Articles

https://doi.org/10.1038/s41590-020-0641-5

Tumor cells suppress radiation-induced immunity

by hijacking caspase 9 signaling
Chuanhui Han1,4, Zhida Liu1,4, Yunjia Zhang2, Aijun Shen1, Chunbo Dong1, Anli Zhang   1,
Casey Moore   1,3, Zhenhua Ren   1, Changzheng Lu1, Xuezhi Cao1, Chun-Li Zhang2, Jian Qiao   1 ✉
and Yang-Xin Fu   1,3 ✉

High-dose radiation activates caspases in tumor cells to produce abundant DNA fragments for DNA sensing in antigen-pre-
senting cells, but the intrinsic DNA sensing in tumor cells after radiation is rather limited. Here we demonstrate that irradiated
tumor cells hijack caspase 9 signaling to suppress intrinsic DNA sensing. Instead of apoptotic genomic DNA, tumor-derived
mitochondrial DNA triggers intrinsic DNA sensing. Specifically, loss of mitochondrial DNA sensing in Casp9−/− tumors abol-
ishes the enhanced therapeutic effect of radiation. We demonstrated that combining emricasan, a pan-caspase inhibitor, with
radiation generates synergistic therapeutic effects. Moreover, loss of CASP9 signaling in tumor cells led to adaptive resistance
by upregulating programmed death-ligand 1 (PD-L1) and resulted in tumor relapse. Additional anti-PD-L1 blockade can further
overcome this acquired immune resistance. Therefore, combining radiation with a caspase inhibitor and anti-PD-L1 can effec-
tively control tumors by sequentially blocking both intrinsic and extrinsic inhibitory signaling.

C
urrent dogma is that radiation induces the fragment of
genomic DNA (gDNA) that increases DNA sensing in
dendritic cells to produce low but detectable type I inter-
feron (IFN) for triggering antitumor immunity1,2. However, irradi-
ated tumor cells can escape immune surveillance and eventually
relapse. As the major component of the tumor microenvironment
(TME), irradiated tumor cells often fail to activate innate sens-
ing to produce type I IFNs. Although previous studies have pro-
posed several strategies to enhance DNA sensing in tumor cells to
improve the therapeutic effect of radiation3,4, these strategies only
slightly increase type I IFN production. Whether major intrin-
sic barriers exist that limit innate sensing, particularly cytosolic
DNA sensing in irradiated tumor cells has been a subject of
debate. Facilitating tumor-derived type I IFN by enhancing DNA
sensing may be a good strategy to enhance radiation-mediated
antitumor immunity.
Intrinsic apoptosis is initiated by mitochondrial permeabiliza-
tion, which promotes the release of cytochrome c to interact with
apoptotic peptidase activating factor 1 (APAF1) and CASP9 to form
the functional apoptosome complex5. Although radiation induces
gDNA damage, leading to mostly intrinsic apoptosis6,7, radiation-
induced mitochondrial permeabilization might also promote
the release of mitochondrial DNA (mtDNA) into the cytosol8–10.
Radiation-induced genome-derived micronuclei DNA can slightly
trigger double-stranded DNA (dsDNA) sensing after radiation
treatment, but it is still largely unclear whether or how the effect
of radiation-induced cytosolic mtDNA in tumor cells can trigger
better intrinsic innate sensing. In our current study, we observed
that blocking radiation-induced CASP9 activation provokes tumor-
intrinsic mtDNA sensing for better T cell priming. Consequently,
however, tumors evolve a counter mechanism to increased innate
sensing by upregulating PD-L1. Therefore, the combination of
radiotherapy with caspase inhibition and anti-PD-L1 were tested
to determine the antitumor efficacy by blocking both intrinsic and
extrinsic inhibitory signaling.
Results
Tumor cells hijack intrinsic apoptosis to restrict type I IFN pro-
duction after radiation. To explore whether and how tumor cells
restrict intrinsic DNA sensing for type I IFN production after
radiation, we first screened US Food and Drug Administration
(FDA)-approved drugs and other inhibitors that may block specific
pathways to break this barrier. Forty-two drugs were selected to treat
irradiated MC38 cells. Drugs were scored by their ability of promot-
ing type I IFN production in tumor cells, which was analyzed by
the RAW-Lucia ISG (RAWISG) reporter system11. We found that
emricasan treatment markedly increased the production of type I
IFNs from irradiated tumors (Fig. 1a). Emricasan is a caspase inhib-
itor that has been approved by the US FDA as an orphan designa-
tion for treatment of liver transplant recipients with re-established
fibrosis and as the first tract designation for treatment of nonalco-
holic steatohepatitis cirrhosis to inhibit liver cell death12,13. We con-
firmed the enhanced production of IFN-β by ELISA (Fig. 1b) and
the expression of IFN-α by quantitative real-time PCR (RT-qPCR)
(Supplementary Fig. 1a,b). Emricasan-alone treatment only slightly
increased type I IFN production, but induced about a thousand-fold
increase of IFN-β (Fig. 1c) and IFN-α (Supplementary Fig. 1a,b)
in tumor cells after radiation treatment. To further confirm the
role of caspases, we also tested another pan-caspase inhibitor,
Q-VD-oph (QVD). Both emricasan and QVD treatment dramati-
cally increased irradiated tumor-derived type I IFN production
(Supplementary Fig. 1c). These data suggest that tumor-intrinsic
caspases are a major barrier to limit production of type I IFN by
irradiated tumor cells.
As pan-caspase inhibitors, emricasan and QVD can block both
intrinsic and extrinsic apoptosis. To further explore which apoptosis

1
Department of Pathology, UT Southwestern Medical Center, Dallas, TX, USA. 2Department of Molecular Biology, UT Southwestern Medical Center,
Dallas, TX, USA. 3Department of Immunology, UT Southwestern Medical Center, Dallas, TX, USA. 4These authors contributed equally: Chuanhui Han,
Zhida Liu. ✉e-mail: Jian.Qiao@UTSouthwestern.edu; Yang-Xin.Fu@UTSouthwestern.edu

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Articles NATure IMMunoLogy

a b c
6,000 5,000 *
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Fig. 1 | Tumor cells hijack intrinsic apoptosis to restrict type I IFN production after radiation. a, MC38 cells (n = 3 per group) were treated with radiation
(IR; 40 Gy) and drugs (10 µM). At 48 h after radiation, supernatants were collected and added to RAWISG to detect type I IFN activity. b, MC38 cells
(n = 3 per group) were untreated (NR, no radiation) or treated with radiation (IR 40 Gy) and/or emricasan (10 µM); 48 h after radiation, supernatants were
collected to detect IFN-β by ELISA. c, MC38 cells (n = 3 per group) were irradiated (IR 15 Gy) or untreated (NR); 48 h later, cells were collected for IFN-β
mRNA detection by RT-qPCR. WT, wild type. d, Indicated cells (n = 3 per group) were irradiated (IR 40 Gy) or untreated (NR) and 48 h later, supernatants
were collected and added to RAWISG to detect type I IFN activity. e, Indicated cells were irradiated (IR 40 Gy) or untreated (NR); 48 h later, cells were
collected and expression of indicated proteins was assayed by western blot. f, MC38 cells (n = 3 per group) were irradiated (IR 40 Gy) or untreated (NR)
with or without drug treatment; 48 h after IR, IFN activity was detected by RAWISG. Data are shown as mean ± s.e.m. and are representative of two
(a,e) or three (b,d,f) independent experiments; c is a pool of three independent experiment. Statistical analysis was performed by an unpaired two-tailed
Student’s t-test in b–d,f. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

pathway is required for restricting tumor-derived type I IFN pro- We then compared the type I IFN production in irradiated tumor
duction, we used the CRISPR–Cas9 system to knock out caspase cells during the blockade of caspases or with CASP9 deficiency.
9 (Casp9, the key mediator of intrinsic apoptosis) and caspase Casp9−/− tumor cells produced a similar level of type I IFNs as wild-
8 (Casp8, the key executor of extrinsic apoptosis) in tumor cells. type tumors under caspase inhibition (Fig. 1f). Moreover, type I
Similarly to wild-type tumor cells, Casp8−/− tumor cells failed to IFN production is detectable 36 h after radiation (Supplementary
produce type I IFNs after radiation treatment (Supplementary Fig. 1f). To address the effect of radiation dose, tumor cells were
Fig. 1d). However, loss of Casp9 in tumor cells dramatically increased treated with different doses of radiation. As shown in Supplementary
type I IFN production after 15 or 40 Gy of radiation treatment (Fig. 1c Fig. 1g, radiation increased type I IFN production in a dose-depen-
and Supplementary Fig. 1d). This indicates that CASP9 signaling dent fashion during blockade of caspases. We also evaluated the
plays a vital role in restricting type I IFN production in irradiated phenotype in TS/A tumor cells. Similarly to MC38 tumor cells,
tumor cells. To further validate the role of the intrinsic apoptosis radiation also promotes Casp9−/− TS/A tumor cells to produce much
pathway, we established APAF1-deficient tumor cells. Similarly, lack more type I IFN (Supplementary Fig. 1h). All these data indicate
of APAF1 also markedly increased type I IFN production in irra- that radiation-mediated activation of CASP9 signaling is the major
diated tumor cells (Fig. 1d). We also investigated the activation of barrier of type I IFN production in tumor cells.
radiation-induced intrinsic apoptosis. Indeed, radiation increased
the activation of CASP3, a key executor of cell apoptosis. However, Tumors evade radiation-induced CD8+ T  cell-mediated antitu-
blockade with caspase inhibitors or loss of CASP9 or APAF1 abol- mor immunity through activation of intrinsic CASP9 signaling.
ished the activation of CASP3 (Fig. 1e and Supplementary Fig. 1e). Type I IFN signaling is required for tumor-specific T cell-mediated

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a b
100 1,000 NR RlgG (n = 5)

Tumor volume (mm3)

IR RlgG (n = 7)

Survival (%)
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0
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Days after tumor inoculation

Fig. 2 | Tumor-intrinsic CASP9 signaling suppresses radiation-induced antitumor immunity. a, C57BL/6J mice were transplanted subcutaneously
(s.c.) with 1 × 106 MC38 or 2 × 106 MC38 Casp9−/− cells. Tumors were untreated (NR) or treated locally with one dose of 15 Gy (IR) radiation on day 8.
b, C57BL/6J mice bearing MC38 Casp9−/− tumors were treated as in a. To deplete T cells, mice were treated with anti-CD4 or anti-CD8 antibody
(200 μg per mouse, intraperitoneally (i.p.) starting on day 7, twice a week). RlgG, rat IgG. c, MC38 Casp9−/− tumor-free mice after IR treatment or naive
mice were rechallenged with 5 × 106 MC38 cells on the left flank. d, BMDCs (n = 3 per group) were exposed to OVA protein (40 µg ml−1) for 12 h. Then
naive CD8+ OT1 T cells, the 20-fold diluted supernatants from IR or NR tumor cells and control IgG or anti-IFNαR1 (50 µg ml−1) were added. At 72 h after
incubation, the level of IFN-γ was determined by cytometric bead array assay. e, C57BL/6J, Ifnar1−/− or Batf3−/− mice were transplanted s.c. with 2 × 106,
1 × 106 or 1 × 106 MC38 Casp9−/− cells, respectively. Tumors were treated as described in a. Data are shown as mean ± s.e.m. and are representative of two
experiments (d,e), a pool of three (c) or a pool of two (a,b) independent experiments. Statistical analysis was performed by unpaired two-tailed Student’s
t-test in d, two-way analysis of variance (ANOVA) in b,c or log-rank Mantel–Cox test in a,e. *P < 0.05, **P < 0.01, ****P < 0.0001.

antitumor effects in radiation, chemotherapy, targeted therapy, anti-
CD47 treatment and anti-Her2 treatment1,14–17. As described above,
we observed that loss of Casp9 in tumor cells also slightly increased
type I IFN production without irradiation. Thus, we first evalu-
ated whether tumor endogenous Casp9 influences tumor growth
and the therapeutic effect of radiation. Casp9 deficiency does
not influence tumor growth in vitro (Supplementary Fig. 2a), but
restrains tumor growth in  vivo (Supplementary Fig. 2b). Notably,
most Casp9−/− tumors completely regressed after radiation treat-
ment (Supplementary Fig. 2c). To exclude the effect of tumor size
on treatment response, we inoculated twice as many Casp9−/− cells
as wild-type cells to ensure that control tumors were the same size
on the day of radiation. Similarly, radiation could only restrict early-
stage parental tumor growth and tumors relapsed soon. However,
most Casp9−/− tumors completely regressed (Fig. 2a). None of
the tumor-free mice relapsed within 60 d after radiation, indicat-
ing that deficiency of tumor-intrinsic CASP9 signaling markedly
increases survival and prevents recurrence (Fig. 2a). To further
test whether adaptive immunity is essential for tumor control, we
inoculated CASP9−/− and wild-type tumor cells in Rag1−/− mice.
The result showed that Casp9−/− tumors grew similarly to wild-type
tumors and are resistant to radiation treatment in Rag1−/− mice
(Supplementary Fig. 2d). This indicates that adaptive immunity
is required in radiation therapy for Casp9−/− tumors. To further
address which T cell population plays the dominant role in limiting
tumor growth, we depleted CD4+ or CD8+ T  cells with anti-CD4
or anti-CD8 antibody, respectively. Casp9−/− tumors grew similarly
as wild-type tumors and resisted radiation in the absence of CD8+
T  cells, whereas depletion of CD4+ T  cells had no effect (Fig. 2b
and Supplementary Fig. 2e). To address whether loss of Casp9 in
tumor cells could generate a strong antitumor memory response
after radiation, tumor-free mice from MC38 Casp9−/− group were
rechallenged with a fivefold higher number of MC38 tumor cells
60 d after radiation. None of the tumors grew out in mice with cured
MC38 Casp9−/− tumors (Fig. 2c). These results suggest that blocking
the intrinsic apoptosis pathway in tumor cells sensitizes the tumor
to radiotherapy, leading to a CD8+ T  cell-dependent antitumor
immune response.
Type I IFN signaling plays an essential role in cross-priming CD8+
T  cells after radiation treatment1,2,18,19. Moreover, tumor-resident
CD103+ dendritic cells are critical in priming tumor-specific T cell
responses during treatment20,21. To further address mechanisms for
enhanced adaptive immunity by irradiated tumors, we performed
a cross-priming assay using tumor supernatants. We observed that
supernatants of irradiated Casp9−/− tumor cells markedly increased
IFN-γ protein levels in a bone marrow dendritic cell (BMDC) and
T  cell co-culture system. To test whether type I IFN is an essen-
tial mediator, we blocked type I IFN signaling with anti-IFNαR1

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antibody and observed abolishment of enhanced cross-priming
activity (Fig. 2d). This indicates that lack of Casp9 in tumor cells
could increase cross-priming of antitumor CD8+T cells by targeting
the type I IFN pathway. To address the role of type I IFN signaling,
we also performed experiments in Ifnar1−/− mice; the radiation can-
not clear Casp9−/− tumor cells in IFNαR1-deficient mice (Fig. 2e).
This indicates that blocking tumor endogenous CASP9 signaling
increases radiation-mediated antitumor immunity by enhancing
type I IFN-mediated cross-priming. Batf3 is highly expressed on
conventional dendritic cells, which are essential for cross-priming22.
Thus, to further validate the role of cross-priming DCs for antitu-
mor immunity, Batf3–/– mice were inoculated with wild-type and
Casp9−/− tumors. Unlike in wild-type mice, loss of tumor-intrinsic
CASP9 signaling cannot limit tumor growth and enhance the thera-
peutic effect of radiation (Fig. 2e and Supplementary Fig. 2f). Taken
together, our results indicate that CASP9 signaling in tumor cells
can limit the radiation-enhanced cross-priming of tumor-specific
T cell immune response.
Blocking CASP9 signaling to facilitate tumor-intrinsic mtDNA
sensing after radiation. Radiation was reported to increase produc-
tion of gDNA-containing micronuclei and activate cyclic GMP–AMP
synthase (cGAS) and stimulator of interferon gene (STING; encoded
by the gene Tmem173) pathway to produce type I IFNs in a DNA-
dependent protein kinase catalytic subunit (DNA-PKcs)-dependent
manner4,23–25. We therefore investigated whether gDNA is required for
production of type I IFNs by irradiated Casp9−/− tumor cells. Indeed,
we observed that radiation increases cytosolic gDNA (Fig. 3a).
However, emricasan treatment reduces cytosolic gDNA levels
regardless of radiation treatment (Fig. 3a). Similarly, radiation also
increases cytosolic mtDNA levels, but emricasan treatment does not
influence the cytosolic mtDNA level (Fig. 3b). It raises an interesting
model that mtDNA might be the major DNA source for sensing. To
further identify the localization of mtDNA, we performed super-
resolution imaging (SIM). Consistently, we observed that emricasan
single treatment does not affect mtDNA releasing and radiation
treatment promotes release of mtDNA into cytosol (Supplementary
Fig. 3a–d). To further investigate the effect of gDNA to type I IFN
production, we observed that blocking the DNA-PK key pathway
for radiation-induced gDNA sensing, does not affect type I IFN
production during caspase inhibition (Supplementary Fig. 3e).
We proposed that mtDNA may play an essential role in irradiated
Casp9−/− tumor-derived type I IFNs. Notably, in viral infection and
hematopoietic cell development, it has been reported that activation
of the intrinsic apoptosis pathway restricts activation of the cGAS–
STING pathway in an mtDNA-dependent manner8,9. However,
the role of tumor-derived mtDNA in radiation treatment is largely
unknown. Thus, to explore whether mtDNA is involved in the pro-
duction of type I IFN in Casp9−/− tumor cells, we depleted mtDNA
with 2′-3′-dideoxycytidine (ddC) treatment (Supplementary Fig. 3f).
Indeed, depletion of mtDNA abolished type I IFN production
after irradiation of Casp9−/− tumor cells or emricasan treatment
(Fig. 3c,d). To investigate the role of the cGAS and STING path-
way, we further constructed Tmem173−/− tumor cells. Indeed, loss
of STING abolishes the activation of interferon regulatory factor 3
(Supplementary Fig. 4a) and type I IFN production when blocking
caspase activity (Fig. 3e and Supplementary Fig. 4b). To confirm the
role of cGAS and STING in CASP9-deficient tumor-derived type
I IFNs, we knocked out cGAS or STING in Casp9–/– tumor cells.
Similarly, lack of cGAS or STING abolished type I IFN production in
irradiated Casp9−/− tumor cells (Fig. 3f and Supplementary Fig. 4c).
Together, these data indicate that tumor cells hijack CASP9 signal-
ing to restrict the radiation-induced mtDNA–cGAS–STING sens-
ing pathway and limit the production of type I IFN.
To validate the role of tumor-intrinsic mtDNA innate sensing
in vivo, we then explore whether loss of Casp9 in tumor cells could
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increase type I IFN production in  vivo. We first measured type I
IFNs after radiation treatment. Compared to wild-type tumors,
radiation treatment increased Ifnb transcription in the setting of
CASP9 deficiency (Fig. 4a). Further knocking out Cgas in Casp9−/−
tumor cells abolished increased IFN-β (Fig. 4a). To confirm the
source of type I IFNs in vivo, we compared type I IFN production
between tumor (CD45–) and immune (CD45+) cells in the TME
after radiation. The results showed that it is Casp9−/− tumor cells
rather than immune cells that produce much more type I IFNs after
radiation in a cGAS-dependent manner (Fig. 4b).
The cGAS–STING pathway in antigen-presenting cells has
been reported to be essential for the therapeutic effect of radia-
tion1. To test whether the host cGAS pathway is required for the
therapeutic effect of radiation on tumors with CASP9 deficiency,
we inoculated CASP9-deficient tumor cells into wild-type or cGAS-
deficient mice. Surprisingly, radiation has a similar therapeutic
effect in wild-type or cGAS-deficient mice (Fig. 4c). To further
address the mechanisms and role of CASP9 deficiency in tumor-
derived type I IFN production and radiation therapy in  vivo, we
inoculated wild-type, Cgas−/− or Casp9−/− single or double knockout
tumor cells into wild-type mice (Fig. 4d). Considering the STING
independent role of cGAS26,27, we also constructed STING–CASP9
(Tmem173−/−Casp9−/−) double knockout tumor cells and compared
them to cGAS–CASP9 double knockout tumor cells (Fig. 4d).
Consistently with previous data, most intrinsic apoptosis deficient
tumors completely regressed after radiation treatment. However,
knocking out Cgas or Tmem173 in the setting of Casp9 deficiency
abolished the enhanced therapeutic effect of radiation (Fig. 4d).
Collectively, these data indicate that lack of CASP9 signaling in
tumor cells provokes radiation-mediated antitumor immunity in a
tumor-derived mtDNA–cGAS–STING-sensing-dependent fashion.
To further confirm the mechanisms for enhanced immunity, we also
performed a cross-priming assay. We observed that the supernatant
of irradiated Casp9−/− tumor cells increased IFN-γ production by
activated T  cells, but cGAS deficiency abolished T  cell activation
(Fig. 4e). Together, these data demonstrate that mtDNA-mediated
cGAS–STING innate sensing in irradiated Casp9–/– tumor cells is
required for enhanced antitumor T cell responses.
Tumors evolve adaptive immune resistance by upregulating
PD-L1. Our studies demonstrated that blocking intrinsic apoptosis
can provoke radiation-mediated antitumor immunity via increased
cross-priming of tumor-specific T cells in a type I IFN signaling-
dependent manner. However, the major challenge of radiotherapy
is the very infrequent rate of systemic antitumor immune response,
termed an abscopal effect28. Previous studies suggest that radiation-
induced adaptive resistance limits the abscopal and/or systemic
antitumor effect29. Thus, we next determined whether CASP9 defi-
ciency in tumor cells could enhance systemic antitumor immunity
of radiation treatment. However, even though irradiated Casp9−/−
tumors regressed, CASP9 deficiency did not further enhance con-
trol of distal tumor growth (Supplementary Fig. 5a,b). That raised
the possibility that a form of adaptive resistance might restrict
systemic antitumor immunity. PD-L1, a negative immune regula-
tor (encoded by gene Cd274), is an IFN-inducible gene. Previous
studies show that IFNs can promote PD-L1 expression to impair
T cell immune response30–32. Thus, the limited abscopal effect might
be induced by increased expression of PD-L1. To address whether
PD-L1 is involved in immune surveillance in radiation treatment,
we analyzed expression of PD-L1 in tumor tissues. Indeed, loss of
tumor-intrinsic CASP9 promotes expression of PD-L1 and radia-
tion further increased expression of PD-L1 in both mRNA (Fig. 5a)
and protein level (Fig. 5b) in CASP9-deficient tumor tissues.
Moreover, upregulation of PD-L1 in Casp9−/− tumors is dependent
on type I IFN signaling in tumor cells (Fig. 5c). Further knocking
out Cgas also abolished the upregulation of PD-L1 in irradiated
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a b
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Fig. 3 | Blocking CASP9 signaling to facilitate tumor-intrinsic mtDNA sensing after radiation. a,b, MC38 cells (n = 3 per group) were irradiated
(IR 40 Gy) or non-irradiated (NR) and treated with or without emricasan. At 48 h after treatment, cytosolic DNA was extracted and real-time PCR was
performed to detect gDNA (a) and mtDNA (b). c, MC38 or ddC-treated MC38 (MC38-ddC) cells (n = 3 per group) were irradiated (IR 15 Gy); 48 h later,
the supernatant was collected and added to RAWISG to detect type I IFN activity. d, The indicated cells (n = 3 per group) with or without ddC treatment
were irradiated (IR 40 Gy) or non-irradiated (NR). At 48 h after radiation, supernatants were collected and added to RAWISG to detect type I IFN.
e, The indicated cells (n = 3 per group) were irradiated (IR 40 Gy) or non-irradiated (NR) and treated with or without QVD; 48 h later, the supernatants
were collected and added to the RAWISG cells to detect type I IFNs. f, The indicated cells (n = 3 per group) were irradiated (IR 40 Gy) or non-irradiated
(NR); 48 h after radiation, cells were collected for IFN-β mRNA detection by RT–qPCR. Data are shown as mean ± s.e.m. and are representative of
three independent experiments. Statistical analysis was performed by unpaired two-tailed Student’s t-test. NS, not significant, *P < 0.05, **P < 0.01,
***P < 0.001, ****P < 0.0001.

tumor cells (Supplementary Fig. 5c). Together, our data indicate that Above all, we observed that irradiated CASP9-deficient tumors
Casp9−/− tumor-derived type 1 IFN plays a vital role in regulation of evolve adaptive immune resistance by upregulating PD-L1, which
PD-L1 expression, which may limit the abscopal effects of radiation limits the abscopal/systemic antitumor immune response in radia-
therapy. To evaluate whether the upregulated PD-L1 contributes to tion therapy.
impaired systemic antitumor immunity, we inoculated wild-type or
Casp9−/− tumor cells on the right flank of mice. On the same day, we Blockade of caspases with emricasan synergizes with immuno-
inoculated wild-type tumor cells in the left flank. Then, tumors in therapy and radiation to promote systemic antitumor immunity.
the right flank were irradiated locally and anti-PD-L1 was adminis- As described above, we found that tumor endogenous CASP9 lim-
tered systemically as indicated (Fig. 5d). We observed that CASP9- its the therapeutic effect of radiation and anti-PD-L1 combination
deficient tumors responded better to anti-PD-L1 treatment and treatment. We then wanted to test the clinically relevant applica-
combining radiation with anti-PD-L1 treatment worked similarly tion of our findings using a caspase inhibitor to block CASP9 sig-
in both wild-type and Casp9−/− primary tumors (Fig. 5e). However, naling. We used the pan-caspase inhibitor, emricasan. To set up a
a combination of radiation with anti-PD-L1 only slightly limited clinically relevant setting for caspase inhibition, we first optimized
distal tumor growth in mice bearing wild-type primary tumors, the timing for caspase inhibitor treatment after radiation in  vivo.
whereas mice bearing primary tumor with CASP9 deficiency We examined type I IFN production over time after simultaneous
showed a much stronger systemic antitumor effect (Fig. 5f). As radiation and caspase blockade in  vitro. To determine the critical
MC38 is sensitive to anti-PD-L1 systemic treatment, we employed window for treating with caspase inhibitors, we treated cells for 12,
another anti-PD-L1 resistant tumor model, TS/A. Similarly, com- 24 or 48 h after radiation. We observed that only blocking caspase
pared to wild-type TS/A tumors, Casp9−/− TS/A tumors generated activity for the first 12 h could not increase type I IFN production.
a stronger systemic antitumor effect after radiation and anti-PD-L1 Compared to 12 or 24 h, treating for 48 h after radiation dramati-
treatment (Supplementary Fig. 5d,e). cally increased type I IFN production (Supplementary Fig. 6a).

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Articles NATure IMMunoLogy

a b c WT NR (n = 5)
300 30 1,000
* WT ****
*** WT IR (n = 5)

Tumor volume (mm3)

Ifnb mRNA (relative)

–/–
Cgas 750 Cgas–/– NR (n = 5) NS

IFN-β (pg ml–1)

200 Casp9 –/–
20 Cgas–/– IR (n = 4)
Casp9 –/–Cgas–/– 500
100
10 250

0 0
0 0 5 10 15 20 25
as C 9 –/– (n 4)
C as –/ R ( 3)

p9 as –N (n )
(n 3)

s –/– (n 3)
(n )

(n )
3)
+ –
4

–/ s – I R = 4

IR = 3
IR CD45 IR CD45
R =
=
p9 IR =
=

ga NR =
Days after tumor inoculation
=
– n
C WT (n
s –/– IR
R

N
N

/–
–/
T
W

– p
ga

p9 ga
g
C

C
as

–
as C
–/
C
C

d WT NR (n = 8) e
1,000
WT IR (n = 12) ****
600 –/– 800
Tmem173 NR (n = 10)

IFN-γ (pg ml–1)

Tmem173 –/– IR (n = 13) 600
Cgas –/– NR (n = 5)
Tumor volume (mm3)

400
400 Cgas –/– IR (n = 10)
*
Casp9 –/– NR (n = 11) 200

Casp9 –/– IR (n = 12) 0

200 Casp9 –/–Cgas –/– NR (n = 7)

– 9 –/–
–
– 9 –/–

–
****

s –/

s –/
C WT

as Ca T
IR R W
ga

ga
p9 sp
p9 asp
Casp9 –/–Cgas –/– IR (n = 13)

I
C

C
–/

–/
**** Casp9 –/–Tmem173 –/– NR (n = 8)

as
C

C
0 Casp9 –/–Tmem173 –/– IR (n = 10)

IR
0 10 20 30
Days after tumor inoculation

Fig. 4 | Casp9−/− tumor-derived dsDNA innate sensing is required for provoking radiation-mediated antitumor immunity. a, C57BL/6J mice (n = 3) were
transplanted s.c. with indicated cells. Tumors were treated as previously described in as Fig. 2a; 72 h after radiation, tumors were removed and RT-qPCR
was performed. b, C57BL/6J mice (n = 3 per group) were transplanted s.c. with indicated cells. Tumors were treated as previously described in Fig. 2a;
24 h after radiation, tumors were removed and live immune cells (CD45+) and tumor cells (CD45−) were sorted and incubated for another 48 h. The level
of IFN-β in supernatants was detected by ELISA. c, C57BL/6J wild-type or Cgas−/− mice were transplanted s.c. with 2 × 106 MC38 Casp9−/− tumor cells.
Tumors were treated as previously described in Fig. 2a. d, C57BL/6J mice were transplanted s.c with 1 × 106 MC38, Cgas−/−, Tmem173−/− or with 2 × 106
Casp9−/−, Casp9−/−Cgas−/−, Casp9−/−Tmem173−/− cells. Tumors were treated as previously described in Fig. 2a. e, BMDCs (n = 3 per group) were exposed to
OVA protein (40 µg ml−1) for 12 h. Then naive CD8+ OT1 T cells and the supernatants from either radiated or non-radiated MC38 tumor cells were added
and incubated for another 72 h. IFN-γ was determined by cytometric bead array assay. Data are shown as mean ± s.e.m. and are representative of two or a
pool of three (d) independent experiments. Statistical analysis was performed by unpaired two-tailed Student’s t-test in a,b,e or two-way ANOVA test in
c,d. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

This suggests that long-term blockade of caspases in tumor tissue results showed that emricasan provoked the therapeutic effect of
is required for provoking tumor-intrinsic mtDNA innate sensing. anti-PD-L1 (Supplementary Fig. 6d).
To determine whether emricasan was directly inhibiting tumor cell However, although emricasan enhanced the therapeutic effect of
proliferation, we treated MC38 tumor cells in vitro and found no radiation, tumors eventually escaped immune surveillance. Similarly
changes in tumor growth during treatment (Supplementary Fig. 6b). to the observation in the Casp9−/− tumor model, we observed that
To study the effect of emricasan on tumor growth in vivo, we first emricasan treatment also increased expression of PD-L1 in irradi-
used a highly immunogenic tumor MC38-ovalbumin (OVA) and ated tumor cells (Supplementary Fig. 6e). Therefore, we investigated
treated tumor-bearing mice with emricasan. Emricasan limited the abscopal effect of emricasan, radiation and PD-L1 combination
tumor growth in vivo (Supplementary Fig. 6c). However, emricasan treatment. Our results showed that a triple combination not only
single treatment could not control the more advanced MC38 tumor generated the best therapeutic effect on irradiated tumors (Fig. 6d),
in vivo (Fig. 6a). We then validated the effect of emricasan in radia- but more importantly also induced an effective systemic antitumor
tion therapy. Similarly to CASP9-deficient tumors CASP blockade effect (Fig. 6e). Taken together, our data indicate that the combi-
also sensitizes the tumor to radiation (Fig. 6a). To confirm the syn- nation of caspase inhibition with anti-PD-L1 and radiation has a
ergistic effect of emricasan and radiation, we employed another potential clinical application for cancer treatment.
tumor model, TS/A. Similarly, emricasan increased the therapeutic
effect of radiation (Fig. 6b). To evaluate the clinical potential of com- Discussion
bining emricasan with radiation, we inoculated human lung can- Our current study revealed that radiation-induced activation of
cer cells, A549, into humanized mice and treated the tumors with CASP9 signaling is the major intrinsic barrier to restrict tumor-
local radiation and emricasan. We observed that the combination derived type I IFN in an mtDNA innate-sensing-dependent man-
therapy showed stronger antitumor effect (Fig. 6c). To investigate ner. Blocking CASP9 causes tumor cells to be the major type I
the antitumor effects of emricasan in combination with anti-PD- IFN source after radiation, leading to enhanced cross-priming of
L1 treatment, we treated mice bearing MC38-OVA tumors and our tumor-specific T cells and radiation-mediated antitumor immunity.

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NATure IMMunoLogy Articles
a b c
* *
10.0
50 ** 30,000
**

PD-L1 (relative MFI)

Pdl1 mRNA (relative)
40 7.5

PD-L1 (MFI)
20,000
30
*** 5.0
20
* 10,000
2.5 *
10
0 0 0

4)
3)
3)

3)

4)
4)

3)
3)

Ifn 9 –/– R
Ifn 9 –/– R

p9 Ca WT R
1 –/ R

ar IR
IR
I
N

N
ar N

/–
=
=
=

=
=

=
=

1–
p9 Ca WT
(n
(n
(n

(n

(n
(n

(n
(n

– p
IR
R
IR

IR

IR
R

R
R

– p

–/ s
N
N

N
N

–/ s
–
–/

–/
T

–
T

–/
–/

T
T

W
W

p9

W
W

p9
p9

p9
as

as
as
as

as
C

as
C
C

C
C
d MC38 or MC38 Casp9 –/–
Right flank
Anti-PD-L1 i.p. every other day

Right flank 2 × 105 MC38 MC38 or MC38

D0 SC
D8 IR15 Casp9 –/–

2 × 105 MC38
Left flank

e IR tumor f NR tumor

800 500 WT NR (n = 12)

WT NR (n = 12)
WT NR anti-PD-L1 (n = 7)
Tumor volume (mm3)

WT NR anti-PD-L1 (n = 7)
Tumor volume (mm3)

400
600 WT IR + anti-PD-L1 (n = 11) WT IR + anti-PD-L1 (n = 11)
Casp9 –/– NR (n = 11) 300 Casp9 –/– NR (n = 11)
400 Casp9 –/– NR anti-PD-L1 (n = 6)
**** Casp9 –/– NR anti-PD-L1 (n = 6)
Casp9 –/– IR + anti-PD-L1 (n = 11) 200 Casp9 –/– IR + anti-PD-L1 (n = 11)
200
100
0
0
0 10 20 30
0 10 20 30
Days after tumor inoculation Days after tumor inoculation

Fig. 5 | Tumors evolve resistance to adaptive immunity by upregulating PD-L1. a, C57BL/6J mice were transplanted s.c. with 1 × 106 MC38 or 2 × 106
MC38 Casp9−/− cells. At 8 d after inoculation, tumors were treated locally with one dose of 15 Gy (IR) or untreated (NR); 72 h later, tumors were removed
and RT-qPCR was performed to validate the mRNA level of PD-L1. b, C57BL/6J mice were transplanted s.c. with 1 × 106 MC38 or 2 × 106 MC38−/− cells.
At day 8 post inoculation, tumors were treated locally with one dose of 15 Gy (IR) or untreated (NR); 3 d post radiation, tumors were removed and the
expression of PD-L1 in tumor cells was validated by flow cytometry. MFI, mean fluorescence intensity. c, MC38 (n = 3 per group) were irradiated (IR) or
untreated (NR) and PD-L1 expression was analyzed by flow cytometry 48 h after radiation. d–f, C57BL/6J mice were transplanted s.c. with 1 × 106 M.C38
or MC38 Casp9−/− tumor cells on the right flank and with 2 × 105 MC38 on the left flank. At 8 d after inoculation, right flank tumors were treated locally
with one dose of 15 Gy (IR) or untreated (NR) with anti-PD-L1. Treatment regimens (d), tumor growth of radiated tumors (e) and non-radiated tumors (f)
are shown, respectively. Data are shown as mean ± s.e.m. and are representative of three (a–c) or a pool of two (e,f) independent experiments. Statistical
analysis was performed by unpaired two-tailed Student’s t-test in a–c or two-way ANOVA in e,f. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

However, deficiency of CASP9 also induces adaptive resistance by type I IFNs in vitro. Another interesting report showed that radiation
upregulating PD-L1. Combination of caspase blockade and anti- increases gDNA leakage into cytosol via micronuclei in a DNA-
PD-L1 increases the abscopal effect of radiation therapy. Moreover, PKcs dependent manner and that cGAS localizes into micronuclei
we observed that blocking caspases in the TME with emricasan can and promotes type I IFN production4,24,36. However, these strate-
synergize with radiation and anti-PD-L1 therapy to achieve effec- gies only achieved a slight increase of tumor-derived type I IFNs.
tive systemic antitumor effects. Therefore, the major intrinsic barrier of type I IFN production
As a key cytosolic dsDNA sensor, cGAS synthesizes cGAMP to in tumor cells still needs to be well defined. Herein, we observed
activate the STING–IFN pathway33. Many studies have reported that that CASP9 deficiency induced about a thousand-fold increase in
cGAS activation in host cells is essential for induction of the anti- tumor-derived type I IFNs upon high dose radiation treatment.
tumor adaptive immune response in radiation, anti-CD47 and anti- This suggests that as one of the major negative regulators of tumor-
PD-L1 treatment1,15,34,35. However, as the major component of TME, derived type I IFNs, CASP9 signaling probably uses a different
tumor cells always failed to produce type I IFNs during treatment. mechanism to restrict tumor-intrinsic dsDNA innate sensing and
Several studies attempted to enhance irradiated tumor-derived type bypass the inhibitory effect of TREX1 during high-dose radiation
I IFNs by different strategies. One study reported that compared treatment. Emricasan treatment does not further increase cytosolic
to low-dose radiation treatment, high-dose radiation treatment can mtDNA levels in irradiated tumor cells. cGAS recognized cyto-
upregulate TREX1 expression and limit tumor-derived type I IFN3. solic DNA depending not only on duplex character and length, but
Compared with high-dose radiation, low-dose multiple radiation also structure37. One possible explanation is that caspase blocking
treatment leads to a twofold to threefold increase of tumor-derived might involve changing the structure of mtDNA to facilitate cGAS

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Articles NATure IMMunoLogy

a b TSA c A549
1,000 NR vehicle 400 NR vehicle (n = 6)
Tumor volume (mm3)

Tumor volume (mm3)

500 NR vehicle

Tumor volume (mm3)

800 NR Emricasan IR vehicle Emricasan (n = 6)
400 300 IR vehicle (n = 10)
IR vehicle Emricasan ****
600
IR Emricasan ** 300 IR + Emricasan IR + Emricasan (n = 9) ****
200
400 200
200 100
100
0 0 0
0 5 10 15 20 25 0 5 10 15 20 25 30 0 10 20 30
Days after tumor inoculation Days after tumor inoculation Days after tumor inoculation

d Primary tumor e Distant tumor

800 NR vehicle + RlgG (n = 4)
400
NR vehicle + RlgG (n = 4) Anti-PD-L1 vehicle (n = 5)

Tumor volume (mm3)

Tumor volume (mm3)

600 Anti-PD-L1 vehicle (n = 5) 300 IR + anti-PD-L1 vehicle (n = 5)

IR + anti-PD-L1 vehicle (n = 5) IR + Emricasan + RlgG (n = 5)
400 IR + Emricasan + RlgG (n = 5) 200 *** Anti-PD-L1 + Emricasan (n = 4)
**** Anti-PD-L1 + Emricasan (n = 4) IR + anti-PD-L1 + Emricasan (n = 5)
IR + anti-PD-L1 + Emricasan (n = 5) 100 IR vehicle + RlgG (n = 5)
200
IR vehicle + RlgG (n = 5) Emricasan + RatlgG (n = 5)
Emricasan + RatlgG (n = 5) 0
0 0 10 20 30
0 10 20 30 Days after tumor inoculation
Days after tumor inoculation

Fig. 6 | Emricasan synergizes with radiation and anti-PD-L1. a, C57BL/6J mice (n = 5 per group) were transplanted s.c. with 1 × 106 MC38 cells. Tumors
were locally irradiated as described previously in Fig. 2a; 24 h post radiation, tumors were treated intratumorally (i.t.) with emricasan (10 mg kg−1) daily
for 3 d. b, BALB/c mice (n = 5 per group) were transplanted s.c. with 5 × 105 TS/A cells. Tumors were irradiated on day 9 and treated as described in a. c,
Human A549 tumor cells were s.c. injected into humanized NSG-SGM3 (Hu-NSG) mice. Tumors were untreated (NR) or treated locally with one dose of
15 Gy radiation (IR) on day 12. At 24 h after radiation, tumors were treated with emricasan as described in a. d,e, C57BL/6J mice were transplanted s.c.
with 1 × 106 MC38 on the right flank on day 0 and with 5 × 105 MC38 cells on the left flank on day 2. The primary tumors on the right flank were treated as
described in a and with or without emricasan treatment (i.t. 10 mg kg−1) on day 8, 9, 10 and 11. Additional anti-PD-L1 was injected (i.p. 100 µg per mouse)
on day 8, 10, 12 and 14 after tumor inoculation. Tumor growth of IR (d) and NR (e) distant tumors was presented. Data are shown as mean ± s.e.m. and
are representative of three (a,d,e), two (b) or a pool of two (c) independent experiments. The statistical analysis was performed by two-way ANOVA.
**P < 0.01, ***P < 0.001, ****P < 0.0001.

recognition or caspase targets downstream of the cGAS–STING caspase blockade to further evoke antitumor immunity. However,
pathway to restrict tumor-derived type I IFNs. emricasan can non-reversibly bind to variously activated caspases,
Our data also raises the question of whether enhanced innate including CASP1, which is a key mediator for inflammasome acti-
sensing or induction of non-apoptotic cell death is most impor- vation and pyroptosis. This might influence the therapeutic effect
tant for our caspase blockade phenotype. There are several cases in of radiation. Furthermore, most of the caspase inhibitors can only
which blocking apoptosis induces different forms of cell death38–40, bind to cleaved caspases but not pro-caspases. Thus, more spe-
which might release damage-associated molecular patterns to evoke cific inhibitors of pro-CASP9 or apoptosome components might
host innate immune response. Along these lines, another interest- increase innate sensing by restricting the CASP9 signal. Such
ing study observed that blocking caspases and BCL-2 members inhibitors might preferentially work on tumors with higher caspase
can trigger caspase-independent cell death and release proinflam- activities. Thus, development of new specific CASP9 signal inhibi-
matory cytokines in the SVEC cell line; however, the role of tumor tors will open a new avenue by combining with various drugs and
intrinsic STING or cGAS for cancer treatment is still unclear41. therapies that increase apoptosis and simultaneously improve innate
One recent report also revealed that tumor-intrinsic CASP3 sup- sensing to potentiate immunotherapy. Tumor cells use immunosup-
presses radiation responses probably through an IFN-independent pressive factors to shape the TME and escape immunosurveillance.
fashion42. However, in our case, knocking out Cgas or Tmem173 in Anticancer treatments, such as radiation, immunotherapy and tar-
Casp9–/– tumor cells abolished the enhanced therapeutic effect of geted therapies can provoke antitumor immunity, but also might
radiation treatment, suggesting the diversity of caspase-mediated induce adaptive immune resistance. We provide evidence for this
intrinsic resistance. paradigm, in that CASP9 deficiency promotes tumor cell-derived
Evading apoptosis is considered to be one of the major mecha- type I IFN production, but also upregulates PD-L1, which then
nisms of tumor resistance to current therapies. Therefore, many limits systemic antitumor immunity. Combination of anti-PD-L1
current antitumor therapies focus on triggering more tumor cell treatment with radiation and CASP9 blockade can achieve effective
apoptosis to reduce tumor burden. However, tumor tissues always systemic antitumor effects. We propose that targeting CASP9 sig-
show higher CASP3 activation and apoptosis in the tumor microen- naling in combination with radiotherapy and immune checkpoint
vironment positively related to the histological grade of cancer43–45. blockade could provide a novel strategy for cancer treatment.
It has been reported that patients with higher cleaved CASP3 have
shorter survival46,47. It is quite interesting to consider why tumor Online content
cells keep continuous proliferation, but also increase the activa- Any methods, additional references, Nature Research reporting
tion of CASP3. Our study suggests that tumor cells seem to hijack summaries, source data, extended data, supplementary informa-
caspase signals to restrict mtDNA innate sensing and further limit tion, acknowledgements, peer review information; details of author
antitumor immunity in the TME. This also suggests that in addition contributions and competing interests; and statements of data and
to local radiation treatment, current antitumor therapies that can code availability are available at https://doi.org/10.1038/s41590-
increase intrinsic apoptosis in tumor tissue might synergize with 020-0641-5.

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NATure IMMunoLogy
Received: 15 September 2019; Accepted: 21 February 2020;
Published: xx xx xxxx
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45. Flanagan, L. et al. Low levels of caspase-3 predict favourable response to
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Articles
Methods
Mice. Female C57BL/6J and BALB/c mice were purchased from UT Southwestern
breeding core or Jackson Laboratory. Rag1−/−, Batlf3−/−, Cgas−/− and C57BL/6-Tg
(TcraTcrb) 1100Mjb/J (OT-I T cell receptor-Tg) mice were purchased from Jackson
Laboratory. Ifnar1−/− mice were kindly provided by A. Chong at the University of
Chicago. All mice were maintained under specific pathogen-free conditions and
all animal procedures were performed in accordance with the animal experimental
guidelines set by the Institutional Animal Care and Use Committee of the
University of Texas Southwestern Medical Center.
Cell lines and reagents. The MC38 cell line was obtained from the American
Type Culture Collection. MC38-OVA cell line was derived from MC38 and
constitutively expressed the chicken OVA antigen. TS/A was kindly provided by
Z. Qin at the Institute of Biophysics, Chinese Academy of Sciences. RAW-Lucia ISG
(RAWISG) was kindly provided by Z. J. Chen at UT Southwestern Medical Center.
A549 cells were adenocarcinomic human alveolar basal epithelial cells, purchased
from the American Type Culture Collection. All cells were cultured in 5% CO2 and
maintained in vitro in Dulbecco’s modified Eagle’s medium supplemented with
10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 100 U ml−1 penicillin and
100 µg ml−1 streptomycin and confirmed to be negative for Mycoplasma. Emricasan
and QVD-oph were purchased from Selleckchem. Anti-PD-L1 (10F.9G2) antibody
was purchased from BioXcell.
CRISPR–CAS9. sgRNAs were cloned into px458 plasmids and then were
transfected into tumor cells by lipofectamine 2000. After 24 h, GFP-positive cells
were sorted and cultured for another week. Then GFP-negative cells were sorted
and seeded onto 96-well plates for single clones. Another week later, GFP-negative
clones were passed onto 12-well plates and western blot was performed to identify
knockout clones. Finally, all knockout clones were pooled together for experiments.
Tumor growth and treatments. Tumor cells were injected s.c. on the right flank
of mice. Mice were randomized to treatment groups when tumors reached certain
sizes. Tumor were treated with local radiation or not, then tumor volumes were
measured by the length (a), width (b) and height (h) and calculated as tumor
volume = abh/2. For the survival curve, if each of length, width or height of tumor
was >1.5 cm or the tumor volume was >500 mm3, the mice were considered
dead. For emricasan treatment, emricasan was dissolved in 2% alginate in PBS
and tumor-bearing mice were treated with emricasan i.t. at 10 mg kg−1, daily for
3 d, or emricasan was dissolved in PBS and tumor-bearing mice were treated
with emricasan i.p. at 20 mg kg−1, twice a day for 3 d. For the PD-L1 blockade
experiments, anti-PD-L1 (clone 10F.9G2) was administered i.p. to mice every 2 d
for a total of three times. For the rechallenge experiment, 60 d after primary s.c.
transplantation, tumor-free mice were inoculated with 5 × 106 MC38 cells
on the left flank.
RAWISG reporter system. RAWISG cells were kindly provided by Z. (James)
Chen at the University of Texas Southwestern Medical Center. We further knocked
out the STING in RAWISG cells by CRISPR–Cas9; the Tmem173–/– RAWISG cells
were used to analyze type I IFN levels in the supernatant. A total of 0.25 million
Tmem173–/– RAWISG cells in 100 µl of RPMI 1640 medium were seeded on a
96-well plate, then 100 μl of supernatants from different treatments were
added to the plate. After 12 h, 40 μl of supernatant was taken out for luciferase
activity analysis.
Cytosolic DNA extraction. After treatment, cells were divided into two equal
aliquots and one aliquot was resuspended in 500 μl of lysis buffer (10 mM Tris-
HCl (pH 8.0), 100 mM NaCl, 25 mM EDTA, 0.5% SDS) and total DNA were
extracted by phenol-chloroform and precipitated with alcohol; these extracts
served as normalization controls for total gDNA. The second equal aliquots were
resuspended in 500 μl of permeabilization buffer (50 mM HEPES (pH 7.4), 150 mM
NaCl, 2 mM EDTA and 50 μg ml−1 digitonin). After 10 min, cells were centrifuged
at 1,000g for 3 min three times. The supernatants were transferred to a fresh
tube and centrifuged at 17,000g for another 10 min. Finally, supernatants were
transferred to a fresh tube and cytosolic DNA was extracted by phenol-chloroform
and precipitated with alcohol and real-time PCR was performed to detect mtDNA
and gDNA. Cytosolic mtDNA and gDNA levels were normalized with total gDNA.
Super resolution imaging. MC38 cells were seeded onto a 24-well plate with
cover glasses, then the cells were treated with radiation (IR 40 Gy) or not (NR) and
emricasan (10 μM) was added. At 48 h after radiation, cells were fixed in 4% PFA/
PBS for 15 min and washed with PBS for 5 min three times, then cell were treated
with 0.25% Triton X-100/PBS for 10 min. After three times washing with PBS,
cells were blocked in 5% BSA/PBS for 1 h. Then cells were incubated with primary
antibodies (rabbit anti-TOM20 and mouse anti-dsDNA) overnight at 4 °C. After
five times washing with PBS, cells were incubated with secondary antibodies anti-
rabbit Alexa Fluor647 (CST 4414) and anti-mouse Alexa Fluor488 (CST 4408) for
1 h at room temperature. After five times washing with PBS, the cover glasses were
mounted with Shandon Immu-Mount (Fisher Scientific, 9990402). Then, super
resolution images were acquired on a Nikon N-SIM microscope using a Nikon
NATure IMMunoLogy
Plan Apo TIRF100 ×1.49 NA objective and a Hamamatsu digital camera C11440
using 488 and 640 nm laser lines. Refractive index-matched immersion oil
(Nikon Instruments) was used for all experiments. For each focal plane, 15
images were collected with Z-stacks (step size of 200 nm). And SIM images
were reconstructed and analyzed with NIS-Elements AR 4.50.00 and NIS offline
deconvolution 4.51 software.
Flow cytometry analysis. Single-cell suspensions of cells were incubated with
anti-CD16/32 (anti-FcγIII/II receptor, clone 2.4G2) for 10 min to block nonspecific
binding and then subsequently stained with antibodies. All fluorescently
labeled antibodies were purchased from BioLegend or eBioscience and detailed
information of antibodies is listed in the Supplementary Table 1. Fixable Viability
Dye eFluor 506 (eBioscience) was used to exclude dead cells. Data were collected
on CytoFLEX (Beckman Coulter) and analyzed with CytExpert (Beckman Coulter)
or FlowJo (Tree Star) software.
Measurement of IFN-γ-secreting T cells by ELISPOT. Spleens from treated mice
were processed to single-cell suspensions and resuspended in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 2 mmol l−1 l-glutamine, 100 U ml−1
penicillin and 100 μg ml−1 streptomycin. A total of 2–4 × 105 spleen cells were used
for assay. Irradiated MC38 cells were used to restimulate tumor-specific T cells.
The ratio of tumor cells and spleen cells was 1:4. IFN-γ production was determined
48 h after incubation with an IFN-γ ELISPOT assay kit according to the
manufacturer’s protocol (BD Biosciences). The visualized spots were enumerated
with CTL-ImmunoSpot S6 Analyzer (Cellular Technology).
Bone marrow dendritic cell generation. BMDCs were generated as described
previously1,15,35. Briefly, bone marrow cells were collected from tibias and femurs
of female C57BL/6/J mice. Bone marrow cells were placed and cultured in 24-
well plates with complete RPMI 1640 medium containing 20 ng ml−1 rmGM-CSF
(BioLegend). Fresh medium with rmGM-CSF was added into culture on day 3. The
immature BMDCs were collected and ready to use on day 7.
T cell isolation. CD8+ T cells were isolated from lymph nodes and spleens of
OT-I T cell receptor-Tg mice with a negative CD8+ T cell isolation kit (Stemcell
Technologies) following the manufacturer’s instructions.
ELISA. Cell culture supernatants were obtained 48 h after radiation treatment
or not. The concentration of IFN-β was measured with VeriKine-HS Mouse
Interferon Beta Serum ELISA kit (PBL Assay Science) in accordance with the
manufacturer’s instructions.
RNA extraction and quantitative real-time PCR. Total RNA from tumor tissues
was extracted with the TRIzol Reagent (Invitrogen) and reversed-transcribed with
iScript gDNA Clear cDNA Synthesis kit (Bio-Rad). Real-time PCR was performed
with ssoAdvanced Universal SYBR Green Supermix (Bio-Rad) according to the
manufacturer’s instructions and different primer sets on CFX Connect Real-Time
PCR Detection System (Bio-Rad). Data were normalized by the level of GAPDH
or 18S rRNA expression in each individual sample. The 2−∆∆Ct method was used to
calculate relative expression changes.
Immunoblotting. Protein sample preparation and immunoblot procedures were
performed as previously described. Briefly, cells were collected and lysed and
run on SDS–PAGE gels for immunoblotting. Proteins were transferred onto a
polyvinylidene difluoride membrane (Millipore), incubated sequentially with
antibodies mentioned above and detected by Clarity Max Western ECL Blotting
Substrates kit and ChemiDoc Touch Gel Imaging system (Bio-Rad).
Statistical analysis. All analysis was performed using GraphPad Prism statistical
software (GraphPad Software). Two-way ANOVA tests were used to analyze
tumor growth data and unpaired two-tailed Student’s t-tests were used to analyze
other data. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001.
Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.
Data availability
All data supporting the findings of this study are available within the article
and its supplementary information files and from the corresponding author
upon reasonable request. A reporting summary for this article is available as a
Supplementary Information file.
Acknowledgements
We thank R. W. Welchselbaum for providing reagents and assisting with experiments
and the UT southwestern Flow Cytometry Facility and Animal Resources Center. YXF
holds the Mary Nell and Ralph B. Rogers Professorship in Immunology. This work was
supported by NCI CA134563, Texas CPRIT grant RR150072 and RR180725 (established
CPRIT scholar in cancer research) to Y.-X. F.
Nature Immunology | www.nature.com/natureimmunology
NATure IMMunoLogy
Author contributions
C.H., Z.L. and Y.-X.F. designed experiments and analyzed data. C.H. and Z.L. performed
experiments. C.H. and Y.-X.F. wrote the manuscript. Z.L. J.Q. and C.M. revised the
manuscript. Y.Z. and C.-L.Z. helped with SIM imaging. A.S., C.D., A.Z., Z.R., C.L. and
X.C. provided mice and reagents. J.Q. gave valuable advice. Y.-X.F. supervised the project.
Competing interests
The authors declare no competing interests.
Nature Immunology | www.nature.com/natureimmunology
Articles
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41590-020-0641-5.
Correspondence and requests for materials should be addressed to J.Q. or Y.-X.F.
Reprints and permissions information is available at www.nature.com/reprints.
Editor recognition statement: Zoltan Fehervari was the primary editor on this article
and managed its editorial process and peer review in collaboration with the rest of the
editorial team.
 nature research | reporting summary
Corresponding author(s): Dr. Yang-Xin Fu last
updated by author(s): 2/1/2020

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Data collection Bio-Rad Image Lab Software 5.2.1, CytExpert, NanoDrop, CFX Connect™ Real-Time PCR Detection System, ChemiDoc™ Touch Gel Imaging
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Data analysis GraphPad Prism software 7.0, CytExpert, FlowJo software v9.3.2, CFX Connect™ Real-Time PCR Detection System, CTL-ImmunoSpot® S6
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Sample size Sample size were determined from the similar experiments in the former publications of the group.

Data exclusions No data were excluded.

Replication The performed replications were successful.

Randomization Mice in this study were matched with age and tumor size, and were randomly allocated to the different groups.

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Antibodies ChIP-seq
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Clinical data

Antibodies
Antibodies used InVivoMab anti-mouse CD4 (GK1.5), BioXcell, BE0003-1; InVivoMab anti-mouse CD8 (YTS169.4), BioXcell, BE0117;
InVivoMab anti-mouse PD-L1 (10F.9G2), BioXcell, BE0101; InVivoMab anti-mouse IFNAR1(MAR1-5A3), BioXcell, BE0241;
InVivoMAb polyclonal rat IgG, BioXcell, BE0094; β-actin antibody, Cell signaling, 5125; InVivoMab anti-mouse CD4 (GK1.5),
BioXcell, BE0117; cGAS(D3080) Rabbit mAb,Cell signaling,31659;STING(D2P2F) Rabbit mAb,Cell signaling,13647;Phosphor-
IRF3(Ser396)(D601M) Rabbit mAb,Cell signaling,83611;IRF-3(D83B9) Rabbit mAb,Cell signaling,4302;

Validation Antibody validations were performed by suppliers and have been published by our group and others

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Cell line source(s) MC38 and A549 are bought from ATCC and TSA was kindly provided by Dr.Zhihai Qin ; RAWISG cell line was kindly provid by Zhijian
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Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research
Laboratory animals Female C57BL/6J , Balb/c and Rag1-/- mice were purchased from UT southwestern mice breeding core. IFNAR1-/-, Mb21d1-/-
Batf3-/- and OT1CD8+ T cell receptor (TCR)-Tg mice in the C57BL/6J background and NSG-SMG3 mice were purchased from The
Jackson Laboratory. Ifnar1-/- mice were provided by Dr. Anita Chong from the University of Chicago

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Methodology
Sample preparation Tumor tissues were Cut into small pieces with scissors (about 1mm*1mm*1mm) and digested in the digested buffer (1mg/ml
collagenase and 0.25mg/ml DNase I in RPMI medium) at 37 degree for 45 minutes with the speed at 80RPM. Add 0.25ml FBS to
stop the digestion, and spin down and resuspend the pellet. Tumor cell suspension was blocked with the anti-CD16/32 antibody
(clone 2.4G2) for 10 min, and then incubated with indicated antibody for 30 min at 4 °C in the dark. Fixable viability Dye eFlour
506 (eBioscience) was used to exclude the dead cells.

Instrument cytoFLEX (Backman coulter),BD FACSMelody,Image Lab™ Software

Software CytExpert, FlowJo software v9.3.2,BD FACSMelody,Image Lab™ Software

Cell population abundance Physical parameter and fixable viability Dye eFlour 506 (eBioscience) was used to exclude the dead cells. Positive populations
were defined using not stained cells as reference. Isotype controls were used to confirm the specificity of the staining.

Gating strategy Physical parameter and fixable viability Dye eFlour 506 (eBioscience) was used to exclude the dead cells. Positive populations
were defined using not stained cells as reference. Isotype controls were used to confirm the specificity of the staining.

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

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