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

Abstract

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 β-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.

Extracellular vesicle proteomics and phosphoproteomics identify pathways for increased risk in patients hospitalized with COVID-19 and type 2 diabetes mellitus

“…To characterize the protein composition of plasma EV preparations purified using Tymora’s non-antibody-based affinity EVtrap™ proprietary technology (designed to quantitatively capture membrane-bound vesicles including exosomes and validated in multiple peer-reviewed publications [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] ), we assessed the enrichment in exosomal proteins curated by the ExoCarta Database. EVs were highly enriched in exosomal proteins, including 87 of the top 100 Exocarta proteins and nanoparticle tracking analysis (NTA) demonstrated size distributions consistent with preparations enriched in small EVs ( Figure 1 A-E).…”

“…EV purification was conducted as described by Nunez Lopez and colleagues [19] , using EVtrap™ (Tymora Analytical, Lafayette, IN, USA), a non-antibody- bead-based affinity technology developed to specifically and quantitatively isolate EVs. The EVtrap™ technology has been further described and validated in detail elsewhere [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] . In short, frozen plasma samples were thawed and large debris removed by centrifugation at 2500×g for 10 min.…”

Analysis of exosomal competing endogenous RNA network response to paclitaxel treatment reveals key genes in advanced gastric cancer

“…Further expression correlation analysis confirmed that ULK2 was involved in cell cycle and meiosis, indicating the importance of ULK2 in disease progression as exosomal ULK2 may exert pro-proliferation function when transported to target cells. However, pancreatic cancer derived exosomes cannot induce changes in distant organ metastases in in vivo models, but alter early murine gut microbiome ( 36 ). The detailed mechanism of ULK2 should be verified experimentally in future.…”

Clinical diagnosis and management of pancreatic cancer: Markers, molecular mechanisms, and treatment options

“…Multi-omics profiling studies can provide new markers for PC diagnosis. Proteomic analysis of EVs derived from co-cultured epithelial and stromal cells in the condition mimicking TME showed that kinesin family member 5B and secreted frizzled related protein 2 had promising values as early PC biomarkers[ 84 ]. PancRISK score evaluated by three urine markers, including lymphatic vessel endothelial hyaluronan receptor 1, regenerating family member 1 beta, and trefoil factor 1, showed reasonable sensitivity and specificity for PDAC detection compared to CA 19-9[ 85 ].…”