Identification of novel early pancreatic cancer biomarkers KIF5B and SFRP2 from "first contact" interactions in the tumor microenvironment.

BACKGROUND: Pancreatic cancer is one of the most difficult cancers to detect early and most patients die from complications arising due to distant organ metastases. The lack of bona fide early biomarkers is one of the primary reasons for late diagnosis of pancreatic cancer. It is a multifactorial disease and warrants a novel approach to identify early biomarkers. METHODS: In order to characterize the proteome, Extracellular vesicles (EVs) isolated from different in vitro conditions mimicking tumor-microenvironment interactions between pancreatic cancer epithelial and stromal cells were analyzed using high throughput mass spectrometry. The biological activity of the secreted EVome was analyzed by investigating changes in distant organ metastases and associated early changes in the microbiome. Candidate biomarkers (KIF5B, SFRP2, LOXL2, and MMP3) were selected and validated on a mouse-human hybrid Tissue Microarray (TMA) that was specifically generated for this study. Additionally, a human TMA was used to analyze the expression of KIF5B and SFRP2 in progressive stages of pancreatic cancer. RESULTS: The EVome of co-cultured epithelial and stromal cells is different from individual cells with distinct protein compositions. EVs secreted from stromal and cancer cells cultures could not induce significant changes in Pre-Metastatic Niche (PMN) modulation, which was assessed by changes in the distant organ metastases. However, they did induce significant changes in the early microbiome, as indicated by differences in α and ß-diversities. KIF5B and SFRP2 show promise for early detection and investigation in progressive pancreatic cancer. These markers are expressed in all stages of pancreatic cancer such as low grade PanINs, advanced cancer, and in liver and soft tissue metastases. CONCLUSIONS: Proteomic characterization of EVs derived from mimicking conditions of epithelial and stromal cells in the tumor-microenvironment resulted in the identification of several proteins, some for the first time in EVs. These secreted EVs cannot induce changes in distant organ metastases in in vivo models of EV education, but modulate changes in the early murine microbiome. Among all the proteins that were analyzed (MMP3, KIF5B, SFRP2, and LOXL2), KIF5B and SFRP2 show promise as bona fide early pancreatic cancer biomarkers expressed in progressive stages of pancreatic cancer.

Modulation of Early Neutrophil Granulation: The Circulating Tumor Cell-Extravesicular Connection in Pancreatic Ductal Adenocarcinoma.

Tumor cells dissociate from the primary site and enter into systemic circulation (circulating tumor cells, CTCs) either alone or as tumor microemboli (clusters); the latter having an increased predisposition towards forming distal metastases than single CTCs. The formation of clusters is, in part, created by contacts between cell-cell junction proteins and/or cytokine receptor pairs with other cells such as neutrophils, platelets, fibroblasts, etc. In the present study, we provide evidence for an extravesicular (EV) mode of communication between pancreatic cancer CTCs and neutrophils. Our results suggest that the EV proteome of CTCs contain signaling proteins that can modulate degranulation and granule mobilization in neutrophils and, also, contain tissue plasminogen activator and other proteins that can regulate cluster formation. By exposing naïve neutrophils to EVs isolated from CTCs, we further show how these changes are modulated in a dynamic fashion indicating evidence for a deeper EV based remodulatory effect on companion cells in clusters.