Gastroi Ntesti Nai Patient-derived Tumor Tissue Xenograft Models And Their Applications In Assessment Of A Novel Anti-VEGF Targeted Agent

Background:Animal models have been used in "front-line" preclinical studies for predicting the efficacy and possible toxicities of anticancer drugs in cancer patients. The lack of general clinic-relevant tumor model for each kind of human cancer or model for a given cancer patient is a major impediment in seeking an general effective anticancer therapy for a certain kind of cancer or selecting the most appropriate therapy for an individual patient. Available in vivo experimental tumor models that are clinically representative of each major human cancer type would largely increase the success in identifying new active agents targetting sectionicular tumors. They can also accelerate the progress, reduce the cost and patient tumor tissue needed for anticancer drug development. Similarly, individualized models of human cancers would greatly facilitate selecting the best therapy for each individual patient as well. The increasingly used patient-derived human tumor tissue (PDTT) xenografts models implanted subcutaneously or in subrenal capsular in immunodeficient mice such as athymic nude mice or severe combined immune deficient (SCID) mice provided a more accurate reflection of human tumor biological characteristics than tumor cell lines. These xenografts models have become increasingly popular as evidence accrues that they are more accurately recapitulate features of patient tumors, such as maintaining the cell differentiation and morphology, the architecture, and molecular signatures of the original patient tumors. The unique feature of PDTT xenografts models is that the implanted tumor tissue still retains most of its normal architecture and function. This model more accurately reflects the in vivo situation than CCL thus could be functional for rapid screening of potential therapeutics.It is believed that PDTT xenografts models possess four general applications. Firstly, it can be used as an in vivo screening tool to test novel drugs with therapeutic potential in cancer treatment. Secondly, it can be used to evaluate key markers of response and resistance to drugs. The ideal characteristics of the PDTT xenograft model might also make it a candidate method to investigate heterogeneity in primary tumor and its corresponding metastases and to evaluate the effect of such heterogeneity on cancer therapy response. Finally, it can be applied to achieve individualized anticancer therapy regimens by preclinically assessing the sensitivity of tumors to registered anticancer agents in vivo. Because of the inherent limitations in the current approaches for personalized cancer therapy, the need for new techniques to realize this goal is urgent. PDTT xenograft model might be an ideal candidate method to help to predict tumor response to therapy and it can be applied to achieve personalized therapy regimens by pre-clinically assessing the therapy-sensitivity of tumors to registered anticancer agents in vivo.Angiogenesis is one of the eight hallmarks of cancer acquired during the multistep development of human tumors. Vascular endothelial growth factor (VEGF) is well established as a central mediator in this process. Furthermore, VEGF is the only angiogenic factor known to be present throughout the entire tumor lifecycle. Based on these evidences, VEGF is considered as a rational target for antiangiogenic drug development. Because anti-VEGF approaches act by blocking tumor-associated angiogenesis, which appears to be widely required by many different types of tumors, these approaches have been proved to be generally useful against a wide assortment of solid tumors. FP3is an engineered protein which contains the extracellular domain2of VEGF receptor1(Flt-1) and extracellular domain3and4of VEGF receptor2(Flk-1, KDR) fused to the Fc portion of human immunoglobulin G1. Previous studies indicated that FP3had promise as a local antiangiogenic treatment of human CNV (choroidal neovascularization)-related AMD (age-related macular degeneration). In subsequent studies, it was demonstrated that FP3has an inhibitory efficacy in VEGF-mediated proliferation, migration and tube formation of human umbilical vein endothelial cells, and in VEGF-mediated vessel sprouting of rat aortic ring in vitro. It was also demonstrated that FP3has an antitumor effect in a non-small cell lung cancer cell line (A549) xenograft model in nude mice. However, little is known of the cellular effects of FP3on tumor vessels and its antitumor effects on gastrointestinal tumors.Section IPurpose:(1) It was the aim of our study to establish a patient-derived tumor tissue (PDTT) xenograft model of gastric carcinoma useful for personalized cancer therapeutic regimen selection as well as testing of novel molecularly targeted agents.(2) In the present studies, we examined the cellular effects of FP3on blood vessels, mainly focused on the endothelial cells and pericytes of tumor vessels in a PDTT xenograft model of gastric carcinoma using large tumors with established vasculature.Methods:(1) A PDTT of primary gastric carcinoma was used to create the xenograft model. After11weeks, xenografts were harvested for serial transplantation. Hematoxylin and eosin staining, immunohistochemical staining and western blotting were used to determine the biological stability of the xenograft during serial transplantation compared with the original tumor tissue. Drug sensitivities of the xenograft to bevacizumab, FP3, and cetuximab were evaluated.(2) In this study, we further investigated the cellular effects of FP3on blood vessels in a PDTT xenograft model of gastric carcinoma using large tumors with established vasculature.Results:(1) A PDTT xenograft model of primary gastric carcinoma was successfully established. Early passages of the PDTT xenograft model of gastric carcinoma revealed a high degree of similarity with the original clinical tumor sample with regard to histology, immunohistochemistry as well as proteins expression. The PDTT xenograft model responded to all the drugs tested, and a higher response rate was observed in bevacizumab in combination with cetuximab-treated group as well as FP3in combination with cetuximab-treated group.(2) Treatment with FP3caused robust and early changes in endothelial cells and pericytes of vessels in the PDTT xenograft model. Vascular density decreased and vascular sprouting was suppressed with treatment of FP3. Pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype.Conclusions:(1) A PDTT xenograft model of gastric carcinoma has been established. It provides an appropriate model for personalized cancer therapeutic regimen selection as well as testing of novel molecularly targeted agents.(2) Our results revealed that FP3has a direct and rapid antiangiogenic effect in tumor vessels, which was achieved mainly via regression of tumor vasculature, inhibition of new and recurrent vessel growth, and normalization of existing tumor vasculature.Section â…¡Purpose:(1) It was the aim of our study to establish PDTT xenograft models of colon carcinoma with lymphatic and hepatic metastasis useful for testing of a novel molecularly targeted agent, FP3.(2) Combining inhibition of VEGF by FP3and chemotherapy by capecitabine might act additive or synergistically.(3) Combining inhibition of VEGF by FP3and EGF signaling by cetuximab might act additive or synergistically.(4) The aim of this study is to investigate whether the heterogeneity in primary tumor and related metastases exists and whether such heterogeneity would result in different response to anti-EGFR and anti-VEGF therapies.Methods:(1) PDTT of primary colon carcinoma, lymphatic and hepatic metastases were used to create xenograft models. Hematoxylin and eosin staining, immunohistochemical staining, genome-wide gene expression analysis, pyrosequencing, qRT-PCR, and western blotting were used to determine the biological stability of the xenografts during serial transplantation compared with the original tumor tissues.(2) In this study, a series of PDTT xenograft models of primary colon carcinoma and lymphatic and hepatic metastases were established for assessment of the antitumor activity of FP3as a monotherapy and in combination with capecitabine. Tumorinoculated nude mice were treated with FP3, capecitabine, alone or in combination, after tumor growth was confirmed and volume and microvessel density in tumors were evaluated. Levels of VEGF, EGFR, and PCNA in the tumor were examined by immunohistonchamical staining, and levels of related cell signalling pathways proteins expression were examined by western blotting.(3) In this study, a series of PDTT xenograft models of primary colon carcinoma and lymphatic and hepatic metastases were established for assessment of the antitumor activity of FP3as a monotherapy and in combination with cetuximab. Tumorinoculated nude mice were treated with FP3, cetuximab, alone or in combination, after tumor growth was confirmed and volume and microvessel density in tumors were evaluated. Levels of VEGF, EGFR, and PCNA in the tumor were examined by immunohistonchamical staining, and levels of related cell signalling pathways proteins were examined by western blotting.(4) In this study, we compared the heterogeneity in primary colon cancer and its corresponding lymphatic and hepatic metastases, focusing on the cell signalling pathways proteins using the methods of immunohistochemical staining and western blotting, the gene status of KRAS using pyrosequencing, and genome-wide gene expression using GeneChip HGU133Plus2.0expression arrays. To investigate whether such heterogeneity would result in different response to anti-EGFR and anti-VEGF therapies, we further evaluate the therapy response of cetuximab in combination with bevacizumab in primary colon cancer and its corresponding lymphatic and hepatic metastases by establishing PDTT xenograft models based on the same tissue samples from above mentioned three tumor sites.Results:(1) Early passages of the PDTT xenograft models of primary colon carcinoma, lymphatic and hepatic metastases revealed a high degree of similarity with the original clinical tumor samples with regard to histology, immunohistochemistry, genes expression, mutation status, mRNA expression as well as proteins expression.(2) FP3and FP3in combination with capecitabine showed significant antitumor activity as a monotherapy in three xenograft models (primary colon carcinoma, lymphatic metastasis, and hepatic metastasis). The microvessel density in tumor tissues treated with FP3and FP3in combination with capecitabine was lower than that of the control. Antitumor activity of FP3in combination with capecitabine was significantly higher than that of each agent alone in three xenograft models (primary colon carcinoma, lymphatic metastasis, and hepatic metastasis).(3) FP3and FP3in combination with cetuximab showed significant antitumor activity as a monotherapy in three xenograft models (primary colon carcinoma, lymphatic metastasis, and hepatic metastasis). The microvessel density in tumor tissues treated with FP3and FP3in combination with cetuximab was lower than that of the control. Antitumor activity of FP3in combination with cetuximab was significantly higher than that of each agent alone in three xenograft models (primary colon carcinoma, lymphatic metastasis, and hepatic metastasis).(4) The expressions of EGFR, VEGF, Akt/pAkt, ERK/pERK, MAPK/pMAPK, and mTOR/pmTOR and the genome-wide gene expression were different in primary colon cancer and matched lymphatic and hepatic metastases. Our results demonstrate that primary colon cancer and its corresponding lymphatic and hepatic metastases have different response rate to anti-EGFR and anti-VEGF therapies.Conclusions:(1) In this study, PDTT xenograft models of colon carcinoma with lymphatic and hepatic metastasis have been successfully established. They provide appropriate models for testing of novel molecularly targeted agents.(2) This study indicated that addition of FP3to capecitabine significantly improved tumor growth inhibition in a series of PDTT xenograft models of primary colon carcinoma and lymphatic and hepatic metastases.(3) This study indicated that addition of FP3to cetuximab significantly improved tumor growth inhibition in a series of PDTT xenograft models of primary colon carcinoma and lymphatic and hepatic metastases with wide-type KRAS gene status. Combination anti-VEGF and anti-EGFR therapy may represent a novel therapeutic strategy for the management of colon carcinoma.(4) Our results indicate that the heterogeneity in primary colon cancer and its corresponding lymphatic and hepatic metastases would result in difference in response to double-inhibition of EGFR and VEGF. PDTT xenograft model could be an ideal in vivo tool to clear whether the primary tumors and corresponding metastases have different response to the same anticancer drugs.