Caspase-8 Contributes to Immuno-Hot Microenvironment by Promoting Phagocytosis via an Ecto-Calreticulin-dependent Mechanism

doi:10.21203/rs.3.rs-1984457/v1

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Research Article

Caspase-8 Contributes to Immuno-Hot Microenvironment by Promoting Phagocytosis via an Ecto-Calreticulin-dependent Mechanism

https://doi.org/10.21203/rs.3.rs-1984457/v1

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Journal Publication

published 12 Jan, 2023

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Background

Caspase-8 play as an initiator caspase of cell apoptosis signaling. However, the role of caspase-8 in tunning tumor immune microenvironment remains controversial due to a complicated crosstalk between immuno-tolerogenic apoptotic cell death and immunogenic cell death (ICD) cascades.

Methods

TCGA and publicly accessible immune checkpoint blockade (ICB)-treated cohort were introduced to investigate the clinical relevance of caspase-8. Tumor-bearing mouse model was used to characterize the change of tumor microenvironment and explore efficacy to ICB treatment in caspase-8 knockout condition.

Results

We showed that the expression level of Casp8 was associated with an immuno-hot microenvironment across various solid tumor types by exploring TCGA dataset. Casp8 deficiency led to decreased CD8⁺ T cell infiltration and resistance to αPD-L1 therapy in mouse model. Mechanistically, Casp8 deficiency or pharmacological disruption resulted in impaired ecto-calreticulin (ecto-CRT) transition on tumor cells, which in turn hampered antigen presentation in draining lymph node. Furthermore, radiotherapy restore the sensitivity to αPD-L1 treatment via elevated surface expression of CRT.

Conclusions

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

Caspase-8

calreticulin

antigen-presentation

dendritic cells

immunotherapy

tumor microenvironment

The application of the immune checkpoint blockade (ICB), such as programmed death-1 receptor (PD-1) and programmed death ligand 1 (PD-L1) antibodies, reinvigorated T cell function and leads to prolonged survival in various cancer types^1–5. However, due to intrinsic or acquired drug resistance, only a minority of patients experience long-term benefits from ICB. Pre-existing cytotoxic T cells in the tumor microenvironment (TME) are a prerequisite for the reinvigoration of T cells and a resulted inflamed circumstance, indicating an immuno-hot TME and the potential to benefit from ICB administration⁶. However, the tumoral intrinsic driven force that leading to an immuno-hot TME remain incompletely understood.

It is known that the form of tumor cell death instructs an immuno-hot or -cold TME. Immunogenic cell death (ICD) supplies an immuno-hot TME by promoting antigen release, antigen presentation, and provoking cytotoxic T cell activation to induce a successful anti-tumor immunity⁷. Strategies aiming at eliciting ICD have been used to overcome resistance in ICB treatments⁸. Necroptosis, pyroptosis, and ferroptosis are the predominant immunogenic forms of cell death, whereas apoptosis is usually regarded as an immune-tolerogenic process^9–11. Furthermore, cells that undergo necroptosis activate the immune system, particularly through antigen presentation and cross-priming of CD8⁺T cells¹². In pyroptosis, Gasdermin proteins are cleaved by inflammatory caspases, leading to inflammatory cytokine release and cell death¹³. In cancer cells, Gasdermin E cleaved by caspase-3 is the essential mediator of pyroptosis, which converts non-inflammatory apoptotic signals into pyroptotic cell death and suppresses tumor growth¹⁴.

Caspase-8 (Casp8) is regarded as a switch for immuno-tolerogenic apoptosis, and immunogenic necroptosis and pyroptosis¹⁵. It has been reported that in the presence of Casp8 malfunctions, the form of cell death could switch to necroptosis¹⁶. It is likely that Casp8 malfunction leads to ICD in cancer cells, which may provoke an adaptive immune response, facilitating CD8⁺T cell infiltration and inducing an inflamed TME, which in turn improves the efficacy to ICB immunotherapies. Consistent with this theory, in a TRAF^−/− melanoma mouse model, tumor cells redirected the TNF signaling pathway to favor RIPK1-dependent necroptosis, enhanced tumor eradiation, and showed a better response to anti-PD-1 therapy compared to the control group¹⁷.

However, evidence also suggests that necrosis-induced inflammation only facilitates tissue repair responses and is not effective enough to induce anticancer immunity^18,19. Moreover, the function of Casp8 could be catalytical activity dependent or independent. Other than the aforementioned role of cleavage-dependent Casp8 function, the expression of catalytically inactive Casp8 is both necessary and sufficient to induce inflammasome formation¹⁵. This implies a complicated role of Casp8 in cell death and adaptive and innate immune responses.

In our research, we explored The Cancer Genome Atlas (TCGA) database and ICB treaded cohorts to determine the role of Casp8 in TME and ICB responsiveness. We further established the Casp8 knock out cell line and animal models to understand its mechanism.

Sources of publicly accessible data

The mRNA transcription profiles of The Cancer Genome Atlas (TCGA) cohorts, including 31 cancer types, were downloaded from TCGA database using the R package TCGAbiolinks. In the immune therapeutic cohorts, including three melanoma cohorts and a clear cell renal cell carcinoma cohort, patients were treated with anti-PD-(L)1 and/or anti-CTLA-4 therapy, and the response and mRNA data were downloaded. According to CASP8 expression, the lowest and highest quartiles were regarded as CASP8-low and CASP8-high, respectively. The IFNγ expended gene signatures²⁰, which predict responsiveness to ICB, were used in the gene set enrichment analysis (GSEA, http://www.broadinstitute.org/gsea/index.jsp) for TCGA cohorts.

Cell line

B16F10 cells were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum (FBS; 10%), penicillin (100 U/mL), and streptomycin (100 mg/mL) in a 37 °C humidified atmosphere containing 5% CO2. The Casp8-knockout B16F10 cell line was generated using (Crispr)/Cas9 technology. The gRNA encoding Casp8 is shown in supplementary Fig. 1.

Animals and animal models

Female C57BL/6 mice, aged 6–8 weeks, were purchased from the Center of Experimental Animals of TMMU. Nude mice were purchased from VitalStar Biotechnology Co., Ltd. (Beijing, China). Mouse handling protocols were approved by the Institutional Animal Care and Use Committee of the TMMU. To establish tumor models, B16F10 cells (2 × 10⁵ in 100 µL PBS) were subcutaneously inoculated into the right flanks of 6–8-week-old female C57BL/6 mice or nude mice. When the tumors became palpable, the tumor volume was monitored twice per week. In immunotherapeutic models, 2 × 10⁵ B16F10 cells were subcutaneously inoculated into the right flanks of 6–8-week-old female C57BL/6 mice. Mice received 200 µg intraperitoneal anti-PD-L1 monoclonal antibody (10F.9G2, Be0101, BioXcell) or the equivalent isotype control antibody (BioXcell, BE0090) on days 4, 7, and 10.

For radiation combined immunotherapeutic models, 2 × 10⁵ B16F10 cells were subcutaneously inoculated into the right legs of C57BL/6 mice. When the tumors reached approximately 50 mm³, the mice were locally irradiated using the Varian Trilogy Stereotactic System with a single dose of 20 Gy. On the same day, 200 µg anti-PD-1 monoclonal antibody (10F.9G2, Be0101, BioXcell) or equivalent isotype control antibody (BioXcell, BE0090) was injected intraperitoneally every 3 days.

RNA-sequencing

Five pairs of control or CASP8-knockout B16F10 subcutaneous tumors from the right flanks of 6–8-week-old female C57BL/6 mice were collected for RNA sequencing. The transcriptome was used for principal component analysis (PCA) [FactoMineR: an R package for multivariate analysis (2008)] and pathway enrichment.

In vitro phagocytosis experiments using bone marrow-derived dendritic cells (BMDCs)

The isolation and culture of BMDCs were performed according to previous publications¹⁸. Briefly, 6-week-old C57BL/6 mice were euthanized and sterilized, then tibiae and femora were removed. Bone marrow was flushed out, after centrifugation cultured in DC medium (RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 1 M HEPES, 50 mM 2-mercaptoethanol) containing 20 ng/mL of GM-CSF. On day 7, non-adherent and loosely adherent cells were harvested for further experiments. B16F10 cells were pre-labeled with 1 µM cell tracker deep red dye (Invitrogen), following the manufacturer’s protocol, and then treated with 25 µM doxorubicin for 24 h. Cells were harvested and adjusted to 5 × 10⁷ cells/mL in PBS. The labeled tumor cells (5 × 10⁶ cells in 100 µL) were injected. BMDCs (1 × 10⁶/mL) were pre-coated in wells of a 24-well plate, and B16F10 cells were added at a ratio of 1:4 and co-cultured either at 37 or 4 °C for 6 h. The cells were then harvested and stained with anti-CD11c (N418) for flow cytometry.

In vivo phagocytosis assay

In vivo phagocytosis followed a previously established protocol²¹. Briefly, the indicated B16F10 cells were stained with 1 µM Cell Tracker Deep Red dye (Invitrogen), following the manufacturer’s protocol and then treated with 25 µM doxorubicin for 24 h. Cells were harvested and adjusted to 5 × 10⁷ cells/mL in PBS. The labeled tumor cells (5 × 10⁶ in 100 µL) were injected into the spleens. After 2 h, the mice were sacrificed, spleens were harvested and stained with anti-mouse CD11c antibody, and phagocytosis was assessed by flow cytometric analysis.

Flow cytometry

In subcutaneous animal models, tumors were harvested on days 18–20. After euthanasia, tumors were collected and filtered through a 70 µm cell strainer to obtain single-cell suspensions. For the analysis of tumor-infiltrating immune cells, the samples were further stained with anti-CD45 (30-F11), anti-CD11b (M1/70), anti-CD3 (17A2), anti-CD8 (53 − 6.7), anti-CD4 (GK1.5), anti-F4/80 (BM8), anti-Gr-1 (RB6-8C5), and Fixable Viability Dye eFluor 780 (65–0865) (eBioscience). For T cell function analysis, samples were cultured with a cell stimulation cocktail (00-4975-03; eBioscience) for 6 h and subsequently stained with anti-CD45 (30-F11), anti-CD3 (17A2), anti-CD8 (53 − 6.7), anti-IFN-γ (XMG1.2), and anti-GZMB(GB11) using the Cytofix/Cytoperm™ Kit (554714, BD).

In the subcutaneous mouse model, draining lymph nodes were harvested for the analysis of dendritic cells. After filtering through a 70 µm cell strainer, the single-cell suspensions were stained with anti-CD11c (N418), anti-CD103 (2E7), anti-CD45 (30-F11), anti-MHCII (M5/114.15.2), and Fixable Viability Dye eFluor 780 (65–0865) (eBioscience). Data were collected using a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software. To detect cell surface and total calreticulin, cells were treated with the caspase-8 inhibitor Z-IETD-FMK or dimethyl sulfoxide (DMSO) and then irradiated using the Varian Trilogy Stereotactic System with a single dose of 20 Gy. After 16 h, the cells were collected for analysis. Staining protocols were adopted from the manufacturers using anti-CRT (EPR3924, Abcam).

Statistics

Comparisons between two groups of continuous variables were performed using an unpaired t-test or Mann-Whitney U test. Comparisons of continuous variables from three or more groups were performed by one-way analysis of variance (ANOVA). The association between responders and the categorical variables CASP8-high and low was compared using the χ2 test or Fisher’s exact test. Tumor growth was compared using one-way ANOVA. Survival was estimated using Kaplan–Meier curves, and the p value and hazard ratio were determined by a log-rank test. Statistical analyses were performed using Prism 6 software (GraphPad, Prism Software Inc., CA, USA) and R V.4.0.0. p < 0.05 was considered statistically significant.

Casp8 indicates an immuno-hot tumor microenvironment in solid tumors

To investigate whether Casp8 contributes to an inflamed TME (favoring an effective anti-tumor immunity, designated as immuno-hot TME) in solid tumor types, expression profiling data from the TCGA dataset were introduced. A normalized gene set enrichment analysis score was used to compare the immuno-cold/hot microenvironment between patients with higher or lower (median expression value as cutoff for higher vs. lower group) expression levels of Casp8 with a well-established IFN-γ-related signature. For all the 31 types of solid tumors (4874 cases) in the TCGA dataset, we observed consistently over-presented (NES > 0) IFN-γ-related signature genes in patients with higher Casp8 expression levels, indicating an immuno-hot microenvironment in these patients (Fig. 1a). Furthermore, when applying this analysis to a merged cohort containing all the above tumor types, we confirmed the significant upregulation of IFN-γ-related signature in patients with higher levels of Casp8 (Fig. 1b). Furthermore, the heatmap clearly illustrated a pattern of upregulated expression of 18 genes in the IFN-γ-related signature panel, further supporting a strong association between Casp8 expression and the immuno-hot microenvironment (Fig. 1c).

Knockout of CASP8 results in an immuno-cold microenvironment

To determine whether Casp8 expression is causative, or merely a concomitant characteristic of an immuno-hot microenvironment, a CASP8 knockout B16F10 cell line (C8KO) was established using the CRISPR/Cas9 strategy. Western blot and sequencing assays demonstrated the effectiveness of our depletion strategy at both the genomic and protein levels (Supplementary Fig. 1a, 1b). Consistently, a decrease in cell apoptosis was observed in vitro or in tumor-bearing immunocompetent mice (Supplementary Fig. 1c, 1d).

To identify the immunological consequences of the CASP8 knockout, B16-C8KO and control cells were subcutaneously inoculated into immunodeficient nude mice and immunocompetent mice. Totally identical tumor volumes were observed in immunodeficient nude mice, demonstrating no intrinsic difference in cell growth rate resulting from the CASP8 knockout (Fig. 2a). For the immunocompetent group, although there was not a statistical difference in tumor size, we identified numerically increased tumor volumes in the C8KO-bearing mice, implicating an immuno-dependent mechanism of Casp8-mediated anti-tumor immunity (Fig. 2b). To comprehensively characterize the TME, homogenates of the tumor mass were subjected to RNA-sequencing profiling. Principle component analysis revealed a pattern of fully distinct transcriptomes between C8KO and control tumors, demonstrating a substantial reprogramming of TME due to CASP8 knockout (Fig. 2c). Focusing on a list of prominent genes in the anti-tumor immune response, a trend of lower expression of these genes was found in the C8KO group (Fig. 2d), which successfully recapitulated our observations in the TCGA cohort (Fig. 1c).

Furthermore, flow cytometry showed significant impaired infiltration of T cells, especially CD8⁺ T cells, in the C8KO microenvironment (Fig. 2e, 2f). Profiling analysis identified comparable infiltration of myeloid-derived suppressor cells and tumor-associated macrophages (Supplementary Fig. 2). Moreover, regarding the cytotoxic function of CD8⁺ T cells, clearly deceased granzyme-B and INF-γ production were identified in C8KO tumor cells (Fig. 2g, 2h).

Poor responsiveness to anti-PD-L1 treatment in the C8KO-bearing model

It is well known that the IFN-γ-related signature is a surrogate measurement of responsiveness to anti-PD-1/PD-L1 treatment. Since the CASP8 knockout significantly reduced CD8⁺ T cell infiltration and inflammatory response in tumor cells, we speculate that the C8KO clone is resistant to immune checkpoint immunotherapy. In the therapeutic mouse model, anti-PD-L1 antibody was successively injected every three days for three times (Fig. 3a). However, this treatment only led to a weak (statistical insignificance) tumor control in the C8KO groups compared to their wild-type counterparts (Fig. 3b, 3c). Accordingly, no survival benefit was found in C8KO-bearing mice, demonstrating less sensitivity to anti-PD-L1 immunotherapy (Fig. 3d). Furthermore, as observed in the control mice receiving this treatment, anti-PD-L1 administration failed to increase the infiltration of CD8⁺ T cells (Fig. 3e, 3f).

Altogether, our findings revealed an impaired CD8⁺ T cell response and poor sensitivity to anti-PD-L1 treatment in CASP8 knockout mice.

Responsiveness to ICB treatment in clinical cohorts

Subsequently, we aimed to test the clinical relevance of caspase-8 in ICB-receiving clinical cohorts. To this end, four cohorts with available outcome follow-up and baseline RNA-sequencing data of tumor biopsies were introduced. Again, 18 genes in the IFN-γ-related signature were used to characterize the immune microenvironment. The heatmap displayed enriched high expression of signature genes in patients with high levels of Casp8, supporting an immuno-hot microenvironment in these patients (Fig. 4a). Furthermore, to monitor the response to ICB administration, we observed a correlation between caspase-8 expression and responder/non-responder category in four cohorts (Fig. 4b, 4c). For the survival outcome, a more favorable overall survival of ICB-treated patients was found in Casp8-high patients compared to that in Casp8-low patients, suggesting higher a sensitivity to ICB treatment in Casp8 high patients (Fig. 4d).

Impaired antigen presentation and ecto-calreticulin translocation in CASP8 knockout mice

To explore the underlying mechanism of caspase-8-mediated anti-tumor immune response, we compared RNA-sequencing data to identify the differentially regulated signaling pathways between CASP8 knockout and control tumors. Gene Ontology analysis showed that the antigen processing and presentation pathway ranked among the top enriched signaling pathways in tumors with wild-type caspase-8 expression (Fig. 5a, 5b), leading us to suspect a phenotype change in dendritic cells in CASP8 knockout mice. Flow cytometry identified significantly fewer antigen-presenting dendritic cells, namely CD103⁺ dendritic cells, from the draining lymph node in the CASP8 knockout group than in the control group (Fig. 5c, 5d). In vivo phagocytosis assays showed that Casp8 inhibitor treatment (Fig. 5e) or knockout (Fig. 5f) could downregulate phagocytosis. Surface-bound calreticulin (ecto-CRT) acts as a bridge for saturable binding sites on phagocytes and favors phagocytosis. CASP8 knockout (Fig. 5g) and pharmacological inhibition (Fig. 5h) blocked CRT translocation from the endoplasmic reticulum to the cell surface without altering the total expression of CRT (Fig. 5g, 5h and Supplementary Fig. 3). In addition, the CASP8 knockout did not affect other well-known danger-associated molecular patterns, such as HSP70 and HMGB1 (Supplementary Fig. 3). Taken together, our findings suggest a CRT-mediated downregulated antigen presentation mechanism in the CASP8-associated immuno-hot microenvironment.

Irradiation rescues ecto-CRT and sensitizes CASP8 knockout tumors to ICB treatment

Radiation can lead to endoplasmic reticulum stress. We found that ecto-CRT in the CASP8 knockout and Casp8 inhibitor-pre-treated B16F10 cells were significantly elevated relative to control group levels when a single dose of 20 Gy irradiation was delivered (Fig. 6a). To determine whether radiotherapy could rescue the sensitivity to anti-PD-L1 treatment for CASP8 knockout tumors in vivo, an additional single dose of irradiation was administered on the same day of ICB initiation (Fig. 6b). Consistently, irradiation attenuated poor growth control in the CASP8 knockout group compared with the control group treated with ICB (Fig. 6c, 6d). In addition, irradiation combined with anti-PD-1 prolonged survival in CASP8 knockout mice compared to those receiving ICB treatment alone, demonstrating the feasibility of a radio-immunotherapy combinational regimen in treating Casp8-deficient patients (Fig. 6e).

ICD can be induced by different stressors, such as chemotherapy, irradiation, targeted anticancer agents, and so on. Anthracycline chemotherapy drugs, such as doxorubicin, induced caspase-dependent ICD by emitting the damage-associated molecular patterns (DAMPs)²². Many chemotherapeutic drugs, such as cisplatin, could induce casp8 expression and lead to apoptosis, but these drugs were not specific inducers²³. Casp8 is a key regulator of cell death¹⁵. As both necrosis and pyroptosis are immunogenic, it is easy to infer that a loss of Casp8 function leads to ICD, which triggers a stronger anti-tumor immune response and might benefit from ICB. However, our findings suggest that the role of Casp8 may be more complex. The clinical data from the TCGA database and ICB-treated cohorts assumed that in certain cancer types, especially melanoma, Casp8 plays a pro-inflammatory role.

Previous studies have revealed that Casp8 may be related to ICB responsiveness. Casp8 mutant cells were found to accumulate in tumors with highly cytotoxic TME²⁴. One explanation is that the loss of Casp8 function leads to resistance to immune cell killing. To prove this theory, tumor cells were treated with the CRISPR library co-cultured with NK cells and T cells^25,26. However, the CASP8 knockout was enriched in MC38/MC38-OVA tumors not B16F10, implying that the role of Casp8 may vary among cancer types. In contrast, in MC38-OVA cells co-cultured with OT-I T cells in vitro, treatment with anti-PD-1 failed to enhance tumor eradiation²⁶.

These results imply that Casp8 function loss may not be immunogenic, as expected; in clinical samples, Casp8 high expression was related to better overall survival and cytotoxicity of T cells in cancer patients²⁷. Further, based on the function of this protein, it is reasonable to infer that this phenomenon occurs because of resistance to inducers of cell death in tumor cells. However, in another study, when Crispr library-treated tumor cells were treated with cytotoxic T cells, CASP8 knockout tumors were not identified. This indicates that resistance might occur ahead of T cell death.

Beyond its role in cell death, Casp8 plays a crucial role in antigen presentation. CRT is a fundamental molecule involved in ICD. When CRT is blocked or knocked down, the immune response is impaired. More precisely, cell surface-bonded CRT is predominant in ICD. We found that Casp8 leads to downregulated ecto-CRT in our B16F10 model, which is consistent with previous studies²⁸ in which CASP8 knockout mice showed a downregulation of CRT and a weak anti-tumor effect in the CT26 mouse model. These reports support our finding that Casp8 is the key regulator of ecto-CRT, which affects the response to ICB. However, in the endoplasmic reticulum stress model, ecto-CRT was not CASP8-dependent; a similar finding was reported in the photodynamic therapy model²⁹. In clinical practice, the detection of Casp8 mutations is practical, implying that such mutations might be independent predictive markers of the response to ICB.

Further, we tried to rescue the resistance to ICB caused by the Casp8 mutation. Irradiation is an important therapeutic method used to overcome low responsiveness to ICBs in clinical practice. As far as immunity was concerned, irradiation was thought to have a dual effect, both inhibiting and promoting immunity^30,31. Lamerton verified that if the whole body of the animals were exposed to radiation 1.76 Gy/day or 0.84 Gy/day, firstly, their immune system responded positively and peripheral blood count increased, but within 20 days, their bone marrow failed to produce platelets, leukocytes, and immune system was destroyed³², which resulted animal death. But more and more studies had shown that local irradiation might enhance anti-tumor immunity. Such as 8.5 Gy × 5 irradiation of tumor had been reported to enhances MHC class I expression, dendritic cell function and improve the efficacy of tumor immunotherapy^33,34. High LET/RBE irradiation, such as particle and heavy ion radiation, could induce single and DNA double-strand breaks³⁵. meanwhile through living tissues generating ROS/RNS and H₂O₂, irradiation damaged DNA, proteins or membranes, and resulted new antigen production and strengthend immune response. The previous study of our group had confirmed that local irradiation of a single 20Gy for tumor could enhanced numbers of tumor-infiltrating CD8 + CTLs into the TME of B16-F10 tumors and the depletion of CD8 + T cells significantly weakened the therapeutic effect of irradiation³⁶. In this study, irradiation combined with ICB improved the ORR and prolonged progression-free and overall survival. In our model, we found that in Casp8-deficient patients, radiation might be an effective approach for overcoming ICB resistance. We explained a new mechanism of SBRT enhancing ICB therapy by inducing CRT expression through inhibiting Casp8, which enriched the immunological theory of SBRT enhancing immunotherapy.

Casp8 deficiency, knockout, and inhibitor treatment lead to an impaired ecto-CRT transition, which in turn resulted in hampered antigen presentation and a cold TME, which are traits associated with ICB resistance. Irradiation can rescue the ecto-CRT of tumor cells and improve phagocytosis to overcome ICB resistance. Consistent with TCGA and ICB-treated cohorts, Casp8 expression was correlated with inflamed TME and a better response to ICB. These results imply that patients with Casp8 function loss mutations may not benefit from the singlet of ICB; however, a radiation combination strategy might sensitize the non-responders to respond and improve clinical outcome.

ICB: immune checkpoint blockade, PD-1: programmed death-1 receptor, PD-L1: programmed death ligand 1, TME: tumor microenvironment, ICD: Immunogenic cell death, Casp8: Caspase-8, TCGA: The Cancer Genome Atlas, CRT: calreticulin, ecto-CRT: Surface-bound calreticulin.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The datasets supporting the conclusions in cohort analysis of this article are available in the TCGA dataset and published article.

Conflict of Interest

The authors declare no potential conflicts of interest.

Funding

This study was supported by National Nature Science Foundation of China (82073147 by QJ and 81902929 by ZG).

Authors’ contributions

YW, BZ and QJ directed the research and obtained funding. ZJ and CH performed data analysis. ZG, YZ and SX performed experiments, statistical analysis and drafted the manuscript.

Acknowledgements

Not applicable

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No competing interests reported.

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published 12 Jan, 2023

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Caspase-8 Contributes to Immuno-Hot Microenvironment by Promoting Phagocytosis via an Ecto-Calreticulin-dependent Mechanism

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Journal Publication

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Sources of publicly accessible data

Cell line

Animals and animal models

RNA-sequencing

In vitro phagocytosis experiments using bone marrow-derived dendritic cells (BMDCs)

In vivo phagocytosis assay

Flow cytometry

Statistics

Results

Casp8 indicates an immuno-hot tumor microenvironment in solid tumors

Knockout of CASP8 results in an immuno-cold microenvironment

Poor responsiveness to anti-PD-L1 treatment in the C8KO-bearing model

Responsiveness to ICB treatment in clinical cohorts

Impaired antigen presentation and ecto-calreticulin translocation in CASP8 knockout mice

Irradiation rescues ecto-CRT and sensitizes CASP8 knockout tumors to ICB treatment

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1