Novel proteomics approach for functional identification of regulators of cancer drug response and apoptosis in ex vivo tissue cultures of human colorectal cancer

Abstract

9658 Background: It is notoriously difficult to study the molecular effects of anticancer drug treatment in vivo. So far, this has been only possible by performing serial biopsies in tumor patients undergoing treatment. An approach to address this problem is to perform ex vivo tissue cultures, where patient tumor tissue is obtained at the occasion of a routine operation and then processed in vitro. The molecular program of apoptosis and the activation status of various signal transduction pathways has been identified as a major determinant of chemotherapy responsiveness in cancer. Methods: To analyze genetic pathways potentially involved in anticancer drug response, we have treated tumor samples from colorectal cancer patients ex vivo with antitumor agents with various mechanisms of action. Cytotoxicity was quantified using a colorimetric dye assay. Proteome profiles of the untreated/treated and normal/cancerous patient samples have been generated by reverse-phase protein microarrays, which are a novel approach to analyze signaling pathways using small numbers of cells microdissected from biopsy specimens. For the feasibility phase of the project, we looked at well established growth factor activation pathways such as p-ERK, p-SAPK/JNK, p-AKT, p-p38, p-JNK, p-IkBa, p-STAT3, proliferation regulatory pathways such as p-histone H3, p-Rb, p-p53, cyclin D1, and pathways involved in apoptosis regulation such as cleaved caspase 3, cleaved PARP, and Bcl-2. Results: We found higher cytotoxic activity of most anticancer agents in cancer tissue as compared to normal tissue of the same organ (colon or liver). There was a prominent down-regulation of p-SAPK/JNK and a less pronounced down-regulation of p-ERK and p-p38 with chemotherapy in tumor samples but not in normal tissue samples. Conclusions: This culture technique allows to treat human tissue with various agents ex vivo to simulate the in vivo treatment situation and to assess cytotoxicity by a simple colorimetric dye assay. With the reverse-phase protein microarray technology activated signalling transduction pathways can be identified on low quantity tissue samples. No significant financial relationships to disclose.