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Although extracellular vesicles (EVs) were discovered over 40 years ago, there has been a resurgence of interest in secreted vesicles and their attendant cargo as novel modes of intracellular communication. In addition to vesicles, two amembranous nanoparticles, exomeres and supermeres, have been isolated and characterized recently. In this rapidly expanding field, it has been challenging to assign cargo and specific functions to a particular carrier. Refinement of isolation methods, well-controlled studies, and guidelines detailed by Minimal Information for Studies of Extracellular Vesicles (MISEV) are being employed to “bring order to chaos.” In this review, we will briefly summarize three types of extracellular carriers - small EVs (sEVs), exomeres, and supermeres - in the context of colorectal cancer (CRC). We found that a number of GPI-anchored proteins (GPI-APs) are overexpressed in CRC, are enriched in exosomes (a distinct subset of sEVs), and can be detected in exomeres and supermeres. This affords the opportunity to elaborate on GPI-AP biogenesis, modifications, and trafficking using DPEP1, a GPI-AP upregulated in CRC, as a prime example. We have cataloged the GPI-anchored proteins secreted in CRC and will highlight features of select CRC-associated GPI-anchored proteins we have detected. Finally, we will discuss the remaining challenges and future opportunities in studying these secreted GPI-APs in CRC.

There has been significant progress in the study of extracellular vesicles (EVs) since the 2017 American Gastroenterological Association-sponsored Freston Conference "Extracellular Vesicles: Biology, Translation and Clinical Application in GI Disorders." The burgeoning interest in this field stems from the increasing recognition that EVs represent an understudied form of cell-to-cell communication and contain cargo replete with biomarkers and therapeutic targets. This short review will highlight recent advances in the field, with an emphasis on colorectal cancer. After a brief introduction to secreted particles, we will describe how our laboratory became interested in EVs, which led to refined methods of isolation and identification of 2 secreted nanoparticles. We will then summarize the cargo found in small EVs released from colorectal cancer cells and other cells in the tumor microenvironment, as well as those found in the circulation of patients with colorectal cancer. Finally, we will consider the continuing challenges and future opportunities in this rapidly evolving field.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Leveraging spatial molecular information to construct a phylogeographic map of tumor evolution can reveal individualized growth trajectories with diagnostic and therapeutic potential. Integrative analysis of spatial multi-omic data from 31 colorectal specimens revealed simultaneous microenvironmental and clonal alterations as a function of progression. Copy number variation served to re-stratify microsatellite stable and unstable tumors into chromosomally unstable (CIN+) and hypermutated (HM) classes. Phylogeographical maps classified tumors by their evolutionary dynamics, and clonal regions were placed along a global pseudotemporal progression trajectory. Cell-state discovery from a single-cell cohort revealed recurring epithelial gene signatures and infiltrating immune states in spatial transcriptomics. Charting these states along progression pseudotime, we observed a transition to immune exclusion in CIN+ tumors as characterized by a novel gene expression signature comprised ofDDR1, TGFBI, PAK4,andDPEP1. We demonstrated how these genes and their protein products are key regulators of extracellular matrix components, are associated with lower cytotoxic immune infiltration, and show prognostic value in external cohorts. Through high-dimensional data integration, this atlas provides insights into co-evolution of tumors and their microenvironments, serving as a resource for stratification and targeted treatment of CRC.

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer’s disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). 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 circulating biomarkers and therapeutic targets for a host of human diseases., Zhang et al. identify and characterize supermeres as extracellular nanoparticles that exhibit unique biological and functional properties with potential prognostic and therapeutic value across distinct diseases.

Alternative splicing (AS) of pre-mRNAs is an essential process that regulates gene expression and functional diversity, yet the underlying regulatory mechanisms that control this process during cancer metastasis are not clear. Here, we uncovered that a long non-coding RNA (lncRNA), MIR99AHG, mediates regulation of AS to alter chromatin remodeler function and promote invadopodia formation in colorectal cancer (CRC). We found that MIR99AHG is highly expressed in metastatic CRC cells and patient tissue samples. In vitro and in vivo assays showed that MIR99AHG potently drove CRC cell motility, invasion, and metastasis. MIR99AHG bound to and stabilized the RNA splicing factor PTBP1, which cooperatively increased cassette exon inclusion of the chromatin remodeling gene SMARCA1. Mechanistically, MIR99AHG acted as an address label for PTBP1 to fine-tune its binding pattern on SMARCA1 pre-mRNA, thereby controlling the skipping-to-inclusion splicing switch of SMARCA1 and favouring the production of a long isoform (SMARCA1-L). Canonical SMARCA1 was found to suppress cell migration and invasiveness by inhibiting invadopodia formation, but SMARCA1-L was functionally inert. Clinicaldata revealed that MIR99AHG was positively correlated with PTBP1 and SMARCA1-L in human CRC specimens and predicted patient outcome. Furthermore, we showed that the crosstalk with cancer-associated fibroblasts triggers TGF-β/SAMD signalling in CRC cells and activates MIR99AHG expression. Our findings establish a novel mechanism for a lncRNA that interacts with PTBP1 to regulate AS process, providing a potential therapeutic target and predictive biomarker of CRC.

Background Metastasis leads to recurrence and death in colorectal cancer (CRC) patients; however, mechanisms underlying CRC metastasis, especially in its initial stages, remain largely unknown. In this study, we aim to identify long noncoding RNAs (lncRNAs) functionally involved in CRC metastasis initiation and their regulation that could lead to targeted therapeutics. Methods We performed whole transcriptome sequencing using CRC cell lines with divergent spontaneous metastatic properties. The biological roles and mechanisms of lncRNA MIR99AHG were characterized with a series of in vitro and in vivo models and molecular analyses. Results MIR99AHG was upregulated in CRC patients and associated with tumor stage and prognosis. MIR99AHG potently drove migration, invasion and metastasis in CRC cells, and these effects were dependent on its role in promoting invadopodia formation. MIR99AHG interacted with RNA splicing factor PTBP1, which cooperatively modulated alternative splicing of chromatin remodeling gene SMARCA1. Mechanistically, MIR99AHG acted as an address label for PTBP1 to direct its binding to the 5’ splice site of SMARCA1 intron 12, thereby facilitating inclusion of the alternative exon 13 to generate a long isoform (SMARCA1-L). The canonical SMARCA1 inhibited cell migration and invasion and suppressed a core set of genes involved in invadopodia, but the SMARCA1-L was functionally inert in metastasis suppression. Clinically, SMARCA1-L levels were positively correlated with MIR99AHG and PTBP1 in patients with metastatic CRC. Conclusions This study identified that invadopodia was regulated by a splicing switch of SMARCA1 due to the interaction between MIR99AHG and PTBP1, highlighting a novel regulatory mode mediated by lncRNA in alternative splicing of chromatin remodeler during CRC metastasis.

Epithelial-to-mesenchymal transition (EMT) is a process linked to metastasis and drug resistance with non-coding RNAs (ncRNAs) playing pivotal roles. We previously showed that miR-100 and miR-125b, embedded within the third intron of the ncRNA host gene MIR100HG, confer resistance to cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, in colorectal cancer (CRC). However, whether the MIR100HG transcript itself has a role in cetuximab resistance or EMT is unknown.The correlation between MIR100HG and EMT was analyzed by curating public CRC data repositories. The biological roles of MIR100HG in EMT, metastasis and cetuximab resistance in CRC were determined both in vitro and in vivo. The expression patterns of MIR100HG, hnRNPA2B1 and TCF7L2 in CRC specimens from patients who progressed on cetuximab and patients with metastatic disease were analyzed by RNAscope and immunohistochemical staining.The expression of MIR100HG was strongly correlated with EMT markers and acted as a positive regulator of EMT. MIR100HG sustained cetuximab resistance and facilitated invasion and metastasis in CRC cells both in vitro and in vivo. hnRNPA2B1 was identified as a binding partner of MIR100HG. Mechanistically, MIR100HG maintained mRNA stability of TCF7L2, a major transcriptional coactivator of the Wnt/β-catenin signaling, by interacting with hnRNPA2B1. hnRNPA2B1 recognized the N6-methyladenosine (mMIR100HG and hnRNPA2B1 interact to control the transcriptional activity of Wnt signaling in CRC via regulation of TCF7L2 mRNA stability. Our findings identified MIR100HG as a potent EMT inducer in CRC that may contribute to cetuximab resistance and metastasis by activation of a MIR100HG/hnRNPA2B1/TCF7L2 feedback loop.

Bert Vogelstein and co-workers identified DPEP1 in 2001 as one of the top six upregulated genes encoding a membrane or secreted protein in colorectal adenomas and cancers (CRCs). DPEP1 is a GPI-linked, zinc-dependent dipeptidase whose enzymatic activity includes processing extracellular glutathione and converting LTD4 to LTE4. Recently, it was reported to act nonenzymatically as a receptor for neutrophils on liver and lung endothelial cells and to influence monocyte migration. DPEP1 has been reported to have roles in cancer proliferation, invasion and metastasis. We recently showed that DPEP1 is the most abundant protein in small extracellular vesicles (sEVs) released from CRC cell lines by mass spectrometry and is present in a subset of EGFR+/CD81+ exosomes along with other clinically relevant biomarkers by fluorescence-activated vesicle sorting (FAVS) (Q Zhang et al., Nature Cell Biology 23:1240-1254, 2021). We also found that DPEP1/CEACAM5 double-positive exosomes are increased in the plasma from three CRC patients compared to normal controls. Immunohistochemical staining for DPEP1 in clinically well-annotated adenoma and CRC tissue microarrays has revealed staining in 27% of adenomas and 71% of CRCs, whereas no staining was observed in normal colonic tissue. Moreover, diffuse cytoplasmic staining in CRCs was associated with a worse progression-free and overall survival. We have now generated adenoma organoids, some of which express endogenous DPEP1 that localizes to the apical plasma membrane in 3D organoids and when cultured as polarized monolayers on Transwell filters. We plan to examine the bi-directional effects of cellular DPEP1 and DPEP1-containing EVs and freshly isolated neutrophils and/or their EVs in a co-culture system that is part of our recently funded Translational and Biological Science in Early Lesions (TBEL) NCI U54 grant. Citation Format: Matthew E. Bechard, Sarah E. Glass, Robert J. Coffey. Dipeptidase-1 is a highly upregulated protein in CRC exosomes: Potential clinical relevance [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B030.

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.