An ER‐Targeting Iridium(III) Complex That Induces Immunogenic Cell Death in Non‐Small‐Cell Lung Cancer

Abstract

Immunogenic cell death (ICD) is a vital component of therapeutically induced anti‐tumor immunity. An iridium(III) complex (Ir1), containing an N,N‐bis(2‐chloroethyl)‐azane derivate, as an endoplasmic reticulum‐localized ICD inducer for non‐small cell lung cancer (NSCLC) is reported. The characteristic discharge of damage‐associated molecular patterns (DAMPs), that is, cell surface exposure of calreticulin (CRT), extracellular exclusion of high mobility group box 1 (HMGB1), and ATP, were generated by Ir1 in A549 lung cancer cells, accompanied by an increase in endoplasmic reticulum stress and reactive oxygen species (ROS). The vaccination of immunocompetent mice with Ir1‐treated dying cells elicited an antitumor CD8⁺ T cell response and Foxp3⁺ T cell depletion, which eventually resulted in long‐acting anti‐tumor immunity by the activation of ICD in lung cancer cells. Ir1 is the first Ir‐based complex that is capable of developing an immunomodulatory response by immunogenic cell death.

Full text

Bioinorganic Chemistry Very Important Paper
An ER-Targeting Iridium(III) Complex That Induces Immunogenic
Cell Death in Non-Small-Cell Lung Cancer
Lili Wang,Ruilin Guan, Lina Xie,Xinxing Liao,Kai Xiong,Thomas W. Rees,YuChen,
Liangnian Ji, and Hui Chao*
Dedicated to the 100th anniversary of Chemistry at Nankai University
Abstract: Immunogenic cell death (ICD) is avital component
of therapeutically induced anti-tumor immunity.Aniridium-
(III) complex (Ir1), containing an N,N-bis(2-chloroethyl)-
azane derivate,asan endoplasmic reticulum-localized ICD
inducer for non-small cell lung cancer (NSCLC) is reported.
The characteristic discharge of damage-associated molecular
patterns (DAMPs), that is,cell surface exposure of calreticulin
(CRT), extracellular exclusion of high mobility group box
1(HMGB1), and ATP, were generated by Ir1 in A549 lung
cancer cells,accompanied by an increase in endoplasmic
reticulum stress and reactive oxygen species (ROS). The
vaccination of immunocompetent mice with Ir1-treated dying
cells elicited an antitumor CD8+Tcell response and Foxp3+T
cell depletion, which eventually resulted in long-acting anti-
tumor immunity by the activation of ICD in lung cancer cells.
Ir1 is the first Ir-based complex that is capable of developing
an immunomodulatory response by immunogenic cell death.
Introduction
Accumulating clinical evidence indicates that the clinical
effects of conventional anticancer drugs not only depend on
direct cell inhibition/cytotoxicity,but also result from the re-
activation of the tumor immune response.[1] Thereby,defects
in the components that engage in the stimulation to the
immune system to promote cancer cell death as immunogenic
limit the clinical outcome among cancer patients.[2] Antitumor
immune response can be primed by immunogenic cell death
(ICD), afunctionally peculiar variant of cellular demise that
involves the activation of an adaptive immune response.ICD
was first dubbed in 2005, when Casares et al. found the
immunogenicity of tumor cells treated with anthracycline was
generated without any adjuvant or co-stimulation.[3] ICD
stimulates an immune response against dead-cell antigens,in
particular when they derive from cancer cells,therefore
enhancing the efficacyofanticancer therapies.[4]
ICD inducers have been found to kill tumor cells while
simultaneously stimulating the spatiotemporal coordinated
release of immunogenic signals.These signals,which are
damage-associated molecular patterns (DAMPs), are nor-
mally inaccessible to the immune system, but released or
exposed to the cell membrane during cell death.[2] As
endogenous danger signals,many DAMPs,which include cell
surface exposure of calreticulin (CRT), migration of high
mobility group box 1(HMGB1) to the extracellular environ-
ment,[5] and the secretion of large amounts of ATP, [6] have
been shown to contribute to immunogenic cell death.[7] The
release of DAMPs enhances the immunogenicity of tumor
cells,promotes the antigen-presenting capacity of dendritic
cells (DCs), and then triggers aseries of Tcell-dependent
immune responses.[8] In addition, Endoplasmic reticulum
(ER) stress and reactive oxygen species (ROS) are important
components of intracellular pathways that control ICD.[9] The
co-existence of ER stress and excess ROSincreases the
number of different DAMPs,which is ultimately critical for
the immunogenicity of dying cancer cells in the process of
ICD.[10]
Based on whether ICD inducers directly act on ER to
induce apoptosis or induce ER stress and apoptosis through
apolymerization but the independent mechanism, the exist-
ing ICD inducers are divided into two categories,type IICD
inducers and type II ICD inducers.[10] Type IICD inducers
induce apoptosis with inducers that are not connected to the
ER, and induce ICD-related immunogenicity through secon-
dary ER stress effects.Type II ICD inducers selectively target
the ER by directly altering endoplasmic reticulum homeo-
stasis and triggering ER stress to induce immunogenic
apoptosis.[10] Most known ICD inducers are of Type I, such
as doxorubicin,[3] oxaliplatin.[11] Afew Type II ICD inducers
such as hypericin Photodynamic therapy,[12] oncolytic virus-
es[13] are found.
Asubstantial body of clinical trials tested ICD-inducing
chemotherapies in oncological indications.However,recent
work reveals only afew chemotherapeutic agents can evoke
ICD,[14] and most of the well-established ICD inducers used in
clinical tumor therapy are non-metallic compounds,such as
doxorubicin,[15] epirubicin,[16] mitoxantrone,[17] and cyclophos-
phamide.[18] Among the classical metallic anticancer drugs,[19]
oxaliplatin is the only one known to induce ICD.[11] Moreover,
recent research by Flieswasser et al. showed the inability of
oxaliplatin to induce ICD in non-small cell lung cancer
(NSCLC).[20]
[*] L. Wang, Dr.R.Guan, L. Xie, X. Liao, K. Xiong, Dr.T.W.Rees,
Dr.Y.Chen, Prof. Dr.L.Ji, Prof. Dr.H.Chao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry
School of Chemistry,Sun Yat-Sen University
Guangzhou, 510275 (P. R. China)
E-mail:ceschh@mail.sysu.edu.cn
Supportinginformation and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202013987.
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Some other metal anticancer agents,such as platinum(IV)
complexes,[21] ruthenium(II) complexes,[22] copper(II) com-
plexes,and manganese oxide nanomaterials (MONs),[23] have
been proven to induce ICD.The Ang group has screened
avariety of platinum complexes to explore their immunomo-
dulatory properties,and found the complex Pt-NHC met the
biochemical requirements to be an ICD inducer.[24] They also
recently identified anew platinum(II) complex, PlatinER an
ER-targeting complex, with ICD performance superior even
to that of Pt-NHC.[25] Huang et al. also found an ER-locating
platinum(II) complex Pt-1 containing an aminophosphate
ester ligand, which, as an ICD inducer, activates the immune
system against solid tumors.[26] Sabbatini et al. found that
aPt
IV prodrug containing a2-(2-propyl) octanol axial ligand-
induced ICD in colon cancer.[27] Kaur et al. reported acopper-
(II) complex containing apolypyridyl ligand and aSchiff base
ligand had the capable of inducing ICD in breast cancer stem
cells.[28] Ding et al. proved MnOx Nanospikes induced ICD by
chemodynamic therapy (CDT) and ferroptosis.[29] Wernitznig
et al. reported that KP1339, aruthenium-based anticancer
drug under clinical trials,can induce ICD.[30] Nevertheless,few
single metal complexes have been reported which evoke ICD
in NSCLC,adisease which represents an estimated 85%of
all lung cancers and accounts for 2.1 million new lung cancer
cases and 1.8 million deaths per year worldwide.[31] Zhu et al.
reported an oxaliplatin-based and photocaged PtIV prodrug,
coumaplatin. Coumaplatin had astrong tumor penetrability
and induced immunogenic death of cisplatin-resistant
NSCLC cells (A549R) and Tcell activation through aunique
photoactivation mechanism.[32] Thefinding of Zhu is inspir-
ing, but the ICD effect of coumaplatin is only shown on the
cellular level and coumaplatin is photoactivated, which means
it is not atypical chemotherapeutic agent.
Theexcellent anti-tumor activity and precise subcellular
localization of IrIII complexes have been well reported,[33] but
until now none have been found to cause ICD.Encouragingly,
immune responses and cancer cell death triggered by
membrane-active iridium(III) complexed oligarginine pep-
tides were reported recently by the Feigroup,showing the
potential of IrIII compounds in immunotherapeutic treat-
ments.However,this eruptive mode of cell death was based
on the maturation of dendritic cells and the accumulation of
inflammatory factors,instead of specific T-cell immune
responses as in ICD.[34] Based on the idea that subcellular
localization determines how the drug initially interacts with
tumor cells,[35] and the groups experience in the design of
organelle-targeted anticancer complexes,[36] the synthesis of
two IrIII complexes which selectively accumulate in the ER is
herein reported. Inspired by the ICD-inducing drug cyclo-
phosphamide,abis(2-chloroethyl)-azane moiety was added to
complex Ir1 only,with Ir2 as the reference compound.
Although both of the complexes target the ER and cause
severe ER stress,only Ir1 generates the multiple character-
istic effects of ICD in NSCLC cells.Invivo vaccination
experiments were carried out on immunocompetent C57BL/6
mice bearing the same syngeneic tumor. Significant tumor
reduction in mice vaccinated by Ir1-treated dying cells was
accomplished by recruiting cytotoxic Tlymphocytes
(Scheme 1). To the best of our knowledge, Ir1 is the first
iridium(III) complex that is capable of developing an
immunomodulatory response by immunogenic cell death,
providing apromising platform for immunotherapy.
Results and Discussion
Ir1 and Ir2 were synthesized by the methods shown in the
Supporting Information, the details and characterization of
which are shown in Scheme S1 and Figure S1–3. Thebasic
photophysical properties of Ir1 and Ir2 were also character-
ized by ultraviolet-visible spectroscopy (UV/Vis), and emis-
sion spectroscopy (Figures S3). Thecomplexes exhibited
strong absorption at 250–280 nm. Theemission maxima of
Ir1 and Ir2 were located at 560 nm and 580 nm, respectively.
Alarge meta-analysis involving 3983 patients across lung
(1432), breast (1115), and ovarian (1436) malignancies
revealed apositive correlation between improved survival
rates in patients with lung,breast, or ovarian malignancies
and immunogenic cancer death.[37] Immunotherapy and
immunogenicity chemotherapy for solid tumors were superior
to hematologic malignancies.[18] Moreover,ICD studies
mainly focus on breast cancer (41%), non-small cell lung
cancer (13%) and colon cancer (8 %).[18] Hence,weselected
cell lines belonging to the above-mentioned solid tumors for
in vitro experiments to study the cytotoxicity of the IrIII
complexes,which include A549 (lung epithelial carcinoma)
and A549R (cisplatin-resistant lung epithelial carcinoma),
LLC (lewis lung carcinoma), MDA-MB-231 (triple-negative
human breast carcinoma), and CT-26 (murine colon carcino-
ma), as well as the normal cell lines HLF (normal human lung
fibroblast cells), BEAS-2B (normal human lung epithelial
cells). Thecells were treated with various concentrations of
Ir1,Ir2,orcisplatin (CDDP,positive control) for 48 h, and
then tested by an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-
Scheme 1. Representation of Ir1-induced immunogeniccell death.
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diphenyl-2-H-tetrazolium bromide) assay.The cytotoxicity
profiles are shown in Table S1. Ir2 displays high cytotoxicity
towards all tested cells,with IC50 values ranging from 2.45 to
5.66 mM. Ir1 was more cytotoxic towards non-small cell lung
cancer cells (IC50 =4.90 mMfor A549 cells,5.00 mMfor
A549R) than breast cancer cells (IC50 =7.73 mMfor MDA-
MB-231). TheIC
50 values of Ir1 towards the three human
cancer cell lines were all lower than that towards the two
normal cell lines (14.31 mMfor HLF cells,12.57 mMfor
BEAS-2B cells), while those of Ir2 were even higher,
indicating abetter selectivity of Ir1 between cancer and
normal cells.Interms of murine cancer cells, Ir1 was more
active in LLC cells than CT-26.
In order to further investigate the cellular toxicity and
bioselectivity,the cellular uptake of Ir1 and Ir2 in different
cell lines was measured by inductively coupled plasma mass
spectrometry (ICP-MS,Figure S4). Low cellular uptake of Ir1
in CT-26 was observed, which could be acontributing factor
towards its low toxicity.Similarly this could explain the lower
uptake and toxicity of Ir1 compared to that of Ir2.The cellular
uptake mechanisms of Ir1 and Ir2 in A549 cells were studied
by confocal microscopy (Figure S5). Thedecrease in phos-
phorescence of the IrIII complexes in cells at low temperature
or with metabolism inhibitors indicated an energy-dependent
mechanism. In cells pretreated with the endocytosis inhibitors
chloroquine and NH4Cl, the cellular uptake was reduced.
These results suggest that Ir1 and Ir2 enter A549 cells by
energy-dependent endocytosis.The stability of Ir1 and Ir2 in
FBS and DMSO/D2O(5:1) is also confirmed by HPLC and
1HNMR (Figure S6 and S7).
Theintracellular distributions of Ir1 and Ir2 were inves-
tigated by colocalization assay with MitoTracker Deep Red
FM (MTDR), LysoTracker Green DND-26 (LTG), and ER-
Tracker Green (ERTG), in A549, A549R, MDA-MB-231,
and LLC cells (Figure 1Aand S8). Thecostain patterns of the
complexes matched poorly with those of LTGand MTDR.
Meanwhile,aconsiderable Pearson correlation coefficient
was observed between the IrIII complexes and ERTG,
indicating that Ir1 and Ir2 selectively localize in the ER. In
the ICP-MS assay,amajority of the IrIII complexes were
shown to be localized in the ER (Figure S9).
Thesubcellular target determines the initial interaction
between therapeutic agents and cells.Damage to the ER
often initiates ER stress.Cells treated with both of the IrIII
complexes at different concentrations were tested by Western
blot for the two ER stress-related proteins,C/EBP homolo-
gous protein (CHOP) and eukaryotic initiation factor 2a
(eIF2a). Up-regulation of CHOP and phosphorylation of
eIF2a(p-eIF2a)occurred which is atypical sign of ER stress
(Figure 1Cand S10). It should also be mentioned that CDDP
displayed some unexpected therapeutic effects including the
induction of ER stress.[38]
Severe ER stress leads to the release of Ca2+from the ER
and promotes the ER-mitochondria Ca2+flux. Mitochondrial
Ca2+overload induces the opening of the mitochondrial
permeability transition pore (mPTP), disrupting the electron
Figure 1. A) Co-localization of Ir1 in A549 cells by confocal spectroscopy.A549 cells were incubated with Ir1 (2 mM, 2h), and then incubated with
either LTG, MTDR, or ERTG for 0.5 hafter being washed with PBS. Scale bar :10mm. B) Confocal images of intracellularCa
2+assays with Fluo-4
AM in A549 cells. A549 cells were treated with Ir1 (5 mM) for 12 hor24h,and then incubated with Fluo-4 AM for 0.5 hafter being washed with
PBS. Scale bar:10mm. C) Representativeimmunoblotting images from acapillary Western blot system. A549 cells were treated with Ir1 (5 mM),
Ir2 (4 mM) and CDDP (10 mM) for 24 h, respectively.D)Flow cytometry results of aJC-1 assay and E) Flow cytometry results of aROS generation
assay.A549 cells were incubated with Ir1 (5 mM, 10 mM) for 24 h.
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transport chain, generating excessive reactive oxygen species
(ROS), and eventually leading to apoptosis.[39] Therefore,
aseries of experiments were performed, namely,intracellular
Ca2+detection, mitochondrial membrane potential assay,
ROSproduction, caspase 3/7 activation assay,[40] and annexin
V/propidium iodide (AV/PI) co-staining assay.Afree Ca2+
fluorescent probe,Fluo-4 AM, was used to monitor changes
in intracellular Ca2+.Atime-dependent increase in the
fluorescence of Fluo-4 clearly indicated the release of Ca2+
from the ER (Figure 1B and S11). As aresult of the opening
of the mPTP,the characteristic green/red (aggregates/mono-
mer) ratio of JC-1 showed the loss of mitochondrial mem-
brane potential after treatment with Ir1 and Ir2 (Figure 1D
and S12). Theclassic ROSprobe 2,7-dichlorodihydro-fluo-
rescein diacetate (DCFH-DA), which could be oxidized to the
fluorescent compound 2,7-dichlorofluorescein (DCF) by
intracellular ROS, was utilized to assess ROSproduction.
An increase in DCF emission was detected by flow cytometry
after 8h of Ir1-treatment, suggesting the elevation of ROS
level occurs in ashort period of time (Figure S13). Subse-
quent experiments of cells treated with various concentra-
tions of Ir1 and Ir2 indicated the over-generation of ROSby
Ir1 and Ir2 in adose-dependent manner (Figure 1Eand S14).
Thesubsequent activation of caspase 3/7 was investigated by
aCaspase-Glo 3/7 assay.Circa 3tocirca 9-fold increase in the
signals of the activity of caspase 3/7 were observed (Fig-
ure S15). Forthe further investigation of cell apoptosis,an
annexin V/PI co-staining assay was carried out. Thegradually
increased population in the AV+PI@and AV +PI+regions in
adose-dependent manner reflected the cell apoptosis induced
by Ir1 and Ir2 (Figure S16). From the aforementioned results,
it can be concluded that the toxicity of Ir1 and Ir2 is attributed
to apoptosis induced by ER-stress and mitochondrial dys-
function.
A549 cells were coincubated with the Ir complexes and
several cell death inhibitors including Z-VAD-fmk (pan-
caspase inhibitor), 3-methyladenine (autophagy inhibitor),
cycloheximide (paraptosis inhibitor), and necrostatin-1 (nec-
roptosis inhibitor). Arise in cell viability after co-incubation
of Z-VAD-fmk was observed, indicating acaspase-dependent
apoptotic pathway (Figure S17). Significantly,the survival
rate also increased after 24 hco-culture with 3-methyladenine
and Ir1 in A549 cells (Figure S17A). Theresult of the
inhibition assay shows that ahigh concentration (10 mM) of
Ir1 causes autophagy in A549 cells,but this was not observed
for Ir2.Importantly,inmost models,one of the key events in
ICD,ATP secretion by cells,requires acomplete autophagy
mechanism.[2,41] Thus,autophagy in A549 cells implies that Ir1
may be an inducer of ICD.
ER stress response constitutes acentral hub in the
signaling cascades leading to ICD.[42] One of the hallmarks
of ER stress is the phosphorylation of eIF2a.The phosphor-
ylation of eIF2acannot be employed as an independent
biomarker of ICD,but it is the basis of the adaptive immune
response induced by ICD.[43] Thereal hallmarks of ICD are
the timely discharge of certain DAMPs,such as the exposure
of CRT, and the release of HMGB1 and ATPinto the
extracellular milieu. As the previous results demonstrated
that Ir1 and Ir2 induce ER stress in A549 cells,the IrIII
complexesQability to evoke ICD in A549 cells by CRT,
HMGB1, and ATPwas investigated.
Although oxaliplatin is normally considered as an ICD-
inducer,studies are suggesting that it cannot induce ICD in
lung cancer cells.[20] Since CRTtranslocates from the cyto-
plasm to the cell surface during ICD,immunofluorescence
analysis of CRTwas performed in cells undergoing the early
stages of apoptosis.Nosurface expression of CRT(green
signals) in oxaliplatin-treated A549 cells was observed by
confocal imaging,which confirms the failure of oxaliplatin to
induce ICD in lung cancer cells (Figure S18). CDDP,achemo-
therapeutic agent without ICD-inducing ability,was therefore
selected as anegative control. Upon treatment with Ir1
(5 mM) for 12 h, CRTonthe cell surface of A549 cells was
identified by green immunofluorescence.CLSM analysis of
cells untreated (control) or treated with CDDP (10 mM)
revealed no surface expression of CRTinA549 cells,neither
did cells treated with Ir2 (Figure 2A,Figure S18). Amore
quantitative evaluation of CRTexposure by flow cytometry
confirmed the dose-dependent effect of Ir1-induced ICD in
A549 cells for 24 h, as the immunofluorescence of CRT
increased (Figure 2B and S19). Interestingly,even though
both Ir1 and Ir2 cause severe ER stress, Ir2 fails to induce the
exposure of CRT(Figure S19), let alone immunogenic cell
death.
In addition, as shown in Figure S13, ROSgeneration
analysis after 8hours of Ir1-treatment, and an increase in the
ROSgeneration was observed. Meanwhile,after 12 hours of
Ir1-treatment with various concentrations,the expression of
CRTwas only aslight increase (Figure S20), while 24 hofIr1-
treatment caused asignificant increase (Figure 2B and S19).
Therefore,the elevation of ROSlevel occurred at avery early
stage (8 h), even before the phosphorylation of elFa(24 h),
the release of Ca2+from the ER (12 h), and the induction of
CRTexpression (Figure 1B,C and S19,20), that is,ROS over-
generation is the upstream event, making Ir1 atype II ICD
inducer.[24]
Themigration of HMGB1 from the nucleus to cytoplasm
then extracellular space is one of the DAMPs associated with
ICD.Asshown in Figure 2C,HMGB1 (red signals) is located
in the nucleus of untreated A549 cells,before translocation to
the cytoplasm after treatment with Ir1 (5 mM) for 30 h. The
release of HMGB1 to the extracellular space was quantified
by analyzing the medium supernatant of Ir1-treated cells,
using acommercially available enzyme-linked immunosorb-
ent assay (ELISA) kit specifically for human or murine
HMGB1. Theextracellular release of HMGB1 from A549
cells treated with Ir1 (10 mM) was 4.54 times as large as the
control cells and the ratio of released HMGB1 from A549
cells increased in adose-dependent manner (Figure 2D).
CLSM and ELISA data show that Ir1 causes significant
HMGB1 extracellular release in NSCLC cells.
Besides CRTexposure and HMGB1 release,ATP secre-
tion to the supernatant after treatment was also measured, by
abioluminescence detection kit. A549 cells were incubated
with Ir1 (5 mM) and CDDP (10 mM) for 24 h, and the
supernatant was analyzed. Ir1 causes the increase of ATP
secretion from 28.05 pmol L@1to 108.81 pmolL@1at 5 mM, and
to 198.92 pmolL@1at 10 mMafter 24 h. Theamount of ATP
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exocytosis of the CDDP group was only 29.78 pmol L@1
(Figure 2E). Therefore,treatment with Ir1 leads to ATP
secretion.
From the above data, it can be concluded that Ir1-induced
cell death displays the three biomarkers of ICD,while the
analogous Ir2 complex does not induce ICD.Itisworth
mentioning that Ir1 also successfully induces CRTexposure,
HMGB1 release,and ATPsecretion in drug-resistant A549R
cells (Figure S21–25). However,nosurface exposure of CRT
in Ir1-treated breast cancer cells (MDA-MB-231) was ob-
served, suggesting Ir1 is unable to induce ICD in MDA-MB-
231 cells (Figure S26).
Currently,the gold standard for determining whether
drugs can induce ICD in cancer cells is vaccination assays
which rely on immunocompetent mice and homogenotypic
tumor cells.[2] In order to validate the results of the in vitro
experiments,vaccination experiments were performed in
C57BL/6J female mice with LLC (murine lung cancer) cells.
Theability of Ir1 to induce ICD in murine lung cancer cells
was first tested by applying the same set of experiments to the
LLC cells as to the A549 cells.CRT exposure,HMGB1
migration, and ATPsecretion occurred after treatment with
Ir1 (Figure S27–31). Thequalitative and quantitative results
demonstrated all three ICD hallmarks in the murine lung
cancer cells in vitro.
In the vaccination study,healthy C57BL/6J female mice
were randomly divided into 3groups with 14 mice in each
group (namely:control group, Ir1 group,and CDDP group).
In the Ir1 group,LLC cells were exposed in vitro to Ir1
(15 mM) for 24 h, then washed with PBS to remove the
complex, and re-suspended in PBS.Atthis point, the treated
cells would be dying due to the cytotoxicity of Ir1,and serve
as the “inactivated vaccine” in subsequent experiments.The
aforementioned dying lung cancer cells were subcutaneously
inoculated to the immunocompetent homogenic mice,as
shown in Figure 3A.Inthecontrol group and CDDP group,
the stimuli were replaced by the same volume of PBS or
CDDP-treated LLC cells (24 mM, 24 h). Aweek later, the
same type of living lung cancer cells was introduced into the
contralateral subcutaneous tissue and the mice were routinely
monitored for palpable neoplastic lesions.The survival rate
was significantly improved in Ir1-treated mice compared with
untreated mice in the 30 days after the re-challenge (Fig-
ure 3B). Tumor growth was monitored over 20 days,asshown
in Figure 3Eand Table S2. Due to the malignant proliferation
of murine LLC cells,rapid tumor growth was observed in the
Figure 2. ICD hallmarksinA549 cells treated with Ir1.A)Confocal images of surface CRT in Ir1-treated A549 cells. A549 cells were incubated with
Ir1 (5 mM) and CDDP (10 mM) for 12 h, and then incubated with Calreticulin (D3E6) XP Rabbit mAb (Alexa Fluor 488 Conjugate) overnight and
Hoechst for 15 min after being washed with PBS. Scale bar:10mm. B) Flow cytometry results of increased CRT expression in Ir1-treated A549
cells. A549 cells were treated with Ir1 (2 mM, 5 mM, 8 mMand 10 mM), compared to untreated A549 cells (control) for 24 h. C) Confocal images of
HMGB1 release in Ir1-treated A549 cells. A549 cells were incubated with Ir1 (5 mM) and CDDP (10 mM) for 30 h, and then incubated with HMGB1
Antibody overnight, Anti-rabbit IgG (H +L) (Alexa Fluor 555 Conjugate) for 2hand Hoechst for 15 min after being washed with PBS. Scale bar :
10 mm. D) Release of HMGB1 in cell culture supernatant. A549 cells were incubated with Ir1 (5 mM, 10 mM) and CDDP (10 mM) for 24 h.
E) Analysis of AT Plevels in cell culture supernatants. A549 cells were incubated with Ir1 (5 mM, 10 mM) and CDDP (10 mM) for 24 h.
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control cohort. Encouragingly,the tumor growth was inhib-
ited in the cell-immunized mice in the Ir1 group.The average
tumor volume of the Ir1 group on the 20th day after
inoculation was 283 mm3,4.59 times smaller than that of the
control group (1328 mm3). In addition, the body weight of all
mice remained stable throughout the whole period (Fig-
ure 3C). Theresults demonstrate that the Ir1-treated-cell
vaccine is effective in enhancing the ability of mice to resist
the attack of living LLC cells. Ir1 achieved the gold standard
for ICD inducing cancer therapeutics.
In order to further understand the potential mechanism of
the Ir1-induced anti-tumor immune response in C57BL/6J
mice,immune cells from mice in the vaccine experiment were
analyzed. Thepresence of cytotoxic Tcells (CTLs,CD8+T
cells) in tumors is considered to be aspecific immune
response and is associated with agood prognosis in many
cancers.Moreover,ICD inducers eventually elicit aTcell-
mediated immune response,including the activation of CD8+
Tcells.[9b] Theproportion of CD8+Tcells (CD3+CD8+T
lymphocytes) and activated CD8+Tcells (CD3+CD8+CD38+
Tlymphocytes) in the tumors and spleens of the mice were
quantified on day 16.[44] As shown in Figure 3F and Gand
Table S2 and Figure S32,33, in the Ir1 group 86.85%ofthe
tumor cells were activated CD8+Tcells,appreciably higher
than that in the control group (60.60%) and the CDDP group
(68.63%). Similarly,the percentage of activated CD8+Tcells
in the Ir1 group was significantly higher than that of the
control group and CDDP group in the spleens of the mice
(Figure S34). In addition, CD4+Tcells (CD3+CD4+T
lymphocytes), whose function is to assist CD8+Tcells and
participate in the cellular immune response were analyzed.
TheCD4 expression in tumor tissue of the Ir1 group (3.06 %)
was also higher than that in the control group (1.51%) and
CDDP group (2.13%), as shown in Figure S35. As Foxp3+T
cells impair antitumor immunity by secreting immunosup-
pressive cytokines and inhibiting cytotoxic cell function
Figure 3. Antitumor vaccination in vivo. A) Schematic diagram of the ICD vaccine experiment. LLC cells were first treated with Ir1 (15 mM), CDDP
(24 mM) or solvent control for 24 h, before the treated LLC cells were subcutaneously injected into the right flanks of C57BL/6 mice (n=14),
which were then re-challenged in the left flanks with untreated LLC cells 7days later.B)Curves showing the survival time of the mice during the
30 days of treatment. Error bars, mean :SD (n=10). C) The weight of the mice throughout the follow-up period. D) Photographs of the tumors
removed from the mice 30 days after treatment. E) Plot of tumor volumes for the different groups versus the time post live LLC cancer cell
inoculation. F–I) Quantitative analyses of the percentages of F) activated CD8+Tcells, G) CD8+Tcells, H) Foxp3+Tcells in the total cells isolated
from tumors of C57BL/6mice treated with PBS, Ir1 or CDDP. I) The tumors were analyzed by flow cytometry to determine the CD8/Foxp3 ratio.
Error bars, mean :SD (n=4), *** P<0.001,** P<0.01 and * P<0.05, in comparison with Ir1 cohort. Control group:PBS; Ir1 group: Ir1
(15 mM, 24 h);CDDP group:CDDP (24 mM, 24 h).
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in late neoplasia,[45] the quantity of Foxp3+Tcells
(CD3+CD4+Foxp3+Tlymphocytes) in the tumors of the
three groups was also determined. As shown in Figure 3H
and Table S2, the percentage of Foxp3+Tcells in the Ir1
group shrank to 4.57%, less than both the control group
(12.66%) and CDDP group (18.13 %). Quantitative assess-
ment of CD8 and Foxp3 using flow cytometry also demon-
strated 4.9- and 15.6-fold higher CD8+/Foxp3+cell ratios in
the Ir1 group,than the control and CDDP groups,respec-
tively (Figure 3I). Theeffect of treatment with CDDP was
negligible.The results of flow cytometry revealed an increase
in the CD8+Tcell component, in parallel with adecrease in
the Foxp3+Tcell component in the tumors of the mice
vaccinated with Ir1-treated cells.Inaddition, CD8 and Foxp3
immunofluorescence staining was performed on tumor and
spleen sites in each group on day 30 to gain amore intuitive
understanding of the physiological distribution of CD8 and
Foxp3 (Figure S36).[46] Theresults indicate that Ir1 induces
ICD in LLC cells by recruiting cytotoxic Tlymphocytesand
down-regulating Foxp3+Tcells,which can lead to the
significant reduction or eradication of tumors.
Histochemical analysis of the main organs stained with
hematoxylin-eosin was carried out. There were some patho-
logical changes in the liver of the control group,b
ut no serious
structural and pathological changes were observed in the
other groups (Figure S37). As an inducer of ICD in lung
cancer cells, Ir1 converts dying cancer cells into therapeutic
vaccines and stimulates an anti-tumor immune response in
vivo.
Conclusion
In this work, we designed two IrIII complexes, Ir1 and Ir2,
both of which localized in the ER and induced ER stress in
cells.Inspired by cyclophosphamide,anICD-inducer with the
derivate bis(2-chloroethyl)-azane as its action-site,wede-
signed Ir1 with the same moiety to evaluate its ICD-induction
ability and found Ir1 as agood ICD inducer in non-small-cell
lung cancer. By setting Ir2 (without the moiety) as the
reference compound, we found that though bis(2-chloroeth-
yl)-azane slightly reduced the cytotoxicity of the compound, it
is essential for the subsequent immune response.Only Ir1
induced the multiple characteristics of ICD in NSCLC cells,
i.e., surface exposure of CRT, extracellular release of
HMGB1, and ATP. We performed avaccination assay using
LLC cells in an attempt to validate our in vitro findings.The
survival rate and tumor inhibition rate in mice confirmed that
the Ir1-treated LLC cell vaccine provokes astrong antitumor
immunity in vivo by triggering an adaptive immune response.
As Type II ICD inducer, Ir1 induced tumor cells that undergo
ROS-induced ER stress released DAMPs,and finally acti-
vated the immune response.
Current anticancer drugs have been developed in pre-
clinical models of immunodeficiencyand clinical trials with-
out any form of immunomonitoring.However,most anti-
cancer drugs exhibit multiple immunological effects as
suggested by clinical experience.Therefore,when we design
and synthesize prospective anticancer drugs,itisnecessary to
select the appropriate cell and animal models to test their
immune effects.Bytapping into ICD and the other immuno-
modulatory functions of metal drugs,c
hemotherapy and
immunotherapy can be combined to enhance the anti-tumor
efficacyofmetal complexes.Inthis work, the immunomodu-
latory properties of IrIII complexes were explored for the first
time.Itishoped that this work paves the way for the future
development of effective metal-based immunochemothera-
puetics.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Nos.21525105, 21778079, and
21907112), the Ministry of Education of China (No.IRT-
17R111) and the Fundamental Research Funds for the
Central Universities of China (No.20lgjc01).
Conflict of interest
Theauthors declare no conflict of interest.
Keywords: bioinorganic chemistry ·immunogenic cell death ·
iridium ·medicinal inorganic chemistry ·metals in medicine
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Manuscript received:October18, 2020
Acceptedmanuscript online: November 20, 2020
Version of record online: January 4, 2021
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