SIGLEC15, negatively correlated with PD-L1 in HCC, could induce CD8+ T cell apoptosis to promote immune evasion

Figures & data

SIGLEC15 demonstrated negative expression patterns with PD-L1 in HCC, which is survival-related and could promote tumor immune evasion through induction of CD8+ T cell apoptosis in tumor microenvironment.

Figure 1. SIGLEC15 was highly expressed in HCC tumor samples and was related to deleterious survival of patients.

a-c. Genetic expression of SIGLEC15 in public and local HCC tissue samples. (a. Expression counts of SIGLEC15 in TCGA-LIHC dataset showed SIGLEC15 expression was higher in HCC in contrast to normal liver samples; b. Expression counts of SIGLEC15 in ICGC-LIRI dataset showed SIGLEC15 was also highly expressed in HCC in comparison to normal liver tissues; c. PCR analysis of local HCC and paired normal tissues demonstrated that SIGLEC15 was highly expressed in tumor samples.) d. Representative SIGLEC15 expression images in IHC analysis of HCC tissue microarray. e-f. SIGLEC15 expression was related to deleterious overall survival and disease-free survival in IHC-examined HCC sample cohort (n = 221). (e. IHC analysis of examined HCC tissues showed high expression of SIGLEC15 was related to poor overall survival and high accumulative death rate; f. in calculation of disease-free survival and related accumulative death rate, high SIGLEC15 expression was also a significant risk factor in examined samples).

Figure 2. SIGLEC15 expression was negatively correlated with PD-L1 expression in HCC samples, relating to a barren immune microenvironment in tumor.

a. Counts expression of SIGLEC15 and PD-L1 in HCC tumor samples demonstrated polarized expression patterns in public datasets of TCGA-LIHC and ICGC-LIRI, showing negative genetic correlations in data. b. Further examination of differentially expressed genes in polarized HCC samples demonstrated that samples with high expression of SIGLEC15 showed poor immune infiltration status across datasets of TCGA-LIHC and ICGC-LIRI, while samples with high expression of PD-L1 demonstrated an activated immune microenvironment with higher expression of immune-related markers. c-d. IHC examination of SIGLEC15 and PD-L1 also demonstrated a negative correlation in HCC samples. (c. Representative images of negatively correlated SIGLEC15 and PD-L1 expression in HCC tumor samples; d. IHC staining scores for SIGLEC15 and PD-L1 demonstrated that tumor samples with high SIGLEC15 expression scores turned to present low levels of PD-L1 expression, and HCC samples examined in IHC analysis could be categorized into four groups according to SIGLEC15 and PD-L1 expression, in which samples classified as SIGLEC15 high/median and PD-L1 no-expression counted for 32.27% in total.) e. Examination of SIGLEC15 and PD-L1 expression in HCC cell lines additionally demonstrated negative correlation in expression.

Figure 3. SIGLEC15, expressed by tumor cells, mainly promoted HCC growth through immune evasion.

a-b. Cell viability analysis of SIGLEC15 knock-down HCC cell lines. (a. SIGLEC15 knock-down in MHCC97H cell models did not affect cell viability in CCK8 examination; b. SIGLEC15 knock-down in Huh7 cell models also did not influence cell growth examined by CCK8 tests.) c-d. Evaluation of subcutaneous tumor grafts in mature T cell deficient BALB/c nude mice (n = 5). (c. Evaluation of subcutaneous tumor growth of SIGLEC15 knock-down MHCC97H cells demonstrated that SIGLEC15 expression did not affect tumor growth in vivo without immune surveillance of T/B cells; d. Knock-down of SIGLEC15 in Huh7 cells also did not affect tumor growth ability in vivo without mature T cells.) e-f. Tumor growth evaluation of Siglec15 knockout Hep1-6 cells in orthotopic (n = 9) and subcutaneous (n = 6) tumor models of immune competent C57 mice. (e. Knock-out of Siglec15 in Hep1-6 cell line could inhibited tumor formation in liver of mouse orthotopic models with competent immune microenvironment; f. Siglec15 knockout Hep1-6 tumor cells demonstrated impaired growth ability in comparison to control tumor cells injected subcutaneously in mice with normal immune activity.) g. Schematic demonstration of a tumor cell and T cell co-cultured system. h-i. SIGLEC15 expression in tumor cells could dampen cytotoxic function of Jurkat cells in vitro after co-culture of 48 h. (h. Co-cultured with SIGLEC15 knock-down Huh7 cells could increase GZMB expression in Jurkat cells through flow cytometry examination; i. Overexpression of SIGLEC15 in Huh7 tumor cells through plasmids transfection could reduce GZMB expression in Jurkat cells after co-culture.) j-l. Functional examination of CD8+ T cells in Siglec15 knockout subcutaneous tumors. (j. Siglec15 knockout in Hep1-6 cells could promote CD8+ T cell GZMB expression in tumor microenvironment of immune competent C57 mice; k. Siglec15 knockout in Hep1-6 cells did not significantly increased perforin expression in CD8+ T cells in tumor in comparison to control models; l. Knock-out of Siglec15 in Hep1-6 tumor cells could also increase IFNg expression in CD8+ T cells in subcutaneous tumor of immune competent C57 mouse models.)

Figure 4. Anti-SIGLEC15 immunotherapy could reduce tumor burden and promote CD8+ T cell cytotoxic functions in mouse models.

a. Schematic demonstration of anti-SIGLEC15 treatment in subcutaneous tumor models of C57 mice. b. Anti-SIGLEC15 treatment inhibited subcutaneous tumor growth (n = 5). c-e. Anti-SIGLEC15 therapy could reinvigorate cytotoxic CD8+ T cell functions in tumor. (c. Anti-SIGLEC15 treatment increased GZMB expression in CD8+ T cells in tumor examined by flow cytometry; d. IFNg expression in CD8+ T cells was also increased after anti-SIGLEC15 treatment; e. Difference of perforin expression in CD8+ T cells was not observed between tumor-bearing mice with or without anti-SIGLEC15 treatment.) f. Anti-SIGLEC15 treatment improved survival of tumor bearing mice (n = 10). g. Schematic demonstration of macrophage deletion in addition to anti-SIGLEC15 therapy in C57 HCC mouse models. h. Mononuclear phagocyte depletion in C57 mice (n = 3) with anionic clophosome (100ul per mouse) could significantly reduce corresponding cell populations. i. Clearance of mononuclear phagocytes did not influence tumor growth in vivo (n = 3). j. Anti-SIGLEC15 treatment could still significantly inhibit tumor growth in mouse model after macrophage depletion (n = 5).

Figure 5. SIGLEC15 could induce CD8+ T cell apoptosis in HCC tumor microenvironment.

a. Anti-SIGLEC15 could increase CD8+ T cell infiltration in HCC tumor microenvironment through flow cytometry examination. b. CD8+ T cell portions were negatively correlated with tumor volume in mouse models. c. Transcriptional difference in magnetically enriched CD8+ T cells of mouse tumors between groups of anti-SIGLEC15 and IgG treatment. d. Biological function annotation of differentially expressed genes showed anti-SIGLEC15 treatment could promote T cell migration and chemotaxis. e-f. scRNA-seq analysis of C57 mouse subcutaneous tumor grafts treated by IgG and anti-SIGLEC15 antibody. (e. Annotated cell clusters of tumor samples after filtration; f. Portions of major cell clusters in tumor samples treated by IgG or anti-SIGLEC15 antibody.) g. Developmental trajectories of CD8+ T cell clusters in tumor demonstrated that main CD8 T2 cluster cells shared a different development trajectory in contrast to CD8 T1 cluster cells. h. Anti-SIGLEC15 treatment could reduce CD8 T2 cluster cells in mouse tumors. i. Signaling analysis of CD8+ T cell clusters in tumor model demonstrated that various signals were differently expressed between CD8 T1 and CD8 T2 clusters, emphasizing different T cell status. j. Among the selected signals, CD8 T1 cluster had lower apoptotic signaling in comparison to CD8 T2 cluster. k-l. Anti-SIGLEC15 or Siglec15 knockout in tumor cell could both increase Ki67 expression in CD8+ T cells in tumor microenvironment. (k. Flow cytometry analysis of Ki67 expression in CD8+ T cells from IgG and anti-SIGLEC15 antibody treated C57 subcutaneous tumors demonstrated anti-SIGLEC15 treatment could increase Ki67 expression in CD8+ T cells; l. Knock-out Siglec15 in Hep1-6 cells could also increase Ki67 expression in CD8+ T cells in tumor microenvironment.).

Data availability statement

Sequencing data for the manuscript were deposited in the Gene Expression Omnibus database under the accession number of GSE218411. Public datasets of LIHC and LIRI projects were downloaded from TCGA and ICGC databases respectively. There was no original code generated in the manuscript.