Inducible nitric oxide synthase (iNOS)-activated Cxcr2 signaling in myeloid cells promotes TGFß-dependent squamous cell carcinoma lung metastasis.

Transforming growth factor beta (TGFß) activity is linked to metastasis in many cancer types, but whether TGFß activity is necessary for squamous cell carcinoma (SCC) lung metastasis has not been studied. Here we used a lung metastatic SCC model derived from keratin 15 (K15). KrasG12D.Smad4-/- SCC and human SCC specimens to identify metastasis drivers and test therapeutic interventions. We demonstrated that a TGFß receptor (TGFßR) inhibitor reduced lung metastasis in mouse SCC correlating with reduced CD11b+/Ly6G+ myeloid cells positive for inducible nitric oxide synthase (iNOS). Further, TGFß activity and iNOS were higher in primary human oral SCCs with metastasis than SCCs without metastasis. Consistently, either depleting myeloid cells with anti-Gr1 antibody or inhibiting iNOS with L-N6-(1-iminoethyl)-l-lysine (L-NIL) reduced SCC lung metastasis. L-NIL treated tumor-bearing mice exhibited reductions in tumor-infiltrating myeloid cells and in plasma Cxcl5 levels, and attenuated primary tumor growth with increased apoptosis and decreased proliferation. Blocking Cxcl5 with an antagonist of its receptor Cxcr2, SB225002, also reduced SCC lung metastasis.

Epidermal growth factor receptor signaling in precancerous keratinocytes promotes neighboring head and neck cancer squamous cell carcinoma cancer stem cell-like properties and phosphoinositide 3-kinase inhibitor insensitivity.

Head and neck squamous cell carcinoma (HNSCC) is commonly associated with tobacco and alcohol consumption that induce a "precancerous field," with phosphoinositide 3-kinase (PI3K) signaling being a common driver. However, the preclinical effectiveness of PI3K inhibitors has not necessarily translated to remarkable benefit in HNSCC patients. Thus, we sought to determine how precancerous keratinocytes influence HNSCC proliferation, cancer stem cell (CSC) maintenance, and response to PI3K inhibitors. We used the NOK keratinocyte cell line as a model of preneoplastic keratinocytes because it harbors two frequent genetic events in HNSCC, CDKN2A promoter methylation and TP53 mutation, but does not form tumors. NOK cell coculture or NOK cell-conditioned media promoted HNSCC proliferation, PI3K inhibitor resistance, and CSC phenotypes. SOMAscan-targeted proteomics determined the relative levels of >1300 analytes in the media conditioned by NOK cells and HNSCC cells ± PI3K inhibitor. These results demonstrated that NOK cells release abundant levels of ligands that activate epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR), two receptor tyrosine kinases with oncogenic activity. Inhibition of EGFR, but not FGFR, blunted PI3K inhibitor resistance and CSC phenotypes induced by NOK cells. Our results demonstrate that precancerous keratinocytes can directly support neighboring HNSCC by activating EGFR. Importantly, PI3K inhibitor sensitivity was not necessarily a cancer cell-intrinsic property, and the tumor microenvironment impacts therapeutic response and supports CSCs. Additionally, combined inhibition of EGFR with PI3K inhibitor diminished EGFR activation induced by PI3K inhibitor and potently inhibited cancer cell proliferation and CSC maintenance.

Cancer-Associated Fibroblasts Facilitate Squamous Cell Carcinoma Lung Metastasis in Mice by Providing TGFß-Mediated Cancer Stem Cell Niche.

Cancer-associated fibroblasts (CAFs) have been shown to enhance squamous cell carcinoma (SCC) growth, but it is unclear whether they promote SCC lung metastasis. We generated CAFs from K15.KrasG12D.Smad4-/- mouse SCCs. RNA expression analyses demonstrated that CAFs had enriched transforming growth factor-beta (TGFß) signaling compared to normal tissue-associated fibroblasts (NAFs), therefore we assessed how TGFß-enriched CAFs impact SCC metastasis. We co-injected SCC cells with CAFs to the skin, tail vein, or the lung to mimic sequential steps of lung metastasis. CAFs increased SCC volume only in lung co-transplantations, characterized with increased proliferation and angiogenesis and decreased apoptosis compared to NAF co-transplanted SCCs. These CAF effects were attenuated by a clinically relevant TGFß receptor inhibitor, suggesting that CAFs facilitated TGFß-dependent SCC cell seeding and survival in the lung. CAFs also increased tumor volume when co-transplanted to the lung with limiting numbers of SCC cancer stem cells (CSCs). In vitro, CSC sphere formation and invasion were increased either with co-cultured CAFs or with CAF conditioned media (which contains the highest TGFß1 concentration) and these CAF effects were blocked by TGFß inhibition. Further, TGFß activation was higher in primary human oral SCCs with lung metastasis than SCCs without lung metastasis. Similarly, TGFß activation was detected in the lungs of mice with micrometastasis. Our data suggest that TGFß-enriched CAFs play a causal role in CSC seeding and expansion in the lung during SCC metastasis, providing a prognostic marker and therapeutic target for SCC lung metastasis.

Distinct immune microenvironment profiles of therapeutic responders emerge in combined TGFß/PD-L1 blockade-treated squamous cell carcinoma.

Transforming growth factor beta (TGFß) and programmed death-ligand 1 (PD-L1) are often overproduced in refractory squamous cell carcinoma (SCC). We examined spatial patterns of PD-L1+ cells in mouse and human SCCs and found that PD-L1 was primarily expressed on infiltrating leukocytes. Although combined TGFß and PD-L1 blockade are undergoing cancer clinical trials, there are no predictive markers for therapeutic responders. To address this, we used both a small molecule TGFß inhibitor in combination with anti-PD-L1 and a bifunctional fusion protein targeting both TGFß and PD-L1 to treat mouse SCCs and found TGFß inhibition enhanced PD-L1 blockade-induced tumor eradication in multiple tumor models. Furthermore, we identified distinct cell populations of responders and non-responders to bintrafusp alfa, with responders showing a shift toward a more immune-permissive microenvironment. The cellular and molecular signatures of responders versus non-responders to combined TGFß and PD-L1 blockade provide important insights into future personalized immunotherapy in SCC.