Exosomes Derived from Human Primary and Metastatic Colorectal Cancer Cells Contribute to Functional Heterogeneity of Activated Fibroblasts by Reprogramming Their Proteome

Abstract

Cancer‐associated fibroblasts (CAFs) are a heterogeneous population of activated fibroblasts that constitute a dominant cellular component of the tumor microenvironment (TME) performing distinct functions. Here, the role of tumor‐derived exosomes (Exos) in activating quiescent fibroblasts into distinct functional subtypes is investigated. Proteomic profiling and functional dissection reveal that early‐ (SW480) and late‐stage (SW620) colorectal cancer (CRC) cell‐derived Exos both activated normal quiescent fibroblasts (α‐SMA−, CAV+, FAP+, VIM+) into CAF‐like fibroblasts (α‐SMA+, CAV−, FAP+, VIM+). Fibroblasts activated by early‐stage cancer‐exosomes (SW480‐Exos) are highly pro‐proliferative and pro‐angiogenic and display elevated expression of pro‐angiogenic (IL8, RAB10, NDRG1) and pro‐proliferative (SA1008, FFPS) proteins. In contrast, fibroblasts activated by late‐stage cancer‐exosomes (SW620‐Exos) display a striking ability to invade through extracellular matrix through upregulation of pro‐invasive regulators of membrane protrusion (PDLIM1, MYO1B) and matrix‐remodeling proteins (MMP11, EMMPRIN, ADAM10). Conserved features of Exos‐mediated fibroblast activation include enhanced ECM secretion (COL1A1, Tenascin‐C/X), oncogenic transformation, and metabolic reprogramming (downregulation of CAV‐1, upregulation of glycogen metabolism (GAA), amino acid biosynthesis (SHMT2, IDH2) and membrane transporters of glucose (GLUT1), lactate (MCT4), and amino acids (SLC1A5/3A5)). This study highlights the role of primary and metastatic CRC tumor‐derived Exos in generating phenotypically and functionally distinct subsets of CAFs that may facilitate tumor progression.

Human Endometrial Extracellular Vesicles Functionally Prepare Human Trophectoderm Model for Implantation: Understanding Bidirectional Maternal‐Embryo Communication

“…For western blotting, proteins were electrophoretically separated (XCell Surelock gel tank (Life Technologies) as described, and electrotransferred onto nitrocellulose membranes using the iBlot 2.0 Dry Blotting System (Life Technologies). The membranes were blocked for 1 h at RT with 5% milk powder in TTBS and probed with primary antibodies according to manufacturer's instructions overnight at 4 °C, including primary mouse TSG101 (BD Biosciences; 1:1000) and mouse Alix (Cell Signaling Technology; 1:1000).…”

“…Briefly, EVs were diluted in ultrafiltered (0.1 µm) PBS (≈8 × 10 8 particles mL –1 ) and loaded into a flow‐cell top plate using a syringe pump. Three separate technical replicates (60 s per video) were recorded for each sample and analyzed by NTA software (Build 3.1.45) …”

“…Raw data were preprocessed as described and processed using MaxQuant (v1.6.0.16) with Andromeda (v1.5.6). For informatic processing, a human‐only (UniProt #71785 entries) sequence database (Feb‐2018) was used.…”

Fat Therapeutics: The Clinical Capacity of Adipose-Derived Stem Cells and Exosomes for Human Disease and Tissue Regeneration

“…All cell types continuously secrete EVs to the extracellular environment. EVs contain select proteins, peptides, RNA species (microRNAs, mRNAs, and long noncoding RNAs), lipids, and DNA fragments, that act locally or disseminate through circulation to act at specific distal sites to pleiotropically modulate cellular responses via paracrine signaling Xu et al, 2018;Rai et al, 2019). The origin, nature, morphology, size and content of EVs are diverse and represent a novel signaling paradigm (Antonyak and Cerione, 2015).…”

Circulating Extracellular Vesicle MicroRNA as Diagnostic Biomarkers in Early Colorectal Cancer—A Review

“…They can act in a paracrine fashion locally within the tumour microenvironment, and in an endocrine manner at distal sites via the circulation [37][38][39]. Pathophysiological functions of EVs in cancer include promotion of tumour invasiveness and growth [40,41], stimulation of stromal accessory cells to tumour-supporting phenotypes [42], and preparation of pre-metastatic niches in distant organs [43].…”