Single cell RNA-sequencing (scRNA-seq) data were obtained from and re-analyzed as described in Puram et al.17 For five samples with matched primary OSCC tumors and metastatic lymph node samples (MEEI5, MEEI20, MEEI25, MEEI26 and MEEI28), differential expression between primary tumor and lymph node niches was analyzed. Equal numbers of cells were sampled from each patient to ensure no bias towards a particular patient’s data, and sampling was done 100 times to calculate a p value. For nine primary tumor samples (MEEI5, MEEI16, MEEI17, MEEI18, MEEI20, MEEI22, MEEI25, MEEI26 and MEEI28), average CXCL14 and MHC class I expression in malignant cells were determined.

C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, Maine, USA). Mice were kept under specific pathogen-free conditions, and mice aged 6–8 weeks were used for all experiments. Mouse oral cavity (MOC)1 and MOC2 murine oral squamous cell carcinoma cell lines, developed from murine oral squamous cell carcinomas induced by topical 7,12-dimethylbenz(a)anthracene administration, were generously provided by Dr Ravindra Uppaluri at Washington University (St. Louis, Missouri, USA). Cells were cultured in complete DMEM/F-12 medium (Mediatech) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

For mouse-derived organoids, tongue tissue was obtained from C57BL/6 mice. Excess fat and muscle tissue were removed to enrich for epithelial cells. The tissue was then finely minced into 2–3 mm pieces on ice, washed twice in DMEM/F-12 (Invitrogen) containing 1X Primocin (InvivoGen), followed by ACK solution (Thermo Fisher Scientific) to remove red blood cell contamination. Minced tissues were washed with DMEM/F-12 media and resuspended with Matrigel (cold Cultrex growth factor reduced Basement Membrane Extract (BME) type 2, Trevigen). Droplets of approximately 40 μL were plated on the bottom of preheated 24-well culture plates (E&K Scientific). Plates were incubated at 37°C for 30 min to allow BME to solidify. Organoid culture media (EN media) containing DMEM/F-12 supplemented with 10% Noggin-conditioned media, nicotinamide (10 mM, Sigma), N-acetylcysteine (1 mM, Sigma), B-27 without vitamin A (1X, Invitrogen), Pen-Strep (1X, Invitrogen) and EGF (50 ng/mL, R&D Systems) was added. The medium was changed every 2–3 days and organoids were split once every 2–4 weeks.

RNA was extracted with the RNeasy mini kit (QIAGEN) and complementary DNA (cDNA) libraries were prepared with the Maxima First Strand cDNA Kit (Thermo Fisher Scientific). Gene expression probes for mouse CXCL14 (Mm00444699_m1, Thermo Fisher Scientific) and mouse HPRT1 (Mm03024075_m1, Thermo Fisher Scientific) were purchased. Quantitative gene expression was performed using the Taqman Gene Expression Assay with recommended primers (Life Technologies). Gene expression was normalized to control for HPRT1 expression and then shown relative to an appropriate control (2ΔCt×100, where ΔCt represents the difference in threshold cycle between the control and target genes).

RNAi Consortium (TRC) Lentiviral short hairpin RNA (shRNA) (pLKO.1-based vector) was purchased from GE Healthcare Dharmacon. A cDNA encoding CXCL14 from ATG codon to the stop codon was PCR cloned (Forward primer: 5′-GAATTC ATGAGGCTCCTGGCGGCCG-3′; reverse primer: 5′-GGATCC CTATTCTTCGTAGACCCT-3′) from pCMV6-cxcl14 (Origene) and subcloned EcoRI/BamHI fragments into the pHIV-Zsgreen lentiviral expression construct (Addgene).

For the production of the lentiviral particles, the 293 T cell line was transfected with the packaging plasmid pCMVR8.74, the envelope plasmid pCMV-VSVG and the lentiviral construct containing the shRNA or the transgene, using Lipofectamine 2000 according to the manufacturer’s instructions. The medium was changed 16 hours after the transfection. Virus-containing culture supernatant was collected after 24 hours and centrifuged for concentration. Virus was used immediately to infect cells, which were seeded at 3×10⁵ cells per well in a 6-well plate 24 hours prior. Polybrene (8 µg/mL) was also added to enhance the lentiviral transduction efficiency. The medium was changed after 24 hours. In the case of the MOC1 cells transduced with the pLKO.1 puro vectors, the cell cultures were treated with 1 µg/mL puromycin for 1 week after media change. For pHIV-Zsgreen transduced MOC2 cells were sorted (GFP-positive) with FACSAria machine (BD Biosciences).

Proliferation experiments were conducted using xCELLigence RTCA DP device (ACEA Biosciences, San Diego, California, USA), which was placed in a humidified incubator at 37°C in 5% CO₂. Cell proliferation experiments were carried out using 96-well plates. Microelectrodes for impedance detection during cell attachment, spreading, and proliferation were attached at the bottom of each well and had electronic connection with the computer software. At the beginning, 100 μL complete growth medium was added to each well, and water was added to the space around the wells to avoid evaporation. Plates were incubated for 30 minutes at room temperature in a laminar chamber. Afterwards, incubation plates were inserted into the device and the background impedance was measured. Next, the MOC1 and MOC2 cells were seeded 1×10⁴ cells/well in 100 µL growth medium per well. Plates were left for 30 minutes at room temperature in a laminar chamber to allow for cell attachment. Finally, the plates were inserted into the device and impedance was automatically monitored and expressed as the Cell Index (CI) value by the software. Cell proliferation experiments were run for 60 hours. CI was monitored every 15 min for the whole experiment duration.

To assess the invasive capacity of the tumor cells, 1×10⁴ cells in 500 μL of serum-free DMEM/F-12 were added into the upper chamber and 500 μL of complete medium was placed into the lower chamber (Corning BioCoatTM Matrigel Invasion Chamber, Corning, New York, USA). Cells were incubated at 37°C for 48 hours, before the non-invading cells were removed from the upper surface of the membrane. After fixation in 95% ethanol for 5 minutes, the cells still on the opposite surface of the filter membrane were stained with 1% crystal violet for 10 minutes. The migratory cells were counted in five microscope fields and averaged.

To assess the expression of MHC class I genes, samples were stained with PE-conjugated antimouse H-2K^b antibody (clone AF6-88.5, Biolegend) and Alexa Fluor 647 antimouse H-2D^b antibody (clone KH95, Biolegend) and analyzed by flow cytometry.

10⁶ cells in each condition were injected into immunocompetent C57BL/6 mice (n=16; control and experimental group cells were injected into the left and right flank, respectively), and tumor volume was measured at the time points shown.

For fluorescent activated cell sorting (FACS) analysis, tumors were harvested, minced and digested in 300 U/mL collagenase and 100 U/mL hyaluronidase (StemCell Technologies) in culture media: DMEM/F-12 medium (Mediatech) with 10% FBS, 2 mmol/L L-glutamine and 1% penicillin-streptomycin-amphotericin B (MP Biomedicals). The tumor digest was pipetted every 15 min and incubated at 37°C for 3 hours, until a single-cell suspension was obtained. The dissociated cells were resuspended in Trypsin-EDTA (StemCell Technologies) for 5 min, then further dissociated with 5 U/mL dispase (StemCell Technologies) and 0.1 mg/mL DNase I (StemCell Technologies) for 1 min. Cells were filtered through a 40 mm cell strainer and erythrocytes were lysed with ACK lysing buffer (Lonza). Suspensions were then pretreated with IgG from mouse serum to block non-specific staining and incubated with anti-CD45 and CD3 antibodies (Biolegend). 4′,6-diamidino-2-phenylindole (DAPI) was used to allow exclusion of non-viable cells. FACS analysis was performed with FACSAria (BD Biosciences).

T cells were depleted using a CD3 antibody. The following antibodies were used: hamster IgG f(ab’) two fragment control antibody (clone BE0091-FAB f(ab’), Bio X Cell) and CD3-depleting antibody (clone 125–2 C11 f(ab’) two fragments). Mice were treated on days −2 and 1 via intraperitoneal inoculation with 200 μg of CD3-depleting antibody diluted in phosphate-buffered saline to a final volume of 200 μL.

H&E slides of nine primary tumors analyzed by scRNA-seq17 were obtained and analyzed in a blinded manner by a dedicated head and neck pathologist (WF). Whole slides were analyzed for stromal lymphocytes adjacent to tumor nests. Stromal lymphocytic infiltrate was qualitatively scored as 1+ (absent or minimal), 2+ (moderate) or 3+ (dense). For each sample, TIL were also assessed as follows: whole slides were analyzed to identify the regions of highest TIL density. In these regions, TIL were calculated on five high power fields (HPFs) with a 40× objective. Slides were visualized on an Olympus BX40CY microscope at 400×. Images were taken at 40× on an Olympus DP27 camera.

Bulk transcriptomic data for 498 OSCC tumors (rnaseqv2-RSEM_genes_normalized) was downloaded from the Broad Firehose website (https://gdac.broadinstitute.org/), along with additional tumor and clinical annotations. Expression data were log₂-transformed and centered for each gene. The relative abundance of TIL in each tumor was estimated as the average normalized expression of CD2, CD3D, CD3E and CD3G. These estimates were evaluated by the Pearson’s correlation with the normalized expression of CXCL14.

Malignant cells in primary tumor and lymph node niches were analyzed for five matched samples previously profiled by scRNA-seq.17 A number of genes were differentially expressed in one or more patients (figure 1). Focusing on genes that demonstrated concordant differential expression in the majority of samples, we found that CXCL14, DUSP1, NFKBIA, FOS and HSPA1B were significantly downregulated and KRT19 and KRT15 were significantly upregulated in the lymph nodes of three out of five samples (figure 1). Of these, CXCL14 was the most highly downregulated gene (2.9-fold change, p=10⁻⁴³) in lymph nodes relative to primary tumors, suggesting it may play a role in suppressing metastasis in HNSCC. Given the previously described associations of CXCL14 with reduced tumor growth in HNSCC,8–10 we focused further investigations on the role of this chemokine as a tumor suppressor and, in particular, on its ability to induce the host immune response.

CXCL14 is expressed constitutively in normal squamous epithelial cells but heterogeneously in most HNSCC and cervical SCC tumors.18 To understand the function of CXCL14 in OSCC, CXCL14 expression was analyzed in MOC1 and MOC2 cell lines relative to housekeeping gene HPRT. MOC1 and MOC2 cell lines were used because of previously demonstrated differences in MOC tumor aggressiveness and immune infiltration. Analysis of transcripts showed that CXCL14 is expressed in both cell lines but at a significantly higher level in indolent MOC1 cells (0.12±0.011), relative to aggressive MOC2 cells (0.03±0.004), with approximately a fourfold change between the cell lines (p<0.05, n=3 biological replicates) (figure 2A). These findings indicate that CXCL14 expression may be correlated with indolent, rather than aggressive, HNSCC tumors.

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<0.05). Relative to control, injection of MOC1 cells after CXCL14 shRNA knockdown resulted in significantly larger tumors (figure 2B, 5.80-fold, p<0.05, n=5), and conversely, injection of MOC2 cells after CXCL14 overexpression resulted in significantly smaller tumors (figure 2C, 5.88-fold, p<0.05, n=5). These results suggest a potential tumor suppressive effect of CXCL14 in vivo.

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.

To assess whether the observed changes in tumor growth with CXCL14 modulation are dependent on the presence of TIL, we depleted T cells using a CD3 antibody and examined tumor growth. Strikingly, this resulted in a significantly diminished effect of CXCL14 modulation on MOC1-derived and MOC2-derived tumor growth (figure 5A,B), further supporting the conclusion that T cell infiltration underlies the tumor suppressive effect of CXCL14.

It was previously shown that CXCL14 expression could restore MHC class I expression that was reduced by HPV in tumor cells.16 To determine the effect of CXCL14 on MHC class I expression in our HPV-negative model, we assessed the impact of CXCL14 modulation on expression of H-2D^b and H-2K^b on MOC1 and MOC2 cells. CXCL14 knockdown in MOC1 cells, which had high baseline expression of CXCL14, reduced the expression of both MHC class I molecules. However, in MOC2 cells, which had very low baseline expression of CXCL14, overexpression of CXCL14 did not affect MHC class I expression (figure 5C,D). To determine whether this association between CXCL14 and MHC class I holds in human samples, we evaluated the correlation between malignant cell CXCL14 and MHC class I genes in our scRNA-seq cohort. We identified a strong correlation in one patient sample (MEEI17), but not in other profiled samples (online supplemental figure S2).

To determine whether the association between CXCL14 expression and lymphocytic infiltrate is recapitulated in human patients, we first assessed stromal lymphocytes by histological analysis in our cohort of nine patients with OSCC analyzed by scRNA-seq and found a strong positive relationship between malignant cell CXCL14 expression and stromal lymphocytes (figure 6). We then assessed CXCL14 expression and TIL in two separate cohorts: (1) the same scRNA-seq cohort of 9 patients and (2) TCGA cohort of 498 patients with HNSCC. In our scRNA-seq cohort, we found a strong significant association of TIL per HPF on H&E with CXCL14 expression in malignant cells (figure 7A, r=0.93, p=0.0003), but not with CXCL14 expression in cancer-associated fibroblasts (figure 7B, r=−0.46, p=0.26) or CXCL14 expression in all non-malignant cells (figure 7C, r=−0.13, p=0.74). Importantly, in the TCGA cohort, there was also no association of bulk CXCL14 expression with TIL as assessed by CD2 and CD3 expression (figure 7D, r=−0.04, p=0.53). Taken together, these results suggested a malignant cell-specific association of CXCL14 with TIL, with CXCL14 from other cellular sources, including fibroblasts, showing no association.