Focal segmental glomerulosclerosis (FSGS) is a frequent glomerular disease, and is the common cause of nephrotic syndrome. However, there is no validated diagnostic blood biomarker for FSGS. Here, we performed a real-time PCR-based high-throughput miRNA profiling to identify the plasma signature for FSGS. We found four miRNAs (miR-17, miR-451, miR-106a, and miR-19b) were significantly downregulated in the plasma of FSGS patients (n = 97) compared with healthy controls (n = 124) in the training, validation, and blinded-test phases. The miRNA panel produced an AUC value of 0.82, and was associated with FSGS severity and histologic classification. A three-miRNA panel, including miR-17, miR-451, and miR-106a was related to FSGS remission. Furthermore, the downregulation of plasma-miRNA signature was not detected in disease controls (n = 119) such as IgA nephropathy (IgAN), mesangial proliferative glomerulonephritis (MSPGN), and membranous nephropathy (MN), and the miRNA panel discriminated between FSGS and disease controls. Pathway analysis showed that the four-miRNA panel may cooperatively regulate the pathways involved in the development of FSGS, such as apoptosis. We identified that phosphatase and tensin homolog (PTEN), Bcl-2-like protein 11 (BCL2L11), and chemokine (C-X-C motif) ligand 14 (CXCL14) were targets of miR-106a in human podocyte. Additionally, miR-106a overexpression suppressed podocyte apoptosis in vitro and the downregulation of four-miRNA panel probably resulted in the enhanced apoptosis in podocyte during FSGS development. Taken together, our data show that the plasma-miRNA panel is a potential independent diagnostic and prognostic factor for FSGS. Above miRNAs are involved in FSGS pathogenesis through regulating podocyte apoptosis.

Herein, we found that serum chemokine ligand 14 (CXCL14) was significantly enhanced in patients with idiopathic pulmonary fibrosis (IPF). In our current study, mouse L929 fibroblasts were stimulated with lipopolysaccharide (LPS) (100 ng/mL). Cell proliferation, the levels of matrix metalloproteinase 2 (MMP2) and MMP9, as well as extracellular matrix (ECM) content were assessed to evaluate the fibrogenesis of L929 cells. Proliferating cell nuclear antigen and cell viability were assessed to evaluate cell proliferation. Hydroxyproline (Hyp), collagen I/III, connective tissue growth factor (CTGF), and phosphorylated Smad2/3 (p-Smad2/3) were assessed to evaluate ECM secretion and deposition. α-Smooth muscle actin (α-SMA) was used to measure the occurrence of differentiation from fibroblast toward myofibroblast. Our data suggested that knockdown of CXCL14 prevented LPS-induced fibrogenesis of L929 cells through inhibiting cell proliferation and decreasing the expression of MMP2/9, Hyp, collagen I/III, CTGF, p-Smad2/3, and α-SMA. Notably, upregulation of protein phosphatase magnesium-dependent 1A (PPM1A) was involved in this process. On the contrary, recombinant CXCL14 protein led to an opposite effect. We first suggested that overexpression of PPM1A ameliorated LPS-induced fibrogenesis. Furthermore, we substantiated that knockdown of CXCL14 exerted an antifibrotic effect in IPF in vitro probably via the upregulation of PPM1A. Besides, evidently enhanced CXCL14, yet reduced PPM1A, was found in bleomycin-induced rat pulmonary fibrosis, confirming the roles of CXCL14 and its potential association with PPM1A in IPF in vivo. In conclusion, CXCL14 could be considered as a therapeutic target for preventing fibrogenesis of mouse L929 fibroblasts.

Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), has been successfully used to treat some patients with colorectal cancer and those with head and neck squamous cell carcinoma (HNSCC). For the effective treatment, it is essential to first identify cetuximab-responsive patients. The level of EGFR expression and/or the presence of mutations in signalling molecules downstream of the EGFR pathway have been reported to be determining factors for cetuximab responsiveness in colorectal cancer patients; however, limited data have been reported for HNSCC patients. We previously reported that the chemokine CXCL14 exhibits tumour-suppressive effects against xenografted HNSCC cells, which may be classified into two groups, CXCL14-expressing and non-expressing cells under serum-starved culture conditions. Here we employed CXCL14-expressing HSC-3 cells and CXCL14-non-expressing YCU-H891 cells as representatives of the two groups and compared their responses to cetuximab and their CXCL14 expression under various conditions. The growth of xenografted tumours initiated by HSC-3 cells, which expressed CXCL14 in vivo and in vitro, was suppressed by the injection of cetuximab into tumour-bearing mice; however, neither the expression of the chemokine nor the cetuximab-dependent suppression of xenograft tumour growth was observed for YCU-H891 cells. Both types of cells expressed EGFR and neither type harboured mutations in signalling molecules downstream of EGFR that have been reported in cetuximab-resistant colon cancer patients. The inhibition of the extracellular signal-regulated kinase (ERK) signalling increased the levels of CXCL14 messenger RNA (mRNA) in HSC-3 cells, but not in YCU-H891 cells. We also observed that the CXCL14 promoter region in YCU-H891 cells was hypermethylated, and that demethylation of the promoter by treatment with 5-aza-2'-deoxycytidine restored CXCL14 mRNA expression and in vivo cetuximab-mediated tumour growth suppression. Finally, we observed in vivo tumour growth suppression when YCU-H891 cells were engineered to express CXCL14 ectopically in the presence of doxycycline. These results indicate that CXCL14 expression may be a good predictive biomarker for cetuximab-dependent tumour suppression.

Obesity is characterized as an accumulation of adipose tissue mass represented by chronic, low-grade inflammation. Obesity-derived inflammation involves chemokines as important regulators contributing to the pathophysiology of obesity-related diseases such as cardiovascular disease, diabetes and some cancers. The obesity-driven chemokine network is poorly understood. Here, we identified the profiles of chemokine signature between human preadipocytes and adipocytes, using PCR arrays and qRT-PCR. Both preadipocytes and adipocytes showed absent or low levels in chemokine receptors in spite of some changes. On the other hand, the chemokine levels of CCL2, CCL7-8, CCL11, CXCL1-3, CXCL6 and CXCL10-11 were dominantly expressed in preadipocytes compared to adipocytes. Interestingly, CXCL14 was the most dominant chemokine expressed in adipocytes compared to preadipocytes. Moreover, there is significantly higher protein level of CXCL14 in conditioned media from adipocytes. In addition, we analyzed the data of the chemokine signatures in adipocytes obtained from healthy lean and obese postmenopausal women based on Gene Expression Omnibus (GEO) dataset. Adipocytes from obese individuals had significantly higher levels in chemokine signature as follows: CCL2, CCL13, CCL18-19, CCL23, CCL26, CXCL1, CXCL3 and CXCL14, as compared to those from lean ones. Also, among the chemokine networks, CXCL14 appeared to be the highest levels in adipocytes from both lean and obese women. Taken together, these results identify CXCL14 as an important chemokine induced during adipogenesis, requiring further research elucidating its potential therapeutic benefits in obesity.

Nano-sized metal oxides are currently the most manufactured nanomaterials (NMs), and are increasingly used in consumer products. Recent exposure data reveal a genuine potential for adverse health outcomes for a vast array of NMs, however the underlying mechanisms are not fully understood. To elucidate size-related molecular effects, differentiated THP-1 cells were exposed to nano-sized materials (n-TiO2, n-ZnO and n-Ag), or their bulk-sized (b-ZnO and b-TiO2) or ionic (i-Ag) counterparts, and genome-wide gene expression changes were studied at low-toxic concentrations (<15% cytotoxicity). TiO2 materials were nontoxic in MTT assay, inducing only minor transcriptional changes. ZnO and Ag elicited dose-dependent cytotoxicity, wherein ionic and particulate effects were synergistic with respect to n-ZnO-induced cytotoxicity. In gene expression analyzes, 6 h and 24 h samples formed two separate hierarchical clusters. N-ZnO and n-Ag shared only 3.1% and 24.6% differentially expressed genes (DEGs) when compared to corresponding control. All particles, except TiO2, activated various metallothioneins. At 6 h, n-Zn, b-Zn and n-Ag induced various immunity related genes associating to pattern recognition (including toll-like receptor), macrophage maturation, inflammatory response (TNF and IL-1beta), chemotaxis (CXCL8) and leucocyte migration (CXCL2-3 and CXCL14). After 24 h exposure, especially n-Ag induced the expression of genes related to virus recognition and type I interferon responses. These results strongly suggest that in addition to ionic effects mediated by metallothioneins, n-Zn and n-Ag induce expression of genes involved in several innate and adaptive immunity associated pathways, which are known to play crucial role in immuno-regulation. This raises the concern of safe use of metal oxide and metal nanoparticle products, and their biological effects.

Aims: The advent of immune checkpoint inhibitor therapy has proven beneficial in a subset of high-grade urothelial carcinomas (HGUC) of the bladder. Although treatment selection is currently largely determined by programmed death-ligand 1 (PD-L1) status, multiple factors in the immune system may modulate the host immune response to HGUC and immunotherapy. In this pilot study, we used a transcriptomic approach to identify the immune milieu associated with PD-L1 expression to enhance our understanding of the HGUC immune evasion network.

Methods: The immune transcriptome of 40 HGUC cystectomy cases was profiled using the NanoString nCounter Human V.1.1 PanCancer Panel. All cases were assessed for associated PD-L1 status (SP263) using whole tissue sections. PD-L1 status was determined as high or low using 25% tumour and/or immune cell staining.

Results: The most significantly differentially expressed gene was PD-L1 messenger RNA (CD274), which strongly correlated with protein expression (r=0.720, p<0.001). The sensitivity, specificity, positive and negative predictive values of CD274 for PD-L1 expression were 85%, 96%, 92% and 93%, respectively. The PD-L1 associated gene signature also included complement components C1QA and CD46 and NOD2 (innate immune system), proinflammatory cytokines CXCL14, CXCL16, CCL3, CCL3L1 and OSM along with the immune response mediator SMAD3, among others. Pathway analysis determined enrichment of these genes in interleukin-10 production, lymphocyte chemotaxis and aberrant IFNγ, NF-κB and ERK signalling networks.

Conclusions: We report key genes and pathways in the immune transcriptome and their association with PD-L1 status, which may be involved in immune evasion of HGUC and warrants further investigation.

Keywords: immunohistochemistry; pathology, molecular; urinary bladder; urologic neoplasms.

Connective tissues (CT) support and connect organs together. Understanding the formation of CT is important, as CT deregulation leads to fibrosis. The identification of CT specific markers has contributed to a better understanding of CT function during development. In developing limbs, Osr1 transcription factor is involved in the differentiation of irregular CT while the transcription factor Scx labels tendon. In this study, we show that the CXCL12 and CXCL14 chemokines display distinct expression pattern in limb CT during chick development. CXCL12 positively regulates the expression of OSR1 and COL3A1, a collagen subtype of irregular CT, while CXCL14 activates the expression of the tendon marker SCX. We provide evidence that the CXCL12 effect on irregular CT involves CXCR4 receptor and vessels. In addition, the expression of CXCL12, CXCL14 and OSR genes is suppressed by the anti-fibrotic BMP signal. Finally, mechanical forces, known to be involved in adult fibrosis, control the expression of chemokines, CT-associated transcription factors and collagens during limb development. Such unexpected roles of CXCL12 and CXCL14 chemokines during CT differentiation can contribute to a better understanding of the fibrosis mechanisms in adult pathological conditions.

Lung cancer is a prime cause of worldwide cancer deaths, with non-small cell lung cancer (NSCLC) as a frequent subtype. Surgical resection, chemotherapy are the currently used treatment methods. Delayed detection, poor prognosis, tumor heterogeneity, and chemoresistance make them relatively ineffective. Genomic medicine is a budding aspect of cancer therapeutics, where miRNAs are impressively involved. miRNAs are short ncRNAs that bind to 3'UTR of target mRNA, causing its degradation or translational repression to regulate gene expression. This study aims to identify important miRNA-mRNA-TF interactions in NSCLC using bioinformatics analysis. GEO datasets containing mRNA expression data of NSCLC were used to determine differentially expressed genes (DEGs), and identification of hub genes-BIRC5, CCNB1, KIF11, KIF20A, and KIF4A (all functionally enriched in cell cycle). The FFL network involved, comprised of miR-20b-5p, CCNB1, HMGA2, and E2F7. KM survival analysis determines that these components may be effective prognostic biomarkers and would be a new contemplation in NSCLC therapeutics as they target cell cycle and immunosurveillance mechanisms via HMGA2 and E2F7. They provide survival advantage and evasion of host immune response (via downregulation of cytokines-IL6, IL1R1 and upregulation of chemokines-CXCL13, CXCL14) to NSCLC. The study has provided innovative targets, but further validation is needed to confirm the proposed mechanism.

Keywords: NSCLC; eigengene; feed-forward loop; module; prognosis.

CXCL14 is a primordial chemokine that plays multiple roles in tumor suppression, autoimmune arthritis, and obesity-associated insulin resistance. However, the underlying molecular mechanisms are unclear. Here, we show that CXCL14 transports various types of CpG oligodeoxynucleotide (ODN) into the endosomes and lysosomes of bone marrow-derived dendritic cells (DCs), thereby activating Toll-like receptor 9 (TLR9). A combination of CpG ODN (ODN2395) plus CXCL14 induced robust production of IL-12 p40 by wild-type, but not Tlr9-knockout, DCs. Consistent with this, ODN2395-mediated activation of DCs was significantly attenuated in Cxcl14-knockout mice. CXCL14 bound CpG ODN with high affinity at pH7.5, but not at pH6.0, thereby enabling efficient delivery of CpG ODN to TLR9 in the endosome/lysosome. Furthermore, the CXCL14-CpG ODN complex specifically bound to high affinity CXCL14 receptors on DCs. Thus, CXCL14 serves as a specific carrier of CpG DNA to sensitize TLR9-mediated immunosurveillance.

OBJECTIVE

Implant surface topography is a key determinant affecting osteoblastic differentiation and cell-cell signaling of implant-adherent cells.

METHODS

To assess the early osteoinductive and cell-cell signaling events in adherent cells, commercially pure titanium implants (2.2 × 5 mm) with nanotopography (HF-treated TiO2 grit-blasted) were compared with micron-scale topography TiO2 grit-blasted (micron-scale, control) implants in vivo. Six implants (n = 3/surface) were placed in 10 systemically healthy subjects and removed by reverse threading at 1, 3, and 7 days. Gene expression profiles of adherent cells were interrogated using low-density RT-PCR arrays.

RESULTS

Osteoinduction was not observed at day 1 on either surface. At 3 days, elevated levels of BMP6, osteopontin, and osterix (OSX) were observed in RNA of cells adherent to both micron-scale and nanotopography surfaces. Both surfaces supported osteoinductive gene expression at 7 days; however, modest elevations of most mRNAs and significantly higher OSX mRNA levels were measured for cells adhered to nanotopography implants. Further, chemokine and cytokine profiles including CXCL10, CXCL14, IL-9, IL-22, and TOLLIP were upregulated on nanotopographic surfaces as compared with microtopographic surfaces.

CONCLUSIONS

Implants with superimposed nanoscale topography generate a greater induction of genes linked to osteogenesis and cell-cell signaling during the early phases of osseointegration.

In the present study, we investigated the distribution of CXCL14 immunoreactive endocrine cells and neurons in mouse alimentary tract by immunohistochemistry. CXCL14 immunoreactive endocrine cells were found as closed-type cells in the stomach and open-type cells in the small intestine. The immunostaining of these endocrine cells corresponded with that of the somatostatin-containing endocrine cells. Only a few CXCL14 immunoreactive endocrine cells were seen in the large intestine. CXCL14 immunoreactive fibers were observed in the muscular layer from the stomach to the rectum with most abundance in the rectum. Many CXCL14 immunoreactive fibers were observed in the lamina propria and submucosal layer from the duodenum to the rectum with most abundance in the rectum; these fibers corresponded to the somatostatin-containing nerve fibers. Some CXCL14 immunoreactive neuronal somata that were also immuno-positive for somatostatin, were noted in the submucosal layer of the rectum. However, the remaining parts of the alimentary tract presented with almost negligible immunoreactive somata. The co-localization of CXCL14 and somatostatin suggests that CXCL14 contributes to the function of somatostatin, which include the inhibition of other endocrine and exocrine cells and the enteric nervous systems.

The term "MELF-pattern myometrial invasion" (MELF pattern) denotes an unusual morphology of myometrial invasion in endometrioid carcinomas, and is associated with frequent lymphovascular invasion and lymph node metastasis. In this study, tumor cells were directly collected from a MELF pattern site, using laser microdissection. Comprehensive microarray analysis of the genes was conducted, and based on the results, expression of a metastasis progression gene, CXCR4, and its ligands CXCL14 and CXCL12, was further investigated. In vitro studies of endometrioid carcinoma cell lines revealed elevated invasion activity in a manner dependent on the CXCL14-CXCR4 or CXCL12-CXCR4 axis. Immunohistochemical analysis of 93 (MELF group, 46; non-MELF group, 47) cases illustrated CXCR4 was expressed in all endometrioid carcinomas, while based on CXCL14 and CXCL12 expression score, high proportions of cells were positive at the sites of the MELF pattern (P<0.01). There was no significant difference in progression-free survival or overall survival between MELF group and non-MELF group by Kaplan-Meier analysis. These findings suggest a possibility that cells at the sites of MELF pattern had acquired increased invasiveness through the function of the CXCL14-CXCR4 and CXCL12-CXCR4 axes.

Cortical interneurons are derived from the subcortical medial ganglionic eminence (MGE), caudal ganglionic eminence (CGE) and preoptic area (POA). CGE-derived cortical interneurons, which comprise around 30% of all cortical interneurons, mainly express Htr3a, calretinin (CR), Reelin (RELN) and vasoactive intestinal polypeptide (VIP). In the present study, we show that transcription factors Sp8 and Sp9 are co-expressed in the subventricular zone (SVZ) of the dorsal CGE. Conditional knockout of Sp8/9 using the Gsx2-Cre transgenic line results in severe loss of CGE-derived cortical interneurons. We observed migration defects of Sp8/9 double mutant CGE-derived cortical interneurons as they had longer leading processes than controls and they ectopically accumulated in the CGE. Dlx5/6-CIE conditional deletion of Sp8/9 also leads to a significant reduction in the CGE-derived cortical interneurons. We provide evidence that Sp8/9 coordinately regulate CGE-derived cortical interneuron development in part through repressing the expression of Pak3, Robo1, and Slit1. Finally, we show that Cxcl14, a member of the CXC chemokine family, is mainly expressed in CGE-derived interneurons in cortical layers I and II, and its expression is critically dependent on SP8.

The potentially different genetics and epigenetics in the primary tumors and metastases affect the efficacy of treatment in breast cancer patients. Nevertheless, the cellular and molecular mechanisms of breast cancer lymph node metastasis still remain elusive. Here, we employed single-cell RNA sequencing (scRNA-seq) to acquire the transcriptomic profiles of individual cells from primary tumours, negative, and positive lymph nodes. We also performed a single-cell assay for transposase-accessible chromatin (ATAC) sequencing (scATAC-seq) of the positive and negative lymph node samples to get the chromatin accessibility profile. We identified a novel cell subpopulation with an abnormally high expression level of CXCL14 in the positive lymph node of breast cancer patients. Cell trajectory analysis also revealed that CXCL14 was increased expressed in the late pseudo-time. Moreover. Based on a tissue microarray of 55 patients and the Oncomine database, We validated that CXCL14 expression was significantly higher in breast cancer patients with lymph node metastasis. Furthermore, scATAC-seq identified several transcription factors (TFs) that may be potential regulation factors for the lymph node metastasis of breast cancer. Thus, our findings will improve our current understanding of the mechanism for lymph node metastasis, and are potentially valuable in providing novel prognosis markers for lymphatic metastasis of breast cancer.

Interleukin 31 (IL-31) is a four-helix cytokine made predominantly by Th2 CD4⁺ T cells. It was initially identified as being associated with the promotion of atopic dermatitis, where increased levels of IL-31 levels have been found and IL-31 induced the expression of proinflammatory cytokines and chemokines in a human bronchial epithelial cell line. However, subsequent study has shown that IL-31RA knockout mice developed exacerbated type 2 inflammation in the lung following infection with Schistosoma mansoni eggs. In this study, we investigated the dynamic expression of IL-31 and IL-31RA during eight consecutive ovalbumin (OVA) challenges and measured the chemokines from lung alveolar epithelial cells induced by IL-31. In addition, we examined the effect deletion of IL-31RA has on lung inflammation and the differentiation of CD4⁺ T cells. Our results demonstrate that the expression of IL-31 and IL-31RA was elevated after each weekly OVA challenge, although slightly less of both observed after the first week of OVA challenge. IL-31 also promoted the expression of inflammatory chemokines CCL5, CCL6, CCL11, CCL16, CCL22, CCL28, CX3CL1, CXCL3, CXCL14 and CXCL16 in alveolar epithelial cells. Migration of macrophages and T cells was enhanced by culture supernatants of IL-31-stimulated alveolar epithelial cells. Lastly, and in contrast to the IL-31 results, mice deficient in IL-31RA developed exacerbated lung inflammation, increased IL-4-positive cell infiltrates and elevated Th2 cytokine responses in draining lymph nodes. The proliferation of IL-31RA^-/- CD4⁺ T cells was enhanced in vitro after anti-CD3/anti-CD28 antibody stimulation. These data indicate that IL-31/IL-31RA may play dual roles, first as an early inflammatory mediator promoting the secretion of chemokines to recruit inflammatory cells, and subsequently as a late inflammatory suppressor, limiting Th2 cytokine responses in allergic asthma.

Ovarian cancer is one of the common malignant tumours of female reproductive organs. Due to early diagnosis difficulties and lack of effective treatment in the late stage, ovarian cancer has the highest mortality rate in female reproductive system malignancies. Therefore, finding reliable early diagnosis indicators and new therapeutic targets for ovarian cancer is an urgent problem to be solved. Chemokine (C-X-C motif) ligand 14 (CXCL14) is a small cytokine belonging to the CXC chemokine family, which has been found to possess multi-effects in tumourigenesis and development. Here, we reported that CXCL14 was preferentially expressed in ovarian cancer. By analysing the TCGA database, we found that CXCL14 was highly expressed in advanced ovarian cancer patients and correlated with poor prognosis. In addition, the abnormal high CXCL14 levels were observed in serum and ovarian tissue of ovarian cancer patients by qRT-PCR and ELISA. In vitro and in vivo experiments both confirmed that overexpression of CXCL14 promoted the ovarian cancer cell proliferation. Moreover, transfection of CXCL14 increased the phosphorylation level of signal transducer and activator of transcription 3 (STAT3), and administration of STAT3 inhibitor III inhibited the tumour-promoting effects of CXCL14. Therefore, our study suggests that CXCL14 could be utilised as a novel adjunct biomarker for early diagnosis of ovarian cancer and provides new targets and ideas for the treatment of advanced ovarian cancer. SIGNIFICANCE PARAGRAPH: CXCL14 could be utilised as a novel adjunct biomarker for early diagnosis of ovarian cancer and provides new targets and ideas for the treatment of advanced ovarian cancer.

Pre-eclampsia (PE) and gestational diabetes mellitus (GDM) are common complications of pregnancy, but the mechanisms underlying these disorders remain unclear. The aim was to identify the extent of altered gene expression in term placentas from pregnant women with late-onset PE and GDM compared to controls. RNAseq identified few significantly differentially regulated genes in placental biopsies between PE, GDM, or uncomplicated pregnancy (n = 10 each group). Five genes were altered in placentas from PE including 4 non-coding genes and Angiopoietin 2 (ANGPT2). No genes were significantly regulated by GDM. In contrast, many genes were significantly regulated by fetal, maternal and delivery-specific variables, particularly spinal and epidural anesthesia. We selected ANGPT2 and Chemokine (C-X-C motif) ligand 14 (CXCL14) to test with qPCR in a larger set of placentas (n = 475) and found no differences between the groups. However, regression analysis revealed a stronger association between placental ANGPT2 and CXCL14 mRNA expression and fetal, maternal and delivery-specific variables than diagnostic group. To conclude, the gene expression in term placentas are highly affected by fetal, maternal and delivery specific variables. Few regulated genes were found in late-onset PE and GDM placentas, which may suggest that these conditions could be more affected by maternal factors.

Objectives: To explore lymphocyte infiltration as a potential mechanism behind CXCL14-mediated tumor growth suppression in oral cavity squamous cell carcinoma (OSCC).

Methods: We analyzed single cell RNA-sequencing (scRNA-seq) data from OSCC to identify expression changes among malignant cells in lymph nodes (LN) versus primary tumors. CXCL14 expression in murine OSCC cell lines was quantified using qRT-PCR. Short hairpin RNA knockdown of CXCL14 was performed in mouse oral cavity (MOC)1 cells, and CXCL14 overexpression was performed in MOC2 cells. Cells in each condition were injected into C57BL/6 mice with and without T cell depletion, and tumor volume was measured. At 30 days, tumors were dissociated and analyzed by flow cytometry for CD45⁺CD3⁺ T cells. CXCL14 expression was correlated with gene expression signatures of tumor infiltrating lymphocytes (TIL) in scRNA-seq data, as well as TCGA tumors.

Results: 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.

Conclusions: Higher CXCL14 expression by tumor cells is associated with reduced tumor growth and increased TIL, supporting immune-mediated suppression of tumor growth in OSCC. Given that CXCL14 is downregulated in LN metastases compared with primary tumors, our data raise the possibility that CXCL14-mediated immune infiltration may discourage invasion and metastasis. In human scRNA-seq data, only malignant cell-specific CXCL14 was associated with TIL, suggesting a critical context-dependent effect of CXCL14 expression.

Keywords: T-lymphocytes; cytokines; gene expression profiling; head and neck neoplasms; lymphocytes; tumor-infiltrating.