TRAMP-C2 Cells Research Articles

Microenvironment M1/M2 macrophages and tumoral progression vary within C57BL/6 mice from same substrain in prostate cancer model

Cancer mice models are critical for immune-oncology research; they provide conditions to explore tumor immunoenviroment aiming to advance knowledge and treatment development. Often, research groups breed their own mice colonies. To assess the effect of C57BL/6 mice breeding nuclei in prostate cancer development and intratumoral macrophage populations, an isotransplantation experiment was performed. C57BL/6J mice from two breeding nuclei (nA and nB) were employed for prostate adenocarcinoma TRAMP-C1 cell implantation; tumor growth period and intratumoral macrophage profile were measured. BL/6nB mice (54%) showed tumor implantation after 69-day growth period while BL/6nA implantation reached 100% across tumor growth period (28 days). No difference in total macrophage populations was observed between groups within several tumoral regions; significantly higher M2 macrophage profile was observed in tumor microenvironments from both mice groups. Nevertheless, BL/6nB tumors showed around twice the population of M1 profile (11–27%) than BL6nA (4–15%) and less non-polarized macrophages. The M1:M2 average ratio was 1:8 for group A and 1:4 for B. Our results demonstrate different tumor progression and intratumoral macrophage populations among mice from the same substrain. Data obtained in this study shows the relevance of animal source renewal for better control of murine cancer model variables.

Chlorin spiro-Tröger's base as a prospective photosensitizer for photodynamic therapy of cancer

Octahydroxy bis-porphyrin Tröger's base (TB) and octahydroxy porphyrin-chlorin spiro-Tröger's base (spiroTB) derivatives were successfully prepared from their Ni(II) complexes utilizing an optimized demetalation method using H2SO4-TFA followed by demethylation via BBr3. The synthesized TB and spiroTB were then in in vitro experiments for their utility in photodynamic therapy (PDT) of cancer. Compared to temoporfin (mTHPC, Foscan®), the chlorin derivative used in PDT of cancer, TB and spiroTB were localized in lysosomes instead of the endoplasmic reticulum and were found to possess enhanced biocompatibility. Cell culture studies were performed on TRAMP-C2 (prostate cancer), HeLa (cervical cancer), and MRC-5 (non cancerous) cell lines. Both TB and spiroTB displayed negligible dark toxicity but increased production of reactive oxygen species (ROS) when illuminated. In addition, spiroTB displayed significant phototoxicity in TRAMP-C2 cells (IC50, light 0.7 μM after 24 h incubation). Despite the phototoxicity of spiroTB being lower than that of mTHPC (IC50, light 0.02 μM), due to the low dark toxicity (IC50, dark > 100 μM), the therapeutic factor of spiroTB (>150) is six times higher than that of mTHPC (25).

Abstract 1353: Potentiating salvage radiotherapy in radiorecurrent prostate cancer through anti-CTLA4 therapy: Implications from a syngeneic model

Abstract Background: Advanced prostate cancer (PCa) is a leading cause in cancer death and can elicit significant morbidity and mortality. A common treatment modality for advanced PCa is radiation therapy (RT). Currently, salvage of local disease recurrence after RT is a major clinical problem. Emerging evidence indicates the importance of the immune system in governing RT response. In support of this, immune checkpoint inhibitors (ICIs), which enhance immune activation, have demonstrated clinical therapeutic promise in combination with RT in certain advanced cancers. Irradiation (IR) combined with ICIs may prime the immune system to recognize and target recurrent cancer. Purpose: Investigate the therapeutic efficacy of ICIs in combination with RT for radiorecurrent PCa in a syngeneic pre-clinical model. Methods: TRAMP-C2 cells were treated with 10 Gy of radiation over 5 fractions (similar to clinical hypofractionated (HF) schedule) to generate TRAMP-C2 HF cells. Immune-competent mice were transplanted subcutaneously with TRAMP-C2 HF cells. Once tumors reached palpable size, mice were administered either ICIs (anti-PDL1 or anti-CTLA4) alone or in combination with RT. Tumor volume was monitored to determine the treatment effect. Correlative studies on excised tumors and secondary lymphoid organs included flow cytometry and NanoString gene expression panel to evaluate immunological mechanisms that contribute to anti-tumor effects. Results: TRAMP-C2 HF cells were validated for radiation resistance and exhibited a more aggressive phenotype (reduction in senescence, increase in clonogenicity) similar to clinically recurrent PCa. Radiation resistance of TRAMP-C2 HF tumors was validated in vivo. Administration of anti-PDL1 and anti-CTLA4 as monotherapy or in combination did not achieve significant tumor growth delay compared to control. IR alone produced an observable tumor growth delay compared to ICIs alone. The combination of anti-PDL1 and IR did not yield additional growth delay compared to IR alone. Strikingly, significant tumor growth delay was seen with the combination of IR and anti-CTLA4 compared to IR alone, while also resulting in complete cure in a third of the mice. mice. Lymph nodes and tumors from mice treated with IR and anti-CTLA4 vs. IR and isotype control demonstrated differential expression of genes in T cell functions and enrichment in both CD4+ and CD8+ T cell populations. Conclusion: We generated the first syngeneic radiorecurrent PCa model and demonstrated that combining anti-CTLA4 and IR results in synergistic tumor response. Combined therapy resulted in augmented immune response, most notably enhancement of CD8 T cell activity. Significance: These findings contribute to our understanding of immunological events associated with RT and ICIs in the context of radiorecurrent PCa and support new avenues for salvage therapy in clinical trials. Citation Format: Hanzhi Wang, Linsey Gong, Xiaoyong Huang, Stephanie D. White, Hanz T. Chung, Danny Vesprini, Tera N. Petchiny, Emmanouil Fokas, Hansen He, Robert S. Kerbel, Stanley K. Liu. Potentiating salvage radiotherapy in radiorecurrent prostate cancer through anti-CTLA4 therapy: Implications from a syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1353.

PEGylated Molybdenum-Iodine Nanocluster as a Promising Radiodynamic Agent against Prostatic Adenocarcinoma.

The combination of photodynamic therapy and radiotherapy has given rise to a modality called radiodynamic therapy (RDT), based on reactive oxygen species-producing radiosensitizers. The production of singlet oxygen, O2(1Δg), by octahedral molybdenum (Mo6) clusters upon X-ray irradiation allows for simplification of the architecture of radiosensitizing systems. In this context, we prepared a radiosensitizing system using copper-free click chemistry between a Mo6 cluster bearing azido ligands and the homo-bifunctional linker bis-dPEG11-DBCO. The resulting compound formed nanoparticles, which featured production of O2(1Δg) and efficient cellular uptake, leading to remarkable photo- and radiotoxic effects against the prostatic adenocarcinoma TRAMP-C2 cell line. Spheroids of TRAMP-C2 cells were also used for evaluation of toxicity and phototoxicity. In vivo experiments on a mouse model demonstrated that subcutaneous injection of the nanoparticles is a safe administration mode at a dose of up to 0.08 g kg-1. The reported results confirm the relevancy of Mo6-based radiosensitizing nanosystems for RDT.

Prostate cancer therapy using immune checkpoint molecules to target recombinant dendritic cells.

We developed immune checkpoint molecules to target recombinant dendritic cells (DCs) and verified their anti-tumor efficacy and immune response against prostate cancer. DCs were generated from mononuclear cells in the tibia and femur bone marrow of mice. We knocked down the programmed death ligand 1 (PD-L1) on monocyte-derived DCs through siRNA PD-L1. Cell surface antigens were immune fluorescently stained through flow cytometry to analyze cultured cell phenotypes. Furthermore, we evaluated the efficacy of monocyte-derived DCs and recombinant DCs in a prostate cancer mouse model with subcutaneous TRAMP-C1 cells. Lastly, DC-induced mixed lymphocyte and lymphocyte-only proliferations were compared to determine cultured DCs' function. Compared to the control group, siRNA PD-L1 therapeutic DC-treated mice exhibited significantly inhibited tumor volume and increased tumor cell apoptosis. Remarkably, this treatment substantially augmented interferon-gamma and interleukin-2 production by stimulating T-cells in an allogeneic mixed lymphocyte reaction. Moreover, we demonstrated that PD-L1 gene silencing improved cell proliferation and cytokine production. We developed monocyte-derived DCs transfected with PD-L1 siRNA from mouse bone marrow. Our study highlights that PD-L1 inhibition in DCs increases antigen-specific immune responses, corroborating previous immunotherapy methodology findings regarding castration-resistant prostate cancer.

Abstract A025: Combination of anti-CTLA4 and radiation induce synergistic tumor control in radiorecurrent prostate cancer

Abstract Background: Advanced prostate cancer (PCa) is a leading cause in cancer death and can elicit significant morbidity and mortality. A common treatment modality for advanced PCa is radiation therapy (RT). Currently, salvage of local disease recurrence after RT is a major clinical problem. Emerging evidence indicates the importance of the immune system in governing RT response. In support of this, immune checkpoint inhibitors (ICIs), which enhance immune activation, have demonstrated clinical therapeutic promise in combination with RT in certain advanced cancers. Irradiation (IR) combined with ICIs may prime the immune system to recognize and target recurrent cancer. Purpose: Investigate the therapeutic efficacy of ICIs in combination with RT for radiorecurrent PCa in a syngeneic pre-clinical model. Methods: TRAMP-C2 cells were treated with 10 Gy of radiation over 5 fractions (similar to clinical hypofractionated (HF) schedule) to generate TRAMP-C2 HF cells. Immune-competent mice were transplanted subcutaneously with TRAMP-C2 HF cells. Once tumors reached palpable size, mice were administered either ICIs (αPDL1 or αCTLA4) alone or in combination with RT. Tumor volume was monitored to determine the treatment effect. Correlative studies on excised tumors and secondary lymphoid organs included flow cytometry and NanoString gene expression panel to evaluate immunological mechanisms that contribute to anti-tumor effects[SL1]. Results: TRAMP-C2 HF cells were validated for radiation resistance and exhibited a more aggressive phenotype (reduction in senescence, increase in clonogenicity) similar to clinically recurrent PCa. Radiation resistance of TRAMP-C2 HF tumors was validated in vivo. Administration of αPDL1 and αCTLA4 as monotherapy or in combination did not achieve significant tumor growth delay compared to control. IR alone produced an observable tumor growth delay compared to ICIs alone. The combination of αPDL1 and IR did not yield additional growth delay compared to IR alone. Strikingly, significant tumor growth delay was seen with the combination of IR and αCTLA4 compared to IR alone, while also resulting in complete cure in a third of the mice. NanoString analysis of lymph nodes from mice treated with αCTLA4 and IR vs. IR alone demonstrated differential expression of genes regulating CD molecules, interleukins, and T-cell functions. Furthermore, enrichment of CD45 and CD8a RNA following αCTLA4 and IR was observed. Validation by flow cytometry is currently ongoing. Conclusion: We generated the first syngeneic radiorecurrent PCa model and demonstrated that combining αCTLA4 and IR results in synergistic tumor response. Combined therapy resulted in augmented immune response, most notably enhancement of CD8 T cell activity. Significance: These findings contribute to our understanding of immunological events associated with RT and ICIs in the context of radiorecurrent PCa and support new avenues for salvage therapy in clinical trials. Citation Format: Hanzhi Wang, Xiaoyong Huang, Stepahnie D White, Linsey Gong, Tera N Petchiny, Hans Chung, Emmanouil Fokas, Robert S Kerbel, Stanley K Liu. Combination of anti-CTLA4 and radiation induce synergistic tumor control in radiorecurrent prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A025.

Anticancer actions of carnosine in cellular models of prostate cancer.

Treatments for organ-confined prostate cancer include external beam radiation therapy, radical prostatectomy, radiotherapy/brachytherapy, cryoablation and high-intensity focused ultrasound. None of these are cancer-specific and are commonly accompanied by side effects, including urinary incontinence and erectile dysfunction. Moreover, subsequent surgical treatments following biochemical recurrence after these interventions are either limited or affected by the scarring present in the surrounding tissue. Carnosine (β-alanyl-L-histidine) is a histidine-containing naturally occurring dipeptide which has been shown to have an anti-tumorigenic role without any detrimental effect on healthy cells; however, its effect on prostate cancer cells has never been investigated. In this study, we investigated the effect of carnosine on cell proliferation and metabolism in both a primary cultured androgen-resistant human prostate cancer cell line, PC346Flu1 and murine TRAMP-C1 cells. Our results show that carnosine has a significant dose-dependent inhibitory effect invitro on the proliferation of both human (PC346Flu1) and murine (TRAMP-C1) prostate cancer cells, which was confirmed in 3D-models of the same cells. Carnosine was also shown to decrease adenosine triphosphate content and reactive species which might have been caused in part by the increase in SIRT3 also shown after carnosine treatment. These encouraging results support the need for further human invivo work to determine the potential use of carnosine, either alone or, most likely, as an adjunct therapy to surgical or other conventional treatments.

Docetaxel radiosensitizes castration-resistant prostate cancer by downregulating CAV-1

Purpose: Docetaxel (DXL), a noted radiosensitizer, is one of the few chemotherapy drugs approved for castration-resistant prostate cancer (CRPC), though only a fraction of CRPCs respond to it. CAV-1, a critical regulator of radioresistance, has been known to modulate DXL and radiation effects. Combining DXL with radiotherapy may create a synergistic anticancer effect through CAV-1 and improve CRPC patients’ response to therapy. Here, we investigate the effectiveness and molecular characteristics of DXL and radiation combination therapy in vitro. Materials and methods: We used live/dead assays to determine the IC50 of DXL for PC3, DU-145, and TRAMP-C1 cells. Colony formation assay was used to determine the radioresponse of the same cells treated with radiation with/without IC50 DXL (4, 8, and 12 Gy). We performed gene expression analysis on public transcriptomic data collected from human-derived prostate cancer cell lines (C4-2, PC3, DU-145, and LNCaP) treated with DXL for 8, 16, and 72 hours. Cell cycle arrest and protein expression were assessed using flow cytometry and western blot, respectively. Results: Compared to radiation alone, combination therapy with DXL significantly increased CRPC death in PC3 (1.48-fold, p < .0001), DU-145 (1.64-fold, p < .05), and TRAMP-C1 (1.13-fold, p < .05) at 4 Gy of radiation. Gene expression of CRPC treated with DXL revealed downregulated genes related to cell cycle regulation and upregulated genes related to immune activation and oxidative stress. Confirming the results, G2/M cell cycle arrest was significantly increased after treatment with DXL and radiation. CAV-1 protein expression was decreased after DXL treatment in a dose-dependent manner; furthermore, CAV-1 copy number was strongly associated with poor response to therapy in CRPC patients. Conclusions: Our results suggest that DXL sensitizes CRPC cells to radiation by downregulating CAV-1. DXL + radiation combination therapy may be effective at treating CRPC, especially subtypes associated with high CAV-1 expression, and should be studied further.

Ascite-solid model of prostate cancer and its morphological characteristics

Targeted therapy and immunotherapy are considered promising novel therapies capable of increasing the effcacy of prostate cancer (PCa) treatment. The purpose of the study was to obtain and characterize TRAMP-C2 subcutaneous and ascite-solid models of prostate cancer in C57Bl/6j mice to study specifc anti-tumor activity of the candidate molecules of targeted drugs and adjust immunotherapy strategies in an evidence-based manner. Material and Methods. We used cultured TRAMP-C2 cells in subcutaneous and ascites mouse prostate cancer models. Histological and immunohistochemical methods were used to study the tumor tissues. Results. The high in vivo growth ability of TRAMP-C2 cells was demonstrated in subcutaneous and intraperitoneal inoculation of C57Bl/6j mice. These tumors are characterized by a high reproducibility and level of PSMA expression. Histological study showed that subcutaneous and carcinomatous TRAMP-C2 tumor nodules had solid structure morphologically corresponding to low differentiated neoplasm, cytomorphological analysis of smears showed that peritoneal fuid containd a large number of rounded tumor cells, macrophages and erythrocytes. Conclusion. The obtained subcutaneous and ascite-solid models of TRAMP-C2 can be useful for the development of new ways to effectively treat cancer, including targeted and immunotherapy, as well as for the experimental study of biotherapeutic effects using PSMA as a target, and photoinduced effects.

Acidity/hydrogen peroxide-responsive PEGylated chitosan-modified polydopamine nanoparticles to realize effective photothermal conversion and intracellular drug delivery

To realize effective photothermal conversion and intracellular drug delivery for application of cancer treatment, an acidity/hydrogen peroxide-responsive nanoparticle system comprising melanin-like polydopamine (PDA) as the hydrophobic core covered by PEGylated chitosan (PEG-CS) conjugates is created herein. The PEG-CS/PDA nanoparticles characterized with a well-dispersed spherical shape exhibited prominent colloidal stability in the milieu of physiological salt concentration. Through π- π stacking and hydrophobic interactions between doxorubicin (DOX) and PDA, DOX molecules were efficiently incorporated into the PEG-CS/PDA nanoparticles. With solution pH being adjusted from 7.4 to 5.0, the ionic complexation of protonated chitosan with negatively-charged PDA enabled the embedding of outer PEG segments of DOX@PEG-CS/PDA nanoparticles into the neutral and gel-like surfaces, thus eliciting interparticle aggregation. The same pH stimulation also accelerated DOX release due to the declined interactions of DOX and PDA combined with enhanced repulsion force between positively-charged chitosan and DOX. Notably, the PEG-CS/PDA nanoparticles not only showed robust photothermal effect and stability, but also were significantly degraded by hydrogen peroxide. After being endocytosed by TRAMP-C1 cells, the DOX@PEG-CS/PDA nanoparticles gradually released drug within acidic organelles, thus promoting drug accumulation in nuclei to kill cancer cells. This work indicates the feasibility for applying such DOX@PEG-CS/PDA nanoparticles in cancer treatment.

Abstract 5139: Chemerin suppresses prostate tumor growth by modulating the recruitment of NK and CD8+ T-cells to tumor microenvironment

Abstract Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of malignant cells, thereby affecting tumorigenesis and tumor progression. While immune checkpoint inhibitors and agonism of costimulatory molecules can act to stimulate leukocytes, the lack of adequate recruitment of effector cells into TME often results in suboptimal immune responses. Chemerin (retinoic acid receptor responder 2, RARRES2) is an endogenous chemoattractant that is widely expressed by many tissues and recruits innate leukocytes through its receptor, CMKLR1 which is expressed on NK cells, some dendritic cells, and macrophages. Previous studies show that RARRES2 is downregulated across multiple tumor types including prostate cancer. Here, using a syngeneic transgenic adenocarcinoma mouse prostate (TRAMP-C1) model, we show that forced overexpression of RARRES2 by tumor cells results in significant tumor suppression and recruitment of NK and CD8+ T-cells within the TME. Male C57/BL/6 mice were inoculated with either RARRES2-overexpressing, vector control (VC) or mixed (50:50; VC: RARRES2) TRAMP-C1 cells. Chemerin overexpression in TRAMP-C1 cells did not alter their phenotypic behavior in vitro, but significantly suppressed tumor growth in vivo in mice. Furthermore, we observed a significantly higher influx of NK (CD3-CD19-NK1.1+) and CD8+ T-cells (CD3+CD8+) in tumor-infiltrating leukocytes (TILs) from mice with mixed tumors compared with VC tumors. Depletion of NK cells, CD4+, or CD8+ T cells showed that NK cells and CD8+ T-cells, but not CD4+ T-cells, were required for chemerin-dependent suppression of TRAMP-C1 tumor growth. We found significant increases in splenic NK and CD8 T cells, and a concomitant significant decrease in splenic G-MDSC in mice with chemerin-expressing tumors compared to controls, suggesting chemerin expression within the TME has an effect on systemic immune responses. Treatment of control tumors with anti-PD1 checkpoint inhibition did not result in significant tumor suppression, while anti-PD1 treatment of chemerin-expressing tumors significantly reduced tumor growth, doubling the complete response rate in these unresponsive tumors. Thus, for the first time we have demonstrated that increasing chemerin expression within the prostate TME can suppress growth by augmenting recruitment of NK and CD8+ T-cells and has the potential to be combined with checkpoint inhibitors in order to improve response rates in non-responsive tumors. Overall, these data suggest that a tumor-targeted chemerin therapeutic may have potential to treat human prostate cancers. Citation Format: Muhammad A. Saeed, Kevin Kim, Russell K. Pachynski. Chemerin suppresses prostate tumor growth by modulating the recruitment of NK and CD8+ T-cells to tumor microenvironment. [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 5139.

Sialic acid-targeted cyclodextrin-based nanoparticles deliver CSF-1R siRNA and reprogram tumour-associated macrophages for immunotherapy of prostate cancer

Prostate cancer remains a serious condition threatening the health of men. Due to the complicated nature of the tumour microenvironment (TME), conventional treatments face challenges including poor prognosis and tumour resistance, therefore new therapeutic strategies are urgently needed. Small interfering RNA (siRNA), a double-stranded non-coding RNA, regulates specific gene expression through RNA interference. Tumour-associated macrophages (TAMs) are a potential therapeutic target in cancer immunotherapy. Colony stimulating factor-1/colony stimulating factor-1 receptor (CSF-1/CSF-1R) signaling pathway plays a crucial role in the polarization of the immunosuppressive TAMs, M2 macrophages. Downregulation of CSF-1R is known to reprogram the immunosuppressive TAMs, M2 macrophages, to the immunostimulatory phenotype, M1 macrophages. Sialic acid is a ligand for Siglec-1 (CD169) which is overexpressed on M2 macrophages with little expression in other phenotypes. Therefore, a sialic acid-targeted cyclodextrin-based nanoparticle was developed to specifically deliver CSF-1R siRNA to M2 macrophages. The nanoparticles were studied in vitro using both human and mouse prostate cancer cell lines. Results show that the targeted nanoparticles achieved cell specific delivery to M2 macrophages via the sialic acid-CD169 axis. The expression of CSF-1R was significantly downregulated in M2 macrophages (29.64% for targeted vs 19.31% for non-targeted nanoparticles in THP-1-derived M2 macrophages and 38.94% for targeted vs 18.51% for non-targeted nanoparticles in RAW 264.7-derived M2 macrophages, n = 4, p < 0.01). The resulting reprograming of M2 macrophages to M1 enhanced the level of apoptosis in the prostate cancer cells in a Transwell model (49.17% for targeted vs 37.68% for non-targeted nanoparticles in PC-3 cells and 69.15% for targeted vs 44.73% for non-targeted nanoparticles in TRAMP C1 cells, n = 3, p < 0.01). Thus, this targeted cyclodextrin-based siRNA drug delivery system provides a potential strategy for prostate cancer immunotherapy.

Remodeling Tumor Immune Microenvironment by Using Polymer-Lipid-Manganese Dioxide Nanoparticles with Radiation Therapy to Boost Immune Response of Castration-Resistant Prostate Cancer.

Despite substantial progress in the treatment of castration-resistant prostate cancer (CRPC), including radiation therapy and immunotherapy alone or in combination, the response to treatment remains poor due to the hypoxic and immunosuppressive nature of the tumor microenvironment. Herein, we exploited the bioreactivity of novel polymer-lipid manganese dioxide nanoparticles (PLMDs) to remodel the tumor immune microenvironment (TIME) by increasing the local oxygen levels and extracellular pH and enhancing radiation-induced immunogenic cell death. This study demonstrated that PLMD treatment sensitized hypoxic human and murine CRPC cells to radiation, significantly increasing radiation-induced DNA double-strand breaks and ultimately cell death, which enhanced the secretion of damage-associated molecular patterns, attributable to the induction of autophagy and endoplasmic reticulum stress. Reoxygenation via PLMDs also polarized hypoxic murine RAW264.7 macrophages toward the M1 phenotype, enhancing tumor necrosis factor alpha release, and thus reducing the viability of murine CRPC TRAMP-C2 cells. In a syngeneic TRAMP-C2 tumor model, intravenous injection of PLMDs suppressed, while radiation alone enhanced recruitment of regulatory T cells and myeloid-derived suppressor cells. Pretreatment with PLMDs followed by radiation down-regulated programmed death-ligand 1 and promoted the infiltration of antitumor CD8+ T cells and M1 macrophages to tumor sites. Taken together, TIME modulation by PLMDs plus radiation profoundly delayed tumor growth and prolonged median survival compared with radiation alone. These results suggest that PLMDs plus radiation is a promising treatment modality for improving therapeutic efficacy in radioresistant and immunosuppressive solid tumors.

Clay nanosheets simultaneously intercalated and stabilized by PEGylated chitosan as drug delivery vehicles for cancer chemotherapy

Montmorillonite (MMT) has been frequently utilized as drug vehicles due to its high specific surface area, excellent cation exchange capacity and biocompatibility. However, the significant flocculation of MMT under physiological condition restricted its application to drug delivery. To conquer this problem, the graft-type PEGylated chitosan (PEG-CS) adducts were synthesized as intercalator to stabilize MMT dispersion. Through electrostatic attraction between the chitosan and MMT, the PEG-CS adducts were adsorbed on MMT surfaces and intercalated into MMT. The resulting PEG-CS/MMT nanosheets possessed PEG-rich surfaces, thus showing outstanding dispersion in serum-containing environment. Moreover, the physicochemical characterization revealed that the increased mass ratio of PEG-CS to MMT led to the microstructure transition of PEG-CS/MMT nanosheets from multilayered to exfoliated structure. Interestingly, the PEG-CS/MMT nanosheets with mass ratio of 8.0 in freeze-dried state exhibited a hierarchical lamellar structure organized by the intercalated MMT bundles and unintercalated PEG-CS domains. Notably, the multilayered PEG-CS/MMT nanosheets showed the capability of loading doxorubicin (DOX) superior to the exfoliated counterparts. Importantly, the DOX@PEG-CS/MMT nanosheets endocytosed by TRAMP-C1 cells liberated the drug progressively within acidic organelles, thereby eliciting cell apoptosis. This work provides a new strategy of achieving the controllable dispersion stability of MMT nanoclays towards application potentials in drug delivery.

Targeting HIC1/TGF-β axis-shaped prostate cancer microenvironment restrains its progression

Prostate cancer (PCa) is a malignant tumor that seriously threatens men’s health worldwide. Recently, stromal cells in the tumor microenvironment (TME) have been reported to contribute to the progression of PCa. However, the role and mechanism of how PCa cells interact with stromal cells to reshape the TME remain largely unknown. Here, using a spontaneous prostate adenocarcinoma (PRAD) model driven by the loss of Pten and Hic1, we found that M2 macrophages markedly infiltrated the stroma of Pten and Hic1 double conditional knockout (dCKO) mice compared with those in control (Ctrl) mice due to higher TGF-β levels secreted by HIC1-deleted PCa cells. Mechanistically, TGF-β in TME promoted the polarization of macrophages into “M2” status by activating the STAT3 pathway and modulating c-Myc to upregulate CXCR4 expression. Meanwhile, TGF-β activated the fibroblasts to form cancer-associated fibroblasts (CAFs) that secrete higher CXCL12 levels, which bound to its cognate receptor CXCR4 on M2 macrophages. Upon interaction with CAFs, M2 macrophages secreted more CXCL5, which promoted the epithelial-mesenchymal transition (EMT) of PCa via CXCR2. Moreover, using the TGF-β receptor I antagonist, galunisertib, significantly inhibited the tumor growth and progression of the TRAMP-C1 cell line-derived subcutaneous tumor model. Finally, we confirmed that the stromal microenvironment was shaped by TGF-β in HIC1-deficient PCa and was associated with the progression of PCa.

Abstract 3711: Modulation and inhibition of prostate cancer cells by microbial metabolites and resveratrol

Abstract The chemotherapeutic compounds resveratrol (RES) and short-chain fatty acids (SCFAs) are potent inhibitors of cancer cell proliferation during in vitro testing. These compounds are naturally occurring and present relatively low toxicity to normal cells and tissues, making them an attractive cancer treatment alternative. SCFAs are the chemical by-products of microbial activity known as fermentation. RES is a polyphenolic compound found in select fruits and vegetables and has been known for its versatility in preventing and reversing many diseases, including several cancers. Butyric and propionic acid are considered the most potent of the SCFAs found within the intestinal lumen. The prime goal of this research is to understand if the RES and SCFA compounds use a shared biological pathway(s) to fight and inhibit cancer. We used TRAMP C1 and C3 prostate cancer cells (transgenic adenocarcinoma mouse prostate) as an experimental model. These cells were treated with the RES and SCFAs both singularly and synergistically for 24hrs. The cells were analyzed to understand the potential cell toxicity and proliferation, the modulation of apoptotic and proapoptotic proteins/genes, and any change in mitochondrial membrane potential pathways. The result demonstrated that the expression of apoptogenic proteins induces programmed cell death in a mitochondria-independent manner. Therefore, the result suggests that RES and SCFAs can facilitate many critical biological functions within the host's body, including cellular respiration, nutrient assimilation, and various immunological responses to inhibit cancer cell proliferation. Citation Format: James Stokes, Rajesh Singh, Manoj K. Mishra. Modulation and inhibition of prostate cancer cells by microbial metabolites and resveratrol [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 3711.

Abstract 1657: Syngeneic tumor development, treatment responses and immune infiltrate in subcutaneous and orthotopic mouse models

Abstract Background: Syngeneic mouse tumor models are widely used and effective tools to evaluate efficacy of novel anti-cancer therapies. They are often implanted subcutaneously in a microenvironment that is unrelated to the site of origin. This can be problematic when evaluating immune modulating therapies as the tumor microenvironment is known to differ substantially between orthotopic and subcutaneous models. Here, we report the development and characterization of several syngeneic mouse tumor models implanted subcutaneously and orthotopically. The models are characterized by immune infiltrate and response to novel and conventional therapies. Methods: Murine MC38 (colon), TRAMPC1 (prostate) and GL261 (glioma) cell lines were implanted subcutaneously or orthotopically in immune competent mice. Tumor growth was monitored at multiple timepoints by external caliper measurements, magnetic resonance (MR) or ultrasound imaging. At confirmed tumor take, mice were treated with external radiation therapy (RT) or immune checkpoint inhibitors. Single cell suspensions were prepared from tumors and tumor draining lymph nodes (TdLNs) at various timepoints after treatment initiation and immuno-profiling was evaluated by multicolor flow cytometry. The tissues were analyzed for proportions of the following cell populations; CD4+ and CD8+ T lymphocytes, myeloid derived suppressor cells (MDSCs), NK cells and macrophages. Results: Subcutaneous and orthotopic tumor volumes developed at a similar pace. However, orthotopic tumor volumes estimated by MR imaging were detectable at earlier timepoints compared to subcutaneous volumes estimated by caliper measurements. Response to therapy was dependent on implantation site. This was further highlighted by the immune infiltration analysis, where CD4+ and CD8+ tumor infiltrating lymphocytes differed markedly between subcutaneous and orthotopic tumors. Also, the temporal profile of tumor infiltrating immune cells up to 14 days following therapy was established in the models. Conclusion: Subcutaneous and orthotopic versions of syngeneic mouse tumor models were successfully established and characterized with respect to their tumor immune microenvironment. The models displayed varying sensitivity towards RT and immune checkpoint inhibitors. Response to therapy was furthermore dependent on implantation site. Altogether, these advanced models are powerful tools for investigating treatment effect within preclinical immuno-oncology. Citation Format: Sigrid Cold, Saif AL-Haidar, Jennifer Solgaard Joergensen, Trine Bjoernbo Engel, Frederikke Petrine Fliedner, Lotte Kellemann Kristensen, Andreas Kjaer, Carsten Haagen Nielsen. Syngeneic tumor development, treatment responses and immune infiltrate in subcutaneous and orthotopic mouse models [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 1657.

Invariant NKT cell-augmented GM-CSF-secreting tumor vaccine is effective in advanced prostate cancer model.

Invariant natural killer T cells (iNKT cells) express a semi-invariant T cell receptor that recognizes certain glycolipids (including α-galactosylceramide, αGC) bound to CD1d, and can induce potent antitumor responses. Here, we assessed whether αGC could enhance the efficacy of a GM-CSF-producing tumor cell vaccine in the transgenicSV40 T antigen-driven TRAMP prostate cancer model. In healthy mice, we initially found that optimal T cell responses were obtained with αGC-pulsed TRAMP-C2 cells secreting GM-CSF and milk fat globule epidermal growth factor protein-8 (MFG-E8) with an RGD to RGE mutation (GM-CSF/RGE TRAMP-C2), combined with systemic low dose IL-12. In a therapeutic model, transgenicTRAMP mice were then castrated at ~ 20weeks, followed by treatment with the combination vaccine. Untreated mice succumbed to tumor by ~ 40weeks, but survival was markedly prolonged by vaccine treatment, with most mice surviving past 80weeks. Prostates in the treated mice were heavily infiltrated with T cells and iNKT cells, which both secreted IFNγ in response to tumor cells. The vaccine was not effective if the αGC, IL-12, or GM-CSF secretion was eliminated. Finally, immunized mice were fully resistant to challenge with TRAMP-C2 cells. Together these findings support further development of therapeutic vaccines that exploit iNKT cell activation.

PD46-08 COMBINED TREATMENT WITH ULTRASOUND AND IMMUNE CHECKPOINT INHIBITORS FOR PROSTATE CANCER

You have accessJournal of UrologyCME1 May 2022PD46-08 COMBINED TREATMENT WITH ULTRASOUND AND IMMUNE CHECKPOINT INHIBITORS FOR PROSTATE CANCER Fuuka Hayashi, Katsumi Shigemura, Koichi Kitagawa, Noriaki Maeshige, Koki Maeda, and Masato Fujisawa Fuuka HayashiFuuka Hayashi More articles by this author , Katsumi ShigemuraKatsumi Shigemura More articles by this author , Koichi KitagawaKoichi Kitagawa More articles by this author , Noriaki MaeshigeNoriaki Maeshige More articles by this author , Koki MaedaKoki Maeda More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002614.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Ultrasound (US) is mostly used for diagnostic purpose but could be used for cancer treatments if the US intensity or frequency fits to such purpose. Prostate cancer (PC) has the highest prevalence in the urological field, but indications of immune checkpoint inhibitors (ICI) for PC are limited to very few cases. Therefore, we verified the antitumor effect of US irradiation alone and the combined use of US and ICI in vitro and in vivo. METHODS: Human-derived PC cell line LNCaP and PC-3, and mouse-derived PC cell line TRAMP-C2 were used in experiments. US (3.0 W/cm2, 3 MHz, irradiation time rate: 20 %) was irradiated with repeated frequency of 1, 10, and 100 Hz. Cell proliferation was evaluated by MTS assay and apoptotic cells were analyzed by flow cytometry. We transplanted LNCaP, PC-3 cells into nude mice, and TRAMP-C2 cells into Balb/c nu/nu mice. US was irradiated on mice at 10 Hz and 100 Hz 3 times a week for 6 weeks. We also administered anti-PD-1 antibody to TRAMP-C2-bearing mice 5 times to verify the effect of combined use of US and ICI. After tumor collection, Immunohistochemical (IHC) stain with Ki-67, caspase-3, CD31 and CD3 were conducted. RESULTS: Cell proliferation assay showed US irradiation suppressed cell growth in LNCaP (at 10 Hz and 100 Hz, p<0.0001, respectively), PC-3 (at 1, 10, and 100 Hz, p=0.0002, p<0.0001, and p=0.0132), and TRAMP-C2 (at 1 Hz and 10 Hz, p<0.0001, respectively). In addition, US irradiation induced apoptosis in LNCaP (at 100 Hz, p=0.0368), PC-3 (at 1 Hz and 10 Hz, p=0.0447, p=0.0137), and TRAMP-C2 (at 1, 10, and 100 Hz, p=0.0129, p=0.0150, p=0.0017). In animal experiments, US irradiation at 100 Hz inhibited tumor growth in both LNCaP and PC-3-bearing mice (p=0.0419, p=0.0064). From IHC analysis, the expression level of Ki-67 was decreased at 100Hz in PC-3 (p=0.0467), and caspase-3 was decreased at 10 Hz and 100 Hz in both LNCaP (p=0.0007, p=0.0297) and PC-3 (p=0.0061, p=0.0115). In addition, the number of vessels was reduced at 10Hz and 100 Hz in both LNCaP (p=0.0290, p=0.0102) and PC3 (p=0.0026, p=0.0007). In TRAMP-C2 in vivo experiments, an antitumor effect was observed at 10 Hz, 100 Hz, and the combined use of ICI and 100 Hz of US (p=0.0365, p=0.0364, p=0.0393) compared to control. IHC stain revealed the number of CD3-positive cells increased at 100 Hz (p=0.0499). CONCLUSIONS: We found that US irradiation induces apoptosis and reduces cell growth. Moreover, tumor growth was suppressed by combined use of US and ICI. Further research on immune system activation will lead to a less invasive and more efficient treatment for PC. Source of Funding: none © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e791 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Fuuka Hayashi More articles by this author Katsumi Shigemura More articles by this author Koichi Kitagawa More articles by this author Noriaki Maeshige More articles by this author Koki Maeda More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement PDF DownloadLoading ...

Combined Treatment with Ultrasound and Immune Checkpoint Inhibitors for Prostate Cancer.

Background: Ultrasound (US) is mostly used for diagnostic purpose but could be used for cancer treatments with a US intensity or frequency fitted to such a purpose. Prostate cancer (PC) has the highest prevalence in the urological field, but indications for immune checkpoint inhibitors (ICIs) for PC are limited to very few cases. In this study, we compared the antitumor effect of US irradiation alone with the combined use of US and ICIs in vitro and in vivo. Methods: PC cell line TRAMP-C2 cells were used in our experiments. TRAMP-C2 cells were irradiated with US with pulse repeated frequencies (PRF) of 1, 10, and 100 Hz. Cell proliferation was evaluated by MTS assay and apoptotic cells were analyzed using flow cytometry. To verify the antitumor effect of US irradiation on PC in vivo, we conducted animal experiments using mice. TRAMP-C2-bearing mice were irradiated with US with PRF of 10 and 100 Hz. Three weeks after the start of US irradiation, anti-PD-1 antibody was administered to the mice. Finally, mice were sacrificed and tumors were collected. Immunohistochemical (IHC) analyses were assessed for cleaved caspase-3 and CD3 in tumor cell extracts. Results: Cell proliferation assays showed that 1 and 10 Hz US significantly inhibited cell survival (p < 0.0001). In addition, US irradiation induced apoptosis at 1, 10, and 100 Hz (p = 0.0129, p = 0.0150, and p = 0.0017, respectively). In animal experiments, a significant tumor growth inhibitory effect was observed at 10 and 100 Hz, and 100 Hz + ICIs (p < 0.05, respectively). Hematoxylin–eosin (H–E) staining showed a significant increase in the necrotic area of the tumor at 100 Hz and 100 Hz + ICIs (p < 0.05, respectively). In addition, under IHC staining the expression level of cleaved caspase-3 and the number of CD3-positive cells increased at 100 Hz (p < 0.05, respectively). Conclusion: US irradiation induced apoptosis in cells and reduced cell viability. In vivo tumor growth was suppressed by combined treatment with US irradiation and ICIs. Further research on immune system activation will lead to less invasive and more efficient treatments for PC.