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. 2023 Jan 12;12(1):7.
doi: 10.1186/s40164-022-00371-1.

Caspase-8 contributes to an immuno-hot microenvironment by promoting phagocytosis via an ecto-calreticulin-dependent mechanism

Zhihua Gong #  1   2 , Qingzhu Jia #  1   2 , Jinming Guo #  1   2   3 , Chongyi Li  4 , Shouxia Xu  1   2 , Zheng Jin  5 , Han Chu  1   6 , Yisong Y Wan  7 , Bo Zhu  8   9 , Yi Zhou  10   11
Affiliations

Caspase-8 contributes to an immuno-hot microenvironment by promoting phagocytosis via an ecto-calreticulin-dependent mechanism

Zhihua Gong et al. Exp Hematol Oncol. .

Abstract

Background: Caspase-8 (Casp8) acts as an initiator in cell apoptosis signaling. However, the role of Casp8 in tuning the tumor immune microenvironment remains controversial due to the complicated crosstalk between immune-tolerogenic apoptotic cell death and immunogenic cell death cascades.

Methods: The Cancer Genome Atlas (TCGA) and publicly accessible immune checkpoint blockade (ICB)-treated cohorts were used to investigate the clinical relevance of Casp8. A tumor-bearing mouse model was used to characterize changes in the tumor microenvironment and to explore the efficacy of ICB treatment under Casp8 knockout conditions.

Results: By exploring TCGA datasets, we showed that the expression level of Casp8 was associated with an immuno-hot microenvironment across various solid tumor types. Casp8 deficiency leads to decreased CD8+ T cell infiltration and resistance to anti-PD-L1 therapy in a mouse model. Mechanistically, Casp8 deficiency or pharmacological disruption results in impaired ecto-calreticulin transition in tumor cells, which in turn hampers antigen presentation in draining lymph nodes. Furthermore, radiotherapy restored sensitivity to anti-PD-L1 treatment via elevated calreticulin surface expression.

Conclusions: Our data revealed a causative role of Casp8 in modulating the immunogenicity of tumor cells and responsiveness to ICB immunotherapies and proposed radiotherapy as a salvage approach to overcome Casp8 deficiency-mediated ICB resistance.

Keywords: Antigen presentation; Calreticulin; Caspase-8; Dendritic cells; Immunotherapy; Tumor microenvironment.

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Conflict of interest statement

The authors declare no potential competing interest.

Figures

Fig. 1
Fig. 1
The correlation of CASP8 transcription and the 18 gene set ICB predictive biomarker. A total of 4874 patients across 31 cancer types from TCGA were enrolled for analysis. a GSEA comparing T cell-inflamed gene sets (measurement of ICB-responsiveness) between CASP8 -low and CASP8-high patients in TCGA. b Enrichment score of the T cell-inflamed gene set across 31 TCGA datasets. c Heatmap of the expression of 18 genes in the T cell-inflamed gene set in CASP8-low and CASP8-high patients. The 1st and 4th quartiles were defined as CASP8-low and CASP8-high, respectively
Fig. 2
Fig. 2
Casp8 knockout B16F10 tumors were associated with a cold TME. a B16-C8KO or control cells (2 × 105 in 100 µL of PBS) were subcutaneously inoculated into the right flanks of 6–8-week-old nude mice. The tumor volume was monitored twice per week. b B16-C8KO or control cells (2 × 105 in 100 µL of PBS) were subcutaneously inoculated into the right flanks of 6–8-week-old C57BL/6 mice. The tumor volume was monitored twice per week. c and d Analysis of the transcriptome of subcutaneous B16F10 tumors in C57BL/6 mice. c Unsupervised principal component analysis based on the 18 ICB responsiveness-related genes. d Heat map of the 18 genes. e and f B16-C8KO or control cells were subcutaneously inoculated into the right flanks of C57BL/6 mice, the TME of subcutaneous xenografts was analyzed. e Representative flow cytometry of CD4+ or CD8+ tumor-infiltrating cells. f Fractions of CD3+, CD4+, or CD8+ cells in CD45+ leukocytes in tumors. g and h Production of granzyme-B and INF-γ by CD8+ T cells was determined by flow cytometry. g Representative flow cytometry of tumor-infiltrating cytotoxic T cell function and h statistical analysis
Fig. 3
Fig. 3
Casp8 knockout induced less CD8+ T cell infiltration and poor response to anti-PD-1 treatment in B16F10 mice models. B16-C8KO or control cells (2 × 105) were subcutaneously inoculated into the right flanks of C57BL/6 mice. Mice received 200 µg of intraperitoneal anti-PD-L1 monoclonal antibody or the equivalent isotype control antibody on days 4, 7, and 10. Tumor volume was monitored twice per week. a Experimental schematic of the B16F10 mouse model. b and c Tumor growth curves of the indicated groups (b) and each tumor (c). d The overall survival in the indicated groups. The mice were sacrificed when the tumor volume reached 2000 mm3. e and f CD4+ and CD8+ tumor-infiltrating cells in subcutaneous xenografts were analyzed by flow cytometry. e Representative flow cytometry of tumor-infiltrating T cells pre-gated for viable CD45+ cells. f Portions of T cells among CD45+ leukocytes in tumors
Fig. 4
Fig. 4
CASP8 expression was related to response to ICB in human cancers. a Heatmap of the expression of 18 genes in the T cell-inflamed gene set in four ICB-treated datasets. b CASP8 transcription level in responders and non-responders. c The response rate in CASP8-high and CASP8-low patients. d Forest plot of the hazard ratio for overall survival. The responders included patients with stable disease for more than 6 months, complete response, and partial response according to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1). CASP8 expression lower than the median of each cohort was regarded as CASP8-low; otherwise, expression was regarded as CASP8-high
Fig. 5
Fig. 5
Casp-8 malfunction hampered ecto-CRT transition and induced antigen presentation in B16F10-bearing mice. a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in genes between Casp8 knockout and control B16F10 cells. Adjusted log10 p-values for the enrichment of KEGG gene sets are presented in color. b Heatmap of enriched genes in the lists of Antigen Processing and Presentation genes. c CD103+MHC-II+ dendritic cells in CD11c+ cells from drained lymph nodes in the subcutaneous mouse tumor model. d Statistical results of CD103+MHC-II+ cells dendritic cells. e and f B16F10 cells pre-labeled with the Deep Red dye were treated with Z-IETD-FMK and doxorubicin for 24 h. Labeled tumor cells were injected into the spleens of naive mice. Spleens were collected 2 h after mice received an intrasplenic injection. Splenocytes were subjected to immunostaining with a CD11c antibody to assess phagocytosis. Representative dot plots of flow cytometric analyses depicting the effect of the Casp8 inhibitor Z-IETD-FMK (e) and Casp8 knockout (f) are shown. Cell surface calreticulin expression (ecto-calreticulin) and total calreticulin in Z-IETD-FMK-treated (g) and Casp8 knockout (h) B16F10 cells
Fig. 6
Fig. 6
Irradiation rescues ecto-CRT and sensitizes B16F10 tumor cells to ICB in vivo. a A single dose of 20 Gy irradiation treatment affected cell surface CRT expression in pre-treated B16F10 cells. b–e B16-C8KO or control cells (2 × 105) were subcutaneously inoculated into the right legs of C57BL/6 mice. When the tumors reached approximately 50 mm3, the mice were locally irradiated with a single dose of 20 Gy. On the same day, 200 µg of anti-PD-1 monoclonal antibody or the equivalent isotype control antibody was injected intraperitoneally every three days three times. b Scheme of irradiation combined with anti-PD-L1 treatment for the B16F10 xenograft mouse model, and tumor growth curves of the indicated groups (c) and each tumor (d). e Overall survival in the indicated groups. When tumor volumes reached 2000 mm3, the mice were sacrificed
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