Time-to-event outcomes such as time to overall survival were summarized with Kaplan-Meier curves, and groups were compared using log-rank assessments. mRNA levels were inversely correlated with overall survival in this group (= 0.0016) (Figure 1A). We also compared the gene expression levels of 3 major galectin family members (Gal1, -3, and -9) in the tumor using the above HNC TCGA cohort with levels in healthy tissues (GTEx cohort, = 44) and found that only Gal1 was significantly overexpressed in tumor samples (Supplemental Physique 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI129025DS1). CIBERSORT analyses revealed a significant inverse relationship between Gal1 expression and CD4+ memory resting T cells and CD4+ follicular T cells, but not CD8+ T cells (Supplemental Physique 1B). This obtaining further highlights the role of Gal1 in tumor Batimastat sodium salt progression in HNC and its relationship to intratumoral T cell infiltration. Open in a separate window Physique 1 Gal1 promotes tumor growth and metastases in a HNC model by causing immune suppression.(A) Kaplan-Meier analysis of overall survival of patients with HNSCC according to Gal1 gene expression (= 518 patients, TCGA data set). = 0.0016. (B) ELISA results for secreted levels of Gal1 in murine HNSCC cells (MOC1, MEERL, and MOC2) after 24 hours of normoxia or hypoxia (0.5% O2). (C) Immunoblots show Gal1 deletion with CRISPR/Cas9 in MOC1, MOC2, and MEERL cells and stable lentiviral overexpression of Gal1 in MOC1 (MOC1 + Gal1) cells. (D) Tumor growth curves Batimastat sodium salt for C57BL/6 mice subcutaneously implanted with 1 106 MOC1 vector control cells (MOC1-Vec) or MOC1 Gal1-overexpressing cells (MOC1-Gal1) (= 5 mice). (E) Batimastat sodium salt Tumor growth curves for C57BL/6 mice subcutaneously implanted with 2.5 105 MOC2 Gal1 WT or Gal1-KO cells (= 5 mice). (F) Tumor growth curves for C57BL/6 mice subcutaneously implanted with 1 106 MEERL Gal1 WT or Gal1-KO cells (= 5 mice/group). (G) Quantification of lung metastases foci after subcutaneous implantation of each cell line. The number of nodules per lung area was quantified by H&E staining (scale bars: 500 m). In the graph, each dot represents 1 mouse, and the bar indicates the mean. (H) Quantification of LN metastases in mice bearing either MOC2 Gal1 WT or Gal1-KO tumors. (I) Quantification and representative histologic images of metastatic foci in lungs after subcutaneous implantation of MOC2 Gal1 WT or Gal1-KO cells, measured at comparable primary tumor sizes. Scale bars: 250 m. (J) Quantification of CD4+ and CD8+ T cells in MOC2 Gal1 WT and Gal1-KO tumors at sizes of approximately 100 mm3 and 300 mm3, after enzymatic dissociation and flow cytometric analyses. (K) Flow cytometric analyses of CD44 and CD62L markers on CD3+ T cells from MOC2 Gal1 WT and Gal1-KO tumors. ** 0.01 and *** 0.001. Overall survival was summarized using Kaplan-Meier curves, and groups were compared using log-rank assessments (A); repeated-measures ANOVA was used for tumor growth measurement over time (DCF); and a 2-tailed Students test was used for comparisons of single treatment with the control (B, G, and ICK). For preclinical studies, we used both HPVC (MOC1 and MOC2) and HPV+ (MEERL) mouse syngeneic HNC models, both of which show high fidelity to human disease in terms of biologic behavior and genomic scenery (22, 23). MOC1 and MOC2 cells show different levels of aggressiveness with regard to tumor growth and metastasis in mice. The highly metastatic and aggressive MOC2 cells secreted high basal levels of Gal1 under normoxia (21%), which was further enhanced in hypoxia (0.5 %). The slow-growing, nonmetastatic MOC1 cells secreted low levels of Gal1 under both conditions. Moderately aggressive MEERL cells showed low Gal1 secretion under normoxia but elevated secretion under hypoxia (Physique 1B). These tumor models thus replicated the prior findings in other malignancy types that correlated Gal1 levels with tumor aggressiveness (24). We next induced overexpression of Gal1 in MOC1 cells Batimastat sodium salt and knocked out Gal1 using CRISPR/Cas9 gene targeting Rabbit Polyclonal to CRHR2 in all 3 cell lines (Physique 1C). Gal1 overexpression in MOC1 cells led to enhanced tumor growth and spontaneous lung metastases in a subcutaneous C57BL/6 tumor model (Physique 1, D and G). In contrast, Gal1 deletion resulted in a significant reduction (~50%, 0.001) in primary growth of MOC2 and MEERL tumors (Figure 1, E and F). Gal1-depleted MOC2 tumorCbearing mice exhibited substantially fewer spontaneous nodal and lung metastases, even when the primary tumors were size matched with their respective parental controls (Physique 1, H.