J Gynecol Oncol. 2023 May;34(3):e25
https://doi.org/10.3802/jgo.2023.34.e25
pISSN 2005-0380·eISSN 2005-0399

Original Article

Interleukin-34 cancels anti-tumor
immunity by PARP inhibitor

Takayoshi Nakamura ,1,2 Nabeel Kajihara ,1 Naoki Hama ,1Takuto Kobayashi ,1 Ryo Otsuka ,1 Nanumi Han ,1 Haruka Wada
Yoshinori Hasegawa ,3 Nao Suzuki ,2 Ken-ichiro Seino 1,1

Division of Immunobiology, Graduate School of Medicine, Institute for Genetic Medicine, Hokkaido
University, Sapporo, Japan2
Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kawasaki, Japan3
Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Japan1

Received: Jun 23, 2022
Revised: Oct 5, 2022
Accepted: Nov 30, 2022
Published online: Dec 21, 2022
Correspondence to
Ken-ichiro Seino
Division of Immunobiology, Graduate Schoolof Medicine, Institute for Genetic Medicine,
Hokkaido University, Kita-15 Nishi-7, Sapporo,
Hokkaido 060-0815, Japan.
Email: seino@igm.hokudai.ac.jp© 2023. Asian Society of Gynecologic
Oncology, Korean Society of Gynecologic
Oncology, and Japan Society of Gynecologic
Oncology
This is an Open Access article distributed
under the terms of the Creative Commons
Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/)
which permits unrestricted non-commercial
use, distribution, and reproduction in any
medium, provided the original work is properly
cited.
ORCID iDs
Takayoshi Nakamura
https://orcid.org/0000-0002-6968-2893
Nabeel Kajihara
https://orcid.org/0000-0003-0822-3391
Naoki Hama
https://orcid.org/0000-0003-4318-8448
Takuto Kobayashi
https://orcid.org/0000-0002-0096-6038
Ryo Otsuka
https://orcid.org/0000-0002-3730-9867
Nanumi Han
https://orcid.org/0000-0001-9524-1808

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ABSTRACT
Objective: Breast cancer susceptibility gene 1 (BRCA1)-associated ovarian cancer patients havebeen treated with A poly (ADP-ribose) polymerase (PARP) inhibitor, extending the progressionfree survival; however, they finally acquire therapeutic resistance. Interleukin (IL)-34 has
been reported as a poor prognostic factor in several cancers, including ovarian cancer, and it
contributes to the therapeutic resistance of chemotherapies. IL-34 may affect the therapeutic
effect of PARP inhibitor through the regulation of tumor microenvironment (TME).
Methods: In this study, The Cancer Genome Atlas (TCGA) data set was used to evaluate theprognosis of IL-34 and human ovarian serous carcinoma. We also used CRISPR-Cas9 genome
editing technology in a mouse model to evaluate the efficacy of PARP inhibitor therapy in thepresence or absence of IL-34.
Results: We found that IL34 was an independent poor prognostic factor in ovarian serous
carcinoma, and its high expression significantly shortens overall survival. Furthermore,in BRCA1-associated ovarian cancer, PARP inhibitor therapy contributes to anti-tumor
immunity via the XCR1+ DC-CD8+ T cell axis, however, it is canceled by the presence of IL-34.
Conclusion: These results suggest that tumor-derived IL-34 benefits tumors by creating an
immunosuppressive TME and conferring PARP inhibitor therapeutic resistance. Thus, we showed
the pathological effect of IL-34 and the need for it as a therapeutic target in ovarian cancer.
Keywords: Interleukin-34; BRCA1-Associated Cancer; PARP Inhibitor; Therapeutic Resistance;
XCR1+ Dendritic Cell; CD8+ T Cell

SynopsisInterleukin (IL)-34 is an independent poor prognostic factor, and its high expression
shortens overall survival in human ovarian serous carcinoma. When tumors express
IL-34, poly (ADP-ribose) polymerase (PARP) inhibitor was not effective even in Brca1deficient ovarian cancer. In the absence of IL-34, PARP inhibitors have anti-tumor
immunity via the XCR1+ DC-CD8+ T cell axis.

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Haruka Wada
https://orcid.org/0000-0002-5526-3647
Yoshinori Hasegawa
https://orcid.org/0000-0003-2904-6611
Nao Suzuki
https://orcid.org/0000-0002-7440-8127
Ken-ichiro Seino
https://orcid.org/0000-0003-4122-1718
Conflict of Interest
No potential conflict of interest relevant to thisarticle was reported.
Author Contributions
Conceptualization: N.T., K.N., H.N., O.R., H.N.,
W.H., S.N., S.K.I.; Data curation: N.T., K.N., K.T.,
O.R., H.Y.; Formal analysis: N.T., K.N., H.N.,
H.Y.; Funding acquisition: S.K.I.; Investigation:
N.T.; Methodology: N.T., K.N., H.N., O.R., H.N.,
W.H., S.N., S.K.I.; Project administration: S.K.I.;
Resources: S.K.I.; Software: N.T., K.T., H.Y.;
Supervision: S.K.I.; Validation: O.R., H.N., W.H.,
S.N., S.K.I.; Writing - original draft: N.T., K.N.;
Writing - review & editing: O.R., H.N., W.H.,
H.Y., S.N., S.K.I.

INTRODUCTION
Cellular deoxyribonucleic acid (DNA) can be exposed to exogenous and endogenous factors
resulting in single- and double-strand breaks that could lead to detrimental consequencessuch as chromosomal aberrations, genomic instability, and cell death [1]. To prevent these
events, cells equip a mechanism to activate poly (ADP-ribose) polymerase (PARP)-1, oneof the DNA repair proteins, when DNA damage occurs [1]. PARP-1 contributes to intranuclear processes, including DNA repair, by catalyzing the protein’s poly-ADP-ribosylation(PARylation) [1]. The reversible ADP-ribosylation of DNA by PARP-1 is responsible for an
efficient DNA damage response [2]. In recent years, PARP inhibitor, which inhibits PARP-1
functions, has attracted attention as a novel therapeutic agent in epithelial ovarian and breast
cancer [3,4]. PARP inhibitor was originally known as a medicine that triggered the synthetic
lethal relationship between breast cancer susceptibility gene 1 and 2 (BRCA1/2) and PARP-1 inhomologous recombination deficient (HRD), resulting in critical genomic instability and cell
death [5]. However, later, it was revealed that PARP inhibitor traps PARP-1 at DNA replication
forks and stabilizes the DNA-PARP-1 complex, resulting in replication fork collapse, finallyleading to double-strand breaks and cell death [6].
PARP inhibitor has been shown to kill cancer cells by the mechanisms mentioned above
and has also improved progression-free survival (PFS) in ovarian cancer [3]. Nevertheless,as well as other anti-cancer drugs, many patients treated with PARP inhibitor finally acquire
therapeutic resistance [7]. Therefore, only a few reports show the prolongation of overall
survival (OS) by PARP inhibitor therapy, and new therapeutic strategies such as combined
use with immunotherapy are required to enhance the therapeutic effect of PAR inhibitor [7].
Indeed, PARP inhibitor increased programmed death-ligand 1 (PD-L1) expression on mouseovarian and breast cancer cells, resulting in a synergistic anti-tumor effect when combinedwith anti-PD-L1 antibody [8,9]. Whereas, it has been reported that PARP inhibitor increased
signal transducers and activators of transcription 3 (STAT3) activation in tumor and tumorassociated immune cells, promoting therapeutic resistance and immunosuppression [9].
Besides, tumor-associated macrophages (TAMs) infiltrating BRCA1-associated cancer tissuechanged to immunosuppressive type by PARP inhibitor therapy, limiting its therapeutic effect[10]. Given these findings, manipulating the tumor microenvironment (TME) can improve theeffects of anti-cancer drugs such as chemotherapeutic and immunotherapeutic agents [11].
The TME is a complex ecosystem of various cellular subsets, including tumor and immune
cells, and changes dynamically during tumor progression [12]. When immunity works
typically, cancer cells are taken up by antigen-presenting cells (APCs), and their information
is presented to T cells, finally eliminated by CD8+ T cells [13]. However, in many stagesof tumor formation, immunity falls to dysfunctional status via immunosuppressive cells
such as regulatory T cells (Tregs), TAMs, and myeloid-derived suppressor cells (MDSCs),
creating a favorable TME for tumor growth [14]. The representative factors that lead tothe generation of immunosuppressive cells are tumor-derived inflammatory cytokinesand chemokines, which potently suppress anti-tumor immunity in most cancer [11,15].
Interleukin (IL)-34, a pro-inflammatory cytokine, is locally expressed in the brain and skinunder physiological conditions. Mouse IL-34 expression was also examined by single-cell
RNA sequencing (scRNA-seq) in the whole mesentery and found to be expressed only in a
subset of fibroblasts, endothelial cells, and mesothelial cells [16,17]. These reports indicatedthat no active IL-34 expression was found in the peritoneal cells under normal conditions,but its expression is increased in various diseases and is involved in cancer pathogenesis

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IL-34 cancels antitumor immunity by PARP inhibitor[16,18]. In particular, it has been reported that IL-34 is an independent poor prognostic
factor in breast cancer and colorectal cancer [18,19]. It has also been shown that high IL-34
expression in TME promotes tumor growth by mediating the induction and proliferation of
immunosuppressive cells and hence contributes to the acquisition of therapeutic resistanceto anticancer drugs [18].
Based on these backgrounds, in this study, we have investigated how IL-34 affects thetherapeutic effect of PARP inhibitor via modulating TME. To investigate the antitumor
efficacy of PARP inhibitors, we selected HM-1 and 4T1 cells in this study, which are highgrade tumors in mice. These cell lines were appropriate for our research because they
are high-grade tumors with high cancerous and metastatic potential, rich in cytokine
release, and frequently used in immunology studies [18,19]. Our results showed that IL-34
strongly limited the anti-tumor effect of PARP inhibitor in BRCA1-associated cancers via
immunosuppressive changes in TME. On the other hand, IL-34 deficiency in cancer cells
dramatically improved the anti-tumor effect of PARP inhibitor on murine BRCA1-associatedovarian cancers via a specific dendritic cell (DC)-CD8+ T cell axis. Here, we propose IL-34 asa responsible molecule of PARP inhibitor therapeutic resistance in BRCA1-associated cancers
and suggest a novel therapeutic strategy.

MATERIALS AND METHODS
1. Cell lines
Mouse ovarian cancer cell line OV2944-HM-1 (HM-1) and breast cancer cell line 4T1 werepurchased from the Japanese Collection of Research Bioresources and the American Type
Culture Collection, respectively. The HM-1 and 4T1 cell lines were maintained in MEMa and
RPMI-1640, respectively (Fujifilm Wako Pure Chemical Industries, Osaka, Japan). All culture
media were supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA),
100 U/mL penicillin (Nacalai Tesque Inc., Kyoto, Japan), 100 µg/ml streptomycin (Nacalai
Tesque Inc.), 0.1 mM MEM nonessential amino acids (Nacalai Tesque Inc.), and maintained
at 37°C with 5% CO2.

2. Generation of BRCA1 and IL-34 knockout (KO) cell line
BRCA1 and IL-34 KO HM-1 cell lines were generated using BRCA1 Double Nickase plasmid(#sc-419362-NIC; Santa Cruz Biotechnology, Dallas, TX, USA) or IL-34 Double Nickase
plasmid (#sc-429354-NIC; Santa Cruz Biotechnology). The plasmids were transfected into
1×105 cells per well using the TransIT-X2 (Mirus Bio, Madison, WI, USA) in a 6-well plate,
and the cells were incubated for 48 hours. After incubation, successful transfection of theplasmid was visually confirmed by detecting green fluorescent protein (GFP) via fluorescent
microscopy ObserverZ1 (Carl Zeiss AG, Oberkochen, Germany). Plasmid-transfected cells
were selected by the GFP expression using FACSAria™ II cell sorter (BD Biosciences, Franklin
Lakes, NJ, USA) 48 hours after transfection. Then, puromycin (6 µg/mL) was added to themedium and cultured for 144 hours to select the transfected cells.

3. Protein expression analysis
Protein expression analysis for BRCA1 KO validation was performed by Western blotting.
Cell lysates were obtained from each cell line by lysis in TNE buffer. The cell lysate wascentrifuged to generate a supernatant sample. Protein concentrations of the supernatantsamples were determined using the Pierce BCA™ Protein Assay Kit (#23225; Thermo Fisher
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Scientific, Waltham, MA, USA). Supernatant samples were added to 6× sodium dodecyl
sulfate (SDS) sample buffer containing 2-mercaptoethanol to achieve a total protein content
of 40–50 µg per applied volume and were separated by electrophoretic migration at 200 V
for approximately 40 minutes using a 10% acrylamide gel. Transcriptions were transferred
to polyvinylidene difluoride (PVDF) membranes by the semidry method at 25 V for 30
minutes. The membrane was blocked with 3% skim milk for 1 hour at room temperature.
The membrane was then probed with the BRCA1 antibody (287. 17) (#sc-135732; Santa
Cruz Biotechnology) at 4°C overnight with gentle shaking. It was also incubated with thecorresponding horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour atroom temperature. Proteins were visualized using Super Signal™ West Femto Maximum
Sensitivity Substrate (#34094; Thermo Fisher Scientific).

4. DNA sequencing analysis
Genomic DNA sequencing analysis was performed to confirm the BRCA1 mutation in
the cell lines. Genomic DNA was isolated from each cell line (1×106 cells) using 0.1%
SDS-TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 10 mM ethylenediaminetetraacetic acid
[EDTA]). Polymerase chain reactions (PCRs) were performed using 1 µg of genomic DNA
according to the Premix Ex Taq™ Hot Start Version (#RR030A; Takara Bio Inc., Kusatsu,
Japan) manufacturer’s instructions. The following primer pairs spanning the target sites
were used in the PCR: Brca1 (Forward: 5′-TGACGCCACCACCACTAGGC-3′ and Reverse:
5′-TGTGCCCATTTCGGACCTGCAT-3′). PCR products were purified using a 2% agarose
gel and FastGene® Gel/PCR Extraction Kit (Nippon Genetics Co., Tokyo, Japan). Sanger
sequencing was then performed. Purified PCR samples were prepared with BigDye®
Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Waltham, MA, USA) and forward
or reverse primers to a final dose of 20 µL and used for sequencing PCR. Samples wereanalyzed by ABI® PRISM 3130 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA).

5. RNA sequencing analysis
Gene expression profiling and sequencing analysis were performed to confirm the major
homologous recombination repair (HRR)-associated genes other than Brca1 in the celllines. RNA samples extracted from cells were transported to Kazusa DNA ResearchInstitute (Kisarazu, Japan) for RNA sequence analysis. RNA samples were adjusted using
the SureSelect Strand-Specific RNA library adjustment kit (G9691A; Agilent Technologies,
Santa Clara, CA, USA) to the library, and these samples were used for sequencing by Illumina
NextSeq500 (Illumina, Inc., San Diego, CA, USA). Mutation detection and annotation were
performed using QIAGEN CLC Genomics Workbench (QIAGEN, Hilden, Germany). All
detected mutation sites were checked against major HRR-associated gene sites other thanBrca1 to confirm the presence or absence of mutations, and changes in expression levels of
those genes in Brca1KO strains were examined. The major HRR-related genes were taken fromthe following references [20,21].

6. Cell proliferation assay
HM-1 and 4T1 cells (1×105) were seeded in 6 cm dishes. The number of cells (total cells
including floating cells) was counted using a hemocytometer on day 3 or 4, and cells wereseeded again after the count. These operations were repeated at least 6 times. Cell numbers
were normalized and were relative to the number of cells seeded initially and were shown by
the common logarithm. When the cells were counted, they were stained with trypan blue,
and the blue-stained cells were excluded from the total cell count.

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7. Cell viability assay
The cell viability of HM-1 and 4T1 cell lines was assessed by the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay using MTT Cell Count Kit (Nacalai TesqueInc.). Cells were seeded at a density of 2×103 in a 96-well plate and stimulated with someconcentrations (0–5 μM) of niraparib (MK-4827; AdooQ BioScience, Irvine, CA, USA), or 5×103
cells were stimulated with niraparib and some concentrations (0–50 μM) of cisplatin. The cell
viability was followed up to 3–4 days. Absorbance at a test wavelength of 570 nm and a reference
wavelength of 650 nm was measured using a Multiskan™ FC (Thermo Fisher Scientific).

8. Gene expression analysis
Gene expression analysis was performed by quantitative PCR (qPCR). Total RNA wasextracted using TRIsure reagent (Nippon Genetics Co.). Complementary DNA (cDNA) was
synthesized from extracted RNA using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka,
Japan). qPCR was performed on cDNA using KAPA SYBR® FAST qPCR Master Mix (2X) ABI
Prism™ (Kapa Biosystems, Wilmington, MA, USA) on a StepOnePlus™ Real-Time PCR
System (Thermo Fisher Scientific). The primers are listed in Table S1.

9. IL-34 quantification
The production of IL-34 from HM-1 and 4T1 cell lines was measured by the enzyme-linked
immunosorbent assay (ELISA) using LEGEND MAX™ Mouse IL-34 ELISA Kit (Biolegend, San
Diego, CA, USA). Cell culture supernatants were collected 48 hours after seeding the cells at adensity of 1×106 in a 6 cm dish.
Protein was extracted from HM-1 Mock or Brca1KO tumors and the concentration of IL-34 ineach tumor was determined using an ELISA assay. For IL-34 expression analysis in tumors,
2×106 HM-1 cells were inoculated subcutaneously into the flank of syngeneic female mice.
Tumors were collected when tumor size reached 10 mm in diameter. From the collected
tumors, 10 mg of tumor tissue was weighed using a high-precision analytical balance (ANDGH-202) and divided into 500 μL of TNE buffer containing a protease inhibitor. Cell lysates
were obtained from the measured tumor tissue which was crushed using a BioMasher® /PowerMasher II (Nippi, Inc., Tokyo, Japan) in TNE buffer. The cell lysate was centrifuged
to generate a supernatant sample. Similarly, the concentration of IL-34 in the supernatantsample was determined by ELISA assay.

10. Mice experiments
Six to nine-week-old female B6C3F1 and BALB/c mice were purchased from Japan SLC orHOKUDO. The mice were maintained under specific pathogen-free conditions and housedin 12 hours light/12 hours dark cycle in the animal facility at Hokkaido University. For in vivo
assay, 1×105 HM-1 or 4T1 cells were inoculated subcutaneously into the right flank of syngeneic
female mice. Niraparib (5 mg/kg) was intraperitoneally administered when tumor size reached
5 mm in diameter. Tumor size was measured by caliper three times a week. The HM-1 and 4T1
tumors were collected 19 days after the cell inoculation. All animal procedures were approvedby the Hokkaido University Animal Care Committee (approval number: 19-0094).

11. Isolation of tumor-infiltrating immune cells from solid tumor
Isolation of tumor-infiltrating immune cells from solid tumors was performed using BD
Horizon™ Dri Tumor & Tissue (BD Biosciences). The recovered tumor-infiltrating cells wereused as samples for flow cytometry.

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12. Analysis of tumor-infiltrating immune cells
Tumor-infiltrating immune cells from solid tumors were analyzed by flow cytometry. Cells
were washed and blocked with FcR Blocking Reagent (CD16/32; BioLegend) and stainedwith 4′,6-diamidino-2-phenylindole (Cayman Chemical Company, Ann Arbor, MI, USA)and the antibodies against to following molecules; Cd3ε, Cd4, Cd8α, Cd11b, Cd11c, Cd45,
Cd69, Cd86, Cd206, F4/80, Gr-1, I-a/i-e, and Xcr1 (BioLegend). Data were acquired using
FACSCelesta™ flow cytometer (BD Biosciences) and analyzed using FlowJo software.

13. FLT3L-driven DC differentiation of bone marrow cultures
Cell suspensions of mouse bone marrow were prepared by centrifugation of femurs, tibias
and humerus with phosphate buffer saline followed by red blood cell lysis and filtering
through a sterile 70 μm filter. The cells were suspended in D-MEM with L-Glutamine(Fujifilm Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum,
100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 mM MEM nonessential amino acids, 55
μM 2-mercaptoethanol and FLT3L 100 ng/mL (#550704; BioLegend) (DC medium). Cells
were seeded at 8×106 cells per well in 4 mL of DC medium with ‘IL-34 100 ng/mL (#577608;
BioLegend) or nothing’ and ‘niraparib 2.5 μM or dimethyl sulfoxide (DMSO)’ in 6-well plateand cultured at 37°C with 5% CO2 for 9 days without replating medium.

14. Ovarian cancer bioinformatics analysis
From the ovarian serous carcinoma dataset (cBioPortal Ovarian Serous
Cystadenocarcinoma—The Cancer Genome Atlas [TCGA], PanCancer Atlas; n=585), we
used mRNA data files (276 patients with ovarian serous carcinoma) for survival analysis
and excluded the other 309 patients because of incomplete RNA, OS, and PFS data. Data
are classified as ‘high’ (138 patients) or ‘low’ (138 patients) bins at 50% (median) stringency
thresholds for levels of gene expression, and Kaplan-Meier survival plot was generated.

15. Statistics
All statistical analysis was performed with JMP14 (SAS Institute, Cary, NC, USA). A two-sided
Student’s t-test was used to compare between 2 groups. Tukey’s test was used to compare
between 3 or more groups. On survival analysis, multivariate analysis was performed for OS
using the Cox proportional hazards model. The Benjamin-Hochberg method was used toevaluate its superiority over the p-value after multiple comparisons. Kaplan-Meier survival
plots were generated using JMP14 and evaluated using the log-rank test. The p-value wasconsidered statistically significant when <0.05.

RESULTS
1. PARP inhibitor did not suppress the BRCA1-associated tumor growth in vivo
PARP inhibitor has been used clinically in ovarian and breast cancers with HRD such as
BRCA1 mutations [3,4]. There are various reports on acquiring therapeutic resistance to
PARP inhibitor, but the mechanisms are still unclear [7]. In this study, we investigated theimmunological aspects that may interfere with the efficacy of PARP inhibitor. To address
this issue, we first established a murine BRCA1 KO ovarian cancer cell line (Brca1KO HM-1) andconfirmed knockout efficiency and indels by western blotting and sequencing analysis (Fig. 1A,
Fig. S1A-C). Furthermore, the presence of mutations in major HRR-associated genes otherthan Brca1 was examined using RNA sequence analysis, and no obvious mutations were foundin these genes (Table S2). The established Brca1KO HM-1 cell line, in vitro, showed equivalent
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IL-34 cancels antitumor immunity by PARP inhibitor

cell proliferation compared to the mock-transfected (Mock) HM-1 cell line and was sensitive
to niraparib (one of the PARP inhibitor) or niraparib with cisplatin (Fig. 1B and C, Fig. S2A),
and similar results were observed in murine BRCA1 KO breast cancer cell line (Brca1KO 4T1)
(Fig. S1D-F, Fig. S2B-D). This result is consistent with previous reports, suggesting that

Brca1

220 kD

α-Tublin

50 kD
Mock

12

Brca1

KO

C
Mock
Brca1KOns

Survival (%)

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Brca1KODays post tumor implantation

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E
Tumor volume (mm3)300Tumor volume (mm3)300Tumor volume (mm3)Tumor volume (mm3)

D

*

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Days post tumor implantation

Il34 conc. (pg/mL)

Normalized to Gapdh

Fig. 1. PARP inhibition does not suppress the BRCA1-deficient ovarian cancer growth in vivo.
F Western blot of Brca1 in Mock and Brca1KO HM-1 cell lines. (B) The number ofGMock and Brca1KO HM-1 cells in cell culture was monitored at the indicated time
(A)
2,000
points0.009(n=3). (C) Cell viability (MTT assay) of Mock and Brca1KO HM-1 cells treated withdifferent concentrations of niraparib for 3 days (n=3). (D) Subcutaneous*
Mocktumor growth of Mock and Brca1KO HM-1 cells (1.0×105 cells, n=4
and n=6). Mice bearing Mock or Brca1*KO HM-1 tumors were treated daily with DMSO or niraparibKO(5 mg /kg, i.p.). (E) Subcutaneous tumor growth of Mock and Brca1KOHM-1 cells (1.0×105 cells, n=4). Mice bearing Mock or Brca1KO HM-1 tumors were treateddaily withDMSO or olaparib (5 mg /kg, i.p.). (F) Inflammatory cytokine (Il34, Il1b, Il6, Il17, Tnfa, Ifng) expression of Mock and Brca1KO HM-1 cells (1.0×106 cells,
0.006n=3) were determined by qPCR. (G) Il34 expression levels of Mock and Brca1KO HM-1 cells (1.0×106 cells, n=3) were determined by ELISA. (H) Mock or Brca1KO HM-1
1,000
cells (1.0×105 cells, n=3) were transplanted subcutaneously into B6C3F1 mice. After 19
days of transplantation, tumor-infiltrating immune cells were isolated fromtumorsandsubjected
toflow
cytometry
analysis(n=3–4).
Shown
arepercentagesof
Cd3e+, Cd11b+F4/80+MhcII+, and Cd11b+Gr-1+ within Cd45+ cells.
0.003
BRCA, breast cancer susceptibility gene; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; i.p., intraperitoneally; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ns = not significant; PARP, poly (ADP-ribose) polymerase; qPCR, quantitative polymerase chain reaction.
*p<0.05.
(continued to the next page)
0
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Il34

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H

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Il1b

Il6

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ns

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Il34

Il1b

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Il17

Tnfa

Cd3+

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Cd11b+Gr-1+*

10% in Cd45+% in Cd45+

2,000

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ns

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Mock Brca1KO Brca1KO(DMSO) (Niraparib) (DMSO) (Niraparib)

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Mock Brca1KO Brca1KO(DMSO) (Niraparib) (DMSO) (Niraparib)% in Cd45+

Normalized to Gapdh

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Tumor vol

100

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ns

50

0

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Mock Brca1KO Brca1KO(DMSO) (Niraparib) (DMSO) (Niraparib)

Fig. 1. (Continued) PARP inhibition does not suppress the BRCA1-deficient ovarian cancer growth in vivo.
(A) Western blot of Brca1 in Mock and Brca1KO HM-1 cell lines. (B) The number of Mock and Brca1KO HM-1 cells in cell culture was monitored at the indicated time
points (n=3). (C) Cell viability (MTT assay) of Mock and Brca1KO HM-1 cells treated with different concentrations of niraparib for 3 days (n=3). (D) Subcutaneoustumor growth of Mock and Brca1KO HM-1 cells (1.0×105 cells, n=4 and n=6). Mice bearing Mock or Brca1KO HM-1 tumors were treated daily with DMSO or niraparib(5 mg /kg, i.p.). (E) Subcutaneous tumor growth of Mock and Brca1KO HM-1 cells (1.0×105 cells, n=4). Mice bearing Mock or Brca1KO HM-1 tumors were treateddaily with DMSO or olaparib (5 mg /kg, i.p.). (F) Inflammatory cytokine (Il34, Il1b, Il6, Il17, Tnfa, Ifng) expression of Mock and Brca1KO HM-1 cells (1.0×106 cells,n=3) were determined by qPCR. (G) Il34 expression levels of Mock and Brca1KO HM-1 cells (1.0×106 cells, n=3) were determined by ELISA. (H) Mock or Brca1KO HM-1
cells (1.0×105 cells, n=3) were transplanted subcutaneously into B6C3F1 mice. After 19 days of transplantation, tumor-infiltrating immune cells were isolated fromtumors and subjected to flow cytometry analysis (n=3–4). Shown are percentages of Cd3e+, Cd11b+F4/80+MhcII+, and Cd11b+Gr-1+ within Cd45+ cells.
BRCA, breast cancer susceptibility gene; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; i.p., intraperitoneally; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ns = not significant; PARP, poly (ADP-ribose) polymerase; qPCR, quantitative polymerase chain reaction.
*p<0.05.

Brca1KO HM-1 cells cause HRD status due to loss of BRCA1, resulting in synthetic lethality by
PARP inhibitor [5,6]. We then subcutaneously inoculated Mock and Brca1KO HM-1 cells to miceand treated them with niraparib daily. Unexpectedly, in vivo, the administration of niraparib
did not affect the growth of not only Mock but also Brca1KO HM-1 tumors, and a similar result
was obtained when we used 4T1 tumors (Fig. 1D, Fig. S3A). As yet another PARP inhibitor, weused olaparib, which was approved before niraparib and has been reported in many clinicaland research studies, but again did not affect the growth of Brca1KO HM-1 tumors (Fig. 1E).
Based on several previous reports [11,22], we have predicted that changes in TME may havecaused this phenomenon and analyzed the expression of several proinflammatory cytokines
produced by Mock and Brca1KO HM-1 cells. In both Mock and Brca1KO HM-1 cells, while mostinflammatory cytokines expressions were low, only IL-34 expression was substantiallyhigh; furthermore, IL-34 expressed significantly high in Brca1KO HM-1 cells compared to
Mock HM-1 cells (Fig. 1F and G). Further, we investigated intra-tumoral IL-34 expression in
vivo. Similar to in vitro results of tumor cell lines, the expression of IL-34 was significantly
increased in Brca1KO HM-1 tumors (Fig. S3B). To determine how the differential expressionof IL-34 alters each immune cell in TME, we evaluated tumor-infiltrating immune cells
using flow cytometry. Mock and Brca1KO HM-1 tumors did not show any change in T cell or
MDSC populations regardless of niraparib administration and were maintained even withniraparib therapy, whereas MHCII+ macrophages notably decreased in Brca1KO HM-1 tumors
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compared to Mock HM-1 tumors and were maintained even with niraparib therapy (Fig. 1H).
This result suggests that IL-34 suppressed the induction of activated-type macrophages.
Collectively, these results suggest that even in BRCA1-associated cancers, the formation of
immunosuppressive TMEs accompanied by high expression of IL-34 cancels the therapeutic
effects of PARP inhibitor.

2. IL-34 expression canceled the anti-tumor effect of niraparib on
BRCA1-associated cancer
From the previous chapter, since PARP inhibitor was not effective even in BRCA1-deficienttumors when tumors express IL-34, we first evaluated the clinical value of IL-34 in humanovarian serous carcinoma by using the TCGA dataset. Clinical data analysis revealed that
IL34 was a factor involved in the OS of ovarian serous carcinoma and was an independent
prognostic factor (Fig. 2A, left, Table 1). Furthermore, supporting Fig. 1H, high IL34
expression and low MHC class II expression in human ovarian serous carcinoma showedworse patient survival (Fig. 2A, right). Thus, targeting IL-34 could help to improve the clinicaloutcome of ovarian cancer patients. We next, to examine the relationship between IL-34 andtherapeutic efficacy of PARP inhibitor therapy, established a murine BRCA1 and IL-34 KO
ovarian cancer cell line (Brca1KOIl34KO HM-1), and confirmed IL-34 expression lost by ELISA
assay (Fig. S4A). The established Brca1KOIl34KO HM-1 cell line, in vitro, showed equivalent cellproliferation compared to the Brca1KO HM-1 cell line and was sensitive to niraparib, the sameas the Brca1KO HM-1 cell line (Fig. S4B and C). We then subcutaneously inoculated Brca1KOand Brca1KOIl34KO HM-1 cells into mice and treated them with niraparib or olaparib daily. The
administration of niraparib or olaparib suppressed the growth of Brca1KOIl34KO HM-1 tumors,and the niraparib anti-tumor effect was more drastic than we had imagined (Fig. 2B). We
have predicted that changes in immune circumstances within TME may have caused thisdramatic anti-tumor effect and then evaluated tumor-infiltrating immune cells in Brca1KOand Brca1KOIl34KO HM-1 tumors. Notably, T cells were increased only in the Brca1KOIl34KO HM-1
tumors treated with niraparib, and the increment was primarily by activated-phenotype(Fig. 2C). This result suggests that the loss of IL-34 leads to the induction of CD8+ T cellsby niraparib therapy, resulting in the creation of anti-tumor immune TME. In summary,these results indicate that IL34 is a potent poor prognostic factor of ovarian serous
cystadenocarcinoma in humans and that IL-34 expression suppresses the anti-tumor effect of
PARP inhibitor therapy.

3. IL-34 cancels the cross-presenting DC-CD8+ T cell axis essential for PARP
inhibitor therapeutic efficacy
To investigate the effect of CD4+ or CD8+ T cells on the anti-tumor effect of niraparib,
Brca1KOIl34KO HM-1 tumors were treated with niraparib in mice pretreated with CD4- orTable 1. Association of IL34 and poor prognostic factors with overall survival
Gene
Exp (coefficient)
95% CI
p-valueIL34
4.10661.7437–9.6718
0.009
OSBPL82.89681.1747–7.1436
0.073
LYPLA22.24990.9264–5.4643
0.171
EED
1.84340.6460–5.2603
0.443
MDM2
1.62350.2923–9.0157
0.770
IMPAD11.53690.2266–10.4253
0.770
CDC42
0.96810.3599–2.6041
0.949
The data was obtained from the ovarian serous carcinoma dataset (276 patients with ovarian serous carcinoma).
Survival analyses were performed using Cox proportional hazards modeling. p-values are used to assess
significance after adjusting for multiple comparisons using the Benjamini-Hochberg method.
CI, confidence interval.

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IL-34 cancels antitumor immunity by PARP inhibitor

IL34

1.0

IL34 high median OS=35.1 mIL34 low median OS=44.8 m0.5

IL34/MHCII

1.0

OS

OS

A

High/high median OS=36.4 m
High/low median OS=34.2 m
Low/high median OS=49.1 m
Low/low median OS=42.0 m

0.5

p=4.31E-40

p=4.17E-5
40

0

80

40

Time (mo)Niraparib

300

200

100

*

5

0

10

* *

15

20

*
100

0

5

10

15

20

Days post tumor implantation
Cd8+

Cd4+Cd69+
4

20

*

15

% in Cd45+% in Cd45+

Cd4+

Brca1KO (DMSO)
Brca1KO (PARPi)
Brca1KOIl34KO (DMSO)
Brca1KOIl34KO (PARPi)

200

Days post tumor implantation

C

Olaparib

300

Tumor volume (mm3)Tumor volume (mm3)B80Time (mo)

10
ns
5
0

Cd8+Cd69+
*

3
2

*

1
0

Brca1KO Brca1KO Brca1KO Brca1KO(DMSO) (PARPi) Il34KO Il34KO(DMSO) (PARPi)

Brca1KO Brca1KO Brca1KO Brca1KO(DMSO) (PARPi) Il34KO Il34KO(DMSO) (PARPi)

Brca1KO Brca1KO Brca1KO Brca1KO(DMSO) (PARPi) Il34KO Il34KO(DMSO) (PARPi)

Brca1KO Brca1KO Brca1KO Brca1KO(DMSO) (PARPi) Il34KO Il34KO(DMSO) (PARPi)

Fig. 2. PARP inhibition suppresses the BRCA1- and IL-34-deficient ovarian cancer growth in vivo.
(A) Kaplan-Meier plot for OS of patients with ovarian serous carcinoma median split for the gene expression of IL34 and the combination with MHCII. The
patients were classified as IL-34 “high” (n=138) or “low” (n=138) in left figure, and as IL-34 and MHCII “high/high” (n=80), “high/low” (n=58), “low/high” (n=58)
or “low/low” (n=80) in right figure. (B) Subcutaneous tumor growth of Brca1KO and Brca1KOIl34KO HM-1 cells treated with niraparib or olaparib (1.0×105 cells,
niraparib group n=6 and n=9, olaparib group n=4 and n=8). Mice bearing Brca1KO or Brca1KOIl34KO HM-1 tumors were treated daily with DMSO or PARP inhibitor(5 mg /kg, i.p.). (c) Brca1KO or Brca1KOIl34KO HM-1 cells (1.0×105 cells, n=3) were transplanted subcutaneously into B6C3F1 mice. After 19 days of transplantation,
tumor-infiltrating immune cells were isolated from tumors and subjected to flow cytometry analysis (n=3). Shown are percentages of Cd3e+Cd4+, Cd3e+Cd8a+,
Cd3e+Cd4+Cd69+, and Cd3e+Cd8a+Cd69+ within Cd45+ cells.
BRCA, breast cancer susceptibility gene; DMSO, dimethyl sulfoxide; IL, interleukin; i.p., intraperitoneally; OS, overall survival; ns = not significant; PARP, poly
(ADP-ribose) polymerase.
*p<0.05.

CD8-depleting antibodies. Mice lacking CD8+ T cells lost the anti-tumor effect of niraparib
therapy on Brca1KOIl34KO HM-1 tumors. On the other hand, mice lacking CD4+ T cells showed
no loss of anti-tumor effect of niraparib therapy (Fig. 3A). These results clearly indicate that
CD8+ T cells are essential for the anti-tumor effect of niraparib and that CD4+ T cells are not
involved. Recently, the presence of DCs that directly cross-present CD8+ T cells without CD4+https://ejgo.org

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IL-34 cancels antitumor immunity by PARP inhibitor

Tumor volume (mm3)A

Brca1KOIl34KO

300

Tumor volume

400

*

*

200
100

0

ns

100

5

10

15

0

20

Brca1KO Brca1KO
Brca1KO
Brca1KO
KO
KO
KO
Il34
Il34
Il34
Il34KO(DMSO (Niraparib (Niraparib (Niraparib
+IgG)
+IgG)
+aCD4)+aCD8)

Days post tumor implantation
Brca1KOIl34KO (DMSO+IgG)
Brca1KOIl34KO (Niraparib+IgG)
Brca1KOIl34KO (Niraparib+aCD4)
Brca1KOIl34KO (Niraparib+aCD8)

B

Xcr1+

4

C

*

30

% in cDC

3% in cDC

*

300

200

2
1

KO

KO

DMSO
Niraparib 2.5 µM

10

Brca1
Brca1
Brca1
Brca1
(DMSO) (Niraparib) Il34KO
Il34KO(DMSO) (Niraparib)
KO

*

20

0

0

Xcr1+

KO

IL-34 (−)

IL-34 (+)

Fig. 3. PARP inhibition activates CD8+ T cells in a specialized dendritic cell-mediated system, resulting in antitumor effects.
(A) Subcutaneous tumor growth of Brca1KOIl34KO HM-1 cells (1.0×105 cells, n=3). Mice were treated with anti-CD4 or anti-CD8 antibodies before 6 or 1 day of tumor
cell transplantation. Mice bearing Brca1KOIl34KO HM-1 tumors were treated daily with DMSO or niraparib (5 mg /kg, i.p.). (B) Brca1KO or Brca1KOIl34KO HM-1 cells(1.0×105 cells, n=3) were transplanted subcutaneously into B6C3F1 mice. After 19 days of transplantation, tumor-infiltrating immune cells were isolated fromtumors and subjected to flow cytometry analysis (n=3). Shown are percentages of Xcr1+ within Cd45+Cd11b+F4/80-MhcII+Cd11c+ cells. (C) The subset compositionof differentiated bone marrow cells. Bone marrow cells were induced to differentiate in vitro by the presence of FLT3L with ‘IL-34 or nothing’ and ‘niraparib or
DMSO’. Shown are percentages of Xcr1+ within Cd45+Cd11b+F4/80-MhcII+Cd11c+ cells.
cDC, conventional dendritic cell; DMSO, dimethyl sulfoxide; IgG, immunoglobulin G; IL, interleukin; ns = not significant; PARP, poly (ADP-ribose) polymerase.
*p<0.05.

T cells have attracted attention in anti-tumor immunity, so we further investigated tumorinfiltrating DCs [23]. Interestingly, only in Brca1KOIl34KO HM-1 tumors treated with niraparibthe percentage of XCR1+ DC was increased (Fig. 3B), suggesting that the cross-presenting
DC-CD8+ T cell axis may be necessary for the anti-tumor effect of PARP inhibitor therapy. To
investigate whether the presence of IL-34 is responsible for the decrease in XCR1+ DC, DCs
were induced from mouse bone marrow cells to differentiate in the presence and absenceof IL-34 and niraparib. In the absence of IL-34, niraparib therapy promoted induction of
XCR1+ DC, whereas it was canceled in the presence of IL-34 (Fig. 3C). These results suggestthat the formation of anti-tumor immune TME via activation of the XCR1+ DC-CD8+ T cellaxis is essential for the anti-tumor effect of PARP inhibitor therapy, but the presence of IL-34
strongly suppresses it.

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IL-34 cancels antitumor immunity by PARP inhibitor

DISCUSSION
Cancer cells constantly promote the acquisition of therapeutic resistance by creating
immunosuppressive TME [11,14]. Unfortunately, a critical factor in acquiring resistance tovarious chemotherapies, including PARP inhibitor therapy, is still unknown [7]. The present
data showed for the first time that the XCR1+ DC-CD8+ T cell axis is crucial for the antitumor
effect of PARP inhibitor therapy, and tumor-derived IL-34 can suppress this effect in ovariancancer. In this section, we explain the value of IL-34 in therapeutic resistance and therationale of IL-34-targeted therapy.
IL-34 is a cytokine that contributes to myeloid cell differentiation and proliferation andis implicated in various diseases, including cancer [18,19]. Indeed, tumor-derived IL-34
contributes to the survival of the tumors themself through autocrine pathways; on the other
hand, it induces the differentiation and recruiting of immunosuppressive TAMs and MDSCs
through paracrine pathways [18,19]. Furthermore, it has been reported that tumor-derived IL34 expression is enhanced when cancer cells’ survival is threatened by chemotherapy [24]. High
IL-34 expression enhances cytokine production, such as IL-6 and IL-10, by activating STAT3
and nuclear factor-κB (NF-κB) signaling pathways, resulting in strong support for the creationof immunosuppressive TME [25]. Notably, IL-34 expression in ovarian cancer was enhanced bychemotherapy, and its high expression shortened PFS [26]. In this study, TCGA dataset analysis
revealed that IL-34 is an independent poor prognostic factor in ovarian serous carcinoma,
and its high expression shortens OS. Although PARP inhibitor therapy has been indicated in
ovarian cancer patients with BRCA1 mutations [3], interestingly, Brca1-deficient tumors were
not responsive to PARP inhibitor therapy in the murine ovarian cancer model. To investigate
the cause of this unexpected result, we screened cytokines secreted from the mouse ovariancancer used in this study and found that IL-34 was dramatical highly expressed. The absenceof functional BRCA1 in tumors has been shown to activate the NF-κB pathway due to increased
reactive oxygen species (ROS) [27]. Furthermore, it has been reported that IL-34 expression is
upregulated upon NF-κB activation due to cellular stresses, such as chemotherapeutic agents[24]. Namely, it suggested that activation of the NF-κB pathway by loss or mutation of BRCA1
could increase IL-34 expression. Thereby, we hypothesized that IL-34 caused resistance to
PARP inhibitor therapy and examined this question. To this end, we disrupted IL-34 from
Brca1-deficient tumors, PARP inhibitor therapy showed a marked antitumor effect by increasing
activated CD8+ T cells within TME. Accumulating evidence indicated that, in BRCA1-associatedovarian cancer, enhancement of CD8+ T cells via activation of the STING pathway is essential
for the antitumor effect of PARP inhibitor therapy [28]. Generally, to present antigens from
APCs to T cells, endogenous antigens are presented to CD8+ T cells by APCs’ MHC class I andexogenous antigens are presented to CD4+ T cells by APCs' MHC class II [13]. Although tumor
antigen-specific CD4+ T cells are known to assist in the activation and proliferation of CD8+ T
cells, thereby enhancing the anti-tumor effect, our data indicate that but not CD4+ T cells, CD8+
T cells are essential for the anti-tumor effect of PARP inhibitor therapy. Therefore, we assumed
the involvement of cells that can present antigen directly to CD8+ T cells.
The cross-presenting DCs can efficiently present exogenous antigens directly to CD8+ T cells via
the MHC class I pathway [23,28,29]. Our data showed that an increase in XCR1+ DC contributes
to an increase in activated CD8+ T cells in PARP inhibitor therapy for ovarian cancer resulting inan anti-tumor effect. Many reports are indicating the importance of this interrelationship in antitumor immunity. In ovarian cancer, it has been reported that tumor-infiltrating DCs, including
cross-presenting DCs, positively correlate with clinical outcomes [30]. Similarly, in melanoma,
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the amount of intra-tumoral cross-presenting conventional DCs (cDCs) correlated with OS [31].
In addition, cross-presenting cDCs were positively correlated with survival in breast cancer,
squamous cell carcinoma of the head and neck, and lung cancer [32]. Thus, this report can beone of the base studies that will boost therapies targeting the XCR1+ DC-CD8+ T cell axis.
Tumor-infiltrating immune cells receive a variety of homeostatic inhibitory stimuli from
TME. For instance, IL-6, which abound in most cancer’s TME, can reprogram precursor
DCs (pre-DCs) into immunosuppressive DCs [33]. Indeed, it has been evident that DCs’functional maturation is strongly inhibited by a positive feedback loop in the STAT3/NF-κB/
IL-6 pathway [34]. PARP inhibitor therapy promotes type I interferon (IFN) production via
the cGAS-STING pathway and the type I IFN signaling is required for the activation of cDCs
[28,35]. However, it has also recently been reported that PARP inhibitor therapy promotes
STAT3 activation in tumor-infiltrating immune cells in ovarian cancer [36]. It suggests that
cDCs differentiation may be inhibited. Most notably, as mentioned above, given that IL-34
enhances cytokine production such as IL-6 and IL-10 via the STAT3/NF-κB signaling pathway[18,25], PARP inhibitor therapy constantly suppresses immune cells’ activation in presenceof IL-34. Our data demonstrated that IL-34 inhibited differentiation into XCR1+ DCs androbustly suppressed anti-tumor immunity during PARP inhibitor therapy in ovarian cancer.
Namely, it considers that IL-34 forcibly suppressed XCR1+ DCs’ inducing via over-enhancing
STAT3/NF-κB/IL-6 pathway during PARP inhibitor therapy.
Three general mechanisms of acquired resistance to PARP inhibitor have been already known[7]: 1) Drug target-related effects; the upregulation of the drug efflux transporter ABCB1[37], or mutations in PARP1 that either reduce the affinity of the PARP inhibitor or preserve
endogenous functions of the enzyme when bound to a PARP inhibitor [38]; 2) Restoration of
HR owing to restoration of BRCA1/2 function or functionally related proteins; such as reversion
mutations or epigenetic alterations that induce the re-expression of a BRCA1 or BRCA2 wildtype protein or result in hypomorphic variants [39]; 3) Loss of DNA end-protection and/or
restoration of replication fork stability; such as depletion of the MLL3/4 complex protein PTIPor the nucleosome remodeling factor CHD4, resulting in fork protection and resistance to
PARP inhibitor in BRCA1/2-deficient cells [40]. However, there have been no previous reportsof tumor immunity, particularly IL-34, acting on PARP inhibitor resistance. In this study, we
revealed a novel mechanism of resistance against PARP inhibitor in which TME is regulated bytumor-derived IL-34. It is possible that IL-34-mediated immunosuppression and the previously
described mechanisms work additively or synergistically to produce overall resistance. Further
studies are required to elucidate this issue. Further studies would also clarify the mechanism of
PARP inhibitor therapy-induced XCR1+ DC increment and whether IL-34 has negative effects on
PARP inhibitor, such as inhibiting or suppressing synthetic lethality or PARP-trapping effects.
IL-34 benefits tumors through various roles and may be a biomarker for therapeutic resistanceand a worse prognosis. In addition, targeting IL-34 for therapy may be beneficial for restoring
chemotherapeutic resistance and increasing sensitivity to immune checkpoint blockade therapy.

SUPPLEMENTARY MATERIALS
Table S1
Primer list for qPCR analysis
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IL-34 cancels antitumor immunity by PARP inhibitor

Table S2
Major HRR-associated gene expression profiling of wild type and Brca1KO HM-1 cells
Click here to viewFig. S1
Sequence trace views of mouse ovarian cancer and breast cancer cell.
Click here to viewFig. S2The effect of PARP inhibition on BRCA1-deficient cancer in vitro.
Click here to viewFig. S3
The effect of PARP inhibition on BRCA1-deficient breast cancer growth in vivo.
Click here to viewFig. S4The effect of PARP inhibition on BRCA1- and IL-34-deficient ovarian cancer in vitro.
Click here to view

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