Identification and characterization of biomarkers reflecting extracellular matrix remodeling in cancer

Abstract

Despite extensive research efforts and validation of numerous new cancer drugs in clinical trials every year, cancer continues to be one of the major causes of death in the western world, emphasizing the need for novel biomarkers. During the last few years, increased attention has been drawn towards precision medicine as a new treatment tool in cancer which strives at matching the right drug(s) to the right patient. To enable this approach, informative biomarkers are needed that can identify patients early, predict who will respond to a certain treatment, give information regarding treatment efficacy and give information about a likely outcome in relation to survival, progression or recurrence. Mounting evidence points towards the extracellular matrix (ECM) playing an active role in cancer progression and not just being a passive bystander. During tumor development and progression, the normal ECM homeostasis is disturbed and excessive ECM remodeling occurs leading to a structure and organization of the tumor-associated ECM that is very different from that of normal tissue. A consequence of this altered remodeling is an increased production of turnover products that are released into the circulation. These small protein fragments hold post-translational modifications giving rise to neo-epitopes that represents unique tissue fingerprints of the combination of the involved proteases and the composition of the ECM. Protein fingerprint biomarkers could provide novel insights into the understanding of cancer pathology as well as to improve cancer treatment and strategy towards a more personalized approach. The hypothesis of this thesis was that the pathologically driven turnover of the ECM results in the release of neo-epitope fragments into the circulation that can serve as diagnostic, prognostic and/or predictive tools in cancer. The overall aim of this thesis was to identify, characterize and validate blood-based neo-epitope biomarkers reflecting ECM remodeling in cancer and their ability to identify patients with cancer and provide prognostic value for the future outcome in cancer patients. The specific aims were as follows: Aim 1 - To investigate the diagnostic and prognostic potential of neo-epitope biomarkers reflecting increased collagen turnover in patients with colorectal cancer (paper I, II and additional results 1) Aim 2 – To identify novel neo-epitope biomarker targets reflecting structural changes in the ECM during cancer progression and develop immunoassays against the neo-epitopes of interest (paper III, IV and additional results 2) To investigate the aims of this thesis we measured the following well validated protein fingerprint biomarkers in serum from patients with different solid tumors: MMP-degraded type I collagen (C1M), MMP-degraded type III collagen (C3M), MMP-degraded type IV collagen (C4M), formation of type I collagen (PINP), formation of type III collagen (PRO-C3), formation of type IV collagen (P4NP7S) and formation of type VI collagen (PROC6). In addition, three novel immunoassays were developed and validated, targeting protease-generated neo-epitopes originating from ECM proteins. The clinical use of the biomarkers were investigated in different clinical studies. The main findings were as follows: Aim 1: We demonstrated that neo-epitope biomarkers reflecting collagen turnover were elevated in patients diagnosed with colorectal cancer, particular in patients with advanced stages. It was not possible to differentiate between cancer patients or healthy controls and subjects with adenomas suggesting that these biomarkers cannot be used for early diagnosis. However, neo-epitope biomarkers reflecting collagen formation showed a great potential as prognostic biomarkers and as a measure of tumor activity. Aim 2: Three highly specific and robust immunoassays were developed and a pathological relevance was shown for two of the assays. These data suggest that structural changes to the ECM plays a pathological role in tumorigenesis and biomarkers reflecting these changes have potential as novel liquid-biopsy biomarkers in cancer. Whether their use is diagnostic, prognostic or predictive needs further evaluation in larger clinical cohorts. The overall conclusion is that neo-epitope fragments reflecting changes of the ECM and the tumor microenvironment, have potential as promising serological biomarkers in cancer. The results strongly indicate that ECM turnover is altered in patients with cancer and protease-generated fragments of proteins originating from the tumor microenvironment are released into the circulation as a result of tumorigenesis. These biomarkers might be able to define a certain ECM phenotype in patients with cancer which may contribute to personalized medicine. For all of the measured biomarkers, further validation are needed to elucidate their exact use in cancer.