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The tumor microenvironment (TME) is a dynamic milieu comprised of various cell types and molecular complexes that eventually evolves to promote cancer cell proliferation, metastasis, and immune evasion. Myeloid cells are a key component of the TME and play key roles in initiating antitumor responses and coordinating with cells of the adaptive immune system. However, these cells are eventually hijacked by tumors and provoke local inflammation that results in chronic cancer-associated inflammation and immune escape. Extracellular adenosine (ecADO) is an immunosuppressive metabolite that is produced in tumors that binds the adenosine 2a receptor (A2aR) which is widely expressed in immune cells. Studies have shown that blocking A2aR signaling has the potential to enhance antitumor immunity by boosting T and NK cell immune responses. However, the relationship between ecADO and tumor-associated myeloid cells is less characterized. We previously reported on the antitumor activity of A2aR and CD73 blockade in preclinical models of Pten-null prostate cancer and their impact on T cell mediated responses. Here, we examine tumor-associated myeloid cell infiltration in models of castration-naïve (CNPC) and castration-resistant prostate cancer (CRPC) the impact of A2aR and CD73 blockade. Transcriptomic, flow cytometric, and immunohistochemical (IHC) profiling studies of CNPC and CRPC from conditional Pten-knockout (KO) and Pten/Trp53-double knockout (DKO) showed that tumor-infiltrating myeloid cells constituted a significant proportion of the prostate tumor microenvironment. CD11b+/Ly6G+ cells representing tumor associated neutrophils (TANs) and myeloid derived suppressor cells (MDSCs) were the most abundant immune cell type comprising 14.9% and 32.1% of the total population, and 46.5% and 69.7% of infiltrating leukocytes in in CNPC and CRPC, respectively. Furthermore, CD11b+/Ly6G+ expressed high levels of CD73. Pte-null prostate tumors were characterized with enriched adenosine signaling signatures which were greater in CRPC compared to CNPC. Treatments with the A2aR inhibitor AZD4635 alone and in combination with CD73 antibody blockade reduced adenosinergic genes as well as genes associated with TAN/MDSC immunosuppressive functions. Additionally, tumors from treated mice exhibited improved T cell mediated cytotoxicity. Together, our findings indicate that tumor associated myeloid cells are an additional source of ecADO contributing to immune suppression which can be targeted with A2aR and CD73 inhibitors. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazutoshi Fujita, Kazuko Sakai, Alwin G. Schuller, Kris F. Sachsenmeier, Masahiro Nozawa, Eri Banno, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Targeting tumor infiltrating myeloid cells in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5155.

Gut microbes are intricately involved in maintaining normal physiology and homeostasis and have become a major area of study to determine their relevance in human disease. In cancer, alterations to gut microbiota has been associated to resistance to chemo- and immuno-therapy, whereas supplementation with specific taxa improves antitumor treatment responses. Mouse models have been essential to study disease biology and drug discovery. However, mice are fundamentally quite different from humans, thus, questions have arisen regarding their utility as pertinent tools to study microbial influences and their impact on human disease. To address these questions, we have performed a cross-species comparative analysis of the fecal microbiota from a human cohort of prostate cancer patients and a preclinical mouse model of prostate cancer. Microbiota composition was determined by 16s RNA gene sequencing on stool samples from tumor-bearing prostate-specific conditional Pten-knockout and disease-free wildtype mice and 16s RNA gene sequencing data from a human cohort of patients with suspected prostate cancer was used. Human cases were assigned to no cancer and cancer groups based on diagnosis from prostate biopsy and as low-risk (negative biopsy of Gleason grade Citation Format: Chisato Wakamori, Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazutoshi Fujita, Kazuko Sakai, Makoto Matsushita, Eri Banno, Yasunori Mori, Masahiro Nozawa, Mitsuhisa Nishimoto, Kazuhiro Yoshimura, Norio Nonomura, Kazuto Nishio, Hirotsugu Uemura. Integrative gut microbiome analysis of human and mouse prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3044.

Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of immature immune cells that expand during cancer and can induce potent immune suppression. These cells play critical roles in supporting tumor growth and progression and their induction has been associated with the inactivation of PTEN. To date, few therapies targeting these cells are available which is partly due to their increasingly complex nature and context-dependent functions. Moreover, there is no clear definition to identify these cells. Enhancing our understanding of this cell population will aid in developing more effective cancer therapies. Here, we used gene expression profiling, flow cytometry, and quantitative immunohistochemistry to characterize tumor-infiltrating and circulating MDSCs in prostate carcinogenesis and progression in transgenic mouse models of prostate cancer driven by the conditional inactivation of Pten and Trp53. Transcriptomic profiling revealed enrichment of various myeloid-derived cell signatures in the early stages of prostate cancer including MDSCs, macrophages, and neutrophils. Flow cytometric analysis confirmed the increased infiltration of CD11b+/Ly6G+ (polymorphonuclear MDSCs (pMDSCs)) and CD11b+/Ly6C+ (monocytic MDSCs (mMDSCs)) cells in prostate tumors. The temporal dynamics of tumor-infiltrating myeloid cells were further profiled and showed that pMDSC and mMDSC abundances were 1.6 and 5.7-fold higher, respectively, in older mice with late-stage locally invasive adenocarcinomas (AdCa) compared to younger mice with early-stage prostatic intraepithelial neoplasia (PIN). In both instances, pMDSCs comprised most of the leukocyte infiltrate, however, mMDSCs tended to be positively correlated with tumor burden in AdCa but not PIN. In peripheral blood, CD44+/CD62L+ immature neutrophils were associated with high tumor burden in aged mice. Treatments targeting the androgen receptor (AR) signaling further exacerbated pMDSC infiltration in tumors and was associated with resistance to second androgen receptor-targeted agent (ARTA) therapy in a model of castration resistant prostate cancer. Our findings indicate that MDSCs are a prominent population in Pten-null prostate cancer and are implicated with resistance to prostate cancer treatment. MDSCs constitute a potentially targetable population and these models serve as viable platforms to further investigate their biological activity and further evaluate novel MDSC-directed therapies. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Eri Banno, Shogo Adomi, Mitsuhisa Nishimoto, Takafumi Minami, Yasunori Mori, Kazutoshi Fujita, Masahiro Nozawa Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Profiling of myeloid-derived suppressor cells in Pten-deficient in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2877.

Studies suggest that certain immune-mediated diseases may be associated with cancer risk beyond local organs. Prolonged systemic inflammation from inflammatory bowel diseases such as ulcerative colitis (UC) and Crohn’s disease (CD) pose a greater risk to colorectal cancer (CRC) and accumulating data suggests that prostate cancer (PCa) risk is higher in patients with UC but not CD and that patients with PCa may be at a greater risk for CRC. A better understanding of the biological interactions between these conditions could have important implications for cancer screening, detection, and prevention in high-risk populations. However, data to establish the relationships and relative contributions of these diseases are lacking. Here, we explored these relationships using preclinical mouse models of dextran sulfate sodium (DSS)-induced colitis (DSS-UC) and DSS/azoxymethane (AOM)-induced CRC (DSS/AOM-CRC) in young and aged healthy wildtype mice and transgenic mice harboring Pten-deficient PCa. In the DSS/AOM-CRC model, aged PCa-bearing conditional Pten-knockout (KO) mice experienced poorer overall survival compared to age-matched wildtype (WT) without PCa because of CRC. DSS/AOM-CRC incidence was not affected by the presence of PCa, however tumor burden was higher in KO mice compared to WT and in KO mice, DSS/AOM-CRC accelerated PCa growth and progression. Prostates from WT DSS/AOM-CRC mice showed higher incidence of hyperplasia and greater myeloid cell infiltration compared to healthy controls. Systemic changes associated with DSS/AOM-CRC in both WT and KO mice included splenomegaly, lymphadenopathy, and immune cell composition. The impact of DSS-UC was investigated conditional Pten/Trp53-double knockout (DKO) mice with DDS only treatments starting at an age prior to the development of PCa. In this setting, cycling DSS led to the development of UC after 10 weeks of treatment and accelerated prostate tumor development characterized by high grade prostatic intraepithelial neoplasia (PIN) grade dysplasia and increased cell proliferation. Prostate tumors from mice with DSS-UC exhibited an altered tumor immune microenvironment characterized by a higher degree of inflammatory cell infiltrate. Changes in immune cell composition were also observed in peripheral blood and secondary lymphoid organs. These preclinical models recapitulated disease relationships observed in humans and provide a platform to further investigate biological mechanisms and therapeutic interventions. Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Naomi Ando, Noriko Sako, Kazutoshi Fujita, Shogo Adomi, Eri Banno, Yasunori Mori, Takafumi Minami, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Exploring the relationships between prostate cancer, colorectal cancer, and ulcerative colitis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 640.

Significant improvements with apalutamide, a nonsteroidal antiandrogen used to treat patients suffering from advanced prostate cancer (PCa), have prompted evaluation for additional indications and therapeutic development with other agents, however, persistent androgen receptor (AR) signaling remains problematic. We used autochthonous mouse models of Pten-deficient PCa to examine the context-specific antitumor activity of apalutamide and profile its molecular responses. Overall, apalutamide showed potent antitumor activity in both early-stage and late-stage models of castration-naïve prostate cancer (CNPC). Molecular profiling by Western blot and immunohistochemistry associated persistent surviving cancer cells with upregulated AKT signaling. While apalutamide was ineffective in an early-stage model of castration-resistant prostate cancer (CRPC), it tended to prolong survival in late-stage CRPC. Molecular features associated with surviving cancer cells in CRPC included upregulated aberrant-AR, and phosphorylated S6 and proline-rich Akt substrate of 40 kDa (PRAS40). Strong synergy was observed with the pan-AKT inhibitor GSK690693 and apalutamide in vitro against the CNPC- and CRPC-derived cell lines and tended to improve the antitumor responses in CNPC but not CRPC in vivo. Upregulation of signal transducer and activator of transcription 3 (STAT3) and proviral insertion in murine-1 (PIM-1) were associated with combined apalutamide/GSK690693. Our findings show that apalutamide can attenuate Pten-deficient PCa in a context-specific manner and provides data that can be used to further study and, possibly, develop additional combinations with apalutamide.

Simple Summary Next-generation antiandrogens have transformed the therapeutic landscape for castration-resistant prostate cancer. Their utility in other indications, such as high-risk castration-sensitive cancers and as combination therapy, are being investigated. Our aim was to profile the in vivo antitumor activity of apalutamide in phenotypically distinct mouse models of Pten-deficient castration-naïve and castration-resistant prostate cancer, using both early- and late-stage disease models, and to profile the molecular responses. We also evaluated the therapeutic potential and characterized the molecular responses of the combined targeted AR/AKT blockade and showed that while this approach was promising in vitro, it was mostly ineffective in vivo, particularly in the castration-resistant setting. Our findings provide evidence that links therapeutic resistance to STAT3 and PIM-1 in the castration-resistant setting and provide insights into the context-specific antitumor activity of apalutamide. Abstract Significant improvements with apalutamide, a nonsteroidal antiandrogen used to treat patients suffering from advanced prostate cancer (PCa), have prompted evaluation for additional indications and therapeutic development with other agents; however, persistent androgen receptor (AR) signaling remains problematic. We used autochthonous mouse models of Pten-deficient PCa to examine the context-specific antitumor activity of apalutamide and profile its molecular responses. Overall, apalutamide showed potent antitumor activity in both early-stage and late-stage models of castration-naïve prostate cancer (CNPC). Molecular profiling by Western blot and immunohistochemistry associated persistent surviving cancer cells with upregulated AKT signaling. While apalutamide was ineffective in an early-stage model of castration-resistant prostate cancer (CRPC), it tended to prolong survival in late-stage CRPC. Molecular features associated with surviving cancer cells in CRPC included upregulated aberrant-AR, and phosphorylated S6 and proline-rich Akt substrate of 40 kDa (PRAS40). Strong synergy was observed with the pan-AKT inhibitor GSK690693 and apalutamide in vitro against the CNPC- and CRPC-derived cell lines and tended to improve the antitumor responses in CNPC but not CRPC in vivo. Upregulation of signal transducer and activator of transcription 3 (STAT3) and proviral insertion in murine-1 (PIM-1) were associated with combined apalutamide/GSK690693. Our findings show that apalutamide can attenuate Pten-deficient PCa in a context-specific manner and provides data that can be used to further study and, possibly, develop additional combinations with apalutamide.

This paper presents a contemporary architectural working method that encompasses digitalization and parameterization of existing buildings and optimization of new buildings designed with ruled surfaces. The method uses parametric modeling and computational structural analysis in order to simplify contemporary building processes. As an example of the application of these techniques, in this paper they are applied to Felix Candela’s Church of St. Joseph the Craftsman, a design which features hyperbolic paraboloids that are considered difficult to design, calculate and build. The optimization method introduced in this paper seeks to explore different possibilities for designing and modifying buildings designed using non-standard geometry allowing them to be built out of simplified elements but also keep construction and visual properties of their shape. This method is also useful for students and young engineers to expand their skills in structural analysis, parametric modeling and optimization methods with contemporary tools.

Prostate tumors are characterized with constitutively activated AKT signaling primarily through the loss of PTEN. Second generation antiandrogens such as abiraterone acetate have improved the survival of prostate cancer patients, however, resistance remains a problem and patients with PTEN-deficient tumors have poorer outcomes to abiraterone treatments. Moreover, PTEN-deficient tumors are also characterized with immunosuppressive tumor microenvironments. In this study we investigate the benefit of adding capivasertib, a potent pan-Akt inhibitor, to abiraterone therapy in a preclinical model of Pten-deficient prostate cancer and examine the antitumor efficacy and its influence on antitumor immunity. The temporal effects of abiraterone and capivasertib alone and as combination therapy were evaluated after one and four weeks of dosing in conditional transgenic mice harboring Pten-deficient prostate tumors. While abiraterone therapy alone reduced genitourinary tract weights, which contain both AR-sensitive normal accessory sex organs and prostate tumor, the treatment had no overall effect on prostate tumor burden. Mice treated with capivasertib monotherapy experienced tumor burden reductions of 8.2% and 15.6% at week 1 and 8, respectively, moreover, the treatment combination of abiraterone + capivasertib significantly improved the therapeutic effect with tumor burden reductions of 35.6% and 37.8% at weeks 1 and 4, respectively versus vehicle control. qRT-PCR analysis of Ar target genes showed decreased expression in mice treated with abiraterone. All treatment combinations showed increased gene expression levels of Casp3 in tumors from mice at week one but were greatest in those treated with abiraterone alone or in combination and increased expression levels were sustained only in the combination cohort at week four. Immunological profiling of tumors from mice treated with abiraterone + capivasertib using qRT-PCR-based panel of immune-related genes revealed enrichments in genes associated with phagocytosis, antigen processing and dendritic cell function. Examination of the tumor draining lymph nodes by flow cytometric analysis showed ~1.5-fold-increases in the abundance of migratory dendritic cells (CD11b+\CD11c+\XCR1+) in all pharmacologically treated mice (versus vehicle control) and remained elevated only in mice receiving capivasertib as monotherapy or combination at week four. Additionally, mice treated with combination therapy also had enrichments in genes sets associated to T cell, NK cell and as well as T cell activation, cytotoxicity, and interferon gamma signature. The findings from this study provide preclinical evidence for the efficacy of combination therapy with abiraterone plus capivasertib and provides insights into its immunomodulatory effects and influence on antitumor immunity. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Keiko Kuroka, Kazutoshi Fujita, Eri Banno, Kazuko Sakai, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Preclinical efficacy of abiraterone plus capivasertib in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5282.

Prostate cancers depend on androgens and the androgen receptor (AR) to drive androgen signaling pathways. Androgen-deprivation therapy (ADT) and second-generation antiandrogens have become the new standard of care for men with prostate cancer. However, resistance to these therapies remains a problem. TP53 mutations and PTEN loss have been associated with patients who fail to respond to anti-androgen therapies and complex interactions between AR signaling and the immune system means that these therapies have the potential to affect antitumor immune responses. Here, we characterize treatment responses of ADT alone and in combination with antiandrogen therapy using apalutamide (Apa) in a mouse model of Pten/Trp53-deficeint castrate-resistant prostate cancer (CRPC). Twenty-eight-week-old conditional Pten/Trp53-double knockout mice were randomized as untreated control or treated with ADT via orchidectomy for four weeks followed by vehicle or Apa therapy for an additional four weeks. While both treatments reduced tumor burden, there was no difference between treatments. Treatment responses in were further evaluated and were judged as favorable or non-favorable, relative to median tumor burden. Analysis of individual treatment responses indicated that 31.3% (5/16) of mice treated with ADT+Apa had tumor burden reductions >20% (relative to the median) compared to 6.3% (1/16) in ADT, and in both instances, 37.5% (6/16) of mice treated with ADT or ADT+Apa had tumor burden increases >20%. IHC showed lower nuclear/cytoplasm expression of AR in ADT+Apa treated mice and the reduction was more pronounced in favorable responders. ADT+Apa treated mice had lower gene expression levels of Ar than ADT alone and AR target genes (Fkbp5, Tmprss2, Timp4 and Nkx3.1) were decreased in ADT+Apa favorable responders whereas these target genes as well as AR-regulating genes (Myc and Igf1r, Igf1) were higher in ADT+Apa non-favorable responders. Cancer cell proliferation did not differ between treatments, but cancer glands in ADT+Apa treated mice had significantly greater IHC expression levels of cleaved caspase 3, a marker for apoptosis, and were greatest in ADT+Apa favorable responders. Levels of p-S6 ribosomal protein, a downstream target of Akt, were higher in the cancer glands of ADT+Apa but were reduced in stromal and infiltrating immune cells. Immune profiling by gene expression and flow cytometry showed enhanced macrophage involvement and reduced MDSC/granulocyte activity in ADT+Apa treated mice. There were no differences in CD8 T cell and NK cell abundance between the two cohorts, but greater cytotoxic activity was noted in all favorable responders. This study provides preclinical data that links therapeutic resistance to anti-AR signal pathways therapies in Pten/Trp53-deficeint CRPC to compensatory pathway activation and immune suppression. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazuko Sakai, Kazutoshi Fujita, Eri Banno, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Therapeutic resistance to anti-AR signal pathway therapies is related to compensatory pathway activation and immune suppression in mouse Pten/Trp53-deficient CRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5211.

Curcumin is a naturally occurring compound found in turmeric and is well-known for its anticancer and anti-inflammatory properties, however, low solubility, poor absorption and fast clearance resulting in low systemic bioavailability has hindered therapeutic applications for free curcumin and has prompted the development of curcumin-based formulations with improved bioavailability. Curcumin monoglucuronide (CMG) is a water-soluble injectable prodrug that shows improved bioavailability over oral free curcumin and has demonstrated anticancer activity. In this study, we examine the therapeutic efficacy and immunomodulatory effects of CMG in mouse prostate cancer models driven by the conditional inactivation of Pten. The acute immunomodulatory effects of CMG were profiled in tumors from 28-week-old conditional Pten/Trp53 double knockout mice one-week after intraperitoneal dosing with CMG and were evaluated using a focused panel of qRT-PCR-based immune related genes, flow cytometry and immunohistochemistry (IHC). Overall, mice receiving CMG showed a higher inflammation profile compared to vehicle controls and was characterized with enrichments in macrophage, dendritic cell, and T cell gene signatures. CMG treated mice showed enhanced dendritic cell activity in tumors including an increased abundance of activated of migratory dendritic cells (CD11b+\CD11c+\XCR1+\MHC-II+\CD80+). Tumors from CMG-treated mice also contained increased T cell infiltration (including CD8+ and CD4+ T cells) as well as enrichment of genes related to T cell differentiation and activation and an increased abundance of CD4+\CD25+ regulatory T cells. While one week of dosing did not result in a significant reduction of tumor burden for CMG-treated mice, there was a 1.9-fold increase in cancer cell apoptosis as measures by cleaved caspase-3 IHC expression (P=0.004) and was correlated to leukocyte infiltration particularly in the ventral lobes of the prostate. The antitumor efficacy and long-term immunomodulatory effects of CMG were further evaluated in 16-week-old conditional Pten knockout after four weeks of dosing. In this model, tumor from mice treated with CMG showed a 10% reduction of tumor burden compared to vehicle controls (P=0.065). Tumors from CMG treated mice also had a 1.7-fold increase in apoptosis compared to vehicle treated mice (P=0.009) and tended to have reduced cancer cell proliferation compared to vehicle controls (P=0.148). Gene expression and flow cytometry analyses showed that tumors from mice treated with CMG for four weeks maintained improved tumor inflammation and showed an increased T cell infiltration over vehicle treated controls including effector cytotoxic T cells (CD8+\Granzyme B+). These findings provide preclinical evidence for the antitumor and immune modulatory activity of CMG in mouse hormone sensitive Pten-deficient prostate cancer. Citation Format: Yurie Kura, Marco A. De Velasco, Naomi Ando, Kazuko Sakai, Kazutoshi Fujita, Eri Banno, Yoshihiko Fujita, Mamoru Hashimoto, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Curcumin monoglucuronide modulates the tumor microenvironment of Pten-null prostate tumors and exhibits antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5952.

Androgen deprivation therapy (ADT) remains the standard care for men with advanced prostate cancer. Although initially effective, many patients fail to maintain durable responses and progress to lethal castration-resistant prostate cancer. Commensal bacteria play important role in maintaining homeostasis and proper immune function. However, ADT alters gut bacterial compositions and the ramifications being investigated. In this study, we used a mouse model of Pten-deficient prostate cancer to characterize differences in bacterial composition of gut bacteria in mice with castration naïve-prostate cancer (CNPC) and gut microbiota from mice three and six weeks after treatment ADT via surgical castration representing castration-sensitive prostate cancer (CSPC) and CRPC, respectively. Fecal samples were collected and processed for 16s rRNA amplicon sequencing. ADT resulted in significant changes to biological diversity and local diversity was highest in CSPC (Shannon index, P=0.04). Community composition also differed significantly different between the groups (Adonis OTU Bray-Curtis, P=0.001, NMDS stress=0.07). Despite dissimilarities between bacterial communities among the groups, CNPC and CRPC shared a greater similarity. Taxa associated with CNPC included Unclassified S247, Prevotella L. reuteri, unclassified Rikenellaceae and unclassified Bacteroidales; taxa associated with CSPC included R. gnavus, Dorea, unclassified Desulfovibrionaceae, Dehalobacterium, A. muciniphilia, Bifidobacterium, and B. producta; and taxa associated with CRPC included Turicibacter, R. flavefaciens, L. garvieae, unclassified Peptostreptococcaceae and unclassified Bacilli. Mice with CSPC and CRPC had an increased abundance genes associated with of catabolic metabolic pathways associated with aromatic compound, steroid Benzoate, and toluene degradation as well as genes associated to pathways corresponding to methane metabolism, phosphonate and phosphinate metabolism, and nitrogen metabolism. Genes enriched in fecal taxa from CNPC and CRPC mice were associated with folate biosynthesis, galactose metabolism, amino sugar and nucleotide sugar metabolism, and carbon fixation pathways in prokaryotes. This study provides preclinical evidence to support the association between the gut microbiome and prostate cancer phenotypes associated with sensitivity to ADT. Citation Format: Chisato Wakamori, Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Naomi Ando, Kazutoshi Fujita, Eri Banno, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuto Nishio, Hirotsugu Uemura. Transformation of the gut microbiome in response to androgen deprivation and the transition to castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5637.

Extracellular adenosine is an immunosuppressive molecule that is generated from ATP by the CD39 and CD73 ectoenzymes and exerts its antitumor effects through its interaction with the adenosine 2A receptor (A2AR) on immune cells. Approaches to curtail the immunosuppressive effects of extracellular adenosine primarily target CD73 and A2aR. In this study, we examined the immunomodulatory effects and antitumor effects of CD73 and A2aR blockade in the context of PTEN-deficient prostate cancer. The immunomodulatory effects of anti-CD73 antibody blockade (aCD73) or A2aR inhibition with the small molecule inhibitor AZD4635 were characterized in transgenic mice bearing Pten-null prostate tumors after one week of dosing. qPCR-based tumor immune profiling revealed distinct differences between the treatment groups that were characterized by the enrichment of gene signatures related to T cell, natural killer cell, and NK T cell in AZD4635 treated mice and macrophage and B cell in mice treated with aCD73. Immunohistochemical analysis showed significant 2.5- and 4.6-fold increases of granzyme B positive immune cells in the cancer glands of aCD73 and AZD4635 treated mice, respectively. Efficacy studies showed that tumors from mice after long-term treatment with AZD4635 were characterized with high adenosinergic signatures and particularly increased levels of Nt5e, the gene encoding CD73. Treatment with aCD73 and AZD4635 alone or in combination led to tumor burden reductions of 4.4%, 15.1% and 16.7%, respectively, after four weeks of treatment and -8.58%, 4.6% and 29.5%, respectively after eight weeks of treatment. Additional analyses showed that androgen deprivation therapy induced an adenosine high signature, and increased Nt5e expression levels and CD73-positive infiltrating MDSCs/neutrophils. The efficacy of AZD4635 versus aCD73 plus AZD4635 was evaluated in an in vivo castration-sensitive model of Pten-null prostate cancer treated with apalutamide plus androgen deprivation therapy via surgical castration. In this model, mice were treated for eight weeks to allow for the progression to castration-resistant prostate cancer (CRPC) and in this setting, the treatment combination of aCD73/AZD4635 improved tumor burden reduction to 34.3% from 23.1% in AZD4635 treated mice. Moreover 75% (6/8) of mice treated with aCD73/AZD4635 had tumor burden reductions greater than 30% relative to vehicle treated controls versus 28.6% (2/7) of mice in the AZD4635 only group. Our findings show that extracellular adenosine is a key immunosuppressive molecule in PTEN-deficient prostate cancer and provide evidence that implicates adenosinergic signaling via CD73 as a contributing factor to decreased efficacy of A2aR blockade and progression to CRPC. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazuko Sakai, Alwin Schuller, Kris Sachsenmeier, Kazutoshi Fujita, Eri Banno, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Targeting CD73 augments the efficacy of A2aR blockade in PTEN-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5604.

Extracellular adenosine impairs immune function in tumors and limits the efficacy of anti-CTAL4 immune checkpoint blockade. We previously showed that adenosine 2A receptor (A2aR) blockade with AZD4635 moderately inhibited tumor growth, in part by improving antigen presentation and cytotoxic T cell (CTL) activity in mouse Pten-deficient prostate cancer. Here, we continue to investigate the antitumor activity of AZD4635 and its effects when combined with CTLA4 blockade (aCTLA4) in preclinical models of Pten-deficient prostate cancer. Prostate tumors from Pten conditional knockout (KO) mice treated with aCTLA4 for five days had increased expression levels of adenosinergic genes. Furthermore, gene expression, flow cytometric and IHC analyses showed increased T regulatory cell differentiation and accumulation that was offset with the addition of AZD4635. Moreover, expression levels of genes associated with Th 1 cell differentiation and T cell-mediated cytotoxicity were increased in AZD4635/aCTLA4 treated tumors. Adding AZD4635 to a four-week regimen of aCTLA4 therapy improved the antitumor response by two-fold in an early-stage intervention model of Pten-deficient prostate cancer. Androgen deprivation (AD) has the potential to promote T cell infiltration in prostate tumors and using the AZD4635/aCTLA4 treatment combination as neoadjuvant therapy to AD via surgical castration led to a greater antitumor response. The antitumor activity of this treatment combination was further examined and confirmed in a subcutaneous syngeneic genome-derived allograft model using tumor-bearing conditional Pten-deficient knockout mice grafted with Pten-deficient CRPC tumor fragments. In a Pten/Trp53 conditional double knockout (DKO) mouse model of advanced prostate cancer, the AZD4635/aCTLA4 treatment combination did not improve overall survival rates. However, when AZD4635/aCTLA4 was used as neoadjuvant therapy to AR inhibition with the anti-androgen apalutamide, it improved median survival time from 21 days in monotherapy treated mice to 34 days in AZD4635/aCTLA4 treated mice. These results provide preclinical evidence to support the rational combination of A2AR blockade with AZD4635 and aCTLA4 immune checkpoint inhibition for PTEN-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Alwin Schuller, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. A2aR inhibition enhances the antitumor activity of CTLA4 blockade in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1568.

The gut microbiome is now known to influence host immune function, malignancies, and response to therapy. We previously showed that sequencing PD-L1/JAK1/2 blockade prior to androgen deprivation therapy (ADT) could promote immune activity to potentiate antitumor responses in mouse Pten-deficient prostate cancer. Here, we investigate the associations between the treatment pairings of PD-L1 antibody (aPD-L1, clone D265A, mouse/IgG1 kappa) blockade and the JAK1/2 inhibitor AZD1480 with ADT in mouse Pten-deficient prostate cancer and fecal gut microbiomes. We used 16S rRNA amplicon sequencing to survey fecal samples of tumor bearing conditional Pten-knockout (KO) mice treated for four weeks with aPD-L1 and AZD14870 alone and as combination therapy in intact mice, and as concurrent (Conc) therapy with ADT (via surgical castration), or sequenced as adjuvant (Adj) or neoadjuvant (NeoAdj) therapy. In this model, PD-L1 blockade was ineffective as monotherapy in all treatment settings and as combination therapy in intact and Conc combination settings and hyperprogression was observed in some mice treated with aPD-L1 monotherapy in intact and Adj settings. In the NeoAdj setting, aPDL1/AZD1480 therapy demonstrated superiority over monotherapy. Significant differences in microbial composition were observed between groups (P Citation Format: Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Naomi Ando, Noriko Sako, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Associations between gut microbiota and PD-L1 immunotherapy/JAK1/2 inhibition in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1780.

The androgen receptor (AR) is a major therapeutic target for human prostate cancer and therapies targeting the AR pathway also impact local and systemic immunity. However, the influences over host immune function vary depending on the type of AR treatment. Androgen deprivation (AD) can improve immune cell infiltration by enhancing T cell trafficking, but it also attracts immunosuppressive myeloid cells and regulatory T cells. We previously showed that chronic dosing of apalutamide, an oral nonsteroidal AR antagonist, was effective in suppressing tumor growth in a preclinical mouse models of Pten-deficient prostate cancer and also changed the composition of the tumor immune microenvironment improving cytotoxic T cell activity. Moreover, we also showed that using AD as neoadjuvant therapy improved the antitumor activity and immune responses of combined JAK/PD-L1 blockade, whereas administered as concurrent or adjuvant therapy was ineffective. Our aim in this study is to determine the potential of using apalutamide in as combination therapy with immunotherapy and immune modulating agents. For this we have characterized the acute immune modulatory activity of apalutamide on mouse Pten-deficient and compared its activity against that of AD. Conditional prostate specific Pten/Trp53 double knockout mice were orchidectomized or treated with apalutamide (30 mg/kg/D) for five days. Tumor samples and lymphoid organs were collected and processed for immune profiling using a qRT-PCR focused panel, flow cytometry and/or immunohistochemistry. Prostates from orchidectomized mice tended to be smaller however there was no decrease in size for apalutamide treated mice. Notably, thymuses were significantly enlarged in both orchidectomized and apalutamide treated mice compared to control. Genes related to antigen presenting/dendritic cell (APC/DC) and activated DC were greater in apalutamide treated mice compared to vehicle treated or surgically castrated mice. This finding was consistent with an increased abundance of MHC-IIhi/CD80+ migratory DCs (CD11c+/XCR1+) in tumors of apalutamide treated mice. Tumor associated macrophage infiltration was also greater in apalutamide treated mice. Granulocyte infiltration was significantly greater after both castration and apalutamide therapy however the proportion of mature granulocytes was higher in castrated mice. T cell infiltration was comparable between orchidectomized and apalutamide treated mice, and regulatory T cell activity was increased in both groups compared to controls, but to a higher degree in orchidectomized mice. In summary, this provides critical data that will be essential when developing rational combinations with additional immunotherapies and immunomodulatory agents. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Kazuko Sakai, Nobutaka Shimizu, Eri Banno, Masahiro Nozawa, Kazuhiro Fujita, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Acute immune responses to apalutamide in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1438.

Prophylaxis using pegfilgrastim is recommended to prevent cabazitaxel-induced neutropenia. We observed GradeB3 neutropenia in a patient after administration of cabazitaxel, despite prophylaxis using pegfilgrastim. To identify the risk factors associated with GradeB3 neutropenia, we retrospectively investigated the records of 10 patients who received prophylaxis with pegfilgrastim after administration of cabazitaxel. They were divided into GradeB3 neutropenia and non-GradeB3 neutropenia groups, and we compared the background data and laboratory values between the 2 groups. A univariate analysis revealed that hypoalbuminemia was significantly observed in patients with cabazitaxel-induced GradeB3 neutropenia. The incidence of GradeB3 neutropenia was significantly high in patients with serum albumin levels3.6 g/dL. Cabazitaxel has a high rate of protein binding; moreover, serum albumin levels3.6 g/dL might be associated with high concentrations of unbound cabazitaxel, and thus an increase in the incidence of GradeB3 neutropenia. Therefore, hypoalbuminemia at the time of administration of cabazitaxel may be a risk factor related to the development of GradeB3 neutropenia.

Inactivation of PTEN occurs frequently in advanced human prostate cancer and can promote an immunosuppressive tumor microenvironment (TME). Extracellular adenosine can be produced by tumors which can in turn modulate anti-inflammatory and immunosuppressive effects via interactions with the A2a receptor (A2aR). Here, we used a transgenic mouse model of Pten-deficient prostate cancer to characterize adenosine-mediated immunosuppression in the TME and evaluate the effects of A2aR blockade. Transcriptomic analysis of normal wildtype prostate and Pten-deficient prostate tumors revealed enriched signatures for genes downregulated after a A2aR knockdown or upregulated after A2aR stimulation. Moreover, androgen withdrawal led to increased expression of the A2aR-stimulated responsive genes Cxcl2/3/5, Il1b, S100a8, Ptgs2 and Thbs1. Immunohistochemical staining of CD73, the ecto-enzyme responsible for producing extracellular adenosine, showed heterogeneous staining ranging from negative to focally strong in epithelial cancer cells and was strongly expressed in immune cells. A2aR was also strongly expressed on immune infiltrates. We used the oral A2aR antagonist AZD4635 to determine the immunosuppressive activity of extracellular adenosine and evaluate its antitumor activity in the context of castration-naïve (CNPC), castration-sensitive (CSPC) and castration-resistant (CRPC) prostate cancer. Four weeks of treatment with AZD4635 led to a 12.1% (P=0.140) reduction of tumor burden compared to control mice. Notably a subset of these mice (4/8) had tumor reductions greater than 20%. For the CSPC/CRPC models, mice were orchidectomized and treated with apalutamide with or without AZD4635 for four or eight weeks to represent CSPC or CRPC phenotypes, respectively. In these settings, treatment with AZD4635 improved the reduction of tumor burden by 16.4% (P=0.165) and 16.8% (P=0.123) in CSPC and CRPC, respectively. Despite similar reduction rates between the cohorts, a greater proportion of AZD4635 treated mice were below the median distribution in the CRPC setting compared to the CSPC setting, 50% (4/8) vs 70% (7/10), respectively. Immunophenotyping showed an increase in the abundance of dendritic cells in tumors and tumor draining lymph nodes of AZD4635-treated mice in the CPNC setting. Whereas a shift in M2 to M1 macrophage, decrease of PD1hi CD8 T cells and an increase in genes related to cytotoxic lymphocyte activity (Gzma, Gzmb, Prf1, Klrg1, and B3gat1) were observed in AZDD4635 treated mice in the CRPC setting. Decreased expressions of immunosuppressive genes related to T regulatory cells (Ctla4, Il2ra, Itga2, Tigit, Foxp3) were noted in both CSPC and CRPC settings. Our findings show that targeting extracellular adenosine with AZD4635 is effective in a subset of mouse Pten-deficient tumors and provides evidence that suggests context-specific immune modulating activity by extracellular adenosine in prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Alwin Schuller, Kazuto Nishio, Hirotsugu Uemura. Targeting A2aR in mouse Pten-deficient prostate cancer [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 1071.

We previously used a transgenic mouse model of prostate cancer to show that TAS-115, a multi-kinase inhibitor that targets c-MET, VEGF-R, and CSF1-R signal pathways, effectively reduced growth in a subset of prostate tumors. In this setting, TAS-115 decreased tumor burden by reducing tumor neovascularization and promoting anti-tumor immune activity, however, this phenomenon was not observed when co-administered with androgen deprivation therapy (ADT) which is the primary treatment option for men with advanced prostate cancer. We have also shown that androgen withdrawal can promote T cell infiltration, however, antitumor immune responses were hampered most likely by the increased prevalence of immune suppressive cells consisting of tumor associated macrophages (TAMs), myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) induced after ADT. Our previous findings revealed that TAS-115 was able to suppress M2 polarized macrophages and promote the recruitment and activity of tumoricidal neutrophils in castration-naïve tumors. We surmised that pretreatment with TAS-115 could potentiate antitumor immunity of ADT-induced inflammation by decreasing immune suppressive cell activity. In this follow-up study, we demonstrate that one-week treatment with TAS-115 modified the composition of immune cells in intact prostate tumors from Pten/Trp53-deficient conditional knockout mice. Immunophenotyping studies showed that mice treated with TAS-115 had reduced levels of tumor and circulating monocytic-MDSCs (m-MDSC, CD11b+/Ly6C+) and TAMs. Expression levels of genes associated with m-MDSCs and M2 polarized macrophages (Arg1, Csf1r, Il10, Nos2, Adgre1, CD163, Cd68 and Tnf) were also decreased in TAS-115-treated mice. Increases of total and CD355+(CRTAM) CD8 T cells indicating enhanced recruitment and activation were observed in both tumors and blood of TAS-115 treated mice. Sequencing TAS-115 with or without surgical castration was evaluated in transgenic mice bearing Pten/Trp53-deificent prostate tumors. Mice treated for four weeks with TAS-115/ADT sequential therapy showed a 13.5% (P=0.0836) reduction of tumor burden compared to vehicle-treated controls. Notably, mice treated with TAS-115/ADT sequential therapy were characterized with a decrease of monocytic and granulocytic MDSCs in tumors. Moreover, neutrophil mediated antitumor activity was increased in TAS-115 treated mice. We also use phenotyping, gene expression and immunohistochemical studies to show that reducing immunosuppressive immune cells led to increased CD8 T cell recruitment and activation. These findings provide evidence to support that TAS-115 sequenced with ADT has the potential to augment innate and adaptive antitumor immunity in prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. The multi tyrosine kinase inhibitor TAS-115 promotes innate and adaptive immune responses of androgen deprivation therapy in mouse prostate cancer [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 5610.

Commensal gut bacteria are essential for maintaining intestinal homeostasis, however, aging, environmental factors and pathological conditions can cause changes in microbial composition resulting in dysbiosis. Specific microbial communities can alter host immune functions and are implicated in promoting carcinogenesis, tumor progression and therapeutic resistance. In this study we use a genetically engineered mouse model of prostate cancer to characterize changes in gut microbiota. 16S marker sequencing data was used to perform a comprehensive profile of the fecal microbiomes of conditional Pten-knockout mice harboring prostate tumors versus wildtype mice with normal prostate. Unsupervised clustering of microbiota was performed by hierarchical clustering. Community composition (beta diversity) was determine by principal components analysis, principal coordinates analysis (PCoA) and Adonis (permutational manova (PERMANOVA)) and the Shannon index was used to determine alpha diversity. The linear discriminant analysis effect size method was used to identify features associated with cancer and multiple linear regression was performed to determine relevant features of fecal bacteria collected from the proximal and distal colon. Predictive biomarkers associated with prostate cancer were identified with the Wilcoxon rank test. Weighted correlation network analysis (WGCNA) was performed to identify clusters of taxa associated with the presence of prostate cancer. Taxon set enrichment analysis (TSEA) was carried out to identify taxon sets associated with host genetic variations, and host intrinsic and extrinsic factors. Overall, the presence of prostate cancer did not affect diversity, however, significant compositional differences were observed between the cohorts (Bray-Curtis Adonis, P=0.003). At the family level S24_7, Odoribacteraceae, and Peptococcaceae were associated with cancer bearing mice while Bifidobacteriaceae, Peptostreptococcaceae, Coriobacteriaceae, Ruminococcaceae and Erysipelotrichaceae were associated with healthy mice (P Citation Format: Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Eri Banno, Naomi Ando, Noriko Sako, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Prostate cancer alters gut microbiota in mice [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 3340.

Modulation of immune response by gut microbiota is believed to play a key role in determining response to anticancer therapy. Conversely, anticancer therapies have the potential to alter resident microbiota and the implications are unknown. We previously showed the antitumor efficacy of targeted JAK1/2 inhibition in a preclinical mouse model of prostate cancer and are now exploring its effectiveness as an immune modulator in prostate cancer therapy. We have also shown that androgen withdrawal can turn a poorly inflamed tumor into an inflamed tumor. In this follow-up study, we examine the effects of targeted JAK1/2 cancer therapy on the gut microbiome. For this we utilize 16S rDNA amplicon sequencing to profile the fecal microbiota (proximal and distal colon) of normal wildtype (WT) and tumor bearing conditional Pten-knockout (KO) mice treated for four weeks with the JAK1/2 inhibitor AZD1480 alone or in combination with orchidectomy. Mice treated with AZD1480 had lower tumor burden compared to control mice. Alpha diversity of fecal microbiota was greater in castrated KO mice compared intact WT and KO mice and mice receiving AZD1480 tended to have lower diversity regardless of castration status. Significant compositional differences were observed between the colonic microbiota of all experimental groups. Overall, mice treated with AZD1480, regardless of castration status resulted in decreased abundance of Clostridiaceae, F16, Gemellaceae, Odoribacteraceae, Rikenellaceae, and Lactobacillaceae and an increase of Paraprevotellaceae, Porphyromonadaceae, Verrucomicrobiaceae, Bifidobacteriaceae bacterial families. In the castration-naïve prostate cancer setting, enrichments of Akkermansia, Coprobacillus, Prevotella and Dehalobacterium and decreases of Lactococcus, Bacilli, Gemella, Lactobacillus, Desulfovibrio, and Odoribacter. Bifidobacterium, Desulfovibrio, Allobaculum, and Lactococcus were enriched in androgen deprived AZD1480-treated mice while Ruminococcus, and unclassified Clostridiaceae and F16 were decreased. We further used weighted correlation network analysis (WGCNA) to identify relevant networks of fecal microbiota associated with systemic targeted JAK1/2 inhibition. Using this approach, we have identified clusters of highly correlated taxa in the context of poorly inflamed and inflamed prostate tumors. Our findings show that systemic anticancer therapies can modify host microbiomes and provides insights into interactions between tumor, host microbiomes and immune modulating cancer therapies. Citation Format: Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Naomi Ando, Noriko Sako, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Systemic targeted JAK1/2 therapy for mouse Pten-deficient prostate cancer model influences the diversity and composition of the gut microbiome [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 3341.

Recently, the tumor suppressor PTEN has been shown to function as an immune modulator and its inactivation is associated with mediating tumor immune evasion. We previously examined the effects of programmed death-ligand 1 (PD-L1) immune checkpoint blockade (ICB) in transgenic mouse models of Pten-null prostate cancer. However, anti-PD-L1 monotherapy was largely ineffective in immunologically cold castration-naïve tumors from intact mice but showed some activity when administered to castrated mice. Our previous immune profiling studies also revealed that while androgen withdrawal increased tumor inflammation, it also mediated the recruitment and accumulation of immunosuppressive cells such as regulatory T cells, M2-polarized macrophages and myeloid-derived suppressor cells (MDSCs). Interleukin-6 (IL-6)/signal transducer and activator of transcription-3 (STAT3) signaling is a key immune modulating pathway that is upregulated in both intact and androgen deprived tumors. Here, we evaluate the effectiveness of targeting PD-L1 and JAK/STAT signaling in preclinical models of Pten-null prostate cancer. Intact tumor bearing conditional Pten-knockout were treated for four weeks with an a-PD-L1 blocking antibody (clone D265A, mouse/IgG1 kappa) and/or the JAK1/2 inhibitor, AZD1480. Pharmacological treatments targeting PD-L1 and JAK1/2 (PJ) combinations were also evaluated as concurrent therapy with androgen deprivation (AD) by orchidectomy (PJ+AD), and as sequential therapy; two weeks AD followed by PJ (AD>PJ), and one week of PT followed by AD (PJ>AD). Combined PD-L1/JAK followed by AD was the only treatment combination that improved antitumor immune responses over monotherapy. Notably, flow cytometry studies showed that combined PD-L1/JAK potently abrogated PD-L1 expression in circulating dendritic cells in all settings. Evidence for enhanced antitumor immune response in PJ>AD was supported by increased numbers circulating effector memory CD8 T cells and CD355+CD8+ T cells (CD355 Class I MHC-restricted T cell-associated molecule (CRTAM) a marker for activated CD8 T cell), and increased CD8 T cell infiltration in tumors and a reduction of CD25+CD4+ regulatory T cells. The efficacy of the PJ>AD treatment combination was further tested and confirmed in a subcutaneous syngeneic allograft model using mice grafted with tumor blocks from Pten-deficient castration-resistant prostate cancer tumors. Together these results indicate that pretreatment with combined PD-L1/JAK blockade can decrease the immunosuppressive effects of androgen withdrawal and have the potential to restore antitumor immune activity in Pten-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Androgen deprivation following JAK1/2 and PD-L1 inhibition improves antitumor efficacy in mouse models of Pten-deficient prostate cancer [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 3416.

Hypomagnesemia caused by panitumumab can often lead to severe adverse effects, such as arrhythmia. However, the risk factors are still controversial. To clarify the risk factors and time to onset of panitumumab-induced hypomagnesemia, we retrospectively investigated the records of 30 patients who had received panitumumab. They were divided into Grade B2 hypomagnesemia and non-Grade B2 hypomagnesemia groups, according to their serum magnesium levels, and we compared patients' backgrounds, laboratory values, and concomitant drugs between the 2 groups. Univariate analysis revealed that hypocalcemia and non-administration of an oral magnesium preparation were significantly associated with Grade B2 hypomagnesemia induced by panitumumab. Incipient grade 1 hypocalcemia was observed after incipient Grade 1 hypomagnesemia in both groups. The occurrence of these complications was significant in the Grade B2 hypomagnesemia group. Thereafter, in all patients of the Grade B2 hypomagnesemia group that exhibited both complications, hypomagnesemia developed to Grade 2 or higher. The development of Grade 3 and Grade 4 hypomagnesemia without the development of Grade 2 hypomagnesemia was observed in 2 patients each. Thus, aggravation of hypomagnesemia can be expected upon the administration of panitumumab, and therefore, not only serum magnesium levels, but also serum calcium levels need to be monitored.

For architect engineers it is very important to know geometric surfaces in order to use them in the design of buildings. Architectural objects, other than functional, should also satisfy the aesthetic criteria which is achieved through design. The existence of different software packages for modeling like Rhinoceros, with additional programs (Grasshopper), allows doing the rational design and construction of these architectural objects. All these facts were noticed by the authors of this paper, which was further followed by giving the students of Faculty of Civil Engineering and Architecture in Nis, an elective subject Geometric surfaces in architecture in the school year of 2009/10. The content of this subject includes study of all geometric surfaces in architecture practice. The importance of studying Geometric surfaces for students of architecture was noticed by the Faculty of Engineering and Design, Hosei University in Tokyo, Japan, which resulted with offering this elective subject to the students in the school year of 2017/18. The students of both faculties have learned to recognize geometric surfaces and how to apply them in complex geometric forms with attractive appearance. In this paper, the work on both faculties will be shown as they are different, the results will be compared, similarities and differences of working on two different continents. With proper analysis it is possible to make a model of study for this subject in different educational systems.

This paper is a study of the spatial representations depicted in the films of the Japanese filmmaker Shinji Somai (1948–2001), who often used long takes. This study examines the characteristics of the long takes in his works. On the one hand, the spaces are rigorously constructed in order to integrate several elements into the scenes, but at the same time, spatial complexities and contradictions, which may be regarded as deconstructed spaces, are considered to appear. Moreover, the analogy between the spaces in films and architecture is discussed.

Epidemiological data has shown that dietary practices can greatly influence cancer rates. Men in East Asian countries, men have significantly lower prostate cancer rates compared to their counterparts the US and Europe. Soybeans are a versatile and rich source of protein and its products constitute a rich portion of Asian diets. Recent interest in healthy eating has expanded the consumption of soy products which also provide a rich source of naturally occurring isoflavones and 17β-estradiol. In this study, we used roasted soybean flour (kinako), which contains high levels isoflavones glycosides and estradiol, as dietary soy source to determine the influence of isoflavones rich diets on prostate cancer. Six-week old conditional Pten/Trp53 double knockout mice were randomized and fed plain AIN-93M (Control) diets or a diets supplemented with kinako ad libitum. Concentrations of kinako were adjusted to for daily intakes of aglycone isoflavones (genistein, daidzein, and glycetein) of 400 (LDI) and 800 (HDI) mg. Mice were sacrificed at 16 and 20 weeks (n=6 mice/group) or maintained for survival assessment (n=8 mice/group). Dietary intake of kinako-supplemented diets did not influence the onset of prostatic intraepithelial neoplasia or tumor burden at the early stages. However, tumors from mice fed the HDI diet experienced reduced tumor proliferation rates. Moreover, mice fed LDI and HDI diets showed reduced androgen receptor (AR) protein expression levels as well as mRNA levels for the AR target genes Fkbp5, Nk3x3.1 and Timp4. Interestingly, mice on the LDI diet, but not the HDI, experienced longer times to disease progression (median times 264, 299 and 250 days for Control, LDI and HDI, respectively, P=0.663), tumor doubling (median times 14, 27 and 14 days for Control, LDI and HDI, respectively, P=0.083), cumulative survival (median times 292, 348 and 320 days for Control, LDI and HDI, respectively, P=0.199), and overall survival times (median times 28, 43 and 35 days for Control, LDI and HDI, respectively, P=0.324). The metastatic incidence was 33%, 14% and 14% for Control, LDI and HDI groups, respectively, P=0.631. We also investigated whether dietary intervention with kinako would impact previously stablished tumors. For this we fed kinako supplemented diets to conditional Pten-knockout mice with established tumors but no changes were observed in tumor burden, proliferation, apoptosis and AR activity. Together our data shows that long-term continuous ingestion of a diet rich in isoflavones may be necessary in order suppress tumor growth. Interestingly, this protective effect appears to be lost with high-doses of the dietary isoflavones. Further studies will need to be performed in order to decipher complex dynamic interplay between survival pathways isoflavones chemoprevention. Citation Format: Yasunori Mori, Marco A. De Velasco, Yurie Kura, Eri Banno, Naomi Ando, Noriko Sato, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Dietary isoflavone decreases prostate cancer progression and improves survival in conditional Pten/Trp53-deficient mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1613.

Increasingly, immunotherapeutic agents are making their way into the clinic for the management of cancer, however, not all patients respond to single agent treatments. Susceptibility to immunotherapy is highly dependent on a tumor’s immune composition. Agents that are able modulate the tumor’s microenvironment (TME) to improve the balance between cytotoxic T cells and immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) M2-polarized macrophages and T regulatory cells (Tregs) are highly sought. We previously evaluated the preclinical activity of the multikinase inhibitor TAS-115 on mouse model of prostate cancer and showed that its antitumor effect was due in large part to its influence on the TME. In this study we examine the consequences of TAS-115 administration alone or in combination with androgen withdrawal via surgical castration or anti-androgen therapy with apalutamide, and focus on its influence on antitumor immunity. Pten/Trp53-double knockout mice were treated with TAS-115 alone or in combination with surgical castration or apalutamide for 4 weeks. Overall, TAS-115 reduced prostate tumor burden by 10.5%, however, 37.5% (3/5) of mice experienced no response (7.5%), while reductions of 14% and 27% were observed in 25% (2/8), 37.5% (3/5) of the remaining mice, respectively. A similar trend was noted in mice treated with the TAS/apalutamide combination but TAS plus surgical castration. Changes in cancer cell proliferation and apoptosis were insignificant with TAS-115 treatment, however, 2 of 4 TAS-115-responsive mice used for immunohistochemical (IHC) analysis showed a ~3-fold increased cleaved caspase-3 expression over non responsive mice. All TAS-115-treated mice showed reduced levels of phosphorylated STAT-3. Flow cytometric analysis of dissociated tumor samples revealed a reduction of tumor associated macrophages (TAMs) particularly when TAS-115 was given in combination with castration or apalutamide. Further analysis by IHC and qRT-PCR showed a M2-M1 polarization switch after TAS-115 therapy accompanied with decreased accumulation of monocytic MDSCs. An increase in tumor infiltrating neutrophils was also observed in mice receiving TAS-115. Although overall T cell infiltration was not improved TAS-115, there was an enhanced of activated CD8+ T cells, which was especially evident in responders. However, Treg infiltration was also increased after the administration of TAS-115. Overall our findings suggest that TAS-115 induced the reduction of immunosuppressive MDSCs and TAMs and may have induced an M2-M1 switch to provide a less immunosuppressive tumor microenvironment resulting in improved T cell mediated responses and provides evidence to support further investigation into using molecular targeting agents such TAS-115 as immune-modulators in combination with other immunotherapies to enhance or restore cytotoxic T cell activity. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sato, Naomi Ando, Kazuko Sakai, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Immunomodulation of the multi-tyrosine kinase inhibitor TAS-115 in a mouse model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5023.

Exciting breakthroughs in tumor immunology have led to the discovery and development of several promising immunotherapeutic strategies for cancer patients. Thus far, immune checkpoint inhibitors have been the most encouraging for solid tumors, however, treatment responses are observed in only a subset of patients and these appear to be primarily dependent on a tumor’s baseline immune profile. Additionally, tumors respond differently to immunotherapy and criteria to assess treatment responses are still being refined. We have identified a set of immune-related genes and developed a scoring system to profile the tumor’s immune status and assess immunological responses. In this study, we used a qRT-PCR-based approach to assess this panel and determine baseline tumor immune-profiles in an immunocompetent mouse model of Pten-null prostate cancer. We also assessed and compared tumor immune responses following androgen withdrawal (via surgical castration) and anti-PD-L1 immune checkpoint blockade. A total of 96 genes were selected for this focused panel which consisted of cell-type, immuno-responsive and housekeeping control genes. Core modules were designated based on functional gene associations which included antigen presentation, tumor inflammation, effector cells, immunomodulatory cells, immunosuppressive signaling and immune checkpoints. Core modules were further subdivided in to appropriately relevant sub-modules. A score for each submodule was calculated and a weighed score was then assigned for each core module. Tumor immunophenotypes based on core and sub-core immune scores (IS) were corroborated with immunohistochemical and/or flow cytometric analyses. Mouse castration-naïve prostate tumors exhibited low scores indicating a ‘”cold tumor” phenotype with little variation between individual mice. Androgen withdrawal induced cancer cell death in these tumors that in turn promoted immune cell infiltration. Core and sub-core IS of individual mice identified varied signatures reminiscent of immune excluded and inflamed “hot tumor” phenotypes. Short-term treatment with anti-PD-L1 blockade (clone D265A, mouse/IgG1 kappa) elicited an increased immune response signature in tumors from surgically castrated mice, but not in tumors from intact mice. Notably, discernable differences were noted among individual responders supporting the notion that responses to immune checkpoint may indeed be dependent on baseline tumor immune-profiles. In summary, androgen withdrawal appears to promote a “hotter” immunological phenotype in this model of prostate cancer, that is increased further by PD-L1 inhibition. Moreover, this investigation provides proof of concept evidence for a qRT-PCR-based biomarker approach for immuno-oncology. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sato, Naomi Ando, Kazuko Sakai, Yasunori Mori, Barry R. Davies, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. A real-time PCR-based approach to quantitatively assess tumor immune profiles and immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4996.

Malakoplakia is a rare acquired disease that can affect many systems but is more common in the urogenital tract. Cutaneous malakoplakia is even rarer. It is far more frequent in immunodeficient patients. We report a case of cutaneous malakoplakia in a kidney transplant patient who had recently stopped receiving immunosuppressive therapy to illustrate a review of the relevant recent literature.

Metastatic breast cancer (MBC) is generally incurable. However, 10-20-year relapse-free survival of MBC is approximately 2%, implying that at least a small subset of MBC patients achieve prolonged survival. We therefore analyzed long-term outcome in a particular subset, i.e., oligometastatic breast cancer (OMBC).Data of OMBC subjects (N = 75) treated in our institution from April 1980 to March 2010 were retrospectively analyzed. OMBC was identified as: one or 2 organs involved with metastatic lesions (excluding the primary lesion resectable by surgery), fewer than 5 lesions per metastasized organ, and lesion diameter less than 5 cm. Patients were generally treated with systemic chemotherapy first, and those who achieved complete response (CR) or partial response (PR) were further treated, if applicable, with local therapy (surgical or radiation therapy) to maintain CR or to induce no evidence of clinical disease (NED), with additional systemic therapy.Median follow-up duration was 103 (6-329) months. Single or 2 organs were involved in, respectively, 44 (59%) and 31 (41%) cases with metastatic lesions, 48% of which were visceral. In cases where effects of systemic therapy, possibly in combination with other treatments, were evaluated (N = 68), CR or PR was achieved in 33 (48.5%) or 32 (47.1%), respectively, with overall response rate (ORR: CR + PR) of 95.6% (N = 65). In cases receiving multidisciplinary treatment (N = 75), CR or NED (CR/NED), or PR was induced in 48 (64.0%) or 23 (30.7%) cases, respectively, with ORR (CR/NED + PR) of 94.7% (N = 71). CR rates (60.5%) with systemic therapy and CR/NED rates (79.5%) with multidisciplinary treatment were significantly better in subjects with a single involved organ than in those with two involved organs (P = 0.047 and 0.002, systemic only or multidisciplinary treatments, respectively). Medians estimated by Kaplan-Meier method were: overall survival (OS) of 185.0 months and relapse-free interval (RFI) of 48.0 months. Estimated outcomes were: OS rates (OSR) of 59.2% at 10 years and 34.1% at 20 years, and relapse-free rates (RFR) of 27.4% at 10 years and 20 years. No disease progression was observed after 101.0 months as RFR. Cases with single organ involvement (N = 44) showed significantly better outcomes (OSR of 73% at 10 years and 52% at 20 years, RFR of 42% at 10 years and 20 years). Those who received local therapies (N = 35) also showed better prognosis: OSR of 82% at 10 years and 53% at 20 years, RFR of 38% at 10 years and 20 years. Three cases (4%) survived for their lifetime without relapse after achieving CR or NED, our definition of clinical cure. Multivariate analysis revealed factors favoring better prognosis as: none for OS, and single organ involvement with metastasis, administration of local treatment, and shorter disease-free interval (DFI) (P = 0.030, 0.039, and 0.042, respectively) for RFR. Outcomes in OMBC in literature were OSR of 35-73% at 10 years and 26-52% at 20 years, and RFR of 27-42% at 10 years and 26-42% at 20 years.The present analyses clearly indicate that OMBC is a distinct subgroup with long-term prognosis superior to MBC, with reasonable provability for clinical cure. Further prospective studies to better characterize OMBC are warranted to improve prognosis in MBC.