LONG TERM CHEMOTHERAPY TREATMENT INDUCES A DUAL IMMUNE RESISTANCE IN COLORECTAL CANCER CELLS

Abstract

Colorectal Cancer (CRC) presents a substantial health burden globally, especially that its prevalence is escalating at an alarming rate. Current therapeutic interventions impart limited efficacy in the patient cure, specifically in progressing metastatic tumors. While recent advancements in immunotherapy have accomplished therapeutic benefit in microsatellite unstable (MSI) CRC, a wider cohort of microsatellite stable (MSS) tumor patients who predominantly present a non-inflamed immune cold phenotype, pose a major clinical challenge. In this context, it would be beneficial to escalate the therapeutic potential of existing conventional chemotherapeutic treatments used in CRC. The study aims to screen the expression of High Mobility Group Box Protein (HMGB1), an immunogenic cell death marker, and Programmed Death Ligand 1 (PD-L1), an immune checkpoint across HT-29, HCT 116, DLD-1, SW480, SW620 and LoVo CRC cell lines upon treatment with 5-Fluorouracil (5-Fu) and Oxaliplatin (Oxp). The cell lines were observed with a maximum cytotoxic response at 72 hours of treatment and the doses of 5µM and 50µM for 5-Fu, and, 5µM and 30µM for Oxp were chosen. HMGB1 expression was analyzed in the cultured cell supernatant and cell lysate of the treated cell lines by using ELISA and western blot analysis, respectively. Despite an inconsistent pattern of expression in the analyzed cell lines, all of them except HT-29 showed a decrease in HMGB1 expression especially at the higher concentration of the drugs. HMGB1 expression was also analyzed in CRC patient plasma samples, where a significant upregulation was observed in the patients as compared to healthy controls. 20 CRC Patients were stratified based on their therapeutic intervention of 5-Fu combinations, FOLFOX and FOLFIRI. Decrease of HMGB1 was seen in the treated patients as compared to non-treated CRC patients though the effect was void of significance. PD-L1 expression was initially analyzed using flow cytometry and further validated with western blot analysis. DLD-1, HCT 116 and HT-29 showed high PD-L1 induction, an effect that was more evident with 5-Fluorouracil than in Oxaliplatin. LoVo exhibited slight PD-L1 induction upon treatment with 5-Fu. The decline of an immunogenic cell death marker and the induction of an immune checkpoint molecule suggests a dual resistance mechanism promoted by the tumors. A combinatorial approach of the cytotoxic agents with immune checkpoint inhibitors, such as anti-PD-1 or anti-PD-L1 could provide durable therapeutic efficiency in CRC patients.

Date of Award	2019
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences