Cathepsin S regulates antitumor immunity through autophagic degradation of PD-L1 in colorectal cancer cells

Abstract

Colorectal cancer (CRC) is a major contributor to cancer-related mortality worldwide, highlighting the need to overcome its immunosuppressive tumor microenvironment. Cathepsin S (CTSS), a cysteine protease essential for MHC class II antigen presentation, has an unclear role in CRC immunity. This study investigated the impact of CTSS on PD-L1 expression and T-cell function in CRC. CTSS expression was analyzed in CRC tumor tissues and CTSS-deficient cell lines using immunohistochemistry, Western blotting, and flow cytometry. T-cell responses were assessed through granzyme B and IL-2 secretion assays, migration analysis, and gene set variation analysis (GSVA) of public datasets. Autophagy activity was evaluated via immunofluorescence, Western blotting, and lysosome isolation assays. An orthotopic CRC mouse model was used to study CTSS function in vivo. Key findings revealed that elevated CTSS expression correlated with higher PD-L1 levels in CRC tissues. CTSS suppression in CRC cells reduced PD-L1 expression while enhancing T-cell cytotoxicity and migration. GSVA further revealed an inverse correlation between CTSS expression and cytotoxic T-cell activity, alongside a strong association with autophagy-related pathways. Mechanistically, CTSS suppression in CRC cells promoted PD-L1 degradation by enhancing autophagic flux. In vivo, CTSS suppression inhibited tumor growth and enhanced CD8⁺ T-cell infiltration and activity. Anti-CD8 antibody treatment promoted tumor growth more significantly in CTSS-proficient CRC cells compared to CTSS-deficient cells. These findings demonstrate that CTSS regulates PD-L1 expression and T-cell cytotoxicity via autophagy-mediated pathways in CRC cells. Given ongoing development of CTSS inhibitors and autophagy modulators, targeting CTSS may offer a promising strategy to improve CRC immunotherapy.

Keywords: Autophagy; CTSS; Colorectal cancer; Immune microenvironment; PD-L1.

Fig. 1

CTSS upregulation in CRC tissues. A Representative IHC images depicting CTSS expression scores of 1 + , 2 + , and 3 + in CRC tumor tissues. B Boxplots indicated that CTSS H-scores were higher in CRC tissues than in matched mucosa tissues. C Correlation of CTSS expression with immune checkpoint expression in CRC tissues, revealing a positive correlation between CTSS expression and PD-L1 levels. D Representative images of PD-L1 staining in CRC tumor cells with TPSs of 0%, 5%–10%, and > 10% shown at 100 × and 400 × magnification. E Boxplots revealing higher CTSS H-scores in CRC tissues with TPS > 0 than in those with TPS = 0. Scale bars and P values are indicated. Data are presented as mean ± standard deviation (SD) and were analyzed using the Wilcoxon rank-sum test. F Western blot analysis revealing lower PD-L1 levels in CTSS-deficient CRC cells compared with CTSS-proficient cells. G and H Flow cytometry histograms displaying membrane PD-L1 expression in HT29 G and SW480 H cells, with MFI reported as mean ± SD from three independent experiments. I Western blot analysis of PD-L1 expression in HT29 cells treated with 10 ng/mL IFN-γ for 24 h. Fold changes in PD-L1 levels were normalized to actin controls and quantified using ImageJ densitometry. IHC immunohistochemical; MFI median fluorescence intensity; TPS tumor proportion score

Fig. 2

CTSS downregulation enhances cytotoxic T-cell activity against CRC cells. A Addition of activated Jurkat cells [effector cell to target cell (E:T) ratio of 20:1] enhanced the cytotoxicity of CTSS-deficient HT29 (left panel) and SW480 (right panel) compared with that of corresponding CTSS-proficient cells. Jurkat cells were activated through PHA treatment for the indicated duration. B Increased cytotoxicity was also observed in CTSS-deficient HT29 (left) and SW480 (right) cells following co-culture with human cytotoxic T-cells at an E:T ratio of 1:1. C and D Representative flow cytometry histograms of granzyme B expression in Jurkat cells cocultured for 24 h with CTSS-proficient or CTSS-deficient HT29 C and SW480 D cells by using a transwell system. Following incubation, T-cells were isolated, and granzyme B levels were assessed. E IL-2 concentrations in the conditioned media from Jurkat cells cocultured with HT29 (left panel) and SW480 (right panel) cells were measured using an ELISA kit after 24 h. F Transwell migration assays indicated higher T-lymphocyte migration toward conditioned media from CTSS-deficient HT29 (left panel) and SW480 (right panel) cells than for the controls. Migrated cells in the lower chamber were collected and quantified after 24 h. All experiments were performed in triplicate. P values are indicated

Fig. 3

GSVA reveals CTSS-associated biological processes in the CRC TME. Immune-related cell functions associated with CTSS expression included the following: A positive regulation of MHC class II biosynthetic process, B positive regulation of lymphocyte anergy, C negative regulation of T-cell-mediated cytotoxicity, and D negative regulation of activated T-cell proliferation. Autophagy-related functions associated with CTSS expression included the following: E negative regulation of autophagosome assembly, F autophagosome organization, G autophagic cell death, H autophagosome maturation, and I processes involving autophagic mechanisms. Spearman correlation coefficients and false discovery rate (FDR) values are indicated. GSVA gene set variation analysis; MHC major histocompatibility complex

Fig. 4

CTSS suppression induces autophagy activation. A HT29 and B SW480 cells with various levels of CTSS expression (control, shCTSS 1, and shCTSS 2) were cultured for 48 h, after which proteins were extracted. Western blot analysis was performed to examine the expression levels of LC3B, SQSTM1, and PD-L1. C HT29 and D SW480 cells with various levels of CTSS expression were seeded in six-well plates (1 × 10⁵ cells/well). After 3 days, the cells were incubated with LysoTracker Deep Red probe for 30 min at 37 °C, and lysosomal activity was visualized using fluorescence microscopy. E HT29 parental cells were seeded in six-well plates (2 × 10⁵ cells/well). After 24 h, the cells were transfected with the ptfLC3 plasmid. Following an additional 24 h, the cells were transfected with si-CTSS and maintained for 48 h, and images were taken using fluorescence microscopy. Protein expression fold changes were normalized to actin and quantified using ImageJ densitometry. P values are indicated. GFP green fluorescent protein; RFP red fluorescent protein

Fig. 5

CTSS downregulation suppresses PD-L1 expression through autophagy in CRC cells. A HT29 and B SW480 cells treated with sh-CTSS1 were seeded in six-well plates (2 × 10⁵ cells/well). After 24 h, the cells were treated with 5 μM chloroquine (CQ) for 48 h. Protein lysates were analyzed through Western blot analysis of SQSTM1 and PDL-1 expression. C HT29 cells and D SW480 cells treated with sh-CTSS1 were seeded in six-well plates (2 × 10⁵ cells/well). After 24 h, the cells were transfected with si-ATG7 for 48 or 72 h. Thereafter, proteins were collected, and Western blot analysis was performed to assess ATG7, SQSTM1, and PDL-1 expression. E HT29 and F SW480 cells (control and sh-CTSS1) were seeded in chamber slides (7000 cells/well). After 24 h, the cells were treated with 5 μM CQ for 24 h. Confocal microscopy was used to evaluate the colocalization of LC3B and PDL-1. G HT29 cells (control and sh-CTSS1) were seeded in 15-cm dishes until 80% confluency. Subsequently, they were treated with 5 μM CQ for 24 h. The cells were then harvested, and lysosomes were isolated through ultracentrifugation. Western blot analysis was performed to assess PD-L1 and LAMP2 expression in the lysosomal fractions. Protein expression fold changes were normalized to actin and quantified using ImageJ densitometry. P values are indicated. CQ chloroquine

Fig. 6

CTSS suppression inhibits MC38 tumor growth in immunocompetent mice. A Schematic of the establishment of an orthotopic CRC mouse model. The mice were sacrificed 4 weeks after MC38 cell injection. B Representative images of tumors excised from C57BL/6 mice orthotopically injected with MC38 cells expressing either shCTSS or shScr. C Tumor volume was measured every 3 days, and D tumor weight was recorded upon rectal excision. E Representative IHC images of tumor tissues stained with antibodies against CTSS, CD8, SQSTM1, LC3B, granzyme B, and PD-L1. Scale bar: 50 μm. F Quantitative IHC analysis of tumor tissues from shScr and shCTSS groups (n = 12). Tumor cell staining was scored as follows: 0 = no expression, 1 = 1–25% positive cells, 2 = 26–50% positive cells, and 3 = 51–100% positive cells. Data are presented as mean ± SD and were analyzed using a two-tailed Student’s t test. P values are indicated. g gram; IHC immunohistochemical; shScr, shScramble

Fig. 7

Effects of CD8 depletion on tumor growth in mice injected with control or CTSS-suppressed MC38 cells. A Schematic of the establishment of the orthotopic CRC mouse model. Anti-CD8 antibodies were administered intraperitoneally on days 7 and 14. B Representative images of tumors excised from C57BL/6 mice orthotopically injected with MC38 cells expressing either shCTSS or shScramble, with or without anti-CD8 treatment. C Tumor volume was measured every 3 days, and D tumor weight was recorded after rectal excision. E The increase in tumor weight induced by anti-CD8 antibody treatment was compared for the shScr group versus shCTSS group. F Representative IHC images of tumor tissues stained with antibodies against CTSS, PD-L1, CD8, and granzyme B. Scale bar: 50 μm. G Quantitative IHC analysis of tumor tissues from shScramble and shCTSS groups treated with anti-CD8 antibodies. Tumor cell staining was scored as follows: 0 = no expression, 1 = 1–25% positive cells, 2 = 26–50% positive cells, and 3 = 51–100% positive cells. Data are represented as mean ± SD and were analyzed using a two-tailed Student’s t test. P values are indicated. ab antibody; g gram; IHC immunohistochemical; shScr shScramble

Fig. 8

Schematic of the proposed mechanism through which CTSS depletion induces autophagy-associated PD-L1 degradation in CRC cells and enhances T-cell-mediated cytotoxicity against tumor cells. CRC colorectal cancer