Abstract
Oral squamous cell carcinoma (OSCC) patients have a dismal survival rate due to distant metastases that
escape primary care. Transforming growth factor beta (TGFß) is a well-known driver of metastasis, modulator of
immune cell activity, and regulator of extracellular matrix (ECM) genes. In breast cancer, a stiff ECM is generally
attributed to excess type I collagen deposition and crosslinking that promotes cancer cell metastasis and cancer
stem cell (CSC) expansion. In OSCC, a consensus has not been reached on whether a stiff or soft ECM
increases metastatic potential and CSC expansion. The impact of OSCC ECM composition on immune cell
trafficking is also unknown. Our laboratory has produced murine OSCC cell lines derived from Keratin15+ stem
cells with Smad4 loss and KrasG12D mutation. Despite having the same genetic background, these cell lines have
different metastatic ability, suggesting microenvironmental factors or cell intrinsic differences may mediate
metastasis. RNAseq analysis revealed that metastatic OSCC cells have increased levels of laminins and laminin-
binding integrins but downregulated type I collagen genes. Additionally, while OSCC cells cultured on stiff ECM
demonstrate increased invasion, those cultured on a soft ECM display increased CSC characteristics. Treating
metastatic OSCC cells with a TGFß inhibitor reduced migration and invasion. Comprehensive immune profiling
using flow cytometry revealed that metastatic tumors have decreased numbers of CD8+ T infiltrating
lymphocytes (TILs) compared to non-metastatic OSCC tumors. These CD8+ TILs are instrumental for a robust
anti-tumor immune response and the success of immune checkpoint inhibitor (ICI) therapy. Identifying
responders to ICI and TGFß inhibitors and sensitizing non-responders to these therapies persist as a major
obstacle. Defining how OSCC cells interact with the ECM to promote their dissemination and enhancing current
prognostic markers are both crucial to improve patient care and prolong survival. The goal of this proposal is
to use unique murine models and human patient samples to define how ECM components and rigidity influence
OSCC CSCs metastasis to improve the efficacy of emerging, targeted therapeutics to inhibit OSCC metastatic
outgrowth. The hypothesis of this proposal is that elevated TGFß signaling in OSCC induces increased
integrin expression and a laminin/collagen imbalance in the ECM, altering ECM stiffness and modulating OSCC
metastasis, OSCC CSCs, CD8+ TIL motility and ICI responsiveness. I will 1) evaluate TGFß-dependency and
function of laminins and associated integrins in OSCC metastasis; 2) determine how laminins contribute to ECM
rigidity and subsequent impact on OSCC CSC characteristics and motility; and 3) assess if inhibiting laminin
deposition enhances ICI. This study will provide insight into how enhanced laminin-binding integrin expression
and laminin deposition facilitates OSCC metastasis, CSC characteristics, and CD8+ TIL exclusion. It will also
identify predictive prognostic biomarkers for ICI and therapeutic targets for treating metastatic OSCC.