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scRNA-seq revealed CXCL14 as the most significantly downregulated gene among malignant cells in LNs relative to primary tumor, supporting a role in preventing invasion and/or metastasis. In a murine immunocompetent model, CXCL14 expression was higher in indolent MOC1 cells than in more aggressive MOC2 cells. Tumor growth in vivo was significantly increased by CXCL14 knockdown in MOC1 cells relative to control, with a corresponding decrease in TIL. In MOC2 cells, tumor growth was significantly reduced by CXCL14 overexpression relative to control and TIL were increased. Both effects were lost with T cell depletion. In a human tumor scRNA-seq cohort, we found that only malignant cell CXCL14, but not non-malignant cell or fibroblast CXCL14, was associated with TIL. Bulk CXCL14 from the TCGA cohort had no association with TIL.

Multiple studies have assessed the impact of CXCL14 on growth. Ozawa et al8 demonstrated that in vitro growth of CXCL14-overexpressing cells is not suppressed, suggesting that tumor suppression is not related to intrinsic cellular growth retardation. Tessema et al12 demonstrated increased cell death in vitro and reduced tumor growth via increased necrosis in vivo of lung tumor xenografts after forced expression of CXCL14 supporting the hypothesis that CXCL14-mediated tumor suppression may be related to anti-angiogenic activity.13

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Here, we examined the relationship between CXCL14 and tumor infiltrating lymphocytes (TIL) in human HNSCC, and using murine immunocompetent models of HPV-negative OSCC, validated the ability of CXCL14 to induce TIL accumulation in the tumor microenvironment and inhibit tumor growth in vivo.

To determine whether CXCL14 expression is associated with in vivo tumor growth, we performed subcutaneous tumor growth assays with MOC1 and MOC2 cell lines in immunocompetent C57BL/6 mice. To examine the specific function of CXCL14 in growth of MOC tumors, CXCL14 expression was inhibited in MOC1 cells by stably transducing an shRNA targeting CXCL14 (MOC1-CXCL14-shRNA) and overexpressed in MOC2 cells by stably transducing a CXCL14 expression construct (MOC2-CXCL14-over) (online supplemental figure S1). The degree to which CXCL14 was expressed in these cell lines was found to correlate with tumor growth kinetics in vivo. Tumor growth from these cells when injected into immunocompetent syngeneic mice was significantly higher with MOC2 cells than with MOC1 cells, at baseline (figure 2B,C, p

To determine whether changes in in vivo tumor growth in response to CXCL14 modulation were attributable to tumor cell-intrinsic changes, proliferation and invasiveness were assessed in vitro. CXCL14-modulated MOC cells showed no significant difference in in vitro cell proliferation capacity (figure 3A,B) or invasiveness (figure 3C,D), suggesting that in vivo tumor growth differences are related to CXCL14 effects on the tumor microenvironment.

We next sought to determine whether modulation of tumor CXCL14-dependent growth may be associated with immune cell infiltration. To this end, we quantified tumor infiltrating T cells after injection of MOC cells into immunocompetent syngeneic mice. Tumors were harvested and analyzed by flow cytometry for CD45+CD3+ T cells. Tumors generated following CXCL14 shRNA knockdown in MOC1 cells demonstrated an 82% reduction of CD45+CD3+ T cells within the tumor relative to control (figure 4A,C, p=0.02, n=3 biological replicates). Conversely, tumors generated following CXCL14 overexpression in MOC2 cells demonstrated a 42% increase in CD45+CD3+ T cells relative to control (figure 4B,D, p=0.006, n=5 biological replicates). Taken together, these results strongly suggest that the expression of CXCL14 in OSCC cells is associated with infiltration of T cells in vivo.

In this study, we demonstrate an association of TIL with CXCL14 expression in in vivo immunocompetent models of HPV-negative oral cavity cancer. In HPV-related HNSCC models, CXCL14 expression was previously associated with increased T cells in primary tumors16 and tumor draining lymph nodes.15 It was also associated with increased T cell activation and tumor cell MHC class I expression, resulting in tumor cell death, providing a mechanism for CXCL14-based tumor suppression.16 These studies lay essential ground work for our own studies, which extend these findings to the distinct pathologic entity of HPV-negative oral cavity cancer. We find concordant changes in tumor growth from CXCL14 shRNA knockdown in indolent MOC1 cells with high baseline expression of CXCL14 and CXCL14 overexpression in aggressive MOC2 cells with low baseline expression of CXCL14 (figure 3), leading to corresponding changes in TIL in both conditions (figure 4). Importantly, we demonstrate that the tumor suppressive effect of CXCL14 is dependent on the presence of TIL, with T cell depletion resulting in the loss of this effect (figure 5).

In analysis of human single cell RNA-seq data, we find that CXCL14 is the most significantly differentially downregulated gene in malignant cells within metastatic lymph nodes, relative to corresponding primary HNSCC tumors. In murine immunocompetent models of HPV-negative OSCC, we demonstrate that CXCL14 expression in vivo is associated with TIL, with overexpression associated with increased TIL and decreased tumor growth and knockdown associated with decreased TIL and increased tumor growth. We find that growth changes in response to CXCL14 modulation are T cell dependent. In human patient samples, we further demonstrate that only malignant cell-specific CXCL14 is associated with TIL, a context dependence that has previously been demonstrated in breast cancer but not in OSCC. These data support the tumor suppressive role of CXCL14 in OSCC via induction of TIL and provide the first direct in vivo patient data to support the malignant cell specificity of this effect. 9af72c28ce