Modeling Fallopian Tube Epithelium Cellular Signaling Reprogramming Driven by Extracellular Vesicles to Define the Ovarian Cancer Precancerous Landscape

Serous tubal intraepithelial carcinomas (STIC lesions) in the human fallopian tube epithelium (hFTE) are theorized to give rise to high grade serous ovarian cancers (HGSOC). Small extracellular vesicles (sEVs) are known to mediate key signaling in both normal and cancerous tissues, but few ex vivo systems exist for studying sEV impact on hFTE tissue. Here, we present a microfluidic tissue culture platform with combined spatial transcriptomic and proteomic readouts that allows us to profile dual responses in tissue exposed to sEV “messages”—capturing both short-term transcriptomic shifts in the tissue and long-term changes in protein cargo of secreted EVs (the “reply”). Using spatial transcriptomics, we show that the short-term 1-day exposure to ovarian cancer-derived sEVs alters expression of 61 transcripts in secretory cells, the progenitor of HGSOC, notably upregulating immune-related mRNA, including CXCL family chemokines, VCAM1, and pro-inflammatory mediators (NFKB1, IL1B, IFNA7/17). Additionally, we observed the long-term 14-day exposure to sEVs alters the expression of 7 transcripts and 25 EV cargo proteins of fallopian tube derived EVs (“secondary release EVs”) following stimulus from cancer EVs. Together, tissue transcriptomics and tissue-derived EV proteomics indicate that ovarian cancer derived sEVs rewire target cell signaling to modify the tubal immune landscape. This study provides insights into the early molecular changes associated with the pathogenesis of ovarian cancer in its tissue of origin, providing a platform to study EV-tissue interactions and identify how sEVs drive cell signaling reprogramming in hFTE. Fallopian tube (FT) tissue explants were cultured on an organ-on-chip tissue culture system. Samples were treated daily with either: 1) PBS (control), 2) FT sEVs (FT240-derived, 1.5x10^9 particles/day), or 3) HGSOC sEVs (OVCAR3-derived, 1.5x10^9 particles/day). To study both short- and long-term consequences of EV insult, we exposed the tissue to sEVs for either 1 day (n = 12 samples) or repeatedly over 14 days (n = 10 samples). At the end of the treatment period, fallopian tissue samples were collected for both 1-day and 14-day conditions and analyzed on the GeoMx Digital Spatial Profiler using the Cancer Transcriptome Atlas (CTA) panel. The GeoMx platform was used to segment the FT epithelium into ciliated and secretory cell types based on staining for PAX8 (secretory cell marker) and FOXJ1 (ciliated cell marker). Final output is transcript expression in ciliated or secretory cells, in each of three treatment conditions (PBS, OVCAR3, FT240), after culture for either 1 day or 14 day.