Poster T9 - Paracrine Interaction among Heterotypic Cell Populations in Oral Squamous Cell Carcinoma (OSCC) Enhances Metastasis

Introduction: Tumors are composed of heterotypic, plastic cell populations that form a complex tumor microenvironment. While we have recently shown that oral epithelia undergo stiffness-mediated epithelial-to-mesenchymal transition (EMT) [1] and exhibit memory of their mechanical when disseminating via AKT and FAK signaling [2], it is not clear to what extent those newly formed mesenchymal cells induce EMT and migration in other epithelial cells. Here, we show that paracrine interaction between mesenchymal (SCC25) or stiffness induced-mesenchymal (simCal27) and their epithelial (Cal27) cell counterparts can induce extrinsic signaling inducing the epithelial cells to undergo EMT and migration in a manner that may mimic the cooperative invasive properties of oral squamous cell carcinoma (OSCC).

[1] Matte, et al, J Cell Sci 2018. [2] Moon, et al, Mol Biol Cell 2023.

Materials and

Methods: Cal27 cells were cultured on soft substrates (0.48 kPa polyacrylamide (PA) gel) to mimic oral stroma and selectively grown in SCC25 or simCal27 conditioned media for 1 to 4 days; time-lapse video microscopy assay was used to measure cell motility. RNA-seq was performed on Cal27 cells after 1- and 3-days conditioned media exposure and relative to control SCC25 and simCal27 cells. Cytokine arrays comparing SCC25 vs Cal27 on soft/stiff-conditioned media were performed with Proteome Profiler Human XL Cytokine Array Kit (R&D Systems). Target cytokines from arrays and sequencing were extrapolated from differentially expressed genes (DEG), and those cytokines were assessed together or individually for their influence on EMT and migration.

Results, Conclusions, and Discussions: SCC25 conditioned media increased cell migration velocity vs. Cal27 cells in normal growth media (Fig. 1A) up to stiffness-induced velocities previously observed in simCal27 cells (dashed line). RNA-seq was used to broadly screen the molecular players involved in this migration change, and we found 134 differentially expressed genes (DEG) resulting from SCC25 conditioned media (Fig. 1B) corresponding to gene ontology (GO) terms that included cell-cell signaling, cytokine response, and response to interleukins; these data were confirmed by marker staining and imply that Cal27’s enhanced motility was stimulated by cytokines released by SCC25 or simCal27 cells. Using a cytokine array (Fig. 1C) and filtering upstream regulators (Fig. 1D; verifying with ELISA), we identified 5 cytokines (IL-1a, IL-6, IL-8, Angiogenin, and PAI-1) that could be necessary or even sufficient for EMT based on enhanced secretion by SCC25 and simCal27 cells. Depletion of IL-8 significantly reduce migration velocity, and conversely, supplementing fresh media with all 5 cytokines enhanced cell migration velocity to a degree that was comparable to Cal27 incubated with SCC25-conditioned media. However, supplementing with each cytokine also enhanced cell migration, albeit it to a more limited degree (Fig. 1E). Thus each cytokine is necessary factor for cell motility improvement via paracrine interaction but not sufficient.

Heterotypic cell populations in OSCC can interact with each other via paracrine signaling. IL-1a, IL6, IL-8, Angiogenin, and PAI-1 are likely involved in this metastasis-inducing cell-cell interaction.

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