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Published OnlineFirst March 10, 2020; DOI: 10.1158/0008-5472.CAN-19-2235 CANCER RESEARCH | TUMOR BIOLOGY AND IMMUNOLOGY 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 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 Ó2020 American Association for Cancer Research. implantation and were cultured in 10% FBS/DMEM with Geneticin AACRJournals.org | OF1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 10, 2020; DOI: 10.1158/0008-5472.CAN-19-2235 Kudo-Saito et al. 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.

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