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We have previously identified that in the absence of MET amplification or mutation, RTK hyperphosphorylation, indicative of its active state, may contribute to resistance to the EGFR-directed therapeutic, cetuximab in colorectal cancer (CRC). In our in vitro 3D cultures and in vivo xenografts, we showed that inhibition of MET (and RON) by crizotinib can overcome cetuximab resistance in CRC lines and xenografts. We now show that MET may get hyperphosphorylated by the autocrine expression and maturation of its ligand, HGF. This non-genetic mode of cetuximab resistance contrasts with the usual genetic modes of CRC cetuximab resistance, with majority of them being RAS/RAF mutations. Exogenous addition as well as autocrine overexpression of HGF led to cetuximab resistance, which could be overcome by MET inhibition by crizotinib. Interestingly, HGF/HGFL are synthesized as inactive precursors and require cleavage by proteases (HGFA, Matriptase, and Hepsin) to be biologically active. A survey of TCGA datasets indicated that HGF/HGFL were overexpressed in several CRC CMS subtypes, while the endogenous inhibitor of HGF proteases, HAI-I, was downregulated across all CRC CMS subtypes. We next expressed human full-length HGF in cetuximab-sensitive CC cells and observed that HGF overexpression imparts cetuximab resistance. Moreover, cetuximab resistance induced by upstream HGF overexpression could be overcome by the downstream MET inhibition. Next, in a subset of KRAS WT CRC patient-derived xenografts (PDXs) we tested and found that at majority of PDXs exhibit elevated pMET/pRON levels. RNA-seq-based principal component analysis showed that the maximal differences in expression among PDXs could be explained by their pMET/pRON phosphorylation status. Next, we generated nude mice xenografts for one PDX with high pMET/pRON levels and showed that cetuximab inhibition alone was not sufficient to inhibit regrowth of tumors after cessation of drug treatment, but a combined cetuximab/crizotinib treatment was, indicating endogenous MET/RON activation contributes to cetuximab resistance. In a parallel tumor stratification strategy, we have optimized conditions to generate [18F]crizotinib and are testing its efficacy as a new non-invasive PET-based imaging probe to identify crizotinib-avid tumors with high MET/RON activity. Together, these studies may provide novel mechanistic insights into RTK cross-talk and provide a positive predictive biomarker for precision therapy in colorectal cancer. Citation Format: Vivian T. Jones, Ramona Graves-Deal, KyuOK Jeon, Adam J. Rosenberg, Zheng Cao, Galina T. Bogatcheva, Sarah J. Harmych, James N. Higginbotham, Kwangho Kim, James W. Janetka, Gary A. Sulikowski, Bhuminder Singh. Role of MET signaling in cetuximab resistance and generation of an imaging biomarker to identify MET-driven cetuximab resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2562.

Cancer-derived extracellular vesicles, including exosomes, and exomere nanoparticles, are under intense investigation for cargo that may serve as clinical biomarkers and therapeutic targets. Here, we report the discovery of a new extracellular nanoparticle, termed supemeres. Supermeres are morphologically different from exomeres as determined by fluid-phase atomic force microscopy and display distinct uptake both in vitro and in vivo. In colorectal cancer (CRC) cell-derived supermeres, TGFBI is the most abundant protein and is linked to CRC progression and survival. Supermeres are enriched with cargo involved in multiple cancers including glycolytic enzymes, AGO2, shed MET and GPC1, and miR-1246. The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab-resistance, and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential cancer-related circulating biomarkers and therapeutic targets. Citation Format: Qin Zhang, Dennis K. Jeppesen, James N. Higginbotham, Sarah E. Glass, Jeffrey L. Franklin, Robert J. Coffey. Supermeres are functional extracellular nanoparticles replete with cancer biomarkers and therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3850.

The heterogeneity of small extracellular vesicles and the presence of non-vesicular extracellular matter is a major obstacle to the study of exosomes. Here we employ two complementary methods, high-resolution density gradient fractionation and direct immunoaffinity capture, to demonstrate separation of small extracellular vesicles from non-vesicular material, and exosomes from other types of small extracellular vesicles. Cells secrete Argonaute 1-4 independently of exosomes. Extracellular RNA and RNA-binding proteins are differentially expressed between exosome and non-vesicle compartments. Exosomes do not possess a cytoskeleton and exclude glycolytic enzymes. Annexin A1 is a novel and specific membrane-associated protein marker of microvesicles shed directly from the cell plasma membrane, distinct from exosomes in vitro and in vivo. Small extracellular vesicles are not vehicles of active DNA release. Instead we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular endosome-dependent but exosome-independent mechanism. A reassessment of exosome composition is necessary. Citation Format: Dennis K. Jeppesen, Jeffrey L. Franklin, James N. Higginbotham, Qin Zhang, Robert J. Coffey. Reassessment of exosome composition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1985.

Epidermal growth factor receptor (EGFR) ligands have been shown to act in an autocrine, paracrine and juxtacrine manner. We report a new mode of signaling - via exosomes. Exosomes are 30-100 nm nano-vesicles that are produced by intralumenal budding into multivesicular bodies (MVB); they are released from many cell types after fusion of the MVB with the plasma membrane. Our studies showed that human breast and colorectal cancer cell lines differ in the composition of EGFR ligands in exosomes isolated from conditioned medium by established methods. By fluorescent-activated vesicle sorting (FAVS; Mol Cell Proteomics 7: 1651-1667, 2008), we show that a single HCA-7-derived exosome contains TGF-α, amphiregulin (AR) and HB-EGF. Ligands localized in exosomes are signaling competent with the ectodomains presented on the outside and cytoplasmic tails on the inside, and exist as stable transmembrane proteins. To explore the biological function of individual ligands, we isolated exosomes from MDCK cells individually stably expressing TGF-α, AR or HB-EGF. We assessed the invasive capacity of these exosomes by incubating them with a breast cancer-derived cell line (LM2-4175) in a Boyden chamber assay and measured the number of invading cells 72 hours later. Exosomes purifed from MDCK cells singly expressing AR induced invasion of LM2-4175 recipient cells 4-fold more than TGF-α or HB-EGF exosomes and 15-30-fold more than recombinant ligands TGF-α, HB-EGF or AR). The number of invading cells exposed to AR exosomes was significantly inhibited by an AR neutralizing antibody, suggesting that the invasive capacity of recipient cells is dependent upon exosomal AR engagement with recipient cell EGFR. Fixed and live cell imaging by confocal microscopy showed that AR exosomes are internalized by recipient cells. Further analysis showed that exosomes isolated from mutant KRAS DLD-1 colon cancer cells increased invasion of LM2-4175 cells 5-fold more than exosomes isolated from cells in which mutant KRAS was eliminated by homologous recombination (DKs-8 cells) and this correlated with increased levels of exosomal AR present in DLD-1 cells compared to DKs-8 cells. We provide the first evidence that EGFR ligands are present in exosomes and demonstrate that AR exosomes confer a greater invasive effect on recipient cells than TGF-α and HB-EGF exosomes or recombinant EGFR ligands. We propose ExTRAcrine signaling as a novel mode of EGFR ligand signaling with important diagnostic and therapeutic implications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 273.