiotherapy, it must be performed after surgery and cannot provide any help for treatment decisions before surgery.

Based on the drawbacks of the eight methods for the diagnosis and efficacy evaluation of rectal cancer in the prior art, an organoid/tissue method has been proposed in recent years.

Organoids/tissues are 3D cultures of cells with stemness potential to form similar organs/tissues of corresponding organs/tissues. Organoids/tissues can simulate the structure and function of in vivo tissues to the greatest extent and can be subcultured for a long time. At present, researchers have begun to study the establishment of organoid/tissue models such as lung, stomach, small intestine, large intestine, liver, pancreas, and prostate. In addition, several studies have shown that the response of normal intestinal organoids to drugs can be used to predict the clinical efficacy of patients. For example, a study on intestinal cystic fibrosis showed (Dekkers JF, Berkers G, Kruisselbrink E, etal. Characterizing responses to CFTR-modulating drugs using rectal organs derived from subjects with cystic fibrosis. Sci Transl Med. 2016, 8( 344):344ra84.), the drug response of organoids is consistent with the published clinical trial data, and for three patients with rare mutations, two patients with good response to organoid drugs achieved better results after drug administration , a patient with a poor organoid response also had a poor clinical outcome. However, in the prior art, the in vitro culture of tumor cells still has certain obstacles to the formation of complete tissue-like tissues and the accuracy of corresponding drug screening and data detection. The basal medium cannot completely simulate the in vivo environment of tumor cells. This is the main reason. For example, Chinese patent CN106190980A discloses a culture medium based on esophageal cancer tissue for culturing tumor tissue in vitro. It still uses ordinary in vitro medium and some cytokines artificially added, and the culture effect is general. Chinese patent CN105358677A discloses a method for forming epithelial organoids from isolated epithelial stem cells, but its source of cells and culture method are different from those of this application.

Prior art studies on tissue-like tissues of rectal cancer, such as (Sato T, Stange DE, Ferrante M, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology. 2011, 141( 5): 1762-72), there are the following problems: 1) The extraction process of biopsy specimen tissue is likely to lead to extraction failure and incomplete digestion; 2) Although the rectal biopsy has intestinal preparation, the biopsy specimens all have different degrees of feces 3) Factors such as inappropriate medium lead to low success rate of culture; 4) The accuracy of preoperative neoadjuvant radiotherapy and chemotherapy for rectal cancer by cultured rectal cancer tissue-like tissue is not high.

To sum up, the prior art still lacks carcinoid tissues and their functions that can be used to evaluate the efficacy of neoadjuvant radiotherapy and chemotherapy, as well as culture media, digestive solutions, and methods that can efficiently culture carcinoid tissues.

Contents of the invention

In view of the above technical problems, the present invention provides a carcinoid tissue and its function that can be used to evaluate the curative effect of neoadjuvant radiotherapy and chemotherapy, as well as a culture medium, a digestive solution, and a method capable of efficiently culturing the carcinoid tissue.

A method for carcinoid tissue prepared from mammalian cancer tissue, comprising the steps of:

A1) placing the cancer tissue isolated from the mammal into the digestive fluid for digestion and separating into cells;

A2) Place the cells in a centrifuge tube rinsed with BSA, centrifuge, and remove the supernatant;

A3) Add special medium to the centrifuge tube to resuspend the cells;

A4) Mix the cell suspension with the thawed Matrigel, inoculate it on a culture plate, wait for the Matrigel to solidify, and add a special medium for culture.

Preferably, the concentration of BSA is 0.1%.

Preferably, the special digestion solution includes: DMEM medium, fetal calf serum, collagenase IV and collagenase II.

Preferably, the concentration of fetal bovine serum is 0.2%-20%; further preferably, the concentration of fetal bovine serum is 0.5%-1%.

Preferably, the concentration of collagenase IV is 200-700U/ml; further preferably, the concentration of collagenase IV is 500-600U/ml.

Preferably, the concentration of collagenase II is 1-5 mg/ml; further preferably, the concentration of collagenase II is 1.5-3.5 mg/ml.

Preferably, the digestion time is 30-50 min; further preferably, the digestion time is 40-45 min.

Preferably, the special medium includes: DMEM/F12 medium, R-spondin 1 protein, epidermal growth factor, prostaglandin E-2; further preferably, the special medium includes: DMEM/F12, R- spondin 1, Noggin, EGF, HEPES, Glutamax, Normocin, Gentamicin/amphoteritin, N2, B27, n-Acetylcysteine, SB202190, Gastrin, Prostaglandin E2.

Preferably, the method of the present invention also includes the following steps:

B1) Wash the cancer tissue isolated from mammals in PBS containing antibiotics; first, after washing with PBS containing antibiotics for 5 min×5 times, mince and digest, and it should be minced as much as possible to facilitate digestion and subsequent separation; for extremely For small and loose specimens, the frequency of shaking and washing should be reduced or no shaking washing should be considered to avoid loss of specimens.

Preferably, the mammal includes human, and more preferably, human cancer tissue includes colon cancer tissue and rectal cancer tissue.

The present invention also includes a carcinoid tissue, which is cultured by the method for preparing carcinoid tissue from mammalian cancer tissue provided by the present invention.

Preferably, the carcinoid tissue is rectal cancer carcinoid tissue. Further preferably, the morphology of the rectal cancer carcinoid tissue includes: cavity type, dense solid type, and differentiated type.

The present invention also includes the use of the carcinoid tissue of the present invention in evaluating the curative effect of rectal cancer neoadjuvant radiotherapy and chemotherapy.

The present invention also includes a method for evaluating the curative effect of neoadjuvant radiotherapy and chemotherapy of rectal cancer by carcinoid tissue, the method comprising: evaluating the sensitivity of carcinoid tissue to radiotherapy and chemotherapeutic drugs.

Preferably, the chemotherapeutic drugs include 5-Fu and CPT-11.

Preferably, the sensitivity of the carcinoid tissue to radiotherapy is evaluated by the survival number of the carcinoid tissue.

Preferably, the radiosensitivity observation time is at least 9 days after radiotherapy.

Preferably, the rectal cancer carcinoid tissue of the present invention can also be used to predict the following three situations: 1) radiotherapy and chemotherapy are required after local recurrence; 2) postoperative adjuvant radiotherapy and chemotherapy; 3) patients with metastatic rectal cancer need radiotherapy chemotherapy.

Compared with the prior art, the technical solution of the present invention has the following advantages:

1. The present invention proposes carcinoid tissue and its function that can be used to evaluate the curative effect of neoadjuvant radiotherapy and chemotherapy, as well as the culture medium, digestive juice and method that can efficiently cultivate carcinoid tissue; the present invention improves the culture method, culture medium, and digestive juice , so that the success rate of carcinoid tissue culture is over 80%.

2. The present invention proposes that the radiotherapy and drug sensitivity of carcinoid tissue can predict the efficacy of preoperative neoadjuvant radiotherapy and chemotherapy, and the diagnostic efficiency is over 80%. According to the sensitivity of carcinoid tissue to radiotherapy and drug, it can guide the rectal Cancer patients choose whether to undergo neoadjuvant chemoradiotherapy; according to the sensitivity of carcinoid tissue to radiotherapy and drugs, patients with advanced rectal cancer can be guided to continue surgical treatment or adopt a wait-and-see strategy after neoadjuvant chemoradiotherapy.

3. The present invention provides a new and more accurate method for assessing radiotherapy sensitivity of carcinoid tissue; CCK8 and other methods for detecting cell activity are used to assess radiotherapy and drug sensitivity of carcinoid tissue, which cannot effectively distinguish sensitivity from tolerance The clinical prediction ability is also poor; under the naked eye light microscope (or taking pictures) to judge whether the quasi-like tissue is alive, or to outline and calculate the change of the quasi-like tissue area, it can effectively distinguish between radiotherapy and drug-sensitive and resistant tissues, and can Accurate prediction of neoadjuvant chemoradiotherapy efficacy in clinical patients with rectal cancer.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the rectal cancer biopsy specimen of 3 patients of the embodiment of the present invention;

Fig. 2 is 3 rectal cancer carcinoid tissues of the embodiment of the present invention;

Fig. 3 is 3 types of rectal cancer carcinoid tissues of the embodiment of the present invention;

Fig. 4 is the rectal cancer carcinoid tissue of the embodiment of the present invention;

Fig. 5 is a relation diagram between X-ray irradiation dose and CCK8 in the embodiment of the present invention;

Fig. 6 is a relationship diagram between the X-ray irradiation dose and the survival number of carcinoid tissue in the embodiment of the present invention;

Fig. 7 is a death and survival graph of the carcinoid tissue of the present invention according to the embodiment of the present invention;

Fig. 8 is the survival situation of different carcinoid tissues in different X-ray irradiation doses according to the embodiment of the present invention;

Fig. 9 is the survival curve of different carcinoid tissues in different X-ray irradiation doses according to the embodiment of the present invention;

Figure 10 is the recovery situation of two types of carcinoid tissues, radiotherapy-resistant and radiosensitive, according to the embodiment of the present invention after receiving 8Gy irradiation;

Fig. 11 is the area change curve after 8Gy irradiation of the embodiment of the present invention;

Figure 12 shows the changes in the area of carcinoid tissue of two types of 5-Fu-resistant and 5-Fu-sensitive over time according to the embodiment of the present invention;

Fig. 13 is the change curve of carcinoid tissue area after 5-Fu treatment of the embodiment of the present invention;

Figure 14 shows the changes in the area of carcinoid tissue of two types of CPT-11 resistance and CPT-11 sensitivity over time in the embodiment of the present invention;

Fig. 15 is the change curve of carcinoid tissue area after CPT-11 treatment of the embodiment of the present invention;

Fig. 16 is a frame diagram of laboratory experiments and clinical trials of the embodiment of the present invention.

Detailed ways

The idea, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings, so as to fully understand the purpose, features and effects of the present invention.

Example 1

The carcinoid tissue of the present invention was prepared by the following method.

1) Carcinoid tissue extraction from biopsy specimens of rectal cancer patients with neoadjuvant radiotherapy and chemotherapy: Take rectal cancer biopsy specimens from 3 patients, as shown in Figure 1, put them into centrifuge tubes, and bring them back to the laboratory in an ice box. Wash in pre-cooled PBS for 5 min X 5 times. Place a sterile petri dish on the surface of the ice basin, pour pre-cooled sterilized PBS, place the rectal cancer tissue in it, cut it into pieces with a sterile blade, move it into 37°C preheated digestion solution, and digest it in a 37°C water bath shaker. After digestion, pipette moderately with a pipette gun, place on ice for a few minutes and the tumor fragments settle, take the supernatant, add pre-cooled PBS and continue pipetting until the tumor fragments disappear. Then centrifuge for 5 minutes to pellet all rectal cancer crypts; then wash with pre-cooled PBS five times, each time for 5 minutes; then centrifuge at 200g to pellet all crypts. Discard the supernatant and prepare to inoculate the crypt 3D culture.

2) Rectal cancer carcinoid tissue culture process: After extracting rectal cancer crypts, add an appropriate amount of Matrigel with a pipette tip, then suspend and mix with a 200ul pipette tip, inoculate in a preheated culture plate, 50ul per well. After inoculation, the culture plate was incubated in a constant temperature incubator at 37°C for 5 minutes and taken out. After adding 500ul of medium to each well, observe the inoculation situation under the microscope, and then place it in the incubator for cultivation. The growth of carcinoid tissue was observed and photographed daily; the medium was replaced every 3 days.

3) Subculture of rectal cancer carcinoid tissue: absorb the culture medium in the culture well, blow off the matrigel with the tip of the pipette, transfer to a 15ml sterile centrifuge tube and beat to blow off the matrigel completely, and centrifuge at 4°C for 5 minutes. Aspirate the supernatant and matrigel precipitated at the bottom; add pre-cooled PBS to resuspend the carcinoid tissue at the bottom of the tube. Continue to culture according to the carcinoid tissue culture procedure; when inoculating, inoculate at the ratio of 4 wells per 1 well.

4) Cryopreservation of rectal cancer carcinoid tissue: at the time of passaging, part of the crypts were collected for cryopreservation before centrifugation and sedimentation.

5) Recovery of rectal cancer carcinoid tissue: the frozen carcinoid tissue was taken out of liquid nitrogen, thawed at 37°C, centrifuged and precipitated, and cultured according to the inoculation procedure.

Finally, the cultured rectal carcinoid tissues of 3 patients are shown in Fig. 2 .

Preferably, the culture medium of this embodiment is selected from the following components.

In one embodiment, the digestion solution is 8 mL of the digestion solution, comprising: 7 mL of DMEM medium, 0.5% fetal bovine serum, 450 U/mL of collagenase IV, and 1.2 mg/mL of collagenase II.

In another embodiment, the digestion solution is 8 mL of the digestion solution, comprising: 7 mL of DMEM medium, 0.8% fetal bovine serum, 500 U/mL of collagenase IV, and 1.3 mg/mL of collagenase II.

In another embodiment, the digestion solution is 8 mL, comprising: 7 mL DMEM medium, 1% fetal bovine serum, 500 U/mL collagenase IV, 1.5 mg/mL collagenase II.

Example 2

After optimizing and improving the rectal cancer biopsy specimen carcinoid tissue extraction and culture system, the present invention successfully established a rectal cancer carcinoid tissue biological sample bank, laying the foundation for subsequent research. The morphology of carcinoid tissue in this sample bank is shown in Figure 3, mainly including the following types: 1) cavity type, as shown in Figure A, that is, the wall of circular carcinoid tissue is thin and the volume of the middle cavity is relatively large; 2) Dense solid type, as shown in Figure B, that is, the round carcinoid tissue has a dense and dense intermediate cavity or no intermediate cavity; 3) Differentiated type, as shown in Figure C, that is, there are more buds around the carcinoid tissue.

As shown in Figure 4, it can be seen that the carcinoid tissue of rectal cancer in this example well retains the heterogeneity of the tumor at the same time, and carcinoid tissue of different shapes is present in the same patient at the same time, wherein the different forms of carcinoid tissue represent their different differentiation abilities.

Example 3

The following is a detailed introduction to the radiosensitivity and drug sensitivity assessment methods of rectal cancer carcinoid tissue.

In the prior art, the result of cell viability detection is used to reflect the survival of carcinoid tissue after radiation exposure. However, the present invention finds that the cell activity detection method cannot truly reflect the survival of the carcinoid tissue after being irradiated by radiation. The reason is shown in Figure 5. In the dose-survival curve, there is no obvious shoulder area in the low-dose area, which is inconsistent with the basic principles of radiotherapy biology.

The present invention finds through multiple experiments that the survival count of the carcinoid tissue can more truly reflect the survival of the carcinoid tissue than the cell activity detection. The dose-survival curve drawn by the present invention according to the survival count of carcinoid tissue conforms to the biological principle of radiotherapy, as shown in Figure 6, there is an obvious shoulder in the low-dose area, and the end point of the survival count time should be from the ninth day to the first day after irradiation Fifteen days is suitable.

Further experiments found that, compared with the clinical results of patients, it was found that in addition to the dose-survival curve of carcinoid tissue can predict the efficacy of neoadjuvant radiotherapy and chemotherapy in clinical patients, the area change of carcinoid tissue after irradiation or drug administration can also be well predicted. Predict the efficacy of treatment in clinical patients. Therefore, the present invention also utilizes the recovery of carcinoid tissue after irradiation or administration as an evaluation method for radiotherapy sensitivity and drug sensitivity.

Therefore, the method of the present invention for evaluating the radiotherapy and drug sensitivity of rectal cancer carcinoid tissue includes: carcinoid tissue radiotherapy dose-survival curve; carcinoid tissue recovery after irradiation and administration. Among them, the survival standard of carcinoid tissue is shown in Figure 7: the picture on the left shows that the complete structure of the carcinoid tissue was destroyed on the 15th day after 16Gy irradiation, and the carcinoid tissue basically died when it formed scattered cell clusters; the picture on the right The picture shows, 15 days after 16Gy irradiation, the carcinoid tissue retained its complete structure, and the carcinoid tissue survived when a clear outline was visible.

Example 4

The following describes in detail the use of the rectal cancer carcinoid tissue produced in Example 1 to conduct a radiotherapy sensitivity experiment using the evaluation method of Example 3. The specific experimental steps are as follows:

1. The well-cultured carcinoid tissue in Matrigel is seeded in a 48-well plate (4 replicate wells for each dose) after passage, with 30ul of Matrigel in each well.

2. Give X-ray irradiation when it grows for 1-3 days to a moderate size and in a good growth state (the X-ray irradiation instrument is RAD-320, PXI, the United States). The irradiation dose includes 0Gy, 2Gy, 4Gy, 8Gy, 12Gy, 16Gy.

3. Observe and take photos from the sixth day after irradiation, and take four photos in the order of upper right, upper left, lower left, and lower right of each hole, which can basically cover completely. Photographs were taken every 3 days.

4. Replace with new medium after taking pictures. After obtaining the photos, Image-Pro Plus 6.0 was used to analyze the photo data, count the number of surviving carcinoid tissues under different doses for the preparation of dose-survival curves, outline and calculate the area of carcinoid tissues at different time points after 8Gy irradiation to evaluate the recovery.

The experimental results are shown in Figure 8-9. On the dose-survival curve of the 15th day after irradiation, the survival of carcinoid tissues from different patients can be clearly divided into two groups. One group is more sensitive, with IC50 below 8Gy; the other group is more resistant. Accepted, IC50 is above 10Gy. According to the experimental results, the following conclusions can be drawn: the difference in the radiotherapy sensitivity of the carcinoid tissue of the present invention can be distinguished through the dose-survival curve of the rectal cancer carcinoid tissue, so as to evaluate the difference in the radiotherapy sensitivity of the original biopsy tissue.

The observation results of the recovery of carcinoid tissue after 8Gy irradiation are shown in Figure 10. As time goes on, there are significant differences in the death and recovery of carcinoid tissue from different patients after irradiation. Some patients completely die after irradiation and cannot regenerate. Some patients have limited recovery, and some patients have relatively complete recovery of radiation resistance. As shown in Figure 11, the standard for judging radiotherapy sensitivity based on the recovery after 8Gy irradiation is: area (day 24)/area (day 0) > 100% is radiation resistance; area (day 24)/area (Day 0) <20% are radiosensitive. According to the experimental results, the following conclusions can be drawn: the difference in the radiotherapy sensitivity of the carcinoid tissue of the present invention can be distinguished through the recovery of the rectal cancer carcinoid tissue after 8Gy irradiation, so as to evaluate the difference in the radiotherapy sensitivity of the original biopsy tissue.

Example 5

The following describes in detail the use of the rectal cancer carcinoid tissue produced in Example 1 to carry out the 5-Fu drug sensitivity test with the evaluation method of Example 3. The specific experimental steps are as follows:

1. The well-cultured carcinoid tissue in Matrigel is seeded in a 48-well plate (4 replicate wells for each dose) after passage, with 30ul of Matrigel in each well.

2. After growing for 1-3 days to moderate size and good growth state, give 10 μM pentafluorouracil (5-Fu) treatment. Begin to observe and take pictures from the sixth day after processing, and the method of taking pictures is the same as above. Photographs were taken every 3 days until the 30th day. After taking pictures, replace with new medium; the results of taking pictures are shown in Figure 12.

3. After obtaining the photos, use Image-Pro Plus 6.0 to analyze the photo data, outline and calculate the area of carcinoid tissue at different time points after 10 μM 5-Fu treatment, and draw the change of carcinoid tissue area over time. The results are shown in Figure 13.

As shown in Figure 13: the carcinoid tissues of some patients were sensitive to 5-Fu, and the area decreased significantly over time; the carcinoid tissues of some patients were resistant to 5-Fu, and the area of carcinoid tissues continued to grow without obvious change over time. According to the experimental results, the following conclusions can be drawn: the rectal cancer carcinoid tissue of the present invention can distinguish the difference in drug sensitivity through the area change after 10 μM 5-Fu treatment, so as to judge the difference in drug sensitivity of the biopsy tissue.

Example 6

The following describes in detail the use of the rectal cancer carcinoid tissue produced in Example 1 to carry out the CPT-11 drug sensitivity test with the evaluation method of Example 3. The specific experimental steps are as follows:

1. The well-cultured carcinoid tissue in Matrigel is seeded in a 48-well plate (4 replicate wells for each dose) after passage, with 30ul of Matrigel in each well.

2. After growing for 1-3 days to moderate size and good growth state, give 10μM CPT-11 treatment. Begin to observe and take pictures from the sixth day after processing, and the method of taking pictures is the same as above. Photographs were taken every 3 days until the 30th day. After taking pictures, replace with new medium; the results of taking pictures are shown in Figure 14.

3. After obtaining the photos, use Image-Pro Plus 6.0 to analyze the photo data, outline and calculate the area of carcinoid tissue at different time points after 10 μM CPT-11 treatment, and draw the change of carcinoid tissue area over time. The results are shown in Figure 15.

As shown in Figure 15: carcinoid tissues of some patients were sensitive to 5-Fu, and the area decreased significantly over time; carcinoid tissues of some patients were resistant to 5-Fu, and the area of carcinoid tissues continued to grow without obvious change over time. According to the experimental results, the following conclusions can be drawn: the rectal cancer carcinoid tissue of the present invention can distinguish the difference in drug sensitivity through the area change after 10 μM 5-Fu treatment, so as to judge the difference in drug sensitivity of the biopsy tissue.

Example 7

The following is a detailed introduction to the comparison of the results of the radiotherapy sensitivity test, 5-Fu and CPT-11 sensitivity test with the rectal cancer carcinoid tissue produced in Example 1, and the results of the neoadjuvant radiotherapy and chemotherapy efficacy evaluation of the patient. The specific methods are as follows:

In the present invention, the locally advanced rectal cancer biopsy specimens were derived from a phase III clinical trial (Fudan University Cancer Hospital) patients. All patients were patients with locally advanced rectal cancer. After receiving neoadjuvant chemoradiotherapy (all patients received X-ray therapy and 5-Fu therapy), most patients continued to receive surgical treatment, and a small number of patients adopted a watch-and-wait strategy .

All patients undergoing surgery will undergo postoperative pathological examination and score the degree of tumor regression (pTRG), which are 0 points (complete response, that is, no viable tumor cells are found), 1 point (moderate response, that is, single or Small clusters of cancer cells remain), 2 points (mild response, that is, residual cancer focus, interstitial fibrosis), and 3 points (poor response, that is, a few or no tumor cells subside, and a large amount of cancer remains). Postoperative pathological evaluation is the gold standard for evaluating the efficacy of neoadjuvant chemoradiotherapy for locally advanced rectal cancer. Patients without pTRG score due to clinical complete response (cCR) without surgery were also considered to be sensitive to treatment.

The methods of Example 4 and Example 5 were used to obtain the radiotherapy sensitivity and drug sensitivity data of the patient's rectal cancer carcinoid tissue, and the postoperative pathological tumor regression score of the patient. Then it was analyzed whether the radiotherapy sensitivity of carcinoid tissue of the present invention could predict the curative effect of neoadjuvant therapy for locally advanced rectal cancer.

1. Comparison of the radiotherapy sensitivity test results of rectal cancer carcinoid tissue produced in Example 1 with the results of neoadjuvant radiotherapy and chemotherapy efficacy evaluation for patients

The experimental results are shown in Table 1. The comparison of 17 samples was carried out. The experimental group is the second column of Table 1, the results of radiotherapy sensitivity experiment of rectal cancer carcinoid tissue; the control group is the third column of Table 1, locally advanced rectal cancer Efficacy of neoadjuvant therapy for cancer.

It can be seen that the radiotherapy sensitivity of carcinoid tissue of the present invention (judged according to the recovery situation and dose-survival curve after 8Gy irradiation) has a sensitivity of 71.4% in predicting clinical curative effect, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value. up to 83.3%, diagnostic efficiency up to 88.2%.

Table 1. Radiosensitivity of rectal cancer organoids and the efficacy of neoadjuvant therapy in patients with locally advanced rectal cancer

2. Comparison of the results of the 5-Fu sensitivity test of the rectal cancer carcinoid tissue produced in Example 1 with the results of the neoadjuvant radiotherapy and chemotherapy efficacy evaluation of the patient

The experimental results are shown in Table 2. The comparison of 17 samples was carried out. The experimental group is the second column of Table 2, and the rectal cancer carcinoid tissue is subjected to the sensitivity test results of 5-Fu; the control group is the third column of Table 2. Efficacy of neoadjuvant therapy in locally advanced rectal cancer.

It can be seen that the sensitivity of the carcinoid tissue of the present invention to 5-Fu predicts the clinical efficacy with a sensitivity of 57.1%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 77.0%, and a diagnostic efficiency of 82.4%.

Table 2 Sensitivity of rectal cancer organoids to 5-Fu and efficacy of neoadjuvant therapy in patients with locally advanced rectal cancer

3. Comparison of the CPT-11 sensitivity test results of rectal cancer carcinoid tissue produced in Example 1 with the results of neoadjuvant radiotherapy and chemotherapy efficacy evaluation in patients

The experimental results are shown in Table 2. For comparison of 17 samples, the experimental group is the second column of Table 3, the results of the CPT-11 sensitivity test on rectal cancer carcinoid tissue; the control group is the third column of Table 3, the local Efficacy of neoadjuvant therapy in advanced rectal cancer.

It can be seen that the sensitivity of the carcinoid tissue of the present invention to CPT-11 in predicting clinical curative effect is 100%, the specificity is 85.7%, the positive predictive value is 85.7%, the negative predictive value is 100%, and the diagnostic efficiency is 92.3%.

Table 3 Sensitivity of rectal cancer organoids to CPT-11 and efficacy of neoadjuvant therapy in patients with locally advanced rectal cancer

4. Comparison of the results of the radiotherapy sensitivity test and drug sensitivity test of the rectal cancer carcinoid tissue produced in Example 1 with the results of the neoadjuvant radiotherapy and chemotherapy efficacy evaluation of the patient

In fact, clinical patients receive comprehensive treatment of concurrent radiotherapy and chemotherapy, that is, they receive 5-Fu or 5-Fu plus CPT-11 concurrent chemotherapy while undergoing radiotherapy. Therefore, a comprehensive analysis of irradiation and drug sensitivity of carcinoid tissues is required.

The results of the comprehensive comparison experiment are shown in Table 4. The comparison of 17 samples is carried out. The experimental group is the second column of Table 4, and the rectal cancer carcinoid tissue is subjected to X-ray and drug sensitivity test results; the control group is the third column of Table 4. , Efficacy of neoadjuvant therapy in locally advanced rectal cancer.

It can be seen that as long as the patient's carcinoid tissue is sensitive to any one of X-rays, 5-Fu or CPT-11, the degree of tumor regression in this patient is almost better (pTRG=0 or 1), that is, the clinical curative effect is good. The comprehensive analysis results show that the sensitivity of rectal cancer carcinoid tissue to predict the efficacy of neoadjuvant therapy for locally advanced rectal cancer is 100%, the specificity is 90%, the positive predictive value is 87.5%, the negative predictive value is 100%, and the diagnostic efficiency is 100%. 94.1%.

Table 4 Sensitivity of rectal cancer organoids to X-rays and drugs and the efficacy of neoadjuvant therapy in patients with locally advanced rectal cancer

In this embodiment, laboratory experiments are compared with clinical treatment results, so as to evaluate the carcinoid tissue of the present invention for evaluating the effect of cancer treatment. The experimental principle and flow chart are shown in Figure 16. The experimental results of this example show that the radiotherapy and drug sensitivity of rectal cancer biopsy specimen carcinoid tissue have high accuracy in predicting the curative effect of neoadjuvant chemoradiotherapy in patients, and have broad clinical application. Application prospects.

The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art shall be within the scope of protection defined by the claims.

Claims (21)

1. a kind of method for preparing carcinoid tissue from mammal cancerous tissue, includes the following steps：

A1 it) will be placed in dedicated digestive juice and digest from the cancerous tissue that mammal separates, be separated into cell；

A2) cell is placed in the centrifuge tube with BSA rinse, is centrifuged, removes supernatant；

A3 special culture media) is added to centrifuge tube, cell is resuspended；

A4 cell suspension is mixed with the matrigel of defrosting), is inoculated in culture plate, is solidified to matrigel, special culture media is added Culture.

2. the method as described in claim 1, dedicated digestive juice include：DMEM culture medium, fetal calf serum, clostridiopetidase A IV and collagen Enzyme II.

3. method according to claim 2, the concentration of fetal calf serum is 0.2%-20%.

4. method as claimed in claim 3, the concentration of fetal calf serum is 0.5%-1%.

5. method according to claim 2, the concentration of clostridiopetidase A IV is 200-700U/ml.

6. method as claimed in claim 5, the concentration of clostridiopetidase A IV is 500-600U/ml.

7. method according to claim 2, the concentration of clostridiopetidase A II is 1-5mg/ml.

8. the method for claim 7, the concentration of clostridiopetidase A II is 1.5-3.5mg/ml.

9. the method as described in claim 1, digestion time 30-50min.

10. the method as described in claim 1, the special culture media include：DMEM/F12 culture medium, R-spondin1 egg White, epithelical cell growth factor, prostaglandin E2.

11. the method as described in claim 1, further comprising the steps of：

B1) cancerous tissue that mammal separates is placed in antibiotic PBS and is washed.

12. the method as described in claim 1, the mammal includes people.

13. method as claimed in claim 12, the human cancer tissue includes colon cancer tissue, rectum cancer tissue.

14. a kind of carcinoid tissue, using method culture described in claim 1-13 any one.

15. carcinoid tissue as claimed in claim 14, the carcinoid tissue is carcinoma of the rectum carcinoid tissue.

16. carcinoid tissue as claimed in claim 15, form include：Cavity type, fine and close solid type, differentiated.

17. the answering in the curative effect of new chemoradiation therapy before assessing rectal cancer of carcinoid tissue described in claim 15 or 16 With.

18. using the method packet of the curative effect of new chemoradiation therapy before the carcinoid tissue assessment rectal cancer of claim 15 or 16 It includes：Carcinoid tissue is assessed to the sensibility of radiation and chemotherapy.

19. method as claimed in claim 18, chemotherapeutics includes 5-Fu, CPT-11.

20. method as claimed in claim 18, carcinoid tissue uses carcinoid tissue survival number to the sensibility of radiation and chemotherapy Assessment.

21. method as claimed in claim 18, radiation sensitivity observing time is at least after radiotherapy 9 days.