Evaluation Of Anti-colorectal Cancer Effect Of Anlotinib Based On PDX Models And Preliminary Exploration Of Its Clinical Application

Colorectal cancer is the third most common malignant tumor in the world,its incidence is second only to breast cancer and lung cancer,and its mortality is second only to lung cancer.With the research on the molecular mechanism of the development of colorectal cancer and the progress of new drug research,colorectal cancer has entered the era of precision medicine,and targeted drugs have occupied a place in the treatment of colorectal cancer.However,due to the high heterogeneity of colorectal cancer,not all patients can benefit from the existing targeted therapy,and with the development of clinical trials of targeted drugs,the problems of primary and secondary drug resistance of targeted therapy are gradually emerging.Nowadays,the choice of drugs for third-line and posterior-line treatment of advanced colorectal cancer is very limited,while the5-year survival rate of advanced colorectal cancer is still less than 15%.Therefore,the research of new drugs for colorectal cancer is extremely urgent.Tumor angiogenesis is an important link in tumor occurrence and development,and targeted angiogenesis has become a hot spot in tumor targeted therapy.Anlotinib is a novel small molecular multi-target receptor tyrosine kinase inhibitor(TKI),which mainly targets vascular endothelial growth factor receptor 2,fibroblast growth factor receptor 1-4,platelet-derived growth factor receptor α / β,stem cell factor receptor(c-Kit)and Ret,that can inhibit tumor angiogenesis and tumor cell proliferation.Anlotinib has been approved for posterior line treatment of non-small cell lung cancer,soft tissue sarcoma and small cell lung cancer currently.And it has also showed promising therapeutic potential in phase II clinical trials of metastatic kidney cancer and medullary thyroid cancer.The phase I non-blind clinical trial of anlotinib has demonstrated that it can reduce the tumor size of patients with colorectal cancer,which makes the application prospect of anlotinib in colorectal cancer began to attract attention.Experiments in vitro have proved that anlotinib can inhibit the proliferation,migration and invasion of colorectal cancer cells,and induce cell apoptosis.A number of phase II clinical trials of anlotinib in combination with chemotherapy in colorectal cancer are also underway,but no clear results have been published yet.Therefore,whether anlotinib is effective in colorectal cancer remains to be verified,and the mechanism of anlotinib against colorectal cancer in vivo remains to be further explored.In view of ethical and other reasons,randomized controlled trials to verify the efficacy of drugs are often difficult to achieve in the population,therefore,it is very important to use appropriate preclinical models to predict the efficacy of various drugs and finally optimize treatment methods to achieve good therapeutic results.The clinical trials of oncology drug development has the lowest success rate among all diseases,mainly because the preclinical model of drug development can’t accurately predict the response of patients to drugs.The patient-derived xenograft(PDX)model is established by inoculating patient tumor tissue directly into immunodeficient mice.Its main advantage is that the heterogeneity and the diversity of molecular characteristics of tumor tissues from patients are retained to a large extent,and the characteristics of the original tumor can still maintain even after generating.Studies have shown that there is a strong correlation between the drug efficacy verified in the PDX models and the clinical response to drug treatment,suggesting the reliability of PDX models as a preclinical model for drug efficacy verification.The National Cancer Institute(NCI)recommends the use of PDX model for tumor clinical transformation research.Nowadays,PDX model has been widely used in the screening of drug-sensitive people,preclinical trials of new anticancer drugs and individualized treatment of tumor patients.Our group has the work foundation of establishing PDX model,which can provide technical support for model establishment.The aim of this study was to establish PDX models of colorectal cancer,and explore the anti-tumor efficacy and mechanism of anlotinib in colorectal cancer by using PDX models.Besides,we aimed to review and analyze the clinical application of anlotinib,and evaluate the application prospect of anlotinib in advanced colorectal cancer comprehensively.Part 1 Establishment of PDX model of colorectal cancerPurpose The aim of this part was to establish the PDX models of colorectal cancer and verify the consistency between the model and the corresponding original tumor tissue,so as to provide a reliable platform for the follow-up preclinical research of new drug development and individualized treatment.Methods We collected fresh tumor tissues from colorectal cancer patients who underwent surgery in our department from February 2017 to May 2020 and were willing to participate in the study.The PDX models of colorectal cancer were established by inoculating fresh tumor tissue subcutaneously in nude mice.The growth rules of each generation of PDX models were observed and recorded,and the consistency between PDX models and tumor tissue was preliminarily identified by hematoxylin-eosin staining and immunohistochemical staining.Results10 out of 14(71.4%)PDX models of colorectal cancer were successfully established.The tumor formation rate of PDX model increased with the increase of passage algebra,and can reach 100% after G2 generation.The growth rate of PDX model accelerated with the increase of passage algebra and remained stable after G3/G4 generation.The established PDX model was consistent with the original tumor tissue in histomorphology which was confirmed by hematoxylin-eosin staining and molecular biological characteristics which was confirmed by immunohistochemical staining(CD34,CDX2,CK7,CK20).Conclusions We finally established 10 colorectal cancer PDX models with high simulation of original tumor tissue,which provide a stable and reliable platform for the evaluating of drug efficacy,screening of sensitive population and exploring of individualized tumor therapy.Part 2 Efficacy evaluation and mechanism exploration of anlotinib based on colorectal cancer PDX modelsPurpose The aim of this part was to initially evaluate the efficacy of the anlotinib monotherapy and combined chemotherapy in colorectal cancer based on PDX models,and to explore the mechanism of antineoplastic effect of anlotinib.Methods We randomly selected 5 PDX models to verify the effect of anlotinib monotherapy and combination chemotherapy,respectively.In the experiment of anlotinib monotherapy,the mice of each model were randomly divided into four groups:(1)control group;(2)low dose group(0.75mg/kg,ig,d1-14,q3w);(3)medium dose group(1.5mg/kg,ig,d1-14,q3w);(4)high dose group(3mg/kg,ig,d1-14,q3w).In the experiment of combination therapy,the mice of each model were randomly divided into seven groups:(1)negative control group,(2)anlotinib group(1mg/kg,ig,d1-14,q3w);(3)OXA group(3mg/kg,ip,biw);(4)anlotinib combined with OXA group(same dose and frequency as single drug);(5)CPT-11 group(5mg/kg,ip,qw);(6)anlotinib combined with CPT-11 group(same dose and frequency as single drug);(7)positive control group(CPT-11 20mg/kg,ip,qw).The body weight of mice,tumor volume and tumor weight were observed and recorded,and the tumor inhibition rate was calculated to evaluate the anti-tumor effect of anlotinib.The clinical and gene sequencing data of patients were analyzed to explore the population characteristics that might benefit from anlotinib.Immunohistochemical staining was used to observe the expression of Ki-67,CD31,VEGFA,VEGFR2,AKT and ERK1/2.Results Anlotinib monotherapy is effective in the PDX model of colorectal cancer.The anti-tumor effect was significant when the concentration of anlotinib was 1.5mg/kg in two PDX models(p < 0.05).Anlotinib also showed significant anti-tumor effect when the concentration was 3mg/kg in other two PDX models(p < 0.01).We did not observe the anti-tumor effect of anlotinib in only one PDX model.Anlotinib combined with oxaliplatin was effective in colorectal cancer.We demonstrated that anlotinib combined with oxaliplatin can significantly inhibit tumor growth in three PDX models(p < 0.05).And we proved that anlotinib combined with oxaliplatin could significantly enhance the anti-tumor effect of oxaliplatin in one PDX model(p < 0.05).Anlotinib combined with irinotecan was also effective in colorectal cancer.We also demonstrated that irinotecan monotherapy had significant anti-tumor effect in two PDX models(p < 0.05),and anlotinib combined with irinotecan could significantly inhibit tumor growth in three PDX models(p < 0.05).Combined with the clinical data of the patients,the patients with left colon adenocarcinoma,non-multiple organ distant metastasis and BRAF V600 E wild-type were more responsive to anlotinib.Immunohistochemical staining showed that the expression of Ki-67 decreased significantly when the concentration of anlotinib was 1.5mg/kg,and the microvessel density decreased significantly when the concentration of anlotinib reached 1.5mg/kg(PDX model 3)or 3mg/kg(model 5).In addition,anlotinib could significantly inhibit the expression of VEGFA,AKT and ERK in PDX models.Conclusions Anlotinib monotherapy could effectively inhibit tumor growth in colorectal cancer PDX models by inhibiting tumor cell proliferation via AKT/ERK signaling pathway and angiogenesis.Alotinib combined with oxaliplatin had a certain synergistic anti-tumor effect.Colorectal cancer patients with tumors in the left colon and BRAF V600 E wild-type were more likely to benefit from anlotinib.Part 3 Clinical application of anlotinib in colorectal cancerPurpose The aim of this part was to analyze the efficacy of anlotinib in advanced colorectal cancer.We aimed to analyze the characteristics of colorectal cancer patients who may benefit from anlotinib,and lay a foundation for the follow-up clinical application of anlotinib in the advanced colorectal cancer.Methods We collected the patients with advanced colorectal cancer who had used anlotinib in our hospital from April 2019 to December 2020.The short-term and long-term efficacy of anlotinib were evaluated by objective response rate,disease control rate and progression free survival.We analyzed the influence of different factors on progression free survival,and evaluated the related adverse reactions during treatment.Results A total of four patients with advanced colorectal cancer were included in this study,two of them responded well to anlotinib,with an average PFS of 6.7 months,and the tumor markers decreased significantly during the treatment.Preliminary analysis showed that patients whose tumor was located in the left colon,and patients with CEA levels much higher than the normal upper limit before treatment had a better response to anlotinib.The adverse reactions of anlotinib were grade 1 or grade 2,and the main adverse reaction reported in this study was hypothyroidism.Conclusions Anlotinib was effective in patients with advanced left-sided colorectal cancer,and the adverse reactions of anlotinib are basically controllable.