Radiotherapy is widely used to treat various types of human cancers (1
). The antitumor effects of radiotherapy rely on the engagement of innate and adaptive immune components (1
). Moreover, the cross-priming capacity of dendritic cells (DCs), induced by radiation treatment, requires cytosolic DNA sensing and subsequent type I interferon (IFN) signaling (4
). Cyclic guanosine monophosphate (GMP)–adenosine monophosphate (AMP) synthase (cGAS) is an essential cytosolic DNA sensor that catalyzes the formation of cyclic GMP-AMP (cGAMP) in response to DNA accumulation in the cytosol (5
). After detection of DNA in the cytosol, cGAS triggers cGAMP binding to stimulator of IFN genes (STING). STING then interacts with TANK-binding kinase 1 (TBK1) and IFN regulator factor 3 (IRF3), leading to the induction of type I IFNs (5
). cGAS is activated by the abundance of cytosolic DNA originating from micronuclei, mitochondria, or viruses in a sequence-independent manner (5
). Regardless of the ability of mitochondrial DNA (mtDNA) in the cytosol to engage cGAS, the prevailing perspective is that chromatin fragments inside micronuclei are essential for cGAS-STING–mediated type I IFN responses and antitumor host immunity after radiation treatment (6
). Moreover, the molecular events that trigger cytosolic DNA sensing by the cGAS-STING pathway in radiation-stressed tumor cells remain unclear.
Necroptosis, which requires receptor-interacting protein kinase 3 (RIPK3) activity and is executed by mixed-lineage kinase domain-like pseudokinase (MLKL) protein, has been considered to be a major modality for immunogenic cell death (12
). However, the role of necroptosis in tumor immune response is controversial. In oncogene-induced spontaneous tumor models, necroptosis was found to contribute to lineage-commitment liver tumorigenesis and pancreatic oncogenesis by accelerating inflammation (13
). By contrast, in transplant tumor models, transduction of active RIPK3 or MLKL constructs into tumor cells was shown to potentiate antitumor adaptive immunity (15
). Among the multiple signal transduction cascades that precipitate necroptosis in response to extracellular signals (e.g., ligand binding to death receptors) or intracellular stimuli (such as nucleic acids), the ZBP1-RIPK3-MLKL axis has been recently documented to aggravate chronic inflammation and influenza pathogenesis (16
). In the tumor microenvironment, there are multiple spatiotemporal barriers preventing the extracellular dissemination of damage-associated molecular patterns (DAMPs) for activation of antitumor immune cell components (20
). It is necessary to avoid potential immune escape while generating robust cross-priming of CD8+
T cells in cancer therapy (20
). However, it remains unknown how the tumor cell–intrinsic necroptotic pathway is elicited and, in turn, activates an antitumor immune response to radiation.
Tumor relapses often occur after radiotherapy in multiple types of human cancers due to immune privilege induced by tumor-intrinsic and tumor-extrinsic factors (21
). Recently, the escape of innate immune sensing was described as a key step for limiting the immunological “cold” to “hot” tumor switch after radiation. For instance, noncanonical nuclear factor κB (NF-κB) activation in tumor-infiltrating DCs was found to counteract STING-mediated type I IFN production following radiation treatment (22
). In addition, monocytic myeloid-derived suppressor cells (MDSCs) can function as feedback circuits to sequester the STING-induced antitumor immune response to radiation therapy (23
). Moreover, tumor cell–intrinsic autophagy and caspase-9 signaling give rise to radiation resistance by inhibiting STING-mediated cytosolic DNA sensing after irradiation (24
). This evidence strongly suggests that prosurvival and prodeath pathways in tumor cells both participate in the reconstitution of the tumor immune environment following radiotherapy (21
). As a master regulator of cell death programs, caspase-8 promotes extrinsic apoptosis by inducing activation of the effector caspases 3, 6, and 7 while preventing ZBP1-dependent necroptosis via cleavage of ZBP1-RIPK3 complex components (26
). In contrast to apoptosis, necroptosis is substantially more potent in triggering inflammation and adaptive immune responses (27
). Thus, tumor cell death reprogramming from apoptosis to necroptosis may be a potential strategy to improve radiotherapy.
In this study, we found that the ZBP1-MLKL necroptotic signaling cascade governs antitumor immune responses after radiation by mutually interacting with STING-mediated cytosolic DNA sensing. Correspondingly, ablation of caspase-8 promoted cytosolic DNA sensing and radiotherapeutic effects by enhancing MLKL activity. Our findings provide new insight to understand the early events that alert the immune system upon radiation and therefore indicate an alternative strategy to improve radiotherapy via cell death reprogramming.
Understanding the immunological aspects of radiotherapy aids the design of new therapeutic combinations. In this study, we found that the ZBP1-MLKL necroptotic cascade facilitated antitumor immunity of radiation through increasing cytosolic DNA sensing. In addition, caspase-8 ablation and radiation treatment synergistically increased mtDNA release and antitumor effects. Our findings highlight the importance of the ZBP1-MLKL necroptotic cascade in radiotherapy and the regulatory module to bridge tumor cell damage and antitumor immunity.
Necroptosis has been considered to be one modality for immunological cell death to cross-prime CD8+
T cells (12
). RIPK3-containing necrosome promotes the release of DAMPs and induces the maturation of DCs (15
). Breaking from the above model, our findings provide a new perspective that the tumor cell–intrinsic STING-mediated DNA sensing relays the action of ZBP1-MLKL necroptotic signaling for the cross-priming of CD8+
T cells after radiation treatment. By preserving the duration of plasma membrane integrity, ESCRT-III–mediated prolonged necroptotic cell death has been shown to promote cross-priming of CD8+
T cells (33
). It is possible that the ESCRT-III machinery regulates cytosolic DNA sensing after radiation by preventing the efflux of mtDNA from the dying cells. A recent study has demonstrated that the accumulation of MLKL at intercellular junctions potentiates necroptosis between neighboring cells (34
). It is therefore possible that necroptotic tumor cells may recruit their neighboring tumor cells to expand the necroptotic signaling and subsequent cytosolic DNA sensing by tight junction proteins after radiation treatment.
ZBP1-MLKL necroptotic signaling plays a critical role in driving chronic inflammation and influenza virus pathogenesis (17
). Besides exerting a necroptotic function, MLKL partly participates in endosomal trafficking mediated by ESCRT machinery in a cell death–independent pattern (29
). We observed the engagement of RIPK3 and MLKL was comparable in IFN-β induction after radiation, suggesting that MLKL mainly functions as the molecular machinery for necroptosis in the context of IFN-β induction after radiation treatment. Although Toll-like receptor 3 (TLR3) ligation is one of regulators involved in initiating the activation of molecular machinery for necroptosis, TLR3 mediates type I IFN production of tumor cells upon chemotherapy in an MLKL-irrelevant pattern (35
). ZBP1 is able to trigger both apoptosis and necroptosis by sensing influenza A virus genomic RNA (36
). We cannot rule out the possibility that ZBP1 simultaneously interacts with molecular machineries of apoptosis and necroptosis after radiation. At least, ZBP1 is sufficient for MLKL-dependent necroptosis regarding type I IFN production in tumor cells after radiation treatment. Of note, it has been demonstrated that MLKL can localize to both nucleus and mitochondria, most likely to induce nuclear envelope rupture and mitochondrial pore formation after radiation (18
). In addition, it will be interesting to determine how ZBP1 is up-regulated and activated by radiation.
gDNA aggregated in micronuclei is essential for cGAS-STING–mediated antitumor immunity following genotoxicity therapy and poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) inhibitor treatment (9
). In this study, we found that both cGAS-positive micronuclei and DNA damage were not affected by MLKL loss after radiation. However, we observed that the removal of mtDNA abrogated type I IFN induction in irradiated tumor cells, indicating that mtDNA may play a paralleled role with gDNA in triggering the cGAS-STING pathway in response to radiation. It has been demonstrated that HMGB/TFAM-bound U-turn DNA is efficient to bind to cGAS by forming protein-DNA ladders (39
). Therefore, TFAM may provide assistance to maintain the stability of mtDNA binding to cGAS. The release of mtDNA is the result of the inner mitochondrial membrane herniation during caspase-inhibited apoptosis (24
). Part of the RIPK3-MLKL–containing necrosome has been observed to translocate to the mitochondria in a dynamic and transient manner (37
). So one possible interpretation is that MLKL is required for the release of mtDNA instead of mitochondrial collapse after radiation; however, the relevant molecular mechanism of how MLKL regulates the release of mtDNA needs to be further determined.
Tumor radiation resistance often occurs in multiple types of human cancers, due to tumor cell–intrinsic or tumor cell–extrinsic factors (21
). Previously, we identified that PD-L1 and MDSCs act as two major barriers of antitumor immunity after radiation (23
). Given the findings in this study, the attenuation of ZBP1-RIPK3-MLKL necroptotic signaling and cGAS-STING signaling may be cell-intrinsic factors for radiation resistance. It is noticeable that caspase-8 is also involved in the regulation of cytosolic mtDNA sensing by inhibiting MLKL activation. According to the reciprocal interaction between MLKL-dependent necroptosis and caspase-8–dependent apoptosis, it would be feasible to interrupt caspase-8 to improve radiotherapy. Despite the additional role of catalytically inactive caspase-8 in modulating pyroptosis (41
), caspase-8 is evidently a major barrier for the ZBP1-RIPK3-MLKL cascade in tumor cells after radiation treatment in terms of STING-mediated type I IFN production. Because MLKL in tumor cells rather than in host cells is required for antitumor effects of radiation treatment, the fine-tuning of MLKL-dependent necroptosis by caspase-8 intervention is appreciated in tumor cells to improve radiotherapy. In addition, ISGs in tumor cells contribute to radiation resistance through limiting both adaptive and innate immune killing (42
). Genetic abolition of type I IFN signaling in tumor cells improves radiation or treatment naïve (42
). These evidences raise a possibility that the ISG signature affects antitumor immune responses of radiation in a cell type–dependent pattern.
Our study does have certain limitations, such as the reason of ZBP1 up-regulation and the localization of MLKL in tumor cells after radiation treatment. Whether MLKL localizes to the mitochondrial membrane to trigger mtDNA release into the cytoplasm after radiation treatment needs to be further determined. Whether ZBP1-RIPK3-MLKL axis and caspase-8 can be an indicator of tumor prognosis in patients with radiotherapy also needs to be investigated. Although the positive correlation between the DNA-sensing signature and ZBP1/MLKL expression is observed in mentioned TCGA-Pan Cancer with treatment naïve, whether the correlation implies that all those tumors should have a better response to irradiation or respond to irradiation through the same pathway needs to be further investigated.
In summary, our results define a previously unrecognized cross-talk between ZBP1-MLKL necroptotic cascade and STING-mediated cytosolic DNA sensing, which governs cross-priming of CD8+ T cells after radiation treatment. Increasing MLKL activity by caspase-8 inhibition greatly augments STING activation and antitumor effects of radiation. Moreover, these data specify a new modality of danger signal recognition that alters immunological aspects of radiotherapy, and indicate an alternative strategy to improve radiotherapy.
MATERIALS AND METHODS
The objective of this study was to identify the potential mechanism of how necroptotic signaling cascade initiates antitumor immune responses to radiation. We generated MLKL-, ZBP1-, cGAS-, or STING-deficient tumor cell lines by CRISPR-Cas9 technology with distinct guide RNAs and implanted these tumor cell lines on the flanks of WT mice. Then, we monitored tumor growth and analyzed the percentage and function of CD8+ T cells in the tumors, spleens, and DLNs after receiving local radiation. To investigate how ZBP1-MLKL axis induces antitumor host immunity after radiation, we performed RNA-seq using tumor cells with or without MLKL. In addition, the association between the expression of ZBP1 or MLKL and the enrichment score of cytosolic DNA–sensing pathway was analyzed in human cancers from TCGA. To investigate whether ZBP1-MLKL cascade is essential for type I IFN responses in tumor cells after radiation, we detected phosphorylation of STING and TBK1 by Western blotting and Ifnb1 mRNA expression by real-time PCR. To determine whether MLKL regulates cytosolic DNA releasing in irradiated tumor cells, cytosolic DNA was extracted and measured by real-time PCR. To further explore whether mtDNA is responsible for radiation-induced type I signaling in tumor cells, we generated mtDNA-depleted tumor cells by culturing MC38 in the presence of EtBr. Last, to assess whether overactivation of MLKL improves therapeutic effects of radiation, we generated caspase-8–deficient tumor cells to enhance the activity of MLKL and STING and antitumor effects after radiation.
In our study, for cell-based experiments, at least biological triplicates were performed in each single experiment, unless otherwise stated. Animals were randomized into different groups after tumor cell inoculation and at least four to six mice were used for each group; then, one fraction of local radiation was administrated for tumor treatment. Three to five mice were used for tumor microenvironment analysis. For cell-based experiments, the number of biological replicates was illustrated in each figure. Whenever possible, the investigators were blinded to group allocation during the experiments and when assessing outcomes. In some cases, selected samples were excluded from specific analyses because of technical flaws during sample processing or data acquisition. Analytical studies were typically performed according to two or three independent experiments.
Six- to 8-week-old female C57BL/6J (WT) mice were purchased from Shanghai Slac Laboratory Animal Co. Ltd., China. MLKL-KO mice were purchased from the National Resource Center of Model Mice (NRCMM), Nanjing, China. All the mice were maintained under specific pathogen–free conditions at the animal facility of Shanghai Jiao Tong University School of Medicine. All the animal studies were conducted in compliance with the protocol approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.
Cell lines and culture conditions
MC38, B16-SIY, and A549 were contributed by the laboratory of Y.-X. Fu; 293T was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) (catalog no. SCSP-502); and E0771 was from the laboratory of B. Zhou. These cell lines were maintained at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium–high glucose medium (HyClone, catalog no. SH30243.01) containing 10% fetal bovine serum (FBS) (Gemini, catalog no. 900-108), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10 mM Hepes (Thermo Fisher Scientific, catalog nos. 15140122 and 15630080). All cell lines were tested to be mycoplasma free. MLKL−/− MC38, ZBP1−/− MC38, RIPK3−/− MC38, cGAS−/− MC38, STING−/− MC38, caspase-8−/− MC38, MLKL−/− E0771, ZBP1−/− E0771, and MLKL−/− B16-SIY cell lines were generated using CRISPR-Cas9 plasmid lenti-crispr-V2 (from the laboratory of X.-Y. Liu). The annealed single-guide RNA (sgRNA) oligos (see table S1) were cloned into pLenti-CRISPR-V2 and packaged in 293T cells (Cell Bank of the Chinese Academy of Sciences, catalog no. SCSP-502). Supernatants containing virus particles were collected 24 and 48 hours after transfection and then added to preplated cells with polybrene (2 μg/ml; Genomeditech, catalog no. GM-040901B). The transduced cells were selected by puromycin (Beyotime Biotechnology, catalog no. ST551) at 2 μg/ml to acquire the gene-deleted stable cell lines. The efficiency of gene knockdown was determined via Western blotting. In addition, tdTomato-expressing Ctrl-MC38 cells and tdTomato-expressing MLKL−/-MC38 cells were generated using pLenti-CMV-tdTomato (from the laboratory of J. Wang). Briefly, Ctrl-MC38 or MLKL−/− MC38 cells were infected by lentivirus supernatants of tdTomato with polybrene (2 μg/ml). tdTomato+ cells were sorted out with a cell sorter (BD Biosciences) and cultured for further experiments.
In vivo tumor models
To explore the effects of radiation on tumor growth, WT mice were subcutaneously inoculated with 2 × 106 Ctrl-MC38, MLKL−/− MC38, ZBP1−/− MC38, STING−/− MC38, or caspase-8−/− MC38, which were resuspended in the mixture of phosphate-buffered saline (PBS) and Matrigel matrix (Corning BioCoat, catalog no. 356234) (100 μl, v/v = 1:1). In B16-SIY model, WT mice were subcutaneously inoculated with 1 × 106 Ctrl-B16-SIY or MLKL−/− B16-SIY tumor cells. To investigate the role of host MLKL in tumor growth, WT and MLKL-KO mice were subcutaneously inoculated with 1 × 106 MC38 cells resuspended in 100 μl of PBS (HyClone, catalog no. SH30256.01). Tumors were measured, randomly grouped, and locally irradiated at a single fraction of 15 Gy on the indicated day, and then tumors were monitored twice a week afterward. For the detection of abscopal effects of radiation, WT mice were subcutaneously inoculated with 1.5 × 106 Ctrl-MC38 or MLKL−/− MC38 cells resuspended in the mixture of PBS and Matrigel matrix on the right flank and with 1 × 106 Ctrl-MC38 cells resuspended in 100 μl of mixture of PBS and Matrigel matrix on the corresponding opposite flank of the same mice. Tumors on the right flank were subjected to local radiation at a single fraction of 15 Gy, while tumors on the left flank were shielded from radiation. Tumors on both flanks were monitored twice a week subsequently.
Quantitative reverse transcription PCR
Total RNA for real-time PCR assay was extracted and purified using the TRIzol Reagent (Thermo Fisher Scientific, catalog no. 15596018). Reverse transcription reactions were performed with ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, catalog no. FSQ-301) following the standard protocol. Quantitative reverse transcription PCR was performed with SYBR Green Realtime PCR Master Mix (Toyobo, catalog no. QPK-201) in the ViiA 7 Real-Time PCR System with 384-well block (Applied Biosystems, Life Technologies). The expression of mRNA was normalized against glyceraldehyde-3-phosphate dehydrogenase (Gapdh) by the change in cycling threshold (ΔCt) method. Primers used in this study are included in table S1.
For whole-cell protein extraction, pretreated cells were washed with cold PBS and lysed in cell lysis buffer for Western blotting and immunoprecipitation (Beyotime Biotechnology, catalog no. P0013). The protein samples were quantified using a BCA protein assay kit (Beyotime Biotechnology, catalog no. P0012), loaded on SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels, electrophoresed, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, catalog no. IPVH00005), and further incubated with the primary and secondary antibodies. For STING dimer detection, the pretreated cells were washed with cold PBS and lysed in cell lysis buffer for 2 hours at 4°C with gentle rocking. Then, the protein samples were diluted with nondenatured gel sample loading buffer [4×; formulation: 40 ml of glycerol, 20 ml of 1 M tris (pH 6.8), 1 ml of 2% bromophenol blue, and 40 ml of 20% SDS] without heating and loaded on SDS-PAGE gels. Last, the blots were developed following protocols as above. The following primary antibodies were obtained from Cell Signaling Technology: anti–phospho-MLKL (Ser345) (D6E3G) (catalog no. 37333), anti-STING (D2P2F) (catalog no. 13647), anti–phospho-STING (Ser365) (D8F4W) (catalog no. 72971), anti-cGAS (D3O8O) (catalog no. 31659), rabbit anti-TBK1 (catalog no. 3013), anti–phospho-TBK1(S172) (D52C2) (catalog no. 5483), and anti–caspase-8 (catalog no. 4927); from Millipore: anti-MLKL (3H1) (catalog no. MABC604); from AdipoGen: anti-ZBP1 (Zippy-1) (catalog no. AG-20B-0010); from Prosci: anti-RIPK3 (catalog no. 2283); and from Proteintech: anti-tubulin (catalog no. 10094-1-AP) and anti-GAPDH (catalog no. 10494-1-AP).
Subcutaneous tumors with or without radiation were removed on indicated days, cut into small pieces with ophthalmic forceps, digested with collagenase I (1 mg/ml; Worthington Biochemical, catalog no. LS004196) and deoxyribonuclease (DNase) I (0.2 mg/ml; Sigma-Aldrich, catalog no. DN25) for 30 min, and filtered with 70-μm cell strainers to obtain the single-cell suspensions followed by cell staining and flow cytometry analysis. Briefly, for surface marker staining, single cells prepared as above were incubated with anti-mouse CD16/32 (2.4G2) (Bio X Cell, catalog no. CUS-HB-197) at 4°C for 15 min, washed with fluorescence-activated cell sorting (FACS) buffer (2% FBS in PBS), and then incubated at 4°C for 30 min with antibody cocktail against anti-CD45–BV785 (104) (BioLegend, catalog no. 109839), anti-CD8–AF700 (53-6.7) (BioLegend, catalog no. 100730), and live/dead (Fixable Viability Stain 780, BD Biosciences, catalog no. 565388). When intracellular staining was needed, single-cell suspensions were fixed with fixation/permeabilization buffer (A:B = 1:3) (Invitrogen, catalog nos. 00-5223-56 and 00-5123-43) at 4°C for 1 hour, washed twice with permeabilization buffer (Invitrogen, catalog no. 00-8333-56), and incubated in corresponding antibody cocktails (in permeabilization buffer) in the dark at 4°C for 40 min to 1 hour. To evaluate the function of T cells in tumor microenvironments, single-cell suspensions were treated with Cell Stimulation Cocktail (plus protein transport inhibitors) (eBioscience, catalog no. 00-4975-03) for 3 hours before cell staining, and the percentages of IFN-γ+TNF-α+CD8+ T cells in tumor microenvironments were determined with flow cytometry. Last, multicolor flow cytometry was performed with either LSR Fortessa or LSR X20, while cell sorting was performed with FACSAria III (BD Biosciences). The data acquired would be further analyzed with FlowJo software (Tree Star).
Single-cell suspensions from tumor tissues were obtained as mentioned above. For the isolation of tdTomato-labeled MC38 tumor cells, single-cell suspensions were blocked with anti-mouse CD16/32 and then stained with anti-CD45 and live/dead. For CD11c+ DC isolation, single-cell suspensions were blocked with anti-mouse CD16/32 and then stained with antibodies anti-CD11c–allophycocyanin (N418) (BioLegend, catalog no. 117310), anti-mouse I-A/I-E-PerCP/Cy5.5 (M5/114.15.2) (BioLegend, catalog no. 107626), anti-CD45.2–AF700 (104) (eBioscience, catalog no. 56045481), and live/dead. The cells were performed with FACSAria III Cell Sorter (BD Biosciences).
For tumor-specific CD8+ T cell functional assay, DLNs or spleens from tumor-bearing mice were removed 7 days after radiation, cut into small pieces, grinded, and filtered through 70-μm cell strainers to obtain the single-cell suspensions. Subsequently, CD8+ T cells were selected by EasySep Mouse CD8a Positive Selection Kit II (STEMCELL, catalog no. 18953). MC38 tumor cells were preexposed to murine IFN-γ (20 ng/ml) for 24 hours before plating with purified CD8+ T cells. CD8+ T cells (2 × 105) were incubated with MC38 at a ratio of 10:1 for 48 hours, and ELISPOT assays were performed to detect IFN-γ spots according to product protocol (BD Biosciences, catalog no. 551083).
Cross-priming activity by DCs
MC38-SIY cells (3 × 105) were plated into a six-well plate overnight, then treated with radiation at 15 Gy, and incubated for 48 hours. For the control group, 1.5 × 105 MC38-SIY cells were plated into a six-well plate. BMDCs were harvested on day 6, and then 1.2 × 106 BMDCs were added and cocultured with MC38-SIY cells in the presence of fresh granulocyte-macrophage colony-stimulating factor (GM-CSF) at 5 ng/ml for additional 12 hours. Subsequently, 2 × 104 CD11c+ cells purified with EasySep Mouse CD11c Positive Selection Kit II (STEMCELL, catalog no. 18780) were incubated with isolated CD8+ T cells from naïve 2C-TCR transgenic mice using EasySep Mouse CD8a Positive Selection Kit II (STEMCELL, catalog no. 18953) for 3 days at a ratio of 1:10. For tumor-infiltrating DCs, CD11c+ DCs were sorted with a BD FACSAria III cell sorter from tumors 3 days after radiation. The sorted CD11c+ cells were cocultured with isolated CD8+ T cells from naïve 2C-TCR transgenic mice for 3 days at a ratio of 1:10. The supernatants were collected for IFN-γ detection using Cytometric Bead Array (CBA) Mouse IFN-γ Flex Set (RUO) (BD Biosciences, catalog no. 558296) according to standard protocols.
Briefly, Ctrl-MC38 (CRISPR-Ctrl) or MLKL−/-MC38 (MLKL-gRNA3) tumor cells were subjected to a single fraction of 15 Gy, and total RNA from each sample was extracted using TRIzol Reagent (Thermo Fisher Scientific) following standard protocols 60 hours later. The concentration and quality of RNA samples were determined with a NanoDrop 2000 spectrophotometer (NanoDrop Technologies) and the Agilent 2100 Bioanalyzer (Agilent Technologies). Qualified RNA samples were used for RNA-seq and data analysis (Novogene, Shanghai, China). Data analysis was performed using R software (R Foundation for Statistical Computing). RNA-seq data generated in this paper were deposited at the Gene Expression Omnibus (accession number GSE168016).
Detection of cytosolic DNA
MC38 cells were cultured in a 10-cm dish and subjected to radiation at 15 Gy. Fifty-four hours later, total DNA and cytosolic DNA were extracted and detected as described previously (45
). Briefly, MC38 cells (2 × 106
to 1 × 107
) were divided into two equal aliquots. One aliquot was resuspended in 300 μl of 50 mM NaOH and boiled for 30 min to solubilize DNA. Thirty microliters of 1 M tris-HCl (pH 8.0) was added to neutralize the pH and then centrifuged at 12,000 rpm for 10 min to pellet intact cells. In addition, these extracts served as normalization controls for total gDNA and mtDNA. The second equal aliquots were resuspended in about 300 μl of buffer containing 150 mM NaCl, 50 mM Hepes (pH 7.4), and digitonin (15 to 25 mg/ml; Sigma-Aldrich, catalog no. 300410). The homogenates were incubated end over end for 10 min on ice to allow selective plasma membrane permeabilization and then centrifuged at 980g
for 3 min three times to pellet intact cells. Last, the cytosolic supernatants were transferred to fresh tubes and spun at 17,000g
for 10 min to pellet any remaining cellular debris, yielding cytosolic preparations free of nuclear, mitochondrial, and endoplasmic reticulum contamination. The cytosolic DNA and whole-cell DNA were purified using DNA Clean & Concentrator-5 (ZYMO RESEARCH, catalog no. D4013). Quantitative real-time PCR was performed on both whole-cell extracts and cytosolic fractions using gDNA primers (Tert and Plog1) and mtDNA primers (Dloop1 and Dloop2), and the Ct
values for whole-cell extracts served as normalization controls for the values obtained from the cytosolic fractions.
MC38 cells were developed in the presence or absence of EtBr (200 to 400 ng/ml) (Sigma-Aldrich, catalog no. E7637) or 50 μM ddC (Sigma-Aldrich) for 6 days. The content of mitochondrial and nuclear DNA in EtBr- or ddC-treated tumor cells was confirmed by real-time PCR. Afterward, tumor cells were collected and cultured without EtBr or ddC followed by radiation treatment.
Analysis of RNA-seq and TCGA datasets
RNA-seq was performed by Illumina NovaSeq 6000 with 150–base paired-end reads. All reads were aligned to the mouse reference genome (mm10) using hisat2 v2.0.5 with the default setting. HTSeq was used to count the read numbers mapped to each gene. DESeq2 was used to normalize the raw counts and identify differentially expressed genes [|fold change| ≥ 1.5; false discovery rate (FDR) < 0.05]. Gene ontology enrichment analysis was performed by R package clusterProfiler, where the differentially expressed genes identified as described above were supplied as the input for genes by function enrichKEGG. Hypergeometric tests were used to calculate P values, which were then subjected to multiple testing adjustments by Benjamini-Hochberg correction. GSEA was performed to test whether interested gene sets are substantially enriched in corresponding conditions.
Gene-level RNA-seq expression data [normalized RSEM (RNA-seq by expectation-maximization) value] of 33 cancer types were downloaded from TCGA data portal (https://portal.gdc.cancer.gov/
). Normalized gene expression values were then log2
-transformed. To generate gene expression signature, we took the mean of log2
-transformed expression values of signature genes. The signature genes of cytosolic DNA–sensing pathway signature (Zbp1
, and Polr3g
) and type I IFN response (Zbp1
, and Shmt2
) are obtained from The Molecular Signatures Database (MSigDB; http://software.broadinstitute.org/gsea/msigdb/
). Gene set variation analysis (GSVA) was performed to calculate the score of these two pathways. The infiltration of CD8+
T cells was determined by CIBERSORT (http://cibersort.stanford.edu/
). We calculated the Spearman correlation between pathway GSVA score or the infiltration of CD8+
T cells and the RNA expression candidate genes, considering |Rs| > 0.2 and FDR < 0.05 for statistical significance.
No statistical method was used to predetermine sample size. Mice were assigned at random to treatment groups for all mouse studies. Experiments were repeated two to three times. Group sizes and number of replications are provided in the figure legends. Statistical analysis was performed using Prism software (GraphPad Prism8 software). Tumor growth monitor, flow cytometry, and quantitative real-time PCR were analyzed using one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test, Brown-Forsythe and Welch ANOVA tests with Tamhane’s T2 multiple comparisons test, two-tailed unpaired t test, unpaired t test with Welch’s correction, or two-way ANOVA with Bonferroni’s multiple comparisons test as indicated in the figure legends. Data were presented as mean values ± SEM. We indicated significance corresponding to the following: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
We thank B. Su, Y.-X. Fu, and S. Wang for helpful scientific discussion. Funding: This work was supported by the National Natural Science Foundation of China (81771682 and 82071741 to L.D., 81702804 to W.L., 31900649 to M.W., and 82030082 to J.Y.), National Thousand Youth Talents Program (to L.D.), Science and Technology Commission of Shanghai Municipality (16JC1406000 to L.D.), Innovative research team of high-level local universities in Shanghai (to L.D. and W.L.), Shanghai Municipal Commission of Health and Family Planning (20194Y0625 to W.L.), and the Interdisciplinary Program of Shanghai Jiao Tong University (YG2021ZD03 to L.D.). Author contributions: Y.Ya. and M.W. performed the experiments and wrote the paper. D.C. and C.Y. performed part of experiments. J.J. and C.Y. aided in the experiments of flow cytometry assay. L.W., X.H., W.L., and J.L. aided in some experiments of real-time PCR and Western blot. Y.Ya., M.W., L.L., X.W., and Y.Ye. analyzed the data. X.M., Z.Z., Y.Ye., H.X., and J.Y. helped in editing the manuscript. L.D. conceived and supervised all experiments and the writing of the manuscript. All authors approved the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.