Proliferation of Mouse Prostate Cancer Cells Inflamed by Trichomonas vaginalis

Our objective was to investigate whether inflammatory microenvironment induced by Trichomonas vaginalis infection can stimulate proliferation of prostate cancer (PCa) cells in vitro and in vivo mouse experiments. The production of CXCL1 and CCL2 increased when cells of the mouse PCa cells (TRAMP-C2 cell line) were infected with live T. vaginalis. T. vaginalis-conditioned medium (TCM) prepared from co-culture of PCa cells and T. vaginalis increased PCa cells migration, proliferation and invasion. The cytokine receptors (CXCR2, CCR2, gp130) were expressed higher on the PCa cells treated with TCM. Pretreatment of PCa cells with antibodies to these cytokine receptors significantly reduced the proliferation, mobility and invasiveness of PCa cells, indicating that TCM has its effect through cytokine-cytokine receptor signaling. In C57BL/6 mice, the prostates injected with T. vaginalis mixed PCa cells were larger than those injected with PCa cells alone after 4 weeks. Expression of epithelial-mesenchymal transition markers and cyclin D1 in the prostate tissue injected with T. vaginalis mixed PCa cells increased than those of PCa cells alone. Collectively, it was suggested that inflammatory reactions by T. vaginalis-stimulated PCa cells increase the proliferation and invasion of PCa cells through cytokine-cytokine receptor signaling pathways.

Involvement of Macrophages in Proliferation of Prostate Cancer Cells Infected with Trichomonas vaginalis

Macrophages play a key role in chronic inflammation, and are the most abundant immune cells in the tumor microenvironment. We investigated whether an interaction between inflamed prostate cancer cells stimulated with Trichomonas vaginalis and macrophages stimulates the proliferation of the cancer cells. Conditioned medium was prepared from T. vaginalis-infected (TCM) and uninfected (CM) mouse prostate cancer (PCa) cell line (TRAMP-C2 cells). Thereafter conditioned medium was prepared from macrophages (J774A.1 cell line) after incubation with CM (MCM) or TCM (MTCM). When TRAMP-C2 cells were stimulated with T. vaginalis, protein and mRNA levels of CXCL1 and CCL2 increased, and migration of macrophages toward TCM was more extensive than towards CM. Macrophages stimulated with TCM produced higher levels of CCL2, IL-6, TNF-α, their mRNAs than macrophages stimulated with CM. MTCM stimulated the proliferation and invasiveness of TRAMP-C2 cells as well as the expression of cytokine receptors (CCR2, GP130, CXCR2). Importantly, blocking of each cytokine receptors with anti-cytokine receptor antibody significantly reduced the proliferation and invasiveness of TRAMP-C2 cells. We conclude that inflammatory mediators released by TRAMP-C2 cells in response to infection by T. vaginalis stimulate the migration and activation of macrophages and the activated macrophages stimulate the proliferation and invasiveness of the TRAMP-C2 cells via cytokine-cytokine receptor binding. Our results therefore suggested that macrophages contribute to the exacerbation of PCa due to inflammation of prostate cancer cells reacted with T. vaginalis.

Osteopontin Deficiency Ameliorates Prostatic Fibrosis and Inflammation

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

PMP(Porphyrin-Micelle-PSMA) Nanoparticles for Photoacoustic and Ultrasound Signal Amplification in Mouse Prostate Cancer Xenografts

Photoacoustic (PA) imaging is used widely in cancer diagnosis. However, the availability of PA agents has not made great progress due to the limitations of the one currently in use, porphyrin. Porphyrin–Micelle (PM), developed by synthesizing porphyrin and PEG-3.5k, confirmed the amplification of the PA agent signal, and added binding affinity in an LNCaP model by attaching prostate-specific membrane antigen PSMA. Compared to the previously used porphyrin, a superior signal was confirmed, and the potential of PMP was confirmed when it showed a signal superior to that of hemoglobin at the same concentration. In addition, in the in vivo mouse experiment, it was confirmed that the signal in the LNCaP xenograft model was stronger than that in the PC-3 xenograft model, and the PMP signal was about three times higher than that of PM and porphyrin.

OSTEOPONTIN DEFICIENCY LEADS TO THE RESOLUTION OF PROSTATIC FIBROSIS AND INFLAMMATION

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

ATM/NEMO signaling modulates the expression of PD-L1 following docetaxel chemotherapy in prostate cancer

BackgroundThe efficacy of docetaxel-based chemotherapy is limited by the development of drug resistance. Recent studies demonstrated the efficacy of anti-programmed death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) immunotherapies in metastatic prostate cancer. The ataxia telangiectasia mutation (ATM) protein plays a crucial role in maintaining genome stability and function of mitosis. Here, we aimed to determine whether PD-1/PD-L1 signaling contributes to the resistance to DTX and to elucidate the mechanism underlying DTX-induced PD-L1 expression.MethodsIn this retrospective study, PD-L1 expression was analyzed in 33 tumor tissue samples from prostate cancer patients. Prostate cell lines were used to perform functional assays and examine underlying mechanisms in vitro. A fully mouse prostate cancer model and a humanized chimeric mouse bearing human prostate tumors and peripheral blood mononuclear cells were used for in vivo assays.ResultsWe have shown that DTX, a chemotherapeutic drug which causing microtubule interference, could significantly induce the expression of PD-L1 in prostate cancer cells. This effect is blocked by the inhibition of ATM, suggesting that it plays an essential role in PD-L1 expression upregulated by DTX. Mechanistic studies have shown that ATM activity in cancer cells enhances the stability of the NF-κB essential modulator (NEMO), which leading to an increase in the NF-κB activity and PD-L1 expression. Using the mouse model, it was further demonstrated that a combination of ATM and NEMO inhibitors along with DTX augmented the antitumor efficacy of chemotherapy, which are comparable to that of PD-L1 antibody.ConclusionsOur findings have revealed that a previously unrecognized ATM-NEMO signaling which induced by DTX is capable of suppressing tumor immunity by activating the expression of PD-L1, suggesting that the ATM-NEMO-NF-κB axis can be exploited to restore the immune balance and overcome cancer resistance triggered by DTX.Graphic Abstract: supplementary file 1

A neuroanatomical mechanism linking perinatal TCDD exposure to lower urinary tract dysfunction in adulthood

Benign Prostatic Hyperplasia / Lower Urinary Tract Dysfunction (BPH/LUTD) is a classic disease of aging which affects nearly all men. Symptoms typically present in the fifth or sixth decade and progressively worsen over the remainder of life. Here, we identify a surprising origin of this disease that traces back to the intrauterine environment of the developing male, challenging existing paradigms about when this disease process begins. We delivered a single bolus dose of a widespread environmental contaminant, present in the serum of most Americans (2,3,7,8 tetrachlorodibenzo-p-dioxin, TCDD, 1 µg/kg), and representative of a broader class of environmental contaminants, to pregnant mice and observed an increase in the abundance of a neurotrophic factor, artemin, in the developing mouse prostate. Artemin is required for noradrenergic axon recruitment across multiple tissues and TCDD rapidly increases prostatic noradrenergic axon density in the male fetus. The hyperinnervation does not resolve, but rather persists into adulthood, when it is coupled to autonomic hyperactivity of prostatic smooth muscle and abnormal urinary function, including increased urinary frequency, a bothersome symptom in men. We offer new evidence that prostate neuroanatomical development is malleable and that intrauterine chemical exposures can permanently reprogram prostate neuromuscular function to cause male LUTD in adulthood.

In silico Computed Clusters of Prostate Luminal Progenitors Match FACS-Enriched LSCmed cells

Several groups recently published single-cell (sc) expression atlases of the adult mouse prostate cells based on RNA sequencing (scRNA-seq) data. All studies identified one computerized cluster of non-secretory luminal progenitor cells enriched in luminal and stemness-related gene transcripts. The actual correspondence between these luminal progenitor cell clusters has not been investigated. In addition, the presence of Krt4 (encoding cytokeratin 4) in these in silico-identified luminal progenitors suggested the overlap with FACS-enriched LSCmed luminal progenitor cells earlier identified as a stem-like, castration-tolerant and tumor-initiating cell population. Here, we used a unified bioinformatics pipeline to re-analyze published prostate scRNA-seq datasets and perform various pan-transcriptomic comparisons including the LSCmed cell signature. Our study demonstrates that i) the mouse prostate luminal progenitor cell clusters identified in the different scRNA-seq studies largely overlap and can be defined by a common 15-gene signature including Krt4, ii) mouse LSCmed cells match both mouse and human luminal progenitors identified by scRNA-seq analysis. Bridging these in silico-identified and ex vivo-characterized prostate luminal progenitor subsets should benefit our understanding of their actual involvement in prostate diseases.

Dietary Tomato Inhibits Tumor Angiogenesis in the TRAMP Model of Prostate Cancer but Is Not Protective With a Western-Style Diet

Objectives The objective of this study was to investigate the interplay between tomato powder (TP), incorporated into control (CON) and obesogenic (OB) diets, and PCa tumor growth and blood perfusion over time in a transgenic model of PCa. We hypothesized that TP would be protective against PCa growth. Methods Diets (either CON [17.2% kcal from fat] or OB [44.6% kcal from fat] both with and without 10% TP) were fed to transgenic adenocarcinoma of the mouse prostate (TRAMP) mice (n = 5/dietary group) from weaning. Tumor growth was monitored by weekly ultrasound scanning, at which time novel ultrasound microvessel images (UMI) were captured to quantitatively measure tumor blood perfusion over time. Animals were euthanized after 5 weeks of tumor growth, and tissues were collected for measurement of protein (HIF-1α, TNFα) and gene (ar, srd5a1, srd5a2) expression. Data were analyzed to determine differences between CON diets (without TP + with 10% TP) and OB diets (without TP + with 10% TP) and to evaluate the impact of 10% TP on outcome measures both independent of TP (No TP vs. TP) as well as within CON and OB diets (interaction effect of diet*TP) by mixed model ANCOVA with body weight as a covariate. Results UMI results showed good agreement with gold-standard immunohistochemistry quantification of endothelial cell density, indicating that this technique can be applied to non-invasively and longitudinally monitor tumor blood perfusion in vivo. Greater body weight (P = 0.029) and OB diets (P = 0.008) were associated with earlier age at tumor detection, and greater body weight was positively associated with tumor growth (P = 0.001). TP significantly inhibited prostate tumor angiogenesis (P = 0.043), but this inhibition differentially affected measured outcomes depending on CON or OB diets. TP led to reduced tumor growth (P = 0.004), intratumoral inflammation (TNFα; P = 0.019), and intratumoral androgen-regulated gene expression (srd5a1, srd5a2; P = 0.030 and P = 0.016, respectively) when incorporated with the CON diet but greater tumor growth and intratumoral gene expression when incorporated with the OB diet. Conclusions Results from this study show that protective benefits from dietary tomato are lost, or may become deleterious, when combined with a Western-style diet. Funding Sources CA was supported by the NIH NIBIB. MRL and PS were partially supported by NIH NCI.

Establishment of An Orthotopic Prostate Cancer Xenograft Mouse Model Using Microscope-Guided Orthotopic Injection of LNCaP Cells Into The Dorsal Lobe of The Mouse Prostate

Background: Orthotopic LNCaP xenograft mouse models closely mimic the progression of androgen-dependent prostate cancer in humans; however, orthotopic injection of LNCaP cells into the mouse prostate remains a challenge.Methods: Under the guidance of a stereoscopic microscope, the anatomy of the individual prostate lobes in male Balb/c athymic nude mice was investigated, and LNCaP cells were inoculated into the mouse dorsal prostate (DP) to generate orthotopic tumors that mimicked the pathophysiological process of prostate cancer in humans. Real-time ultrasound imaging was used to monitor orthotopic prostate tumorigenesis, contrast-enhanced ultrasonography (CEUS) was used to characterize tumor angiogenesis, and macroscopic and microscopic characteristics of tumors were described.Results: The DP had a trigonal bipyramid-shape and were located at the base of the seminal vesicles. After orthotopic inoculation, gray scale ultrasound imaging showed progressive changes in tumor echotexture, shape and location, and tumors tended to protrude into the bladder. After 8 weeks, the tumor take rate was 65% (n= 13/20 mice). On CEUS, signal intensity increased rapidly, peaked, and decreased gradually. Observations of gross specimens showed orthotopic prostate tumors were well circumscribed, round, dark brown, and soft, with a smooth outer surface and a glossy appearance. Microscopically, tumor cells were arranged in acini encircled by fibrous septa with variably thickened walls, mimicking human adenocarcinoma.Conclusions: This study describes a successful approach to establishing an orthotopic LNCaP xenograft Balb/c athymic nude mouse model. The model requires a thorough understanding of mouse prostate anatomy and proper technique. The model represents a valuable tool for the in vivo study of the biological processes involved in angiogenesis in prostate cancer and preclinical evaluations of novel anti-angiogenic therapies.