Naoki Horikawa

IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer

Background:PD-L1 (programmed cell death 1 ligand 1) on tumour cells suppresses host immunity through binding to its receptor PD-1 on lymphocytes, and promotes peritoneal dissemination in mouse models of ovarian cancer. However, how PD-L1 expression is regulated in ovarian cancer microenvironment remains unclear.Methods:The number of CD8-positive lymphocytes and PD-L1 expression in tumour cells was assessed in ovarian cancer clinical samples. PD-L1 expression and tumour progression in mouse models under conditions of altering IFN-γ signals was assessed.Results:The number of CD8-positive cells in cancer stroma was very high in peritoneally disseminated tumours, and was strongly correlated to PD-L1 expression on the tumour cells (P<0.001). In mouse models, depleting IFNGR1 (interferon-γ receptor 1) resulted in lower level of PD-L1 expression in tumour cells, increased the number of tumour-infiltrating CD8-positive lymphocytes, inhibition of peritoneal disseminated tumour growth and longer survival (P=0.02). The injection of IFN-γ into subcutaneous tumours induced PD-L1 expression and promoted tumour growth, and PD-L1 depletion completely abrogated tumour growth caused by IFN-γ injection (P=0.01).Conclusions:Interferon-γ secreted by CD8-positive lymphocytes upregulates PD-L1 on ovarian cancer cells and promotes tumour growth. The lymphocyte infiltration and the IFN-γ status may be the key to effective anti-PD-1 or anti-PD-L1 therapy in ovarian cancer.

Chemotherapy Induces Programmed Cell Death-Ligand 1 Overexpression via the Nuclear Factor-κB to Foster an Immunosuppressive Tumor Microenvironment in Ovarian Cancer

Emerging evidence has highlighted the host immune system in modulating the patient response to chemotherapy, but the mechanism of this modulation remains unclear. The aim of this study was to analyze the effect of chemotherapy on antitumor immunity in the tumor microenvironment of ovarian cancer. Treatment of ovarian cancer cell lines with various chemotherapeutic agents resulted in upregulated expression of MHC class I and programmed cell death 1 ligand 1 (PD-L1) in a NF-κB-dependent manner and suppression of antigen-specific T-cell function in vitro. In a mouse model of ovarian cancer, treatment with paclitaxel increased CD8(+) T-cell infiltration into the tumor site, upregulated PD-L1 expression, and activated NF-κB signaling. In particular, tumor-bearing mice treated with a combination of paclitaxel and a PD-L1/PD-1 signal blockade survived longer than mice treated with paclitaxel alone. In summary, we found that chemotherapy induces local immune suppression in ovarian cancer through NF-κB-mediated PD-L1 upregulation. Thus, a combination of chemotherapy and immunotherapy targeting the PD-L1/PD-1 signaling axis may improve the antitumor response and offers a promising new treatment modality against ovarian cancer.

Expression of Vascular Endothelial Growth Factor in Ovarian Cancer Inhibits Tumor Immunity through the Accumulation of Myeloid-Derived Suppressor Cells.

Purpose: High VEGF expression in ovarian cancer is an unfavorable prognostic factor. However, the role of VEGF in tumor immunity remains unclear. Here, we examined the impact of VEGF on local immunity, including induction of myeloid-derived suppressor cells (MDSC), in ovarian cancer. Experimental Design: High-grade serous ovarian cancer (HGSOC) cases were analyzed by gene expression microarray and IHC for VEGF, CD8, and CD33. VEGF receptor (VEGFR) 1 and VEGFR2 expression levels on MDSCs were analyzed in a mouse model, and the direct effects of VEGF-A on MDSC expansion were investigated. Gr1+ MDSCs and lymphocyte frequencies were analyzed in control tumors and tumors derived from cells harboring short hairpin RNA targeting Vegf-a. In addition, the therapeutic effects of anti-Gr-1 antibodies were examined. Results: Microarray analysis revealed the upregulation of several myeloid cell chemoattractants and the downregulation of lymphocyte-related pathways in cases with high VEGF expression. In immunohistochemical analysis, VEGF expression in peritoneal dissemination correlated with MDSC infiltration. Cases with high MDSC infiltration, which was inversely correlated with intratumoral CD8+ T-cell infiltration, exhibited shorter overall survival. In a mouse model, intratumoral MDSCs expressed both VEGFR1 and VEGFR2. MDSC migration and differentiation were augmented by VEGF signaling. Vegf-a knockdown in tumor cells resulted in decreased MDSC infiltration and increased CD8+ T-cell infiltration. Moreover, treatment with anti-Gr-1 antibodies delayed the growth of control tumors, whereas Vegf-a-knockdown tumors were unaffected by anti-Gr-1 antibody treatment. Conclusions: VEGF expression in ovarian cancer induced MDSCs, inhibited local immunity, and contributed to poor prognosis. Clin Cancer Res; 23(2); 587–99. ©2016 AACR.

Snail promotes ovarian cancer progression by recruiting myeloid-derived suppressor cells via CXCR2 ligand upregulation

Snail is a major transcriptional factor that induces epithelial-mesenchymal transition (EMT). In this study, we explore the effect of Snail on tumor immunity. Snail knockdown in mouse ovarian cancer cells suppresses tumor growth in immunocompetent mice, associated with an increase of CD8+ tumor-infiltrating lymphocytes and a decrease of myeloid-derived suppressor cells (MDSCs). Snail knockdown reduces the expression of CXCR2 ligands (CXCL1 and CXCL2), chemokines that attract MDSCs to the tumor via CXCR2. Snail upregulates CXCR ligands through NF-kB pathway, and most likely, through direct binding to the promoters. A CXCR2 antagonist suppresses MDSC infiltration and delays tumor growth in Snail-expressing mouse tumors. Ovarian cancer patients show elevated serum CXCL1/2, which correlates with Snail expression, MDSC infiltration, and short overall survival. Thus, Snail induces cancer progression via upregulation of CXCR2 ligands and recruitment of MDSCs. Blocking CXCR2 represents an immunological therapeutic approach to inhibit progression of Snail-high tumors undergoing EMT.Snail is a transcription factor that induces epithelial-mesenchymal transition. Here the authors show that, in the mesenchymal subtype of ovarian cancer, Snail expression promotes tumorigenesis by inducing immune evasion through CXCR2-ligands-mediated recruitment of myeloid-derived suppressor cells.

Genomic profile predicts the efficacy of neoadjuvant chemotherapy for cervical cancer patients

BackgroundNeoadjuvant chemotherapy (NAC) using platinum and irinotecan (CPT-11) followed by radical excision has been shown to be a valid treatment for locally advanced squamous cervical cancer (SCC) patients. However, in NAC-resistant or NAC-toxic cases, surgical treatment or radiotherapy might be delayed and the prognosis may be adversely affected. Therefore, it is important to establish a method to predict the efficacy of NAC.MethodsGene expression microarrays of SCC tissue samples (nu2009=u200912) and UGT1A1 genotyping of blood samples (nu2009=u200923) were investigated in terms of their association with NAC sensitivity. Gene expression and drug sensitivity of SCC cell lines were analyzed for validation.ResultsMicroarray analysis revealed that the glutathione metabolic pathway (GMP) was significantly up-regulated in NAC-resistant patients (pu2009<u20090.01), and there was a positive correlation between 50xa0% growth inhibitory concentrations of CPT-11 and predictive scores of GMP activation in SCC cells (ru2009=u20090.32, pu2009<u20090.05). The intracellular glutathione (GSH) concentration showed a highly positive correlation with GMP scores among 4 SCC cell lines (ru2009=u20090.72). UGT1A1 genotyping revealed that patients with UGT1A1 polymorphisms exhibited significantly higher response rates to NAC than those with the wild-type (79.5 vs. 49.5xa0%, respectively, pu2009<u20090.05).ConclusionsThese results indicate that GMP scores of cancerous tissue combined with UGT1A1 genotyping of blood samples may serve as highly potent markers for predicting the efficacy of NAC for individual SCC patients.

Clinical efficacy of neoadjuvant chemotherapy with irinotecan (CPT-11) and nedaplatin followed by radical hysterectomy for locally advanced cervical cancer

Objective To investigate the clinical efficacy of neoadjuvant chemotherapy (NAC) with irinotecan (CPT-11) and nedaplatin (NED) followed by radical hysterectomy. Methods Patients with locally advanced cervical cancer (stage Ib2–IIb) were treated with NAC followed by surgery, primary surgery or primary radiotherapy. NAC was usually performed using transuterine arterial chemotherapy (TUAC) or intravenous CPT-11/NED. Survival rates were analysed in the three treatment groups; response rates and adverse events associated with NAC, TUAC and CPT-11/NED were compared, along with previously reported adverse events of chemoradiotherapy. Results A total of 165 patients with cervical cancer were recruited. Of these, 70 were treated with NAC followed by surgery (48 with CPT-11/NED, 18 with TUAC and four with other types of chemotherapy), 73 were treated with primary surgery and 22 with primary radiotherapy (including chemoradiotherapy). There were no significant differences in progression-free survival or overall survival rates between the three treatment groups. The response rates for the NAC regimen of CPT-11/NED and TUAC were high (75% and 78%, respectively). The frequency of severe thrombocytopenia was lower in patients receiving CPT-11/NED compared with TUAC, and the incidence of severe anaemia, vomiting and cystitis was lower in patients receiving CPT-11/NED compared with chemoradiotherapy. Conclusions The use of CPT-11/NED as a NAC regimen shows favourable activity, with lower toxicity compared with NAC using TUAC or chemoradiotherapy, for the treatment of locally advanced cervical cancer.

Abstract A56: Inhibition of Snail-induced EMT promotes anti-tumor immune response in ovarian cancer.

Background: Peritoneal dissemination is a common mode of tumor progression in ovarian cancer, and one of the most important unfavorable prognostic factors. Epithelial-mesenchymal transition (EMT) has been indicated to be a key process in tumor invasion and metastasis. Recent studies demonstrate that EMT suppresses anti-tumor immunity in some types of cancer. The aim of the current study is to elucidate the relationship between EMT-related gene Snail and local immunity in ovarian cancer. Methods: The expression of Snail and patient survival was analyzed in two microarray datasets, TCGA, and KOV75 (75 ovarian cancer cases operated in our department). Using mouse ovarian cancer cell line HM-1, the influence of Snail expression on EMT, peritoneal dissemination, survival, and local immunity was analyzed using both in vitro and in vivo models. Snail-silenced cell line, HM1-sh-snail was established, and compared with the control HM-1. Results: Among 4 subtypes of high grade serous ovarian cancer, Mesenchymal subtype, which show marked stromal reaction in histopathology, shows the worst prognosis (TCGA, KOV75). Snail expression was significantly high in Mesenchymal subtype (TCGA, p In HM1-sh-snail, expression of vimentin and fibronectin was decreased in RT-PCR analysis. Cell migration was inhibited in wound-healing assay (p Flow cytometric analysis of mouse tumors revealed that infiltration of CD4+ T cells and CD8+ T cells were all increased and MDSCs were decreased in HM1-sh-snail group. Conclusion: Snail-induced EMT inhibits local immune reaction and promotes peritoneal dissemination in ovarian cancer. Snail may be a potential therapeutic target in ovarian cancer. Citation Format: Mana Taki, Kaoru Abiko, Noriomi Matsumura, Tsukasa Baba, Junzo Hamanishi, Naoki Horikawa, Ikuo Konishi. Inhibition of Snail-induced EMT promotes anti-tumor immune response in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A56.

Abstract B63: Expression of VEGF in ovarian cancer suppresses tumor immunity through recruitment of myeloid derived suppressor cells.

Background and Objective: High expression of VEGF in ovarian cancer is a known unfavorable prognostic factor. However, its role in tumor immunity remains unclear. In this study, the impact of VEGF on ovarian cancer local immunity, including myeloid derived suppressor cells (MDSC), was explored. Methods: High grade serous ovarian cancer (HGSOC) cases were analyzed by gene expression microarray and immunohistochemistry for VEGF and CD33. Mouse HM-1 sh-Vegfa cells were generated and inoculated into immunocompetent mice subcutaneously. The proportions of lymphocytes and MDSC in tumors and spleens were analyzed by flow cytometry. Spleen cells from these mice were stimulated with CD3/CD28 antibody and Granzyme B+ CTLs were analyzed. The effects of Vegfa on differentiation and migration of MDSC were investigated. Results: Microarray analysis of 32 clinical HGSOC samples revealed that, in VEGF-high expressing cases, several chemoattractants for myeloid cells were significantly up-regulated, whereas lymphocyte-related pathways were down-regulated. In immunohistochemical analysis of 56 HGSOC samples, the number of CD33+MDSC positively correlated with the intensity of VEGF staining (r=0.43, p In an immunocompetent mouse ovarian cancer model, silencing Vegf resulted in diminished infiltration of CD11b+/Gr-1+MDSC (p In the mouse ex vivo assay, generation of MDSC from mouse bone marrow cells was augmented by adding recombinant VEGF, whereas this was attenuated by adding an anti-VEGF antibody. In chemotaxis assays, inhibition of VEGF signaling attenuated the migration of MDSC. Conclusion: High expression of VEGF correlates with infiltration of MDSC and poor prognosis in ovarian cancer clinical samples. VEGF promotes the generation, migration, and immunosuppressive function of MDSC in mouse models. It is likely that VEGF expressed in ovarian cancer inhibits local immunity through recruitment of MDSC and thus contributes to poor prognosis. Citation Format: Naoki Horikawa, Kaoru Abiko, Noriomi Matsumura, Junzo Hamanishi, Susan Murphy, Tsukasa Baba, Ken Yamaguchi, Masafumi Koshiyama, Ikuo Konishi. Expression of VEGF in ovarian cancer suppresses tumor immunity through recruitment of myeloid derived suppressor cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B63.

Abstract A83: Induction of PD-L1 expression by cytotoxic agents through activation of NF-kB signal

Recently, growing evidence has shown that the therapeutic efficacy of chemotherapy largely depends on the host immune system. We analyzed gene expression microarray dataset GSE13525 to examine how cytotoxic agents influence the immune system. Expression of programmed cell death 1 ligand 1 (PD-L1) was increased following carboplatin (CBDCA) treatment in the human ovarian cancer cell line 36M2 from GSE13525 (p Pathway analysis of gene expression microarray data from clinical ovarian cancer samples collected before and after chemotherapy (GSE15622) showed that both paclitaxel (PTX) (n=20) and CBDCA (n=14) treatment activated the NF-kB signaling pathway (single-sample Gene Set Enrichment Analysis; p In order to determine if PD-L1 expression on ovarian cancer cells induced by cytotoxic agents could serve as a molecular target in ovarian cancer, we performed in vivo experiments. We performed lentiviral transduction into ID8 cells using PD-L1 cDNA (ID8-PDL1), vector control (ID8-control) and microRNA targeting PD-L1 mRNA (ID8-mirPDL1). Following intraperitoneal injection of these ID8 cells into syngeneic C57/BL6 mice, we injected PTX or vehicle intraperitoneally (6 groups, n=12 in each group). The ID8-PD-L1 mice without PTX showed the worst prognosis, while ID8-mirPDL1 mice with PTX showed the best prognosis (p In summary, we found chemotherapeutic agents upregulate PD-L1 expression through activation of NF-kB signaling. Immunotherapeutic targeting of the PD-L1/PD-1 signal combined with chemotherapy may be a promising treatment modality against ovarian cancer. Citation Format: Junzo Hamanishi, Noriomi Matsumura, Peng Jin, Kaoru Abiko, Naoki Horikawa, Ken Yamaguchi, Tsukasa Baba, Susan K. Murphy, Ikuo Konishi, Masaki Mandai. Induction of PD-L1 expression by cytotoxic agents through activation of NF-kB signal. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A83.