Platform for screening chemotherapy combined anti-tumor natural product medicine and application thereof

Abstract

The invention provides a screening platform for chemotherapy combined anti-tumor natural products and application of the screening platform. PTC has tumor cells, interstitial cells, fibroblasts and immune cells from primary epithelium, is rich in cell components, and has advantages in the aspect of tumor microenvironment simulation. Meanwhile, PTC can reproduce tumor heterogeneity, the culture success rate and clinical consistency can reach 90% or above, and the detection period is within two weeks. And the detection result can be read through AI intelligent software, so that the accuracy can be further improved. The screening period can be shortened, the cost is reduced, and the accuracy is improved; according to the platform, high-throughput screening can be carried out in a short time, and the treatment effect of a chemotherapy drug combined anti-tumor natural product drug scheme can be predicted.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a platform for screening chemotherapy combined with anti-tumor natural product drugs. Background Art

[0002] Despite improvements in diagnostic techniques, identification of new disease predictive markers, and approval of numerous new therapies, cancer remains one of the leading causes of death worldwide. Surgical resection, radiotherapy, and chemotherapy remain the cornerstones of treatment, but each approach has its limitations. Chemotherapy has two major limitations: more than 90% of patients develop drug resistance and it also has a killing effect on normal cells. Radiotherapy also causes healthy tissue damage, inflammation, and oxidative stress, with up to 85-95% of patients experiencing skin damage and 60% of patients experiencing lung damage. Recently, cancer immunotherapy has attracted attention as a new type of treatment for specific cancers. Immunomodulation is also emerging as an effective strategy to improve efficacy. Natural phytochemicals with known anticancer properties have been reported to mediate their effects by modulating traditional cancer pathways and immunity. The mechanism of phytochemical-mediated immunomodulatory activity may be attributed to the remodeling of the tumor immunosuppressive microenvironment and the sensitization of the immune system. This allows the immune system to better recognize and target cancer cells and to synergize with chemotherapeutic drugs.

[0003] Approximately half of clinically used cancer chemotherapeutics, ranging from topoisomerase inhibitors, cancer chemotherapeutic antibiotics to microtubule stabilizers, are derived from or inspired by natural sources. As new therapeutic targets emerge, interest in novel biomolecules as adjuncts to conventional therapies continues to grow. While initial research focused on dietary compounds with potential cancer prevention effects through their antioxidant and anti-inflammatory properties, pro-apoptotic and anti-proliferative effects have also been identified, leading to clinical trials. As attention has shifted toward the development of immunomodulatory cancer chemotherapies, the identification of anti-inflammatory and immunomodulatory phytochemicals has followed. Collateral damage can be reduced by targeting inflammation, which is a double-edged sword for cancer, as chronic inflammation itself promotes tumorigenesis and cancer progression. At the same time, harnessing the power of the immune system by stimulating cancer cell recognition and inducing immunogenic cell death by stimulating host defenses is also an attractive approach.

[0004] Checkpoint inhibitors are designed to reactivate T cells and re-establish cytotoxicity and are becoming the most important immunomodulators in cancer chemotherapy. They have been successful in metastatic melanoma, urothelial carcinoma, and non-small cell lung cancer, but are associated with immune-related adverse events in 70-90% of patients. This requires the development of new drugs with improved safety. It has been reported that natural biomolecules, including berberine, triptolide, epigallocatechin gallate (EGCG), and curcumin, have been shown to reduce the expression of PD-1 ligand PD-L1 in cancer cells. Whether these phytochemicals can become new checkpoint inhibitors with higher safety requires further study. Patient-derived tumor-like cell clusters (PTC) are an emerging 3D cell model for in vitro drug sensitivity testing after PDO and PDX. The PTC model is composed of multiple cells such as tumor stem cells, epithelial cells, fibroblasts, macrophages, etc., which simulates the tumor microenvironment to a certain extent. By using the PTC model drug sensitivity test, the tumor condition in the patient's body after the drug action can be more objectively reflected, thereby achieving the purpose of screening natural products with new checkpoint inhibitor functions. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for screening chemotherapy combined with anti-tumor natural product drug regimens using a microtumor PTC in vitro model in view of the shortcomings of the existing technology. This method can shorten the screening cycle, improve accuracy and reduce costs.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a platform for screening chemotherapy combined with anti-tumor natural product drugs and its application, the screening platform is a tumor-like cell cluster derived from a patient - a micro-tumor PTC in vitro model; the anti-tumor natural products include all anti-tumor natural products; the tumors include four major tumors: gastrointestinal tumors, breast cancer, and lung cancer.

[0007] The above-mentioned microtumor PTC in vitro model has advantages in simulating the tumor microenvironment, including tumor-derived epithelial cells, stromal cells, and immune cells, with tissue specificity and stem cell characteristics.

[0008] The above-mentioned microtumor PTC in vitro model is consistent with the original tumor tissue in terms of epigenetic level and genetic characteristics, and its clinical consistency and culture success rate can reach more than 90%.

[0009] The above natural product can be curcumin. The curcumin is a turmeric extract.

[0010] The above-mentioned chemotherapy drug can be oxaliplatin. The oxaliplatin is the active ingredient of oxaliplatin.

[0011] The above tumors include gastrointestinal tumors, breast cancer, and lung cancer.

[0012] The above antitumor activity can be demonstrated by PTC microsphere cell viability assay.

[0013] The effectiveness of the above drug regimen is manifested by the degree of regression of tumor-like cell clusters.

[0014] The above-observed tumor-like cell cluster retraction degree can be counted by the self-installed microscope AI intelligent software.

[0015] The advantages of the present invention compared with the prior art are:

[0016] This invention uses the microtumor PTC in vitro model for the first time to screen the effectiveness of chemotherapy combined with anti-tumor natural products. The final results of the platform can be statistically analyzed by AI intelligent software, which can shorten the screening cycle, reduce costs and improve accuracy. The platform can perform high-throughput screening in a short period of time and can predict the therapeutic efficacy of chemotherapy drugs combined with anti-tumor natural product drug regimens. DETAILED DESCRIPTION

[0017] The technical solution of the present invention will be further described below, but the present invention is not limited thereto.

[0018] Example 1

[0019] The patient-derived tumor-like cell clusters described in this embodiment are colorectal cancer cell clusters, the natural product is curcumin, and the method is to dissociate the colorectal cancer experimental samples into single tumor cells by physical shearing and enzymatic digestion. After 7 days, the cells can form PTC microspheres through proliferation and self-assembly; curcumin combined with oxaliplatin can effectively kill lung cancer PTC microspheres, significantly reducing their number and size.

[0020] The colorectal cancer tissue samples were collected and placed in a culture dish, cut into pieces, digested with collagenase, and washed. The digestion time was measured, and the samples were observed under a microscope and placed in an incubator to observe the digestion status regularly.

[0021] After digestion was terminated, the intestinal cancer PTC culture medium was added and placed in a constant temperature incubator for 3-7 days, and the cells formed PTC microspheres through self-assembly and proliferation. During the culture process, the cells were photographed and counted under a microscope at D0, D3, and D7.

[0022] 100ul of the pre-treated cell fluid was taken for HE staining identification; first permeabilization was performed with tritonX 100, and then the nucleus was stained with hematoxylin, and then rinsed with running water after 1 to 3 minutes, and then differentiated with 1% hydrochloric acid alcohol for a few seconds, and then rinsed with running water, and then blued with ammonia water for 1 minute and rinsed with running water, and eosin stain was used for 0.5 to 1 minute and rinsed with running water, and finally, 75% ethanol for 2 minutes, 85% ethanol for 2 minutes, anhydrous ethanol for 5 minutes, anhydrous ethanol for 5 minutes, and xylene for 5 minutes were used for step-by-step dehydration, and the slides were sealed with neutral resin and identified as tumor cells under a microscope.

[0023] After the culture, the PTC microspheres were plated in a 96-well plate. The experimental groups were divided into a curcumin group, an oxaliplatin group, and a curcumin combined with oxaliplatin group. The experimental group was plated with 3 replicate wells, and a control group (3 replicate wells) was set up. The degree of tumor regression, including changes in quantity and volume, was observed at 24 hours, 48 ​​hours, 72 hours, and on the 7th day. Photos were taken and counted using AI.

[0024] After the PTC microspheres and drugs were co-incubated, the 96-well plate was slightly tilted and stood up, 100ul of supernatant was carefully sucked off along the liquid surface, 80ul of cellititor was added, and then the mixed liquid was transferred from the transparent plate to the white plate. Before transfer, the mixture was fully washed and mixed, and the shaker was set to 20 peaks and the speed was medium to shake well. After transfer, the cell viability value was measured with a spectrophotometer.

[0025] The results of the present invention show that the curcumin combined with oxaliplatin group can significantly reduce the number of PTC microspheres, significantly reduce the volume, and affect cell viability compared with other groups, proving that it has a better anti-tumor effect.

Claims

1. A screening platform for chemotherapy combined with anti-tumor natural products and its application, wherein: The screening platform for chemotherapy combined with anti-tumor natural products only includes screening using the PTC platform, and is characterized by comprising the following steps: Step 1: Collect available experimental specimens from patients with malignant tumors and construct an in vitro microtumor PTC model; Step 2: Screen chemotherapy combined with natural products with anti-tumor effects based on the microtumor PTC in vitro model.

2. The method according to claim 1, characterized in that The malignant tumors include four major tumors: gastrointestinal tumors, breast cancer, and lung cancer.

3. The method according to claim 1, characterized in that The experimental specimens in step 1 include surgical tissue, puncture tissue, biopsy tissue or pleural effusion.

4. The method according to claim 1, characterized in that The natural products in step 2 include but are not limited to celastrol, resveratrol, berberine, curcumin, quercetin, etc., all natural products with anti-tumor activity.

5. The PTC construction method according to claim 1, characterized in that: The following steps are involved: Step 1: Using physical shearing or enzyme digestion, the experimental specimens from patients with malignant tumors are dispersed into single cells; Step 2: In the specific culture medium of different cancer types, single cells self-assemble to form cell clusters to obtain the PTC model, which contains cell types such as epithelial tumor cells, stromal cells, fibroblasts, and immune cells.

6. The screening method according to claim 1, characterized in that The drug sensitivity test was carried out using the microtumor PTC in vitro membrane type, and the killing efficiency percentage was calculated using AI intelligent software to screen natural products with anti-tumor activity.