Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma

Bidirectional
communication between cells and their microenvironment
is crucial for both normal tissue homeostasis and tumor growth. During
the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated
fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional
crosstalk with cancer cells, mediated by classically secreted factors
and various nanometer-sized vesicles, termed as extracellular vesicles
(EVs). To better understand the role of CAFs within the tumor stroma
and elucidate the mechanism by which secreted proteins contribute
to OTSCC progression, we isolated and characterized patient-derived
CAFs from resected tumors with matched adjacent tissue fibroblasts
(AFs). Our strategy employed shotgun proteomics to comprehensively
characterize the proteomes of these matched fibroblast populations.
Our goals were to identify CAF-secreted factors (EVs and soluble)
that can functionally modulate OTSCC cells in vitro and to identify
novel CAF-associated biomarkers. Comprehensive proteomic analysis
identified 4247 proteins, the most detailed description of a pro-tumorigenic
stroma to date. We demonstrated functional effects of CAF secretomes
(EVs and conditioned media) on OTSCC cell growth and migration. Comparative
proteomics identified novel proteins associated with a CAF-like state.
Specifically, MFAP5, a protein component of extracellular microfibrils,
was enriched in CAF secretomes. Using in vitro assays, we demonstrated
that MFAP5 activated OTSCC cell growth and migration via activation
of MAPK and AKT pathways. Using a tissue microarray of richly annotated
primary human OTSCCs, we demonstrated an association of MFAP5 expression
with patient survival. In summary, our proteomics data of patient-derived
stromal fibroblasts provide a useful resource for future mechanistic
and biomarker studies.