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The topographical organization of collagen within the tumor microenvironment has been implicated in modulating cancer cell migration and independently predicts progression to metastasis. Here, we show that collagen matrices with small pores and short fibers, but not Matrigel, trigger a conserved transcriptional response and subsequent motility switch in cancer cells resulting in the formation of multicellular network structures. The response is not mediated by hypoxia, matrix stiffness, or bulk matrix density, but rather by matrix architecture-induced β1-integrin upregulation. The transcriptional module associated with network formation is enriched for migration and vasculogenesis-associated genes that predict survival in patient data across nine distinct tumor types. Evidence of this gene module at the protein level is found in patient tumor slices displaying a vasculogenic mimicry (VM) phenotype. Our findings link a collagen-induced migration program to VM and suggest that this process may be broadly relevant to metastatic progression in solid human cancers., Extracellular matrix plays a central role in driving cancer development. Here the authors using an in vitro approach show that confining collagen architectures induce fast and persistent cell migration and the formation of multicellular network structures linked to vascular mimicry observed in tumours from patients.

SUMMARYMammals progress through similar physiological stages during life, from early development to puberty, aging, and death. Yet, the extent to which this conserved physiology reflects conserved molecular events is unclear. Here, we map common epigenetic changes experienced by mammalian genomes as they age, focusing on evolutionary comparisons of humans to dogs, an emerging model of aging. Using targeted sequencing, we characterize the methylomes of 104 Labrador retrievers spanning a 16 year age range, achieving >150X coverage within mammalian syntenic blocks. Comparison with human methylomes reveals a nonlinear relationship which translates dog to human years, aligns the timing of major physiological milestones between the two species, and extends to mice. Conserved changes center on specific developmental gene networks which are sufficient to capture the effects of anti-aging interventions in multiple mammals. These results establish methylation not only as a diagnostic age readout but as a cross-species translator of physiological aging milestones.

The successful implementation of miRNA (miR) therapies in humans will ultimately rely on the use of vehicles with improved cellular delivery capability. Here we tested a new system that leverages extracellular vesicles (EVs) laden with a tumor suppressor miRNA (miR-335) produced in B cells by plasmid DNA induction (iEVs). We demonstrate that iEVs-335 efficiently and durably restored the endogenous miR-335 pool in human triple negative breast cancer cells, downregulated the expression of the miR-335 target gene SOX4 transcription factor, and markedly inhibited tumor growth in vivo. Remarkably, iEVs-335 mediated transcriptional effects that persisted in tumors after 60 days post orthotopic implantation. Genome-wide RNASeq analysis of cancer cells treated in vitro with iEVs-335 showed the regulation of a discrete number of genes only, without broad transcriptome perturbations. This new technology may be ideally suited for therapies aimed to restore tumor suppressor miRNAs in cancer cells, disrupting the oncogenic program established after escape from miRNA control.

All mammals progress through similar physiological stages throughout life, from early development to puberty, aging, and death. Yet, the extent to which this conserved physiology reflects underlying genomic events is unclear. Here, we map the common methylation changes experienced by mammalian genomes as they age, focusing on comparison of humans with dogs, an emerging model of aging. Using oligo-capture sequencing, we characterize methylomes of 104 Labrador retrievers spanning a 16-year age range, achieving >150× coverage within mammalian syntenic blocks. Comparison with human methylomes reveals a nonlinear relationship that translates dog-to-human years and aligns the timing of major physiological milestones between the two species, with extension to mice. Conserved changes center on developmental gene networks, which are sufficient to translate age and the effects of anti-aging interventions across multiple mammals. These results establish methylation not only as a diagnostic age readout but also as a cross-species translator of physiological aging milestones.

Cancer genomics has enabled the exhaustive molecular characterization of tumors and exposed hepatocellular carcinoma (HCC) as among the most complex cancers. This complexity is paralleled by dozens of mouse models that generate histologically similar tumors but have not been systematically validated at the molecular level. Accurate models of the molecular pathogenesis of HCC are essential for biomedical progress; therefore we compared genomic and transcriptomic profiles of four separate mouse models [MUP transgenic, TAK1-knockout, carcinogen-driven diethylnitrosamine (DEN), and Stelic Animal Model (STAM)] with those of 987 HCC patients with distinct etiologies. These four models differed substantially in their mutational load, mutational signatures, affected genes and pathways, and transcriptomes. STAM tumors were most molecularly similar to human HCC, with frequent mutations in Ctnnb1 , similar pathway alterations, and high transcriptomic similarity to high-grade, proliferative human tumors with poor prognosis. In contrast, TAK1 tumors better reflected the mutational signature of human HCC and were transcriptionally similar to low-grade human tumors. DEN tumors were least similar to human disease and almost universally carried the Braf V637E mutation, which is rarely found in human HCC. Immune analysis revealed that strain-specific MHC-I genotype can influence the molecular makeup of murine tumors. Thus, different mouse models of HCC recapitulate distinct aspects of HCC biology, and their use should be adapted to specific questions based on the molecular features provided here.

The Sequence Read Archive (SRA) contains over one million publicly available sequencing runs from various studies using a variety of sequencing library strategies. These data inherently contain information about underlying genomic sequence variants which we exploit to extract allelic read counts on an unprecedented scale. We reprocessed over 250,000 human sequencing runs (>1000 TB data worth of raw sequence data) into a single unified dataset of allelic read counts for nearly 300,000 variants of biomedical relevance curated by NCBI dbSNP, where germline variants were detected in a median of 912 sequencing runs, and somatic variants were detected in a median of 4,876 sequencing runs, suggesting that this dataset facilitates identification of sequencing runs that harbor variants of interest. Allelic read counts obtained using a targeted alignment were very similar to read counts obtained from whole-genome alignment. Analyzing allelic read count data for matched DNA and RNA samples from tumors, we find that RNA-seq can also recover variants identified by Whole Exome Sequencing (WXS), suggesting that reprocessed allelic read counts can support variant detection across different library strategies in SRA. This study provides a rich database of known human variants across SRA samples that can support future meta-analyses of human sequence variation.

An initial step in solid tumor metastasis involves the migration of tumor cells through extracellular matrix. Several cancer cell migration strategies exist in vivo, and the local properties of collagen fibers are implicated in modulating migration behaviors. Yet, individual tumor cells also display heterogeneity in their intrinsic ability to migrate and metastasize. It remains unclear to what extent intrinsic and extrinsic heterogeneity contribute to the emergence of distinct migration phenotypes and whether certain migration phenotypes contribute more to metastasis than others. To study this, we generated 3D collagen matrices of varying densities and monitored single cancer cell migration in these matrices with time-lapse microscopy. We observed a collagen density threshold at 2.5mg/ml, above which 86% of MDA-MB-231 breast cancer cells transition from single mesenchymal migration to collective cell migration, with a 50% increase in persistence after cell division. After seven days, these collectively migrating cells created networks coated with basement membrane molecules resembling a clinical phenotype known as vascular mimicry (VM).The remaining 14% of cells migrated randomly and eventually formed spheroids. HT-1080 fibrosarcoma cells also responded similarly, migrating persistently and forming cellular networks. Next we sought to identify the physical feature of high-density collagen driving VM. Neither hypoxia or matrix stiffness was sufficient to induce VM. However, PEG-induced matrix confinement triggered VM network formation. RNA sequencing revealed collectively migrating cells up-regulated a conserved transcriptional program significantly enriched for annotations of vascular development and motility regulation processes. This gene module predicted survival in human tumor transcriptome datasets. Our results suggest that the VM phenotype arises in a subpopulation of cells from a conserved transcriptional and migratory response to confinement in 3D collagen. Note: This abstract was not presented at the meeting. Citation Format: Daniel Ortiz, Brian Tsui, Tyler Goshia, Colleen Ricker, Hannah Carter, Stephanie I. Fraley. 3D matrix confinement triggers vascular mimicry through a conserved migratory and transcriptional response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1888. doi:10.1158/1538-7445.AM2017-1888