IL33 Is a Key Driver of Treatment Resistance of Cancer Chie Kudo-Saito1, Takahiro Miyamoto1,2, Hiroshi Imazeki1,2, Hirokazu Shoji2, Kazunori Aoki1, and Narikazu Boku2

Published OnlineFirst March 10, 2020; DOI: 10.1158/0008-5472.CAN-19-2235

CANCER RESEARCH | TUMOR BIOLOGY AND IMMUNOLOGY

ABSTRACT ◥ Recurrence and treatment resistance are major causes of cells, which promoted tumor progression and metastasis directly cancer-associated death. There has been a growing interest in and indirectly via induction of immune exhaustion and dysfunc- þ better understanding epithelial–mesenchymal transition, stem- tion. Blocking IL33 with a specificmAbinmurineIL33 ness of cancer cells, and exhaustion and dysfunction of the metastatic tumor models abrogated negative consequences and immune system for which numerous genomic, proteomic, micro- successfully elicited antitumor efficacy induced by other com- environmental, and immunologic mechanisms have been dem- bined treatments. Ex vivo assays using tumor tissues and periph- onstrated. However, practical treatments for such patients have eral blood mononuclear cells of patients with cancer validated the not yet been established. Here we identified IL33 as a key driver clinical relevancy of these findings. Together, these data suggest of polyploidy, followed by rapid proliferation after treatment. that targeting the IL33-ST2 axis is a promising strategy for IL33 induction transformed tumor cells into polyploid giant cells, diagnosis and treatment of patients likely to be resistant to showing abnormal cell cycle without cell division accompanied treatments in the clinical settings. by Snail deregulation and p53 inactivation; small progeny cells were generated in response to treatment stress. Simultaneously, Significance: These findings indicate that the functional role of soluble IL33 was released from tumor cells, leading to expansion IL33 in cancer polyploidy contributes to intrinsic and extrinsic þ þ of receptor ST2-expressing cells including IL17RB GATA3 mechanisms underlying treatment failure.

þ Introduction EMT-governing transcriptional factor snail (designated F10-snail ), tumor growth and metastasis were adversely promoted by ICI treat- Recurrence and treatment resistance are the major causes of cancer- ment just like hyperprogression reported in the clinical settings (6). In associated death. The topics that most resonated has been epithelial– this study, we harvested and biologically and immunologically ana- mesenchymal transition (EMT) that enables tumor escape by confer- lyzed B16-F10 cells obtained from the metastatic bone marrow of the ring mesenchymal and stem properties such as high motility and implanted mice, and attempted to deﬁne the molecular mechanisms dormancy (1), and numerous genomic, proteomic, microenvironmen- underlying the adverse effect on tumors. tal and immunologic mechanisms have been demonstrated (2, 3). However, little is known about the precise mechanisms underlying the metastatic colonization of cancer stem cells (CSC) after terminating Materials and Methods EMT within the niche, and thus practical treatment strategies targeting Mice CSCs have not been established yet. Although its reversal MET has Five-week-old female C57BL/6 mice were purchased from Charles been believed as the sequential step in the mechanism (4), relapsed and River Laboratories, and were maintained under pathogen-free condi- metastatic tumors appear more aggressive than primary tumors, tions. The mice were used according to the protocols approved by the implying a possible different mechanism. Animal Care and Use Committee at the National Cancer Center We have been investigating the interplay between cancer EMT and Research Institute (Tokyo, Japan). host immunity, and previously demonstrated that tumor metastasis (lung and bone marrow) is a possible risk factor of resistance to Cell lines immune checkpoint inhibitors (ICI) using mouse metastasis mod- Human breast cancer cell lines (MCF7 and MDA-MB-231) were els (5). Particularly in the bone metastasis models, which were purchased from ATCC, and were authenticated by short tandem implanted with murine melanoma B16-F10 cells transduced with an repeat proﬁling. Murine melanoma B16-B10 cells were purchased from the Cell Resource Center for Biomedical Research at Tohoku University in Japan. We used B16-F10 cells transfected with plasmid 1Department of Immune Medicine, National Cancer Center Research Institute, 2 vector pcDNA3.1(þ) encoding neomycin-resistant gene with or with- Tokyo, Japan. Division of Gastrointestinal Medical Oncology, National Cancer þ Center Hospital, Tokyo, Japan. out murine snail (F10-snail or F10-mock) that we established before (7). All tumor cells were tested for Mycoplasma negativity Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). using a Hoechst-staining Detection Kit (MP Biomedicals) and were expanded and frozen in liquid nitrogen to avoid changes occurred by a Corresponding Author: Chie Kudo-Saito, Department of Immune Medicine, National Cancer Center Research Institute, Tokyo 1040045, Japan. Phone: 813- long-term culture until used in experiments. 3542-2511; Fax: 813-3547-5137; E-mail: [email protected] Establishment of B16-F10 subclones Cancer Res 2020;80:1–10 F10-mock cells were harvested from subcutaneous tumors (F10- doi: 10.1158/0008-5472.CAN-19-2235 primary) and femur bone marrow (F10-BM) of mice 25 days after 2020 American Association for Cancer Research. implantation and were cultured in 10% FBS/DMEM with Geneticin

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þ (Merck) to select tumor cells. The details were described in the cells was validated by flow cytometry. The IL17RB cells (1 106/mL) Supplementary Methods. were cultured in 10%FBS/RPMI1640 at 37C for 3 days, and the supernatant fluid was harvested and filtrated (ø ¼ 0.22 mm) before Establishment of IL33 transfectants stock at 4C. IL13 in the supernatant was measured using an ELISA þ B16-F10 cells and MCF7 cells were transfected with plasmid vector Kit (R&D Systems). The IL17RB supernatant or IL13 (1 ng/mL; pCMV6-ENTRY (OriGene Technologies) encoding murine or human PeproTech) were added to a CTL induction system with splenic þ il33 by electroporation (0.4 kV, 25 mFD), and were cultured in 10% CD3 T cells, gp70 peptide (MBL), antigen-presenting cells (inacti- FBS/DMEM with Geneticin. The details were described in the Sup- vated bulk SPCs) in the presence or absence of anti-mouse IL13 þ plementary Methods. mAb (1 mg/mL; R&D Systems). Six days later, the sorted CD8 T cells were tested for IFNg production (24 hours) and cytotoxic þ Functional analysis of tumor cells activity (ET ratio ¼ 25:1, 4 hours) as described before (7). The IL17RB We assessed cellular functions: cell proliferation (2 days) by cell cells (3 105) were coinjected with B16-F10 cells (3 105) in mice, count or WST-1 assay (Takara), cell adhesion (1 hour) using fibro- and tumor volume was measured. In a setting, anti-mouse IL13 mAb nectin-coated multiwell plates (Corning), and cell invasion (8 hours) (20 mg) was intratumorally (i.t.) injected in the mice on days 4 and using a transwell chamber with a Matrigel-coated membrane (Corn- 8 after coinjection. ing) as described before (7). To determine cell cycle, after fixation with ethanol, cells were treated with propidium iodide (PI; 50 mg/mL) and In vivo therapy RNase A (200 mg/mL) for 30 minutes, and were analyzed by flow To evaluate the antitumor efficacy on both subcutaneous tumor cytometry. Cells were stimulated with IL13 (1 ng/mL; PeproTech) for growth and metastasis mimicking metastatic cancer patients, tumor 2 days, and were tested for adhesion and invasion. To determine cells were both subcutaneously (3 105 cells) and intravenously (3 chemosensitivity, tumor cells were treated with paclitaxel (Wako), 105 cells) implanted in mice. The mice were intraperitoneally treated 5-fluorouracil (5-Fu; Wako) or gemcitabine (Wako) for 3 days (0–100 with PBS or 5-Fu (20 mg/kg; Wako) on days 4 to 8 after tumor mg/mL, two-fold serial dilution), and the data were indicated as the implantation, or were intratumorally treated with anti-mouse PD1 percentage of untreated control (100%). Cell death was analyzed by mAb (BioLegend), anti-mouse IL33 mAb (R&D Systems), or the flow cytometry after staining with PI and Annexin V. For tracking cell isotype control (R&D Systems) on day 5 (n ¼ 5–10 per experiment). division, PKH67-labeled cells were used. In the in vivo experiments, Tumor volume was measured twice a week. Metastatic nodules in lung tumor cells were subcutaneously (3 105 cells) and intravenously (3 were counted, and subcutaneous tumors and spleens were harvested 105 cells) implanted into mice, and tumor volume (0.5 length for assays on days 14 to 18. width2,mm3) was measured. For a convenience of observation and quantification, we assessed lung metastasis by counting the number of Clinical analysis tumor metastatic nodules in lung, albeit tumor metastases in many For IHC analysis, we purchased paraffin-embedded tissue sections tissue organs of the mice. (normal mammary tissues, and primary and metastatic tumor tissues) of stage II–III patients with breast cancer from SuperBioChips, and siRNA transfection stained with immunofluorescence-conjugated anti-human IL33 mAb For il33 knockdown, we used two siRNAs targeting distinct il33 (R&D Systems), anti-human IL17RB mAb (R&D Systems), or the sequences or one scrambled sequence as a negative control (Invitrogen; isotype IgG (BioLegend) as described before (5). The immunofluo- Supplementary Methods). The siRNAs were complexed with jetPEI rescence intensity was automatically measured as pixel counts at (PolyPlus) according to the manufacturer's instruction before trans- two fields per section using a LSM700 Laser Scanning Microscope fection. The transfection efficacy was validated by RT-PCR 1 to 2 days (Carl Zeiss), and the average was plotted in graphs. For flow cyto- after transfection. metric analysis, EDTA-added peripheral blood was collected from healthy donors (n ¼ 4) and patients with stage IV metastatic colorectal Flow cytometric analysis cancer (n ¼ 9; age 57–79) after receiving written informed consent After flow cytometric blocking, cells were stained with the (November 2018 and June 2019), according to the protocol approved immunofluorescence-conjugated antibodies (Supplementary Meth- by the Institutional Review Board at the National Cancer Center ods). For intracellular staining, cells were treated with Cytofix/ (Tokyo, Japan). PBMCs were isolated by Ficoll (Nacalai), and were Cytoperm solution (BD Biosciences) before the staining. In mouse stained with the specific mAbs (Supplementary Methods). All activities study, data were acquired using the FACSCalibur cytometer (Becton were conducted in accordance with the ethical principles of the Dickinson), and were analyzed by Cellquest software (BD Bio- Declaration of Helsinki. sciences). In clinical study, data were acquired using a BD LSR Fortessa X-20 cytometer (Becton Dickinson), and were analyzed by Statistical analysis FlowJo software (BD Biosciences). Before defining the specific Data indicate mean SD unless otherwise specified. Significant molecular expressions, debris was firstly excluded by forward differences (P < 0.05) were statistically evaluated using GraphPad þ scatter/side scatter, followed by gating CD45 leukocytes, and Prism 7 software (MDF). Data between two groups were analyzed by immunofluorescence intensity was compared to the isotype control the unpaired two-tailed Student t test. Data among multiple groups (Supplementary Fig. S1). were analyzed by one-way ANOVA, followed by the Bonferroni post hoc test for pairwise comparison of groups based on the normal þ Functional analysis of IL17RB cells distributions. Nonparametric groups were analyzed by the Mann– þ IL17RB cells were sorted from spleen cells (SPC) of mice 7 days Whitney test. Mouse survival was analyzed by the Kaplan–Meier after 5-Fu treatment using a BD IMag System (BD Biosciences) with method and ranked according to the Mantel–Cox log-rank test. anti-mouse IL17RB mAb (R&D Systems) and the secondary anti-rat Ig Correlation between two factors was evaluated by the nonparametric þ magnetic beads (BD Biosciences). The purity (> 90%) of the IL17RB Spearman rank test.

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IL33 in Evolutional Transformation of Cancer

Results PBS treatment; cytotoxicity, P ¼ 0.001; Fig. 2D). These suggest that IL33 is involved in chemoresistance of tumor cells IL33 induction confers a progressive property to tumor cells via not only biological self-transformation, but also immunologic damage To compare tumor properties between primary sites and metastatic þ possibly mediated by ST2 cells. sites, B16-F10 cells were both subcutaneously and intravenously implanted into mice, and tumor cells were harvested from the sub- IL33 regulates cancer polyploidy cutaneous tumors (F10-primary) and the metastatic bone marrow We next examined whether IL33 could be associated with poly- (F10-BM) on day 25 after implantation. The F10-BM cells slightly ploidy because nuclear IL33 is known but its functional roles remain expressed an epithelial marker E-cadherin, but highly expressed a unclear (14), and IL33 overexpression transformed tumor cells into a mesenchymal marker Snail in the cytoplasm, but not in the nucleus, giant size (Fig. 2A) that is a representative feature of polyploidy, by implying deregulation state (Fig. 1A). The F10-BM cells showed which giant cells are generated through misregulation of canonical significant lower proliferative activity and higher resistance to treat- G1–S–G2–M cell cycle without cell division followed by unrestrained ment with 5-Fu with a slight increase at low doses as compared with þ propagation (15). F10-TR cells contained significantly more 3 4N F10-primary cells and F10-snail cells derived from bone marrow (P < DNA contents (P < 0.001 vs. F10-mock; Fig. 3A), and generated 0.05; Fig. 1B). In the in vivo setting, the F10-BM tumor also signif- smaller progeny cells by stimulation with a low dose of 5-Fu albeit cell icantly slowly grew than F10-primary tumor (P ¼ 0.002), but the death in F10-mock cells (Fig. 3B). Aurka (16) and Aurkb (17) regulate growth was adversely promoted by 5-Fu treatment (days 4–8; P ¼ þ polyploidization followed by inactivation of p53 (18). Indeed, Aurka/b 0.002 vs. PBS treatment), although F10-snail tumors were just increased and phosphorylated p53-Ser15 (p53-p) decreased in the unresponsive to the treatment (Fig. 1C). These suggest that the F10-TR cells (P < 0.002 vs. F10-mock), and IL33 knockdown reduced F10-BM cells acquire a distinct property from that of the F10-primary Aurka/b expression but increased p53-p expression in the F10-TR cells after metastasis. The F10-BM tumors showed significant increase in þ þ þ (Fig. 3C). These suggest that IL33 expression, potentially in the ST2/ST2L cells including c-kit FceRIa mast cells (P ¼ 0.002 vs. PBS þ þ nucleus, plays a key role in polyploidization followed by rapid pro- treatment), CD11b Gr1 myeloid-derived suppressor cells (MDSC; P þ þ liferation in response to treatment stress. ¼ 0.005), and IL17RB GATA3 type 2 innate lymphoid cells (ILC2s; P ¼ 0.005) after the treatment (Fig. 1D). This observation raised a þ IL33-inducible IL17RB cells promote tumor progression possibility of its ligand IL33 release from the progressing F10-BM þ ST2 cells remarkably increased in the F10-TR–implanted mice tumors. Indeed, higher IL33 production was observed in the F10-BM particularly after treatment. We further pursued the observation cells compared with other clones, and IL33 knockdown with the þ þ because the ST2 cells would likely play important roles in the IL33 specific siRNAs significantly reduced the chemoresistance (P < 0.05 vs. tumor progression mechanisms. Because ST2 is expressed not only in control siRNA; Fig. 1E). Similar results were observed using highly þ ILC2s (10, 11), mast cells, and MDSCs (19) but also in NK (20) and bone-metastatic breast cancer IL33 MDA-MB-231 cells after siRNA- Tregs (21), we conducted antibody-mediated ablation experiments to il33 transfection (P < 0.02 vs. control siRNA; Supplementary Fig. S2). determine the key effector cells using immunodeficient nu/nu mice We chose breast cancer cell lines because bone metastasis is most treated with anti-asialo GM1, indicating absence of functional T cells frequently seen in breast cancer in clinical settings (8, 9). These results and NK/NKT cells. 5-Fu–induced F10-TR tumor progression was suggest IL33 is involved in the treatment-induced tumor progressive þ þ partly retarded in the absence of FceRIa cells and CD11b cells, but mechanisms. not NK/NKT cells and T cells (Supplementary Fig. S4B). In coinjection experiments, however, greater impact on tumor progression was þ IL33 drives tumor progression after treatment provided by IL17RB cells derived from F10-TR–implanted mice þ IL33 is a member of the IL1 family expressed in some types of cells (designated TR-IL17RB cells) compared with FceRIa cells and þ such as endothelial cells and fibroblasts, and is associated with diseases CD11b cells (P < 0.001; Supplementary Fig. S4C). These suggest þ þ including allergy, infection, and cancer (10, 11). However, the role of ST2 cells, especially ILC2s, could play important roles in the IL33 IL33 in cancer is still controversial, albeit IL33 upregulation in many tumor progression. The TR-IL17RB cells highly produced IL13, types of cancers (12). To address this issue, we established IL33 suggesting these cells could be ILC2s (Fig. 4A). Stimulation with a transfectants using B16-F10 cells (F10-TR; Fig. 2) and human breast low dose of IL13 that did not affect F10-mock cells remarkably cancer MCF7 cells (MCF7-TR; Supplementary Fig. S3). IL33 over- enhanced F10-TR invasion (Fig. 4B). Anti-IL13 mAb injection into expression generated large cells with low proliferative property (P < the TR-IL17RB–coinjected tumors significantly suppressed the growth 0.001 vs. F10-mock) and high 5-Fu–resistant property in vitro (P ¼ 0.010; Fig. 4C). These suggest IL13 is involved in the ILC2- (Fig. 2A) and in vivo (Fig. 2B). The 5-Fu treatment remarkably induced tumor progressive mechanisms. promoted F10-TR tumor growth and metastasis, and significantly In the in vitro CTL induction, addition of the TR-IL17RB shortened mouse survival (P ¼ 0.0001 vs. F10-mock; Fig. 2B). These supernatant generated exhausted and dysfunctional CTLs expres- suggest IL33 plays a key role in the treatment-induced tumor pro- sing PD1, Tim3, and Tigit (Fig. 4D). However, anti-IL13 mAb gression, albeit likely a better prognostic factor under tranquility. In the addition to the culture-generated potent CTLs, suggesting ILC2- þ F10-TR–implanted mice, ST2 cells, particularly ILC2s, were signif- induced IL13 could impair CTLs (P < 0.004; Fig. 4D). Because icantly expanded not only in the tumor tissues (P ¼ 0.003 vs. PBS IL13R is not generally expressed on T cells except some cases (22), treatment) but also in the spleen (P < 0.0001) after the treatment the CTL dysfunction observed might be indirectly induced by other þ þ (Fig. 2C; Supplementary Fig. S4A). ST2 increase was also observed IL13R cells such as M2 macrophages and MDSCs contained in within F10-mock tumor tissues regardless of treatment. This might be the antigen-presenting cells used. Anti-IL13 mAb injection signif- potentially mediated by chemokines such as CXCL12 for CXCR4 that icantly suppressed 5-Fu–induced F10-TR tumor progression and is commonly expressed in mast cells, MDSCs and ILC2s (13). CTLs metastasis (P < 0.02 vs. 5-Fu only; Fig. 4E). These suggest that þ were impaired in the F10-TR–implanted mice, and the CTL activities ILC2s contribute to IL33 tumor progression via immune exhaus- were further reduced by the treatment (IFNg production, P ¼ 0.002 vs. tion and dysfunction partly mediated by IL13.

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Figure 1. IL33 expression is associated with chemoresistance of tumors. A, Morphology and EMT-related molecular expressions of tumor cells. B16-F10 cells were isolated from subcutaneous tumors (F10-primary) or bone marrow (F10-BM) of mice 25 days after both subcutaneous and intravenous implantation. As another control, snail- þ transduced B16-F10 cells (F10-snail ) were isolated from bone marrow. Scale bar, 50 mm. B, Chemoresistance of the F10-BM cells. Tumor cells were cultured with or without 5-Fu for 3 days. Data are depicted as the percentage of untreated proliferation (n ¼ 3). C, Enhancement of in vivo F10-BM tumor growth by chemotherapy. Mice were intraperitoneally injected with PBS (open bars) or 5-Fu (20 mg/kg; closed bars) on days 4 to 8 after tumor implantation (n ¼ 5). Tumor volume on day 14. þ þ D, Increase of ST2L cells in the F10-BM tumors after chemotherapy. Tumor-infiltrating cells were analyzed for ST2L cells by immunostaining (scale bar, 50 mm) and flow cytometry (n ¼ 5) on day 14. E, Enhancement of chemosensitivity by IL33 knockdown. F10-BM cells were transfected with il33-specific siRNAs (#1 and #2), and were cultured with or without 5-Fu. The knockdown efficacy was validated by RT-PCR (photos) and ELISA (IL33 production; n ¼ 3). Open circles, F10-primary. Closed squares, F10-BM þ control siRNA. Open triangles, F10-BM þ siRNA-il33. , P < 0.01, , P < 0.05. Graphs show mean SD. Representative data of five independent experiments.

þ Blocking IL33 elicits antitumor immunity in mice with IL33 progression, and the mouse survival was slightly but significantly tumors prolonged (P ¼ 0.004 vs. control; Fig. 5A). In the anti-IL33–treated þ Unexpected hyperprogression is a serious problem in cancer ther- mice, tumor-specific CD8 T cells increased within tumors, and apy with ICIs (6). In the mouse models implanted with F10-BM splenic CTL activities were significantly elevated (Fig. 5). Combination tumors, which is similar to the F10-TR tumors (Supplementary of anti-PD1 treatment synergistically enhanced the anti-IL33 efficacy Fig. S5), anti-PD1 treatment significantly promoted tumor growth, on tumor growth (P ¼ 0.0003 vs. anti-IL33 monotherapy) and mouse and shortened mouse survival (P < 0.0001 vs. control), albeit signif- survival (P ¼ 0.0036). The combinatory regimen was also significantly icantly effective in the F10-primary models (P ¼ 0.0017; Fig. 5A). In effective in the F10-primary models (P ¼ 0.047 vs. anti-PD1 mono- the anti-PD1–treated mice, cell infiltration was hardly seen in the F10- therapy). These results provide a potential of blocking IL33 for BM tumors (Fig. 5B and C), and splenic CTLs were still dysfunctional successfully eliciting antitumor immunity in combination with other þ (Fig. 5D and E). In contrast, anti-IL33 treatment did not cause tumor therapeutics in the treatment of IL33 tumors.

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IL33 in Evolutional Transformation of Cancer

Figure 2. IL33 drives tumor progression after treatment. A, IL33 overexpression confers chemoresistance. B16-F10 cells were transduced with murine il33 (F10-TR1 and F10-TR2) and were tested for IL33 receptor expressions (scale bar, 50 mm),IL33production(3days;n ¼ 3), and cell proliferation with or without 5-Fu (3 days; n ¼ 3). B, Chemotherapy worsens the F10-TR tumor progression. Mice were treated with PBS or 5-Fu on days 4 to 8 after tumor implantation (n ¼ 5). Lung metastasis on day 14 (n ¼ 5). Mouse survival (n ¼ 10). C, Possibly ST2Lþ cells are expanded in the F10-TR–implanted mice after 5-Fu treatment. TILs and SPCs were analyzed by ﬂow cytometry on day 14 (n ¼ 5). D, Chemotherapy impairs CTLs in the F10-TR–implanted mice (n ¼ 3). In the cytotoxic assay, F10-mock cells were used as a target (ET ratio ¼ 25:1). , P < 0.01, , P < 0.05. Graphs show mean SD. Representative data of three independent experiments.

Clinical relevancy of the IL33-ST2/IL17RB axis obtained from patients with metastatic colorectal cancer because We next validated the findings using clinical samples. We firstly of only one available for us. It has been demonstrated that IL33 conducted IHC analysis using tumor tissues obtained from patients upregulation is associated with colorectal cancer pathogenesis (23), with breast cancer. IL33 expression was upregulated in primary and and hyperprogression is seen in patients with colorectal cancer þ metastatic tumor tissues compared with noncancerous portions (P < after anti-PD1/PDL1 treatment albeit a few in number (24). ST2 þ þ 0.05; Fig. 6A), and the IL33 intensity was correlated with IL17RB cell cells (P ¼ 0.008) and IL17RB cells (P ¼ 0.021) were significantly infiltration particularly in the stage III patients with tumor metastasis increased in patients with metastatic colorectal cancer compared þ (P ¼ 0.0005) rather than the stage II patients (P ¼ 0.0886). IL17RB with healthy donors, and the increase of these cells, particularly þ þ þ cells were hardly seen in the tumors if IL33 was expressed only in the ST2 cells (P ¼ 0.005), was correlated with decrease in CD3 CD8 nuclei. These suggest a potential causal relationship between IL33 T cells (Fig. 6B). These suggest a potential causal relationship þ þ þ positivity in tumors and increase in IL17RB cells within the tumor between ST2/IL17RB expansion and CD8 T-cell reduction in microenvironment. systemic immunity. Taken together, targeting IL33 and the conse- þ þ þ WefurtheranalyzedPBMCsforST2 cells and IL17RB cells by quent ST2/IL17RB cellsmaybeapromisingstrategyofdiagnosis flow cytometry, because the cells could potentially play critical roles and treatment of patients who are likely resistant to treatments in in systemic immunity as well as local immunity. We used PBMCs the clinical settings.

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Figure 3. IL33 expression induces polyploidy in tumor cells. A, Abnormal cell cycle in the F10-TR cells. PI-stained DNA contents were quantified by flow cytometry (n ¼ 3). Photo shows IL33 expression in a large þ DAPI nucleus of F10-TR cells. Scale bar, 20 mm. B, Chemotreatment generates small progeny cells of the F10-TR cells. PKH67-labeled tumor cells were treated with 5-Fu (5 mg/mL) for 24 hours and analyzed for cell division and Annexin Vþ apoptosis by flow cytometry. C, Expression of polyploidy-associated molecules, Aurka, Aurkb, and p53. Total p53 and phosphorylated p53-ser15 (p53-p) expressions were analyzed by flow cytometry (n ¼ 3) and immunostaining (photos). , P < 0.01. Bar graphs show mean SD. Data in each panel are representative of three independent experiments.

þ þ Discussion IL17RB cells is correlated with reduction of CD8 T cells in PBMCs. We identified IL33 as a determinant of treatment resistance of This suggests clinical relevancy of the basic findings. Thus, this study cancer. IL33 induction in tumor cells transforms into polyploid giant reveals the functional role of IL33 in cancer polyploidy that could be cells showing abnormal cell cycle without cell division accompanied by intrinsic tumor biological and extrinsic immunological mechanisms Snail deregulation and p53 inactivation, and its small progeny cells are underlying treatment failure. generated in response to treatment stress. Simultaneously, soluble IL33 Although many studies demonstrated the roles of IL33 in cancer, it þ is released from the tumor cells, and expands ST2 cells including is still controversial (12). For example, treatments with mAbs specific þ þ IL17RB GATA3 ILC2s that promote tumor progression and metas- for IL33 (25), ST2 (26), and IL1RAP (27) significantly suppressed tasis directly and indirectly through induction of immune exhaustion tumor progression through reduction of tumor-associated macro- þ and dysfunction partly mediated by IL13. In the mouse IL33 met- phages and Tregs in mouse tumor models. On the other hand, IL33 astatic tumor models, however, blocking IL33 with the specific mAb induction in melanoma suppressed tumor progression (28). We found abrogates the negative consequences, and successfully elicits antitumor that IL33 expression in tumor cells retards proliferation and growth, efficacy induced by other treatment combined. In clinical samples, whereas aberrant mitotic progression is caused by treatment stress. In IL33 positivity in tumors is significantly correlated with increase of the contrast, stable silencing of IL33 in tumor cells restricts cell prolifer- þ þ IL17RB cells within the tumor milieu, and increase of ST2 cells and ation, resulting in poor engraftment and growth of the tumors in mice

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Figure 4. IL33-inducible IL17RBþ cells promote tumor progression. A, IL13 production from IL17RBþ cells (n ¼ 3). IL17RBþ cells were sorted from SPCs of F10-mock–implanted mice (Mock-17RB) and F10-TR–implanted mice (TR-17RB) 7 days after 5-Fu treatment. B, High sensitivity of F10-TR cells to IL13. Tumor cells were tested for adhesive and invasive properties after treatment with IL13 (1 ng/mL; n ¼ 3). Photos show morphologic changes. Scale bar, 50 mm. C, IL17RBþ cells promote tumor progression. Tumor cells were coimplanted with IL17RBþ cells (1:1) in mice, and anti-IL13 mAb or control IgG (20 mg) was intratumorally injected on days 4 and 8 after coimplantation (tumor volume on day 14; n ¼ 5). D, IL17RBþ cells impair CTLs partly via IL13 released. TR-17RB supernatant or IL13 was added to the þ CTL induction system in the presence of anti-IL13 mAb or control IgG (1 mg/mL). The cells were analyzed by ﬂow cytometry, and the sorted CD8 T cells were tested for cellular functions (n ¼ 3). E, Blocking IL13 suppresses 5-Fu–induced F10-TR tumor progression. Anti-IL13 blocking mAb or control IgG (20 mg) was intratumorally injected in the mice on days 4 and 8 (5-Fu treatment ¼ days 4–8). Data on day 14 (n ¼ 5). , P < 0.01, , P < 0.05. Bar graphs show mean SD. Representative data of three independent experiments.

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Figure 5. Antitumor immunity induced by blocking IL33 in mice. A, Anti-IL33 treatment enhances anti-PD1–induced efﬁcacy. Mice were intratumorally injected with anti-PD1 mAb and/or anti-IL33 mAb (50 mg) on day 5 after tumor implantation (tumor growth, n ¼ 5; mouse survival, n ¼ 10). B, The number of TILs and SPCs of the mice on day 18 (n ¼ 5). C–E, Anti-IL33 treatment increases tumor- speciﬁc CD8þ T cells within tumors (C), following reduction of immune exhausted CD8þ T cells (D) and generation of potent CTLs (E; n ¼ 3) in spleen of the F10-TR– implanted mice. , P < 0.01, , P < 0.05. Graphs show mean SD. Representative data of two independent experiments.

þ probably because of damaging cells not only internally via reduction IL1RAP knockdown enhanced chemosensitivity of IL33 tumor of Aurka/b that is required for mitotic control, but also externally cells (Supplementary Fig. S2). The receptor coexpression may þ þ via loss of ST2 cell support. Therefore, the tumor suppressive intensify the treatment resistance of IL33 tumor cells in an activity reported might be explained by the low proliferative prop- autocrine manner. erty due to polyploidization. The authors did not conduct thera- Cancer polyploidy has been demonstrated as cancer stemness peutic experiments, in which tumor progression might be seen as because of producing unrestrained propagation that undermines shown in our study. IL33 overexpression simultaneously induced genomic stability (15, 29). Polyploid giant cancer cells are patholog- ST2 and IL1RAP expressions (Fig. 2A; Supplementary Fig. S3), and ically observed in clinical tumor tissues, and the incidence is associated

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IL33 in Evolutional Transformation of Cancer

Figure 6. Clinical relevancy of the IL33-ST2/IL17RB axis. A, Significant correlation between IL33 positivity and IL17RBþ cell accumulation within tumor microenvironment. Primary tumor tissues (stage II, n ¼ 23; stage III, n ¼ 14), metastatic lymph nodes (n ¼ 9) and the neighboring noncancerous mammary tissues (stage III, n ¼ 8) obtained from patients with breast cancer were analyzed by IHC for IL33 and IL17RB expressions. Immunofluorescence intensity is depicted as pixel counts (IL33 < 76, IL17RB < 443 in normal tissues). Representative photos are shown. Scale bar, 100 mm. , P < 0.01, , P < 0.05 versus normal tissues. B, Significant reversal correlation between ST2þ/IL17RBþ cells and CD8þ T cells in peripheral blood. PBMCs obtained from healthy donors (n ¼ 4, open circles) and patients with stage IV metastatic colorectal cancer (n ¼ 9; closed circles) were analyzed by flow cytometry. Bar graphs show mean SD. P values in the scatter plot graphs were analyzed by the nonparametric Spearman rank test.

with chemoresistance (30) and poor prognosis of patients with can- Disclosure of Potential Conflicts of Interest cer (31). However, there is no practical management of the polyploi- No potential conflicts of interest were disclosed. dization and the rapid proliferation. Our findings that IL33 plays a critical role in cancer polyploidy provided a possible biomarker and a potentially druggable target in cancer therapy. Combination with Authors’ Contributions polyploidy-associated Aurka/b inhibitors (32) may potentially Conception and design: C. Kudo-Saito enhance the anti-IL33 therapeutic efficacy. Development of methodology: C. Kudo-Saito Acquisition of data (provided animals, acquired and managed patients, provided This study provides new insights into the IL33 roles in recurrence facilities, etc.): C. Kudo-Saito, T. Miyamoto, H. Imazeki, N. Boku and treatment resistance of cancer. Targeting IL33-driven poly- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, ploidy could, at least in part, overcome treatment failure in clinical computational analysis): C. Kudo-Saito, T. Miyamoto settings. Writing, review, and/or revision of the manuscript: C. Kudo-Saito

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Kudo-Saito et al.

Administrative, technical, or material support (i.e., reporting or organizing data, The costs of publication of this article were defrayed in part by the constructing databases): H. Imazeki, H. Shoji, K. Aoki payment of page charges. This article must therefore be hereby marked Study supervision: C. Kudo-Saito, N. Boku advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments This work was financially supported by Grants-in-Aid for Scientific Research KAKENHI (21590445 and 26430122 to C. Kudo-Saito) and Japan Agency for Received July 19, 2019; revised October 10, 2019; accepted March 4, 2020; Medical Research and Development AMED-P-CREATE (106209 to C. Kudo-Saito). published first March 10, 2020.

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OF10 Cancer Res; 80(10) May 15, 2020 CANCER RESEARCH

IL33 Is a Key Driver of Treatment Resistance of Cancer

Chie Kudo-Saito, Takahiro Miyamoto, Hiroshi Imazeki, et al.

Cancer Res Published OnlineFirst March 10, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-2235

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