Tumor Cells In Prostate Cancer Research Articles

Integrated machine learning algorithms reveal a bone metastasis-related signature of circulating tumor cells in prostate cancer

Bone metastasis is an essential factor affecting the prognosis of prostate cancer (PCa), and circulating tumor cells (CTCs) are closely related to distant tumor metastasis. Here, the protein-protein interaction (PPI) networks and Cytoscape application were used to identify diagnostic markers for metastatic events in PCa. We screened ten hub genes, eight of which had area under the ROC curve (AUC) values > 0.85. Subsequently, we aim to develop a bone metastasis-related model relying on differentially expressed genes in CTCs for accurate risk stratification. We developed an integrative program based on machine learning algorithm combinations to construct reliable bone metastasis-related genes prognostic index (BMGPI). On the basis of BMGPI, we carefully evaluated the prognostic outcomes, functional status, tumor immune microenvironment, somatic mutation, copy number variation (CNV), response to immunotherapy and drug sensitivity in different subgroups. BMGPI was an independent risk factor for disease-free survival in PCa. The high risk group demonstrated poor survival as well as higher immune scores, higher tumor mutation burden (TMB), more frequent co-occurrence mutation, and worse efficacy of immunotherapy. This study highlights a new prognostic signature, the BMGPI. BMGPI is an independent predictor of prognosis in PCa patients and is closely associated with the immune microenvironment and the efficacy of immunotherapy.

Combating anoikis resistance: bioactive compounds transforming prostate cancer therapy.

The study aims to discuss the challenges associated with treating prostate cancer (PCa), which is known for its complexity and drug resistance. It attempts to find differentially expressed genes (DEGs), such as those linked to anoikis resistance and circulating tumor cells, in PCa samples. This study involves analyzing the functional roles of these DEGs using gene enrichment analysis, and then screening of 102 bioactive compounds to identify a combination that can control the expression of the identified DEGs. In this study, 53 DEGs were identified from PCa samples including anoikis-resistant PCa cells and circulating tumor cells in PCa. Gene enrichment analysis with regards to functional enrichment of DEGs was performed. An inclusive screening process was carried out among 102 bioactive compounds to identify a combination capable of affecting and regulating the expression of selected DEGs. Eventually, gastrodin, nitidine chloride, chenodeoxycholic acid, and bilobalide were selected, as their combination demonstrated ability to modulate expression of 50 out of the 53 genes targeted. The subsequent analysis focused on investigating the biological pathways and processes influenced by this combination. The findings revealed a multifaceted and multidimensional approach to tumor regression. The combination of bioactive compounds exhibited effects on various genes including those related to production of inflammatory cytokines, cell proliferation, autophagy, apoptosis, angiogenesis, and metastasis. The current study has made a valuable contribution to the development of a combination of bioactive natural compounds that can significantly impede the development of treatment resistance in prostate tumor while countering the tumors' evasion of the immune system. The implications of this study are highly significant as it suggests the creation of an enhanced immunotherapeutic, natural therapeutic concoction with combinatorial potential.

The functions and mechanisms of RNA modification in prostate: Current status and future perspectives.

The increasing incidence and mortality of prostate cancer worldwide significantly impact the life span of male patients, emphasizing the urgency of understanding its pathogenic mechanism and associated molecular changes that regulate tumor progression for effective prevention and treatment. RNA modification, an important post-transcriptional regulatory process, profoundly influences tumor cell growth and metabolism, shaping cell fate. Over 170 RNA modification methods are known, with prominent research focusing on N6-methyladenosine, N7-methylguanosine, N1-methyladenosine, 5-methylcytidine, pseudouridine, and N4-acetylcytidine modifications. These alterations intricately regulate coding and non-coding RNA post-transcriptionally, affecting the stability of RNA and protein expression levels. This article delves into the latest advancements and challenges associated with various RNA modifications in prostate cancer tumor cells, tumor microenvironment, and core signaling molecule androgen receptors. It aims to provide new research targets and avenues for molecular diagnosis, treatment strategies, and improvement of the prognosis in prostate cancer.

CB307: A Dual Targeting Costimulatory Humabody VH Therapeutic for Treating PSMA-Positive Tumors.

CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease. We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumor microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse antitumor pharmacology studies. Nonclinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. CB307 provides effective CD137 agonism in a PSMA-dependent manner, with antitumor activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 IHC assay demonstrated a higher prevalence of CD137-positive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in nonhuman primates and exhibited a plasma half-life supporting weekly clinical administration. CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T-cell activation, thereby alleviating toxicologic concerns against unrestricted CD137 agonism.

Abstract 170: Targeting of macrophage PI3Kγin prostate cancer using Eganelisib (IPI-549) reprograms immune-suppressive infiltrating macrophages to enhance anti-tumor immune responses and promote immunologically mediated tumor growth

Abstract Background: Prostate cancer (PCa) is traditionally considered an immunologically 'cold’ indication, as evidenced in part by significant infiltration of immunosuppressive tumor associated macrophages (TAMs) and few activated effector CD8+ T cells. Attempts to (re)invigorate an anti-prostate cancer immune response with immune checkpoint blockade (ICB) such as anti-CTLA-4 and anti-PD-1 have been largely ineffective clinically, despite promising results in other cancers. This is likely due to a multitude of immunosuppressive cell types and secreted factors within the tumor microenvironment (TME) that diminish T cell activation and trafficking. Reducing the immunosuppressive burden intratumorally by targeting TAMs is a clinically attractive approach and offers opportunity to sensitize PCa to immune checkpoint blockade. While PI3K signaling is ubiquitous in most cell types, specific catalytic isoforms and regulatory elements are differentially expressed depending on cell type, with PI3Kγ being significantly enriched in myeloid cells and therefore offering a unique therapeutic opportunity for specific targeting. Methods: Utilizing novel syngeneic prostate cancer tumors, PI3Kγ deletion and antagonism using Eganelisib (a small molecule inhibitor for PI3Kγ), we tested our hypothesis that targeting PI3Kγ signaling in PCa TAMs would lead to tumor growth inhibition and anti-tumor immune responses. Results: We show here that PCa tumor cells do not express the PI3Kγ isoform and, therapeutic inhibition of PI3Kγ with Eganelisib reduces the TAM suppressive phenotype. We also show PI3Kγ signaling is essential to monocyte anti-apoptotic signaling, and inhibition of PI3Kγ can enhance anti-tumor immune responses in preclinical models of PCa. Specifically, inhibition of PI3Kγ alone is sufficient to enhance anti-tumor immune responses in the less aggressive B6CaP PCa model, but insufficient in the more aggressive RM-1 PCa model. However, treatment of RM-1 tumors with Eganelisib produces improved tumor control when combined with anti-PD-1 therapy, relative to Eganelisib or anti-PD1 as a monotherapy. Conclusions: Altogether, these data highlight the integral role of PI3Kγ signaling in TAMs and the potential for therapeutic targeting with Eganelisib to sensitize PCa to ICB. Citation Format: Kenneth M. Adusei, Thomas R. Nirschl, Alex J. Lee, Fan Shen, Xiaoxu Wang, Monali Praharaj, Jade Alvarez, Iyana Gross, Kara A. Lombardo, Tamara L. Lotan, Judith A. Varner, Jelani C. Zarif. Targeting of macrophage PI3Kγin prostate cancer using Eganelisib (IPI-549) reprograms immune-suppressive infiltrating macrophages to enhance anti-tumor immune responses and promote immunologically mediated tumor growth [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 170.

Abstract 2988: TUG1 exosomal lncRNA sponges let-7 miRNA family members and advances prostate cancer

Abstract Exosomal long-non coding RNAs (lncRNAs) play an important role in cancer progression and have potential prognostic biomarker potential in several cancers including prostate cancer (PCa). Taurine-upregulated gene 1 (TUG1) is an oncogenic lncRNA overexpressed in PCa. This exosomal lncRNA has been reported to promote PCa migration, invasion and Epithelial-Mesenchymal Transition (EMT). However, TUG1 PCa prognostic mechanisms remain to be elucidated. This study aimed to profile differentially expressed lncRNAs in PCa high-grade tumor cells (PC-3) cells compared to low-grade PCa tumor cells (LNCaP). This was achieved by using a 384-well plate of PCa lncRNA gene panel. A human lncRNA database, LncSEA and other bioinformatics tools were used for annotation and enrichment analysis. RT-qPCR was used to confirm the gene array data. Up or down-regulation of ±2 and p <0.05 were considered significant. Forty two (42%) percent and 14% of lncRNAs were shown to be up and downregulated, respectively. Notably, TUG1 was shown to be upregulated in PC-3 cells from PCR array and RT-qPCR results. Bioinformatics analysis revealed that TUG1 is involved in several cancer hallmarks including apoptosis evasion, migration, invasion and EMT. Furthermore, lncRNA/miRNA complexes regulate a series of oncogenic signaling pathways and this study demonstrated that TUG1 sponges the let-7 miRNA family members (a highly conserved tumor suppressor miRNA family) and promotes PCa progression. Precise mechanisms of TUG1/let-7 axis in PCa prognosis need to be largely elucidated as this may hold potential to understanding lncRNA/miRNA-mediated PCa progression and thus be targeted for novel therapeutics. Citation Format: Rahaba Makgotso Marima, Mandisa Mbeje, Jeyalakshmi Kandhavelu, Clement Penny, Zodwa Dlamini. TUG1 exosomal lncRNA sponges let-7 miRNA family members and advances prostate cancer [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 2988.

BACCHARIN FROM GREEN PROPOLIS: A REVIEW OF ITS ANTIPROLIFERATIVE EFFECTS

Baccharin (3-prenyl-4-dihydrocinnamoyloxy-cinnamic acid) is a prenylated phenolic compound found in the extract of the Baccharis dracunculifolia plant, as well as in green propolis. It is one of the main components of this resinous material, which results from the interaction between bees and plants. This substance has been extensively studied for its biological effects, mainly its anti-inflammatory and antitumor activities. In this literature review, we have compiled several articles available between 1999 and 2022 that discuss scientific research carried out on baccharin. The objective is to explore the already evaluated beneficial effects of this compound in different types of tumors. The cancer begins with a genetic mutation that transforms a normal cell into a mutated cell. Subsequently, important mechanisms for its development and progression occur, including increased proliferation, evasion of apoptosis, angiogenesis, invasion, and metastasis. In this regard, numerous studies are being conducted to identify natural compounds that can act on these stages of carcinogenesis, including the components of green propolis. It has already been demonstrated that baccharin can act at different stages of tumor development, exhibiting antimitotic, antiangiogenic, pro-apoptotic, and immunomodulatory activities. Additionally, baccharin has been found to interfere with carcinogenic metabolic pathways and exhibit genotoxic effects. Baccharin has demonstrated a significant antitumor activity when applied to melanoma cells, colon cancer, gastric cancer, hepatocarcinoma, sarcoma, prostate cancer, breast cancer, and leukemia tumor cells. Therefore, the studies cited in this review provide evidence of baccharin's ability to inhibit relevant steps in the carcinogenic process of different types of tumors.

Analysis of androgen receptor dynamics through immunofluorescent staining of circulating tumor cells in castration-resistant prostate cancer.

e17021 Background: Prostate adenocarcinomas are initially dependent on the androgen receptor (AR) for growth and proliferation. Androgen synthesis inhibitors and AR antagonists are the most commonly used first line treatments in metastatic prostate cancer, but these agents are not curative and virtually all tumors progress to a castration resistant state often associated with AR amplification (ARamp) and overexpression. Small molecule AR degraders designed to overcome ARamp/overexpression and other AR alterations associated with resistance are now in clinical development. In parallel, it is important to identify and validate biomarkers that predict sensitivity and inform treatment selection. Methods: CTC samples collected at progression from 114 men with metastatic castration resistant prostate cancer were stained for CK, DAPI, CD45, and AR. AR cRatio (ratio between a cell’s immunofluorescent [IF] intensity and the median IF intensity across all cells) was quantified for each CTC. Low-pass whole genome sequencing (lpWGS) was performed on candidate CK-positive (CK+) CTCs to generate copy number profiles. Cohen’s kappa was used to evaluate the relationship between AR copy number and IF at the single cell level. Dynamics in high AR expressing (ARhigh) CTCs were analyzed separately in a longitudinal cohort of patients (n = 18) from whom CTCs were collected at baseline, early on-treatment (4-12 weeks), and at progression on their 1st AR signaling inhibitor (1L-ARSI). Results: lpWGS was performed on 726 CK+ CTCs, of which 23% (170) were ARamp and 27% (195) were ARhigh (AR cRatio ≥ 3). Fifty-six percent (96/170) of CTCs with ARamp were ARhigh. Overall, a fair agreement was observed between AR copy number and IF (76.1% agreement; k = 0.37, 95%CI = 0.29 – 0.44). At baseline prior to 1L-ARSI, the median CK+ CTC burden was 7.9 CTC/mL (IQR: 1.3 – 10.5). One or more ARhigh CTC was detected in 28% (5/18) of patients, but no patient had more than 0.7 ARhigh CTCs per mL of blood. ARhigh CTCs were not observed in early on-treatment samples. At progression, the median CTC burden was 2.3 CTC/mL (IQR: 0.9 – 9.45) and, despite lower CTC burden, the emergence of a substantial (≥1 CTC/mL) ARhigh CTC subpopulation was observed in 17% (3/18) of patients. In samples with ≥1 detected CTC, the mean AR cRatio was 1.106 and 1.777 at baseline and progression, respectively. Conclusions: AR protein can be measured in CTCs with IF staining and is supported by the agreement with data from lpWGS. ARhigh CTC subpopulations can be detected at baseline for 1L-ARSI, but these cells were all cleared from circulation after 4-12 weeks of therapy. In a subset of patients at progression, resistance appears at least partially attributable to selection for ARhigh CTC subclones. [Table: see text]

High-throughput method to analyze the cytotoxicity of CAR T Cells in a 3D tumor spheroid model using image cytometry

Abstract Chimeric antigen receptor (CAR)-T cell therapy is an antigen-dependent cellular therapy that has gained considerable traction in the field of cancer immunotherapy. Currently, there are six FDA-approved CAR-T cell therapies, which all target the CD19 or BCMA antigens for hematologic B cell malignancies. In recent years, a strong focus has been placed on CAR T cell therapy discovery for solid tumors, which may better recapitulate physiological conditions, thereby potentially improving the selection of CAR construct candidates. Therefore, it is critical to develop novel methodologies for high-throughput phenotypic and functional assays using 3D tumor spheroid models to better assess CAR-T cell therapies against solid tumors. Recently, plate-based image cytometry has emerged as a method to investigate and characterize CAR T cell functions in a high-throughput manner. Image cytometry has demonstrated capabilities in analyzing transduction efficiency, cell proliferation, and cytotoxicity for CAR T cell therapy. With the development of 3D spheroid models, image cytometry may provide the necessary tools and applications for CAR T cell therapy discovery geared towards solid tumors. In this work, we discuss the use of CAR-T cells targeted towards PSMA, an antigen that is found on prostate cancer tumor cells, the second most common cause of cancer deaths among men worldwide. Herein, we demonstrate the use of high-throughput image cytometry to characterize PSMA CAR-T cell-mediated cytotoxic potency against 3D prostate tumor spheroids and simultaneously monitor location of the T cells in vitro. The proposed method can effectively assess cell-mediated 3D tumor spheroid cytotoxicity and generate time- and dose-dependent results.

Zn2+-interference and H2S-mediated gas therapy based on ZnS-tannic acid nanoparticles synergistic enhancement of cell apoptosis for specific treatment of prostate cancer

Zn2+ and H2S are essential to maintain normal prostate function, and sometimes can evolve into weapons to attack and destroy prostate cancer (PCa) cells. Nevertheless, how to achieve the targeted and effective release of Zn2+ and H2S, and reverse the concentration distribution within PCa tumor cells still highly challenging. Herein, combined with these pathological characteristics of prostate, we proposed a tumor microenvironment (TME) responsive Zn2+-interference and H2S-mediated gas synergistic therapy strategy based on a nanoplatform of tannic acid (TA) modified zinc sulfide nanoparticles (ZnS@TA) for the specific treatment of PCa. Once the constructed pH-responsive ZnS@TA internalized by cancer cells, it would instantaneously decomposed in acidic TME, and explosively release excess Zn2+ and H2S exceeding the cell self-regulation threshold. Meanwhile, the in situ produced Zn2+ and H2S synergistic enhancement of cell apoptosis, which is evidenced to increase levels of Bax and Bax/Bcl-2 ratio, release of Cytochrome c in cancer cells, contributing to inhibit the growth of tumor. Moreover, the TA in cooperation with Zn2+ specifically limits the migration and invasion of PCa cells. Both in vitro and in vivo results demonstrate that the Zn2+-interference in combination with H2S-mediated gas therapy achieves an excellent anti-tumor performance. Overall, this nanotheranostic synergistic therapy provides a promising direction for exploring new strategies for cancer treatment based on specific tumor pathological characteristics, and provides a new vision for promoting practical cancer therapy.

Epigenetic modulation and prostate cancer: Paving the way for NK cell anti-tumor immunity.

Immunoepigenetics is a growing field, as there is mounting evidence on the key role played by epigenetic mechanisms in the regulation of tumor immune cell recognition and control of immune cell anti-tumor responses. Moreover, it is increasingly acknowledgeable a tie between epigenetic regulation and prostate cancer (PCa) development and progression. PCa is intrinsically a cold tumor, with scarce immune cell infiltration and low inflammatory tumor microenvironment. However, Natural Killer (NK) cells, main anti-tumor effector immune cells, have been frequently linked to improved PCa prognosis. The role that epigenetic-related mechanisms might have in regulating both NK cell recognition of PCa tumor cells and NK cell functions in PCa is still mainly unknown. Epigenetic modulating drugs have been showing boundless therapeutic potential as anti-tumor agents, however their role in immune cell regulation and recognition is scarce. In this review, we focused on studies addressing modulation of epigenetic mechanisms involved in NK cell-mediated responses, including both the epigenetic modulation of tumor cell NK ligand expression and NK cell receptor expression and function in different tumor models, highlighting studies in PCa. The integrated knowledge from diverse epigenetic modulation mechanisms promoting NK cell-mediated immunity in various tumor models might open doors for the development of novel epigenetic-based therapeutic options for PCa management.

Abstract 1807: CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors

Abstract Introduction: CD137 (HGNC:TNFRSF9, 4-1BB) is a TNF receptor superfamily costimulatory receptor expressed by T cells and NK cells. Upon engagement, CD137 enhances immune cell survival, proliferation, cytokine production and memory formation. CD137 agonists in the clinic have shown tantalizing anti-tumor effects, however, hepatic toxicity has hampered clinical utility. To circumvent this limitation, we developed CB307, a half-life extended heavy-chain only Humabody® that selectively agonizes CD137 only in the presence of prostate specific membrane antigen (PSMA, HGNC:FOLH1). PSMA is highly upregulated on tumor cells in prostate cancer and tumor neovasculature in other solid tumors including lung (NSCLC), kidney (clear cell RCC), bladder and colorectal cancers. CB307 enhances immune cell activation in a PSMA-dependent manner. We present here preclinical mechanistic differentiators of CB307 with respect to non-targeted CD137 agonists. Methods: CD137 agonism, immune cell activation and cytokine secretion were evaluated in vitro using coculture assays in the presence/absence of PSMA-expressing tumor cells and in vivo with a transgenic mouse syngeneic tumor model. The ability of CB307 or non-targeted CD137 agonists to enhance tumor cell killing was additionally characterized in 3D spheroid cocultures. In vitro cocultures were used to determine the impact of PSMA expression level on CB307 activity. CB307 was evaluated as a single agent and in combination with PD-1 or PD-L1 inhibitors; tumoral checkpoint contribution was investigated using isogenic PD-L1 positive vs. PD-L1 CRISPR-deleted tumor cells. Results: CB307 or PD-1/PD-L1 inhibition alone induced IL-2 secretion in PBMC/PSMA-expressing tumor cell cocultures; this effect was synergistically enhanced when CB307 was combined with either PD-1 or PD-L1 inhibition. CB307 augmented tumor cell killing in 3D spheroids containing PSMA-expressing cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB307 in combination with PD-1/PD-L1 inhibition. In in vivo pharmacology studies in a syngeneic mouse model, CB307 demonstrated statistically significant inhibition of tumor growth versus control without peripheral T cell activation - consistent with pharmacology restricted to the PSMA+ tumor microenvironment, whereas an anti-CD137 monoclonal antibody induced tumor growth inhibition accompanied by peripheral immune cell proliferation and cytokine production. Summary: CB307 is a novel CD137 agonist that selectively enhances immune cell activity only in the presence of PSMA-positive cells. Our data demonstrate PSMA-targeted CB307 immunological pharmacology both in vitro and in vivo which has supported the ongoing Phase 1 ‘POTENTIA’ clinical trial (NCT04839991). Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Clare Song, Anca-Giuilia Cionca, Joanne Fernando, Phillip D. Bartlett, Philip Bland-Ward. CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors [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 1807.

Abstract 4575: High-throughput method to analyze the cytotoxicity of CAR T cells in a 3D tumor spheroid model using image cytometry

Abstract Chimeric antigen receptor (CAR)-T cell therapy is an antigen-dependent cellular therapy that has gained considerable traction in the field of cancer immunotherapy. CAR-T cell therapy involves specifically engineering T cells to attack tumor cells by binding a tumor antigen and inducing T cell activation resulting in intracellular signaling and cytokine release. Currently, there are six FDA-approved CAR-T cell therapies, which all target the CD19 or BCMA antigens for hematologic B cell malignancies. In the recent years, a strong focus has been placed on CAR T cell therapy discovery for solid tumors, which may better recapitulate physiological conditions, thereby potentially improving the selection of CAR construct candidates. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. Therefore, it is critical to develop novel methodologies for high-throughput phenotypic and functional assays using 3D tumor spheroid models to better assess CAR-T cell therapies against solid tumors. Recently, plate-based image cytometry has emerged as a method to investigate and characterize CAR T cell functions in a high-throughput manner. Image cytometry has demonstrated capabilities in analyzing transduction efficiency, cell proliferation, and cytotoxicity for CAR T cell therapy. With the development of 3D spheroid models, image cytometry may provide the necessary tools and applications for CAR T cell therapy discovery geared towards solid tumors. In this work, we discuss the use of CAR-T cells targeted towards PSMA, an antigen that is found on prostate cancer tumor cells, the second most common cause of cancer deaths among men worldwide. Herein, we demonstrate the use of high-throughput plate-based image cytometry to characterize PSMA CAR-T cell-mediated cytotoxic potency against 3D prostate tumor spheroids and simultaneously monitor location of the T cells in vitro. We were able to kinetically evaluate the efficacy and therapeutic value of PSMA CAR-T cells by analyzing the cytotoxicity against prostate tumor spheroids. Furthermore, the T cells are fluorescently labeled with a tracer dye to visually locate the cells on the tumor spheroids. The proposed image cytometry method can overcome limitations placed on traditional methodologies to effectively assess cell-mediated 3D tumor spheroid cytotoxicity and efficiently generate time- and dose-dependent results. Citation Format: Samir Patel, David Zurowski, Daniel Hui, Mignane Ka, Charles Hernandez, Andrea Love, Bo Lin, Andrea Moore, Leo Li-Ying Chan. High-throughput method to analyze the cytotoxicity of CAR T cells in a 3D tumor spheroid model using image cytometry. [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 4575.

High-throughput method to analyze the cytotoxicity of CAR-T Cells in a 3D tumor spheroid model using image cytometry.

Solid tumors account for approximately 90% of all adult human cancers. As such, the development of novel cellular therapies has become of increasing importance to target solid tumor malignancies, such as prostate, lung, breast, bladder, colon, and liver cancers. One such cellular therapy relies on the use of chimeric antigen receptor T cells (CAR-T cells). CAR-T cells are engineered to target specific antigens on tumor cells. To date, there are six FDA-approved CAR-T cell therapies that have been utilized for hematologic B cell malignancies. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. Therefore, it is critical to develop novel methodologies for high-throughput phenotypic and functional assays using 3D tumor spheroid models to assess CAR-T cell products against solid tumors. In this manuscript, we discuss the use of CAR-T cells targeted towards PSMA, an antigen that is found on prostate cancer tumor cells, the second most common cause of cancer deaths among men worldwide. We demonstrate the use of high-throughput, plate-based image cytometry to characterize CAR-T cell-mediated cytotoxic potency against 3D prostate tumor spheroids. We were able to kinetically evaluate the efficacy and therapeutic value of PSMA CAR-T cells by analyzing the cytotoxicity against prostate tumor spheroids. In addition, the CAR-T cells were fluorescently labeled to visually identify the location of the T cells as cytotoxicity occurs, which may provide more meaningful information for assessing the functionality of the CAR-T cells. The proposed image cytometry method can overcome limitations placed on traditional methodologies to effectively assess cell-mediated 3D tumor spheroid cytotoxicity and efficiently generate time- and dose-dependent results.

Prognostic impact of circulating tumor cells in oligometastatic hormone-sensitive prostate cancer following metastasis-directed therapy.

199 Background: Contained within the spectrum of metastatic cancer is an oligometastatic state where metastases are limited in number. Recent prospective data have shown that metastasis directed therapy (MDT) can alter the natural history of oligometastatic disease. In hormone-sensitive prostate cancer (HSPC), the positive clinical effect of MDT has been observed by the phase II STOMP and ORIOLE trials. Circulating tumor cells (CTCs) are a likely origin of the formation of macroscopic metastases. CTCs thus may provide an approach for identifying subgroups of patients with oligometastatic HSPC (oligoHSPC) that would benefit most from MDT. Our main goal was to evaluate the association between presence of CTCs at baseline or day 180 with clinical outcomes in the ORIOLE trial. Methods: ORIOLE was a phase II trial randomizing men with recurrent oligoHSPC with 1-3 metastases to observation (Obs) versus stereotactic ablative radiotherapy (SABR) MDT. Blood samples were prospectively collected at baseline and day 180. Progression-free survival (PFS) was a composite endpoint including any of the following: a PSA rise of at least 2 ng/dL and 25% above nadir; radiologic progression by CT, MRI or bone scan (RECIST v1.1); symptomatic progression of disease; initiation of ADT; or death. Patient blood samples were shipped for analysis on Epic Sciences liquid biopsy platform (Epic Sciences, San Diego, CA). Machine learning algorithms identified CTCs and characterized androgen receptor (AR) expression. Comparisons of patient and tumor characteristics between the groups were performed by two-sample t-tests. Survival curves were generated using the Kaplan-Meier method and p-values were calculated using log-rank test. Analysis was performed using SPSS version 28. Results: A total of 82 samples were collected: 70 from the SABR arm (35 baseline and 35 on day 180) and 12 Obs (7 baseline and 5 on day 180). CTCs were detected in 30/42 samples at baseline (71%, AR+= 7) and in 26/40 samples on day 180 (65%, AR+= 9). In the SABR group there was no difference between CTC+ versus CTC- and AR + versus AR- groups for PSA evaluated at baseline, day 90 or day 180 or Gleason score. With a median follow-up of 41.7 months, PFS was significantly lower in the patients with AR+ versus AR- CTCs at baseline in the SABR arm (p = 0.011, mean PFS: AR+ = 9.3 months, AR- = 27.1 months). The mean biochemical failure-free survival was AR+= 12.9 versus AR-= 29.2 months (p = 0.058). Conclusions: Our preliminary results demonstrate an association between AR+ CTCs at baseline and Day 180 with clinical outcomes following SABR MDT in oligoHSPC. This is the first report examining baseline and dynamic presence of CTCs in oligoHSPC treated from a prospective randomized trial of SABR. Longer follow-up, further analysis and a greater number of patients are needed for a more comprehensive conclusion.

Efficacy of cabazitaxel and androgen splicing variant-7 status in circulating tumor cells in Asian patients with metastatic castration-resistant prostate cancer

Androgen receptor splice variant-7 (AR-V7) expression in circulating tumor cells (CTCs) in metastatic castration-resistant prostate cancer (mCRPC) is associated with abiraterone and enzalutamide resistance. We determine whether cabazitaxel (CBZ) is equally effective in AR-V7-positive and -negative CRPC and whether AR-V7-positive patients retain CBZ sensitivity. This is the first prospective, open-label, Asian validation study of CBZ in Japanese patients with mCRPC after docetaxel (n = 48; four CBZ cycles; 2017–2020, Juntendo University Hospitals). Primary endpoint was prostate-specific antigen response rate (PSA-RR); secondary endpoints included overall survival (OS), bone scan index (BSI) PSA-RR (≥ 50% decline from baseline) for CTC−/ARV7−, CTC+ /ARV7−, and CTC +/ARV7+ groups. PSA-RR ≥ − 30% was 38% (18/48) and ≥ − 50% was 26% (12/48). BSI-change rate ≥ − 30% was 19% (9/41) and ≥ − 50% was 17% (8/41). Median OS was 13.7(12.2–18.9) months. PSA decline in early CBZ treatment associated with OS (p = 0.00173). BSI decline associated with OS (p = 0.0194). PSA-RR(≥ 50%) was 43%(6/14) in CTC−/ARV7−, 19%(5/26) in CTC+ ARV7−, and 12%(1/8) in CTC+/ARV7+ ( p > 0.05). AR-V7 in CTCs at baseline not associated with OS. AR-V7 was not associated with CBZ resistance in CTCs. Reductions in BSI and PSA in early stages of CBZ treatment may predict OS.

Ribonuclease 4 is associated with aggressiveness and progression of prostate cancer

Prostate specific antigen screening has resulted in a decrease in prostate cancer-related deaths. However, it also has led to over-treatment affecting the quality of life of many patients. New biomarkers are needed to distinguish prostate cancer from benign prostate hyperplasia (BPH) and to predict aggressiveness of the disease. Here, we report that ribonuclease 4 (RNASE4) serves as such a biomarker as well as a therapeutic target. RNASE4 protein level in the plasma is elevated in prostate cancer patients and is positively correlated with disease stage, grade, and Gleason score. Plasma RNASE4 level can be used to predict biopsy outcome and to enhance diagnosis accuracy. RNASE4 protein in prostate cancer tissues is enhanced and can differentiate prostate cancer and BPH. RNASE4 stimulates prostate cancer cell proliferation, induces tumor angiogenesis, and activates receptor tyrosine kinase AXL as well as AKT and S6K. An RNASE4-specific monoclonal antibody inhibits the growth of xenograft human prostate cancer cell tumors in athymic mice.

Protein Kinase A (PKA) as a Master Regulator of the Early Metabolic Reprogramming in Bone Metastatic Prostate Cancer Cells

PURPOSE The arrival of metastatic prostate cancer (PCa) tumor cells to the bone niche requires a metabolic adaptation. We sought to identify metabolic dysregulations fueling PCa metastasis, modulated by bone secreted factors. METHODS By an indirect co-culture system of PCa (PC3) and bone progenitors (MC3T3, pre-osteoblasts, or Raw 264.7, pre-osteoclasts) we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. We validated the transcriptional profile of metabolic genes in open-access transcriptomic datasets. We performed an Ingenuity Pathway Analysis (IPA) to delineate the regulators of these metabolic genes. Bone secretome was profiled on the conditioned media (CM) by ESI-MS/MS. RESULTS PC3 cells co-cultured with bone progenitors displayed an activation of lipidic categories, including PPAR-signaling and fat absorption/digestion. Principal Component and Unsupervised Clustering analyses using transcriptomic data from human PCa and bone metastatic samples (GSE74685) showed that the metabolic genes deregulated in PC3 accurately clustered samples in primary tumor or bone metastasis. Moreover, four lipid-associated genes, PPARA, VDR, SLC16A1 and GPX1, were associated with a shorter survival time (SU2C-PCF dataset), and were independent risk-predictors of death ( P < .05). An IPA revealed that these genes are regulated by the Protein Kinase A (PKA). Accordingly, PC3 cells treated with the CM of the co-culture presented a decreased ATP content compared to the treatment with the CM of PC3 grown alone, which was restored upon PKA inhibition. Finally, the secretome analysis revealed soluble factors secreted by bone progenitors (Col1a1, Fn1) which could regulate PKA activity. CONCLUSION We identified a novel lipid-associated gene signature important for metastatic PCa, triggered by the dialogue with bone cells. Moreover, PKA could regulate this signature in response to bone-secreted factors reprogramming the metabolic phenotype of metastatic cells, emerging as a potential druggable target for this disease.

Combined spinal and general anesthesia attenuate tumor promoting effects of surgery. An experimental animal study

BackgroundRadical prostatectomy, a standard management approach for localized Prostate Cancer (PC), may cause a stress response associated with immune modulating effects. Regional anesthesia was hypothesized to reduce the immune effects of surgery by minimizing the neuroendocrine surgical stress response, thus mitigating tumor cells dissemination. Our primary objective was to investigate whether the use of spinal blocks attenuates PC tumor cells dissemination on an animal model. We also assessed the number of circulating NK cells and the amount of inflammatory and anti-inflammatory cytokines. Materials and methodsA subcutaneous tumor model, with PC-3M cell line transfected with a luciferase-producing gene (PC-3M-luc-C6) was used. After proper tumor establishment and before tumors became metastatic, animals were submitted to tumor excision surgeries under general or combined (general and spinal) anesthesia. A control group was only anesthetized with general anesthesia. ResultsThe subcutaneous tumor model with PC-3M-luc-C6 cells was effective in causing distant metastasis after 35 days. The number of circulating tumor cells increased in animals that underwent surgery under general anesthesia alone compared to the group submitted to combined anesthesia. Interleukin 6 levels were different in all groups, with increase in the general anesthesia group. ConclusionOur results suggest that combination of spinal and general anesthesia may attenuate the suppression of innate tumor immunity and it might be related to a reduction in the neuroendocrine response to surgery. Institutional protocol numberAnimal Ethics Committee 1332/2019.

Macrophages connection: aggressive nanomechanical phenotypes of circulating tumor cells in prostate cancer

Metastasis is the major cause of cancer patients' death. The process of metastasis starts with intravasation of tumor epithelial cells into a blood stream through which they reach their receptive destinations as circulating tumor cells (CTCs) to seed new cancer sites. Since CTCs are poorly prepared to survive the shear stress of blood flow, they develop a range of strategies to survive the hostile environment. We show that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy.