Purpose Recent advances in immunotherapy highlight the antitumor effects of immune- checkpoint inhibition despite a relatively limited subset of patients receiving clinical benefit. The selective class I histone deacetylase inhibitor (HDACi) entinostat has been reported to have immunomodulatory activity including targeting of immune suppressor cells in the tumor microenvironment. Thus, we decided to assess whether entinostat could enhance anti-PD-1 treatment and investigate those alterations in the immunosuppressive tumor microenvironment that contribute to the combined anti-tumor activity. Experimental design We utilized syngeneic mouse models of lung (LLC) and renal cell (RENCA) carcinoma, and assessed immune correlates, tumor growth and survival following treatment with entinostat (5 or 10 mg/kg, P.O.) and a PD-1 inhibitor (10 and 20 mg/kg, s.c.). Results Entinostat enhanced the antitumor effect of PD-1 inhibition in two syngeneic mouse tumor models by reducing tumor growth and increasing survival. Entinostat inhibited the immunosuppressive function of both PMN- and M-MDSC populations. Analysis of MDSC response to entinostat revealed significantly reduced arginase-1, iNOS and COX-2 levels, suggesting potential mechanisms for the altered function. We also observed significant alterations in cytokine/chemokine release in vivo with a shift from an immunosuppressive to a tumor suppressive microenvironment. Conclusions Our results demonstrate that entinostat enhances the antitumor effect of PD-1 targeting through functional inhibition of MDSCs, and a transition away from an immune suppressive tumor microenvironment. These data provide a mechanistic rationale for the clinical testing and potential markers of response of this novel combination in solid tumor patients.

LNCaP lineage‐derived human prostate cancer cell lines C4‐2 and C4‐2B4 acquire androgen independence and osseous metastatic potential in vivo. Using C4‐2 and C4‐2B4 the goals of the current investigation were 1) to establish an ideal bone xenograft model for prostate cancer cells in intact athymic or SCID/bg mice using an intraosseous route of tumor cell administration and 2) to compare prostate cancer metastasis by administering cells either through intravenous (i.v.) or intracardiac administration in athymic or SCID/bg mice. Subsequent to tumor cell administration, prostate cancer growth in the skeleton was assessed by radiographic bone density, serum prostate‐specific antigen (PSA) levels, presence of hematogenous prostate cancer cells and histopathologic evaluation of tumor specimens in the lymph node and skeleton. Our results show that whereas LNCaP cells injected intracardially failed to develop metastasis, C4‐2 cells injected similarly had the highest metastatic capability in SCID/bg mice. Retroperitoneal and mediastinal lymph node metastases were noted in 3/7 animals, whereas 2/7 animals developed osteoblastic spine metastases. Intracardiac injection of C4‐2 in athymic hosts produced spinal metastases in 1/5 animals at 8–12 weeks post‐injection; PC‐3 injected intracardially also metastasized to the bone but yielded osteolytic responses. Intravenous injection of either LNCaP or C4‐2 failed to establish tumor colonies. Intrailiac injection of C4‐2 but not LNCaP nor C4‐2B4 cells in athymic mice established rapidly growing tumors in 4/8 animals at 2–7 weeks after inoculation. Intrafemoral injection of C4‐2 (9/16) and C4‐2B4 (5/18) but not LNCaP (0/13) cells resulted in the development of osteoblastic bone lesions in athymic mice (mean: 6 weeks, range: 3–12 weeks). In SCID/bg mice, intrafemoral injection of LNCaP (6/8), C4‐2 (8/8) and C4‐2B4 (8/8) cells formed PSA‐producing, osteoblastic tumors in the bone marrow space within 3–5 weeks after tumor cell inoculation. A stepwise increase of serum PSA was detected in all animals. Reverse transcription‐polymerase chain reaction (RT‐PCR) to detect hematogenously disseminated prostate cancer cells could not be correlated to either serum PSA level or histological evidence of tumor cells in the marrow space. We have thus established a PSA‐producing and osteoblastic human prostate cancer xenograft model in mice. Int. J. Cancer 77:887–894, 1998.© 1998 Wiley‐Liss, Inc.

An androgen-repressed human prostate cancer cell line, ARCaP, was established and characterized. This cell line was derived from the ascites f luid of a patient with advanced metastatic disease. In contrast to the behavior of androgen-dependent LNCaP and its androgen-independent C4-2 subline, androgen and estrogen suppress the growth of ARCaP cells in a dose-dependent manner in vivo and in vitro. ARCaP is tumorigenic and highly metastatic. It metastasizes to the lymph node, lung, pancreas, liver, kidney, and bone, and forms ascites f luid in athymic hosts. ARCaP cells express low levels of androgen receptor mRNA and prostate-specific antigen mRNA and protein. Immunohistochemical staining shows that ARCaP cells stain intensely for epidermal growth factor receptor, c-erb B2͞neu, and c-erb B3. Staining is negative for chromogranin A and positive for bombesin, serotonin, neuron-specific enolase, and the c-met protooncogene (a hepatic growth factor͞scatter factor receptor). ARCaP cells also secrete high levels of gelatinase A and B and some stromelysin, which suggests that this cell line may contain markers representing invasive adenocarcinoma with selective neuronendocrine phenotypes. Along with its repression of growth, androgen is also found to repress the expression of prostatespecific antigen in ARCaP cells as detected by a prostatespecific antigen promoter-␤-galactosidase reporter assay. Our results suggest that the androgen-repressed state may be central to prostate cancer progression and that advanced prostate cancer can progress from an androgen-independent to an androgen-repressed state.