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The scattergram and real number of pearson correlations between expression levels (RPKM) of immune-related genes and CD274 or PDCD1LG2 in BLCA, GC, HNSC, LUAD and LUSC.

Purpose: Invasion and metastasis are key steps in the progression of urothelial cancer (UC) into a critical disease. Foxo3a is a member of the Foxo transcription factor family that modulates the expression of various genes. We aimed to elucidate the role of Foxo3a in UC invasion.Experimental Design: Foxo3a mRNA and protein expressions in UC samples were investigated by gene expression assays and immunohistochemistry, respectively. Foxo3a expression was compared with clinicopathologic characteristics and patient prognoses based on UC samples. Quantitative real-time polymerase chain reaction, Western blotting, and migration assays were also conducted in UC cells.Results: Foxo3a expression decreased in invasive UC; patients with low Foxo3a expression had poor disease-free survival, cancer-specific survival, and overall survival; Foxo3a knockdown in UC cells increased cellular motility. Foxo3a negatively regulated Twist1 and Y-box–binding protein 1 (YB-1), and positively regulated E-cadherin in KK47 and TCCsup cells that expressed Twist1, but not in T24 cells that did not express Twist1. Foxo3a-associated acetyltransferase p300 and Foxo3a acetylation status also affected UC motility.Conclusion: The results of this study indicate that Foxo3a regulates motility of UC through negative regulation of Twist1 and YB-1, and through positive regulation of E-cadherin. This suggests that Foxo3a could act as an independent prognostic factor in UC and could represent a promising molecular target for cancer therapeutics. Clin Cancer Res; 16(23); 5654–63. ©2010 AACR.

In vivo tumor growth treated with anti-PD-1 and/or anti-PD-L2 mAb in immunocompetent mice.

Immune-related gene list.

In vivo tumor growth in nude, CD4+ T cell or CD8+ T cell-depleted mice.

Effector CD8+ T cells detected by CD44+CD62L-CD8+ T cells, PD-1+CD8+ T cells, and cytokine-producing CD8+ T cells in TILs from MC-38 tumors.

In vitro PD-L1 expression in MC38, CT26-NY-ESO-1, B16-F10, Renca, CMS5a and A20 treated with or without IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, IL-4, TGF-β1 or GM-CSF.

Supplementary Tables S1-S2.

In vitro PD-L2 expression in MC38, CT26-NY-ESO-1, B16-F10, Renca, CMS5a and A20 treated with or without IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, IL-4, TGF-β1 or GM-CSF.

In vivo tumor growth treated with PD-1 signal blockades in nude, CD4+ T cell or CD8+ T cell-depleted mice.

Legends for supplementry figures.

Purpose: The progression of prostate cancer to metastatic and castration-resistant disease represents a critical step. We previously showed that the transcription factor Twist1, which promotes epithelial–mesenchymal transition, was involved in castration-resistant progression. Similarly, protein kinase C (PKC) has been implicated in both metastatic progression and castration resistance in prostate cancer.Experimental Design: In this study, we aimed to elucidate the role of PKC/Twist1 signaling in castration resistance, and to apply this information to the development of a novel therapeutic concept using PKC inhibitor Ro31-8220 against prostate cancer using various prostate cancer cell lines.Results: Androgen deprivation and the next-generation antiandrogen enzalutamide induced PKC activation and Twist1 expression, which were reversed by the PKC inhibitor Ro31-8220. Ro31-8220 suppressed cell proliferation in androgen-dependent prostate cancer LNCaP cells, which was augmented by its combination with androgen deprivation or enzalutamide. The favorable anticancer effects of the combination of Ro31-8220 and enzalutamide were also observed in castration-resistant C4-2 and 22Rv1 cells. Furthermore, PKC phosphorylation was elevated in castration-resistant and enzalutamide-resistant cells compared with their parental cells, leading to persistent sensitivity to Ro-31-8220 in castration- and enzalutamide-resistant cells.Conclusions: Taken together, these findings indicate that PKC/Twist1 signaling contributes to castration resistance as well as enzalutamide resistance in prostate cancer, and suggest that therapeutics targeting PKC/Twist1 signaling, such as PKC inhibitors, represent a promising novel therapeutic strategy for prostate cancer, especially castration-resistant prostate cancer, when combined with enzalutamide. Clin Cancer Res; 20(4); 951–61. ©2013 AACR.

Clinicopathological features in patients with RCC (n = 29).

Effector CD8+ T cells detected by CD44+CD62L-CD8+ T cells, PD-1+CD8+ T cells, and cytokine-producing CD8+ T cells in TILs from MC-38-L2 tumors.

In vitro PD-L1 and PD-L2 expressions by MC-38-L1KO and MC-38-L1KO-L2 treated with or without IFN-γ and in vivo tumor growth of those tumors.

The relationship between CD274 or PDCD1LG2 expressions and survival in RCC and LUSC.

XLSX file 9K, Taget sequense of siRNAs

Purpose:To evaluate the detailed immunosuppressive role(s) of PD-L2 given that its detailed role(s) remains unclear in PD-1 signal blockade therapy in animal models and humans.Experimental Design:We generated mouse cell lines harboring various status of PD-L1/PD-L2 and evaluated the tumor growth and phenotypes of tumor-infiltrated lymphocytes using several PD-1 signal blockades in animal models. In humans, the correlation between immune-related gene expression and CD274 (encoding PD-L1) or PDCD1LG2 (encoding PD-L2) was investigated using The Cancer Genome Atlas (TCGA) datasets. In addition, PD-L1 or PD-L2 expression in tumor cells and CD8+ T-cell infiltration were assessed by IHC.Results:In animal models, we showed that PD-L2 expression alone or simultaneously expressed with PD-L1 in tumor cells significantly suppressed antitumor immune responses, such as tumor antigen–specific CD8+ T cells, and was involved in the resistance to treatment with anti-PD-L1 mAb alone. This resistance was overcome by anti-PD-1 mAb or combined treatment with anti-PD-L2 mAb. In clinical settings, antitumor immune responses were significantly correlated with PD-L2 expression in the tumor microenvironment in renal cell carcinoma (RCC) and lung squamous cell carcinoma (LUSC).Conclusions:We propose that PD-L2 as well as PD-L1 play important roles in evading antitumor immunity, suggesting that PD-1/PD-L2 blockade must be considered for optimal immunotherapy in PD-L2–expressing cancers, such as RCC and LUSC.

In vivo PD-L2 expression in APCs from MC-38 tumor.

Clinicopathological features in patients with NSCLC (n = 27).

Effector CD8+ T cells detected by CD44+CD62L-CD8+ T cells, PD-1+CD8+ T cells, and cytokine-producing CD8+ T cells in TILs from MC-38, MC-38-GFP, or MC-38-L2 tumors.

Background: The correlation between PD-L1 expression on tumor-infiltrating immune cells and prognosis in renal cell carcinoma (RCC) has been reported in several studies. Methods: A multicenter retrospective study was carried out to investigate the prognostic value of PD-L1 expression (by the VENTANA SP142 immune cell [IC] scoring method) in primary RCC tumors among patients administered systemic therapy for recurrent or metastatic RCC. The primary endpoint was overall survival (OS), defined as the time from the initiation of first-line treatment to the date of death from any cause. The difference in OS between patients of positive PD-L1 status (IC ≥1%) and those of negative status was compared using a stratified log-rank test at a two-sided level of 0.1, stratified by MSKCC risk and liver metastases. Stratified hazard ratio (HR) and its 90% confidence interval (CI) was estimated by the Cox proportional hazard model. An unstratified analysis and an analysis adjusted by propensity score weighting (PSW) were also conducted. Results: We examined 777 RCC surgical specimens, 770 of which were included for primary analysis (7 were excluded owing to indeterminate PD-L1 status). PD-L1 positive was observed in 315 (40.9%). Immune phenotype that describe the level of T-cell presence and activity within the tumor microenvironment differed between PD-L1-positive vs. -negative groups: Excluded (74.3% vs. 31.6%), Inflamed (16.5% vs. 1.5%), and Desert (9.2% vs. 66.8%). PD-L1-positive status was more common in patients with higher clinical stage, higher Fuhrman grade, and presence of sarcomatoid component. A higher proportion of PD-L1-positive patients had poor prognostic features at the start of first-line therapy: 17.5% vs. 7.3% with ‘poor' MSKCC risk; 29.2% vs. 14.9% with ‘poor' IMDC risk. In line with this, fewer PD-L1-positive patients had good prognostic features at the start of first-line therapy: 11.4% vs. 29.7% with ‘favorable' MSKCC risk; 10.8% vs. 26.2% with ‘favorable' IMDC risk.The median OS in PD-L1-positive vs. -negative patients was 30.9 months (95%CI: 25.5-35.7) vs. 37.5 months (95%CI: 34.0-42.6). The stratified HR was 1.04 (90%CI: 0.89-1.22, P=0.65), the unstratified HR was 1.21 (90%CI: 1.04-1.40), and the PSW-adjusted HR was 0.99. Subgroup analysis based on MSKCC risk at the start of first-line therapy revealed that there was no significant difference in mOS based on PD-L1-positive vs. -negative status for ‘favorable' (58.6 vs. 59.8 months; HR: 0.92; 95%CI: 0.54-1.56), ‘intermediate' (31.6 vs. 33.5; HR: 1.06; 95%CI: 0.85-1.32) or ‘poor' (6.6 vs 12.8.; HR: 0.89; 95%CI: 0.55-1.43) categories. Conclusion: This study suggested that PD-L1 status on tumor-infiltrating immune cells was not an independent prognostic factor in recurrent or metastatic RCC patients, although positive PD-L1 status was associated with poor prognostic factors. Citation Format: Hirotsugu Uemura, Motohide Uemura, Naoto Sassa, Noboru Nakaigawa, Katsunori Tatsugami, Kenichi Harada, Toshinari Yamasaki, Nobuaki Matsubara, Tamaki Fukuyama, Takuya Yoshimoto, Yuki Nakagawa, Mototsugu Oya, Nobuo Shinohara, Toyonori Tsuzuki. Prognostic value of PD-L1 expression on tumor-infiltrating immune cells in renal cell carcinoma (ARCHERY study) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-153.

Nonmyeloablative allogeneic stem cell transplantation (SCT) has been used for various malignancies, although detailed mechanisms of antitumor effects remain unclear. We showed that a nonmyeloablative allogeneic SCT regimen, which consists of mixed chimerism induced by an injection of donor spleen and bone marrow cells followed by cyclophosphamide treatment and a donor lymphocyte infusion (DLI), exerted antitumor effects on established murine bladder tumor, MBT-2. An expansion of donor CD4 T cells accompanied by transient but vigorous IFN-γ production was detected shortly after DLI. In vivo neutralization of IFN-γ or depletion of CD4 T cells from DLI abolished the antitumor effects, indicating an indispensable role of donor CD4 T cells producing IFN-γ. Donor as well as host CD8 T cells accumulated in the tumor region with time. Importantly, depletion of CD8 T cells from DLI did not reverse the suppression of tumor growth, indicating that CD4 T cells play a more essential role in mediating early antitumor effects. Furthermore, tumor-specific response of host CD8 T cells was suggested. These results not only provide the first evidence of nonmyeloablative allogeneic SCT for the treatment of bladder tumor but also elucidate detailed mechanisms of antitumor effects provoked by DLI. [Cancer Res 2009;69(12):5151–8]

Purpose: Much attention has been paid to nonmyeloablative allogeneic stem cell transplantation for the treatment of renal cancer. We recently proposed a cyclophosphamide-using nonmyeloablative cell therapy in which donor lymphocyte infusion (DLI) was carried out after the tolerance induction to donor cells. In considering the clinical application of the cyclophosphamide-using cell therapy, attempts to reduce graft-versus-host disease (GVHD) are crucial. The aim of the present study was to modify the cyclophosphamide-using cell therapy to reduce the risk of GVHD while preserving the antitumor activity against renal cancer.Experimental Design: We assessed whether a delay in performing DLI from day 1 to day 5 after the cyclophosphamide treatment could reduce the risk of GVHD while preserving antitumor activity against RENCA, a murine carcinogen-induced renal cell carcinoma, in the cyclophosphamide-using cell therapy.Results: Regarding the in vivo antitumor effect, there was no difference between DLI on day 1 and day 5 after the cyclophosphamide treatment, whereas the histologic findings of the small intestine showed that the cyclophosphamide-using cell therapy with DLI on day 5 decreased the risk of GVHD. In addition, the acquired immunity against RENCA was also observed in the RENCA-rejected mice that had been treated with DLI on day 5.Conclusions: Our results show that a delay in DLI during cyclophosphamide-using nonmyeloablative cell therapy can dissociate graft-versus-tumor effects from GVHD by reducing the risk of GVHD.

Recently, much attention has been paid to nonmyeloablative allogeneic stem cell transplantation for the treatment of metastatic renal cancer. Mature donor T cells cause graft-versus-host disease (GVHD) although they are also the main mediators of the beneficial graft-versus-tumor activity associated with this treatment. Hence, the segregation of the graft-versus-tumor activity from GVHD is an important challenge in managing the clinical course of treatment. We previously reported a series of studies regarding the allograft tolerance induced by allogeneic spleen cells (with bone marrow cells) and cyclophosphamide in mice. Here, we show a modified cyclophosphamide-induced tolerance system for the treatment of murine renal cell carcinoma, RENCA, by shifting the equal balance between graft-versus-host and host-versus-graft reactions toward graft-versus-host reaction with donor lymphocyte infusion. Our results clearly show the antitumor activity against RENCA with only low levels of mixed chimerism in the periphery and the in vivo and in vitro acquired immunity against RENCA even when mixed chimerism is almost undetectable. Because the withdrawal of mixed chimerism reduces the risk of GVHD, the antitumor activity is thus sequentially segregated from the initial GVHD in our model. We believe that this is the first unique model system of nonmyeloablative allogeneic hemopoietic cell transplantation to ever be reported for the treatment of renal cancer.