Combination of novel intravesical xenogeneic urothelial cell immunotherapy and chemotherapy enhances anti-tumor efficacy in preclinical murine bladder tumor models.

BACKGROUND: Immune checkpoint inhibitors induce robust and durable responses in advanced bladder cancer (BC), but only for a subset of patients. Xenovaccination has been proposed as an effective immunotherapeutic approach to induce anti-tumor immunity. Thus, we proposed a novel intravesical xenogeneic urothelial cell immunotherapy strategy to treat advanced BC based on the hypothesis that implanted xenogeneic urothelial cells not only provoke xeno-rejection immune responses but also elicit bystander anti-tumor immunity. METHODS: Mouse advanced bladder cancer models were treated with vehicle control, intravesical xenogeneic urothelial cells, cisplatin + gemcitabine, or the combination and assessed for tumor responses to treatments. Tumors and spleens samples were collected for immunohistological staining, cellular and molecular analysis assessed by antibody staining, ELISA, cytotoxicity, and flow cytometry, respectively. RESULTS: The combination treatment of xenogeneic urothelial cell immunotherapy with chemotherapy was more efficacious than either single therapy to extend survival time in MBT-2 graft bladder tumor model and to suppress tumor progression in murine carcinogen BBN-induced bladder tumor model. The single-cell immunotherapy and combined therapy increased more tumor-infiltrating immune cells in MBT-2 graft tumors compared to vehicle control and chemotherapy treatment groups. The activated T-cell proliferation, cytokine production, and cytotoxicity capacities were also higher in mice with xenogeneic urothelial cell immunotherapy and combination treatments. CONCLUSIONS: Our results suggest the potential for a novel xenogeneic urothelial cell-based immunotherapy alone and synergy with chemotherapy in the combination therapy. Therefore, our study supports developing xenogeneic urothelial cells as an immunotherapeutic agent in combination with chemotherapy for BC treatment.

Xenogeneic cell therapy provides a novel potential therapeutic option for cancers by restoring tissue function, repairing cancer wound and reviving anti-tumor immune responses.

Conventional cancer treatments such as surgery, radiotherapy, chemotherapy and targeted therapy, not only destruct tumors, but also injure the normal tissues, resulting in limited efficacy. Recent advances in cancer therapy have aimed at changing the host milieu of cancer against its development and progression by targeting tumor microenvironment and host immune system to eradicate tumors. To the host body, tumors arise in tissues. They impair the normal healthy tissue physiological function, become chronically inflamed and develop non-healing or overhealing wounds as well as drive immuno-suppressive activity to escape immunity attack. Therefore, the rational therapeutic strategies for cancers should treat both the tumors and the host body for the best efficacy to turn the deadly malignant disease to a manageable one. Xenogeneic cell therapy (i.e. cellular xenotransplantation) using cells from non-human source animals such as pigs has shown promising results in animal studies and clinical xenotransplantation in restoring lost tissue physiological function and repairing the wound. However, the major hurdle of xenogeneic cell therapy is the host immunological barriers that are induced by transplanted xenogeneic cells to reject xenografts. Possibly, the immunological barriers of xenogeneic cells could be used as immunological boosters to activate the host immune system. Here, we hypothesized that because of the biological properties of xenogeneic cells to the recipient humans, the transplantation of xenogeneic cells (i.e. cellular xenotransplantation) into cancer patients' organs of the same origin with developed tumors may restore the impaired function of organs, repair the wound, reduce chronic inflammation and revive the anti-tumor immunity to achieve beneficial outcome for patients.

The anti-tumor effect of intravesical administration of normal urothelial cells on bladder cancer.

BACKGROUND AIMS: Urothelial bladder cancer (UBC) is the second most common cancer of the genitourinary tract and for advanced forms of the disease it has a high mortality rate. There are no approved new molecularly targeted agents or chemotherapeutics for advanced UBC beyond cisplatin-based chemotherapy except the recently approved anti-programmed death ligand 1 (anti-PD-1/PD-L1) antibody. With complex genetic and epigenetic alterations in tumors, despite several druggable targets identified, to cure UBC is still a challenging unmet medical need. Like other cancers, UBC to the host body is considered as a wound, aging stem cell disease and immunosuppressive disorder. Therefore, we proposed a novel cellular approach to target the host body by intravesical instilling of normal urothelial cells that could repair the injury and reduce inflammation by activating body-reparative capacity and because non-self cells are transplanted, host body immune responses could be induced in the tumor microenvironment of UBC to restrain and even eliminate tumor cells. METHODS: In this study, we isolated and expanded normal male murine urothelial cells and intravesically administered them into the bladders of female mice of two orthotopic bladder tumor models and one urothelial injury model. RESULTS: We showed that the instillation of normal urothelial cells containing stem/progenitor cell population into bladders could have anti-tumor effect in orthotopic tumor models, possibly by activating immune responses and helping injured urothelium tissue recovery in a chemically induced urothelial injury model. CONCLUSIONS: Our findings could lead to an innovative and revolutionary cell therapy modality with normal urothelial cells as an effective and safe therapeutic option for UBC.

Anabolic androgens affect the competitive interactions in cell migration and adhesion between normal mouse urothelial cells and urothelial carcinoma cells.

The urothelium is constantly rebuilt by normal urothelial cells to regenerate damaged tissues caused by stimuli in urine. However, the urothelial carcinoma cells expand the territory by aberrant growth of tumor cells, which migrate and occupy the damaged tissues to spread outside and disrupt the normal cells and organized tissues and form a tumor. Therefore, the interaction between normal urothelial cells and urothelial carcinoma cells affect the initiation and progression of urothelial tumors if normal urothelial cells fail to migrate and adhere to the damages sites to regenerate the tissues. Here, comparing normal murine urothelial cells with murine urothelial carcinoma cells (MBT-2), we found that normal cells had less migration ability than carcinoma cells. And in our co-culture system we found that carcinoma cells had propensity migrating toward normal urothelial cells and carcinoma cells had more advantages to adhere than normal cells. To reverse this condition, we used anabolic androgen, dihyrotestosterone (DHT) to treat normal cells and found that DHT treatment increased the migration ability of normal urothelial cells toward carcinoma cells and the adhesion capacity in competition with carcinoma cells. This study provides the base of a novel therapeutic approach by using anabolic hormone-enforced normal urothelial cells to regenerate the damage urothelium and defend against the occupancy of carcinoma cells to thwart cancer development and recurrence.

Decreased tumorigenesis and mortality from bladder cancer in mice lacking urothelial androgen receptor.

Much fewer mice lacking androgen receptor (AR) in the entire body develop bladder cancer (BCa). However, the role of urothelial AR (Uro-AR) in BCa development remains unclear. In the present study, we generated mice that lacked only Uro-AR (Uro-AR(-/y)) to develop BCa by using the carcinogen BBN [N-butyl-N-(4-hydroxybutyl)-nitrosamine] and found that Uro-AR(-/y) mice had a lower incidence of BCa and a higher survival rate than did their wild-type (WT; Uro-AR(+/y)) littermates. In vitro assay also demonstrated that Uro-AR facilitates the neoplastic transformation of normal urothelial cells to carcinoma. IHC staining exhibited less DNA damage, with much higher expression of p53 and its downstream target protein PNCA in Uro-AR(-/y) than that found in WT urothelium, which suggests that Uro-AR may modulate bladder tumorigenesis through p53-PCNA DNA repair signaling. Indeed, Uro-AR(-/y) mice with the transgene, simian vacuolating virus 40 T (SV40T), in the urothelium (Uro-SV40T-AR(-/y)) had a similar incidence of BCa as did their WT littermates (Uro-SV40T-AR(+/y)), and p53 was inactivated by SV40T in both genotypes. Use of the AR degradation enhancer ASC-J9 led to suppression of bladder tumorigenesis, with few adverse effects in the BBN-induced BCa mouse model. Together, these results provide the first direct in vivo evidence that Uro-AR has an important role in promoting bladder tumorigenesis and BCa progression. Targeting AR with ASC-J9 may provide a novel approach to suppress BCa initiation.

Anti-tumor activity of intratumoral xenogeneic urothelial cell monotherapy or in combination with chemotherapy in syngeneic murine models of bladder cancer.

Advanced bladder cancer is still an area of high unmet need even with the use of immune checkpoint inhibitors and antibody drug conjugates. Therefore, transformatively novel therapeutic approaches are needed. Xenogeneic cells are capable of inducing potent innate and adaptive immune rejection responses, which properties could turn xenogeneic cells into an immunotherapeutic agent. Here, we investigated the anti-tumor effects of intratumoral xenogeneic urothelial cell (XUC) immunotherapy alone and in combination with chemotherapy in two murine syngeneic models of bladder cancer. In both bladder tumor models, intratumoral XUC treatment suppressed tumor growth, and the efficacy was enhanced with chemotherapy. The experiments on mode of action for intratumoral XUC treatment found that the remarkable local and systemic anti-tumor effects were achieved with significant intratumoral immune cell infiltration and systemic activation of immune cell cytotoxic activity, cytokine IFNγ production and proliferation ability. The intratumoral XUC alone and combined treatment increased T cell natural killer cell infiltration into tumors. In the bilateral tumor model with intratumoral XUC monotherapy or combined therapy, the uninjected tumors at the other side also simultaneously demonstrated significant tumor growth delay. Consequently, intratumoral XUC treatment alone and the combination resulted in elevated chemokine CXCL9/10/11 levels. These data suggest that intratumoral XUC therapy may be useful in the treatment of advanced bladder cancer as a local therapy that injects xenogeneic cells into either primary or distant tumors. By exerting both local and systemic anti-tumor effects, this new treatment would complete the comprehensive cancer management along with systemic approaches.

Immunotherapeutic Agents for Intratumoral Immunotherapy.

Immunotherapy using systemic immune checkpoint inhibitors (ICI) and chimeric antigen receptor (CAR) T cells has revolutionized cancer treatment, but it only benefits a subset of patients. Systemic immunotherapies cause severe autoimmune toxicities and cytokine storms. Immune-related adverse events (irAEs) plus the immunosuppressive tumor microenvironment (TME) have been linked to the inefficacy of systemic immunotherapy. Intratumoral immunotherapy that increases immunotherapeutic agent bioavailability inside tumors could enhance the efficacy of immunotherapies and reduce systemic toxicities. In preclinical and clinical studies, intratumoral administration of immunostimulatory agents from small molecules to xenogeneic cells has demonstrated antitumor effects not only on the injected tumors but also against noninjected lesions. Herein, we review and discuss the results of these approaches in preclinical models and clinical trials to build the landscape of intratumoral immunotherapeutic agents and we describe how they stimulate the body's immune system to trigger antitumor immunity as well as the challenges in clinical practice. Systemic and intratumoral combination immunotherapy would make the best use of the body's immune system to treat cancers. Combining precision medicine and immunotherapy in cancer treatment would treat both the mutated targets in tumors and the weakened body's immune system simultaneously, exerting maximum effects of the medical intervention.

Effects of hepatocyte growth factor on porcine mammary cell growth and senescence.

Background: The porcine mammary glands share morphological and physiological similarities with human ones, making primary porcine mammary cells (PMC) suitable for biomedical research and a potential cellular therapeutic for breast cancer xenogeneic cell immunotherapy. Primary cells isolated from tissues remain the physiological functions of origin tissues but their self-renewal ability is restricted and cells acquire senescence during in vitro expansion. To overcome these drawbacks, here we sought to establish an approach to efficiently increase PMC's in vitro growth. We studied the effects of the hepatocyte growth factor (HGF) to maintain the expansion capacity of porcine mammary cells and identify the possible mechanisms. Purpose: HGF could allow for the increase in vitro proliferation capacity of primary epithelial cells isolated from tissue samples. To effectively produce cells for biomedical research and xenogeneic cell therapy, we planned to study the effects of HGF and its potential mechanisms of action to stimulate cell growth for PMC expansion. Methods: After HGF treatment, the growth, cell cycle, senescence and the cell marker gene expression of PMCs were analyzed in standard 10% FBS and low serum 1% FBS containing medium. Results: HGF significantly enhanced the cell proliferation by shifting the cell cycle population from G1 phase into S phase to increase cell division, reduced the senescent cells and reprogrammed gene expression profiles. Conclusion: We demonstrated that HGF could maintain the expansion capacity of PMCs by increasing cell growth and anti-senescence capability, suggesting its potential application in optimizing the long-term culture of primary cells. Adding a specific growth factor such as HGF in culture allows enhanced expansion of heterogeneous cell populations from normal porcine mammary glandular tissues in vitro. We believe that this cell culture approach will efficiently provide cells for studying mammary cell function and supply cells for therapeutic uses.

Increased acetylation in the DNA-binding domain of TR4 nuclear receptor by the coregulator ARA55 leads to suppression of TR4 transactivation.

The nuclear receptor TR4 is a key regulator for many physiological processes, including growth, development, and metabolism. However, how the transcriptional activity of TR4 is regulated in the absence of ligand(s) remains largely unknown. Here we found that an androgen receptor (AR) coactivator, ARA55, might function as a corepressor to suppress TR4 transactivation. Molecular mechanistic dissection with mutation analysis found that ARA55 could enhance TR4 acetylation at the conserved acetylation sites of lysine 175 and lysine 176 in the DNA-binding domain via recruiting proteins with histone acetyl transferase activity, which might then reduce significantly the TR4 DNA binding activity that resulted in the suppression of TR4 transactivation. These results are in contrast to the classic ARA55 coactivator function to enhance AR transactivation partially via increased AR acetylation in the hinge/ligand-binding domain. Together, these results not only provide a novel functional mechanism showing that acetylation of different nuclear receptors at different domains by coregulator may lead to differential receptor transactivation activity but also provide a new way for small molecules to control TR4 transactivation via altering TR4 acetylation levels, and such small molecules may have potential therapeutic applications in the future.

Altered prostate epithelial development in mice lacking the androgen receptor in stromal fibroblasts.

BACKGROUND: Androgens and the androgen receptor (AR) play important roles in the development of male urogenital organs. We previously found that mice with total AR knockout (ARKO) and epithelial ARKO failed to develop normal prostate with loss of differentiation. We have recently knocked out AR gene in smooth muscle cells and found the reduced luminal infolding and IGF-1 production in the mouse prostate. However, AR roles of stromal fibroblasts in prostate development remain unclear. METHODS: To further probe the stromal fibroblast AR roles in prostate development, we generated tissue-selective knockout mice with the AR gene deleted in stromal fibroblasts (FSP-ARKO). We also used primary culture stromal cells to confirm the in vivo data and investigate mechanisms related to prostate development. RESULTS: The results showed cellular alterations in the FSP-ARKO mouse prostate with decreased epithelial proliferation, increased apoptosis, and decreased collagen composition. Further mechanistic studies demonstrated that FSP-ARKO mice have defects in the expression of prostate stromal growth factors. To further confirm these in vivo findings, we prepared primary cultured mouse prostate stromal cells and found knocking down the stromal AR could result in growth retardation of prostate stromal cells and co-cultured prostate epithelial cells, as well as decrease of some stromal growth factors. CONCLUSIONS: Our FSP-ARKO mice not only provide the first in vivo evidence in Cre-loxP knockout system for the requirement of stromal fibroblast AR to maintain the normal development of the prostate, but may also suggest the selective knockdown of stromal AR might become a potential therapeutic approach to battle prostate hyperplasia and cancer.

Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis.

BACKGROUND: Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. METHODS: We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. RESULTS: We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. CONCLUSIONS: Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

Intratumoral xenogeneic tissue-specific cell immunotherapy inhibits tumor growth by increasing antitumor immunity in murine triple negative breast and pancreatic tumor models.

Low immunogenicity in tumors and the immunosuppressive tumor microenvironment (TME) represent major obstacles to the full success of immunotherapy in cancer patients. A novel intratumoral xenogeneic tissue-specific cell immunotherapeutic approach could overcome the obstacles. Murine 4T1 triple negative breast cancer (TNBC) cells and Pan18 pancreatic ductal adenocarcinoma (PDAC) cells were used for establishing syngeneic graft tumor models to evaluate antitumor effect of intratumoral injection of xenogeneic tissue-specific cells. Responses to treatment were assessed by measuring tumor growth and tumor weight of the tumor-bearing mice. To investigate the mechanisms of action, tumor histology and immunohistochemistry and cytokine gene expression were measured. Splenic lymphocytes proliferation, cytokine production and cytotoxicity activities were also assessed. The findings showed that intratumoral injection of xenogeneic tissue-specific cells in monotherapy and combination with chemotherapy inhibit tumor growth. The therapeutic efficacy of intratumoral xenogeneic cells was significantly enhanced by the addition of cytotoxic chemotherapeutic agents. Mice that received combined treatment showed maximal attenuation in tumor growth rate. The antitumor immunity was explained by altered immune cell infiltration in tumors and immune cell functions. Our findings demonstrate that xenogeneic tissue-specific cells given intratumorally, provide a potent antitumor effect in murine breast and pancreatic tumor models by enhancing recruitment and activation of immune cells in tumors for local and systemic antitumor effects. Moreover, intratumoral xenogeneic cell treatment turns immunologically "cold" tumors to "hot" ones, generates systemic antitumor immunity, and synergizes with chemotherapy. Thus, the intratumoral xenogeneic tissue-specific cell immunotherapy may represent a useful therapeutic option to difficult-to-treat cancers.

The reduced trabecular bone mass of adult ARKO male mice results from the decreased osteogenic differentiation of bone marrow stroma cells.

Male mice with androgen receptor knock-out (ARKO) show significant bone loss at a young age. However, the lasting effect of AR inactivation on bone in aging male mice remains unclear. We designed this study to evaluate the effect of AR on bone quality in aging male mice and to find the possible causes of AR inactivation contributing to the bone loss. The mice were grouped according to their ages and AR status and their trabecular bones were examined by micro-CT analysis at 6, 12, 18, and 30 weeks old. We found that bone mass consistently decreased and the bone microarchitectures continuously deteriorated in male ARKO mice at designated time points. To determine the cause of the bone loss in ARKO mice, we further examined the role of AR in bone cell fate decision and differentiation and we conducted experiments on bone marrow stromal cells (BMSC) obtained from wild type (WT) and AR knockout (KO) mice. We found that ARKO mice had higher numbers of colony formation unit-fibroblast (CFU-F), and CD44 and CD34 positive cells in bone marrow than WT mice. Our Q-RT-PCR results showed lower expression of genes linked to osteogenesis in BMSCs isolated from ARKO mice. In conclusion, AR nullification disrupted bone microarchitecture and caused trabecular bone mass loss in male ARKO mice. And the fate of BMSCs was impacted by the loss of AR. Therefore, these findings suggest that AR may accelerate the use of progenitor cells and direct them into osteogenic differentiation to affect bone metabolism.

Utilizing Xenogeneic Cells As a Therapeutic Agent for Treating Diseases.

The utilization of biologically produced cells to treat diseases is a revolutionary invention in modern medicine after chemically synthesized small molecule drugs and biochemically made protein drugs. Cells are basic units of life with diverse functions in mature and developing organs, which biological properties could be utilized as a promising therapeutic approach for currently intractable and incurable diseases. Xenogeneic cell therapy utilizing animal cells other than human for medicinal purpose has been studied as a new way of treating diseases. Xenogeneic cell therapy is considered as a potential regenerative approach to fulfill current unmet medical needs because xenogeneic cells could be isolated from different animal organs and expanded ex vivo as well as maintain the characteristics of original organs, providing a versatile and plenty cell source for cell-based therapeutics beside autologous and allogeneic sources. The swine species is considered the most suitable source because of the similarity with humans in size and physiology of many organs in addition to the economic and ethical reasons plus the possibility of genetic modification. This review discusses the old proposed uses of xenogeneic cells such as xenogeneic pancreatic islet cells, hepatocytes and neuronal cells as a living drug for the treatment of degenerative and organ failure diseases. Novel applications of xenogeneic mesenchymal stroma cells and urothelial cells are also discussed. There are formidable immunological barriers toward successful cellular xenotransplantation in clinic despite major progress in the development of novel immunosuppression regimens and genetically multimodified donor pigs. However, immunological barriers could be turn into immune boosters by using xenogeneic cells of specific tissue types as a novel immunotherapeutic agent to elicit bystander antitumor immunity due to rejection immune responses. Xenogeneic cells have the potential to become a safe and efficacious option for intractable diseases and hard-to-treat cancers, adding a new class of cellular medicine in our drug armamentarium.

Tumor suppressor PAX6 functions as androgen receptor co-repressor to inhibit prostate cancer growth.

BACKGROUND: PAX6, a transcription factor, has currently been suggested to function as a tumor suppressor in glioblastoma and to act as an early differentiation marker for neuroendocrine cells. The androgen receptor (AR) plays a pivotal role in prostate cancer development and progression due to its transcriptional activity in regulating genes involved in cell growth, differentiation, and apoptosis. To determine the role of PAX6 in prostate cancer, we investigated whether PAX6 interacts with AR to affect prostate cancer development. METHODS: We used immunostaining, RT-PCR, and Western blotting assays to show the expression status of PAX6 in prostate tissue and human prostate cancer cell lines. The role of PAX6 in cell growth and colony regeneration potential of LNCaP cells were evaluated by MTT assay and soft agar assay with PAX6-overexpressed LNCaP cells. Mammalian two-hybrid and co-immunoprecipitation (Co-IP) assays were used to demonstrate the interaction between PAX6 and AR. Reporter gene and Q-RT-PCR assays were performed to determine the effects of PAX6 on the function of AR. RESULTS: In prostate cancer tissues, PAX6 expression was stronger in normal epithelial cells than cancer cells, and decreased in LNCaP cells compared to that of DU145 and PC3 cells. Enforced expression of PAX6 suppressed the cell growth of LNCaP cells and also inhibited the colony formation of LNCaP cells. PAX 6 interacted with AR and repressed its transcriptional activity. PAX6 overexpression decreased the expression of androgen target gene PSA in LNCaP cells. CONCLUSIONS: In this study, we found that PAX6 may act as a prostate cancer repressor by interacting with AR and repressing the transcriptional activity and target gene expression of AR to regulate cell growth and regeneration.

Tumor-associated Macrophages Facilitate Bladder Cancer Progression by Increasing Cell Growth, Migration, Invasion and Cytokine Expression.

BACKGROUND/AIM: Interactions between stromal and tumor cells in tumor microenvironment contribute to tumor progression. In bladder cancer (BCa), infiltration of macrophages in tumors correlates with cancer progression. Herein, the aim was to study the paracrine effects of tumor-associated macrophages (TAM) on BCa cells. MATERIALS AND METHODS: The correlation between TAMs and tumor grade and stages was examined in tumor tissue microarrays. In addition, a conditioned media (CM) model was employed to investigate the paracrine effects of macrophages on BCa cell growth, migration, and invasion, as well as on the cytokine profile of each cell line. RESULTS: The correlation of tumor-infiltrating macrophages with high-grade and muscle-invasive BCa was demonstrated in human bladder tumor tissue microarrays. CM from co-cultures of macrophages and BCa cells increased BCa cell growth, migration and invasion. Moreover, higher mRNA and protein expression levels of CCL5 and IL-8 were found in cells and CM from co-cultures, respectively. CONCLUSION: The paracrine interaction between BCa cells and TAMs led to enhanced BCa cell growth, migration, and invasiveness, and moreover, increased IL-8 and CCL5 cytokine production in tumor microenvironment.

Androgen dihydrotestosterone (DHT) promotes the bladder cancer nuclear AR-negative cell invasion via a newly identified membrane androgen receptor (mAR-SLC39A9)-mediated Gαi protein/MAPK/MMP9 intracellular signaling.

While androgens may function via nuclear androgen receptor (nAR) to increase bladder cancer (BCa) progression, the impact of androgens on muscle invasive BCa, which contains nearly 80% nAR-negative cells, remains unclear. To dissect the androgens potential impacts on these nAR-negative muscle invasive BCa, we first found that the androgens, dihydrotestosterone (DHT) might function via a novel membrane AR (mAR-SLC39A9) to increase nAR-negative BCa cell migration and invasion. Mechanism dissection revealed that DHT/mAR-SLC39A9 might function by altering Gαi protein-mediated MAPK/MMP9 intracellular signaling to increase nAR-negative BCa cell migration and invasion. Preclinical studies using multiple in vitro nAR-negative BCa cell lines and an in vivo mouse model all demonstrated that targeting this newly identified DHT/mAR-SLC39A9/Gαi/MAPK/MMP9 signaling with small molecules mAR-SLC39A9-shRNA or Gαi-shRNA, and not the classic antiandrogens including enzalutamide, bicalutamide, or hydroxyflutamide, could suppress nAR-negative BCa cell invasion. Results from human clinical samples surveys also indicated the positive correlation of this newly identified DHT/mAR signaling with BCa progression and prognosis. Together, these results suggest that androgens may not only function via the classic nAR to increase the nAR-positive BCa cell invasion, they may also function via this newly identified mAR-SLC39A9 to increase the nAR-negative/mAR-positive BCa cell invasion.

Intravesical Administration of Xenogeneic Porcine Urothelial Cells Attenuates Cyclophosphamide-Induced Cystitis in Mice.

The urothelium of the bladder, renal pelvis, ureter and urethra is maintained through the regulated proliferation and differentiation of urothelial stem and progenitor cells. These cells provide a rich source of a novel urothelial cell therapy approach that could be used to protect, regenerate, repair and restore a damaged urothelium. Urothelial injury caused by physical, chemical and microbial stress is the pathological basis of cystitis (bladder inflammation). The loss of urothelial integrity triggers a series of inflammatory events, resulting in pain and hematuria such as hemorrhage cystitis and interstitial cystitis. Here we investigate a novel cell therapy strategy to treat cystitis by protecting the urothelium from detrimental stresses through intravesically instilling porcine urothelial cells (PUCs) into the bladder. Using a chemical-induced urothelial injury mouse model of cyclophosphamide (CPP)-induced hemorrhagic cystitis, we determined how the intravesical instillation of PUCs could protect the urothelium from toxic attack from CPP metabolites. We show that intravesical PUC instillation protected the bladder from toxic chemical attack in mice receiving CPP with reduced inflammation and edema. Compared with the vehicle control mice, the proliferative response to chemical injury and apoptotic cells within the bladder tissues were reduced by intravesical PUC treatment. Furthermore, the urothelium integrity was maintained in the intravesical PUC-treated group. After xenogeneic PUCs were introduced and adhered to the mouse urothelium, immunological rejection responses were observed with increased neutrophil infiltration in the lamina propria and higher immune-related gene expression. Our findings provide an innovative and promising intravesical PUC cell therapy for cystitis with urothelial injury by protecting the urothelium from noxious agents.

Targeted inactivation of testicular nuclear orphan receptor 4 delays and disrupts late meiotic prophase and subsequent meiotic divisions of spermatogenesis.

Testicular orphan nuclear receptor 4 (TR4) is specifically and stage-dependently expressed in late-stage pachytene spermatocytes and round spermatids. In the developing mouse testis, the highest expression of TR4 can be detected at postnatal days 16 to 21 when the first wave of spermatogenesis progresses to late meiotic prophase. Using a knockout strategy to delete TR4 in mice, we found that sperm production in TR4(-/-) mice is reduced. The comparison of testes from developing TR4(+/+) and TR4(-/-) mice shows that spermatogenesis in TR4(-/-) mice is delayed. Analysis of the first wave of spermatogenesis shows that the delay can be due to delay and disruption of spermatogenesis at the end of late meiotic prophase and subsequent meiotic divisions. Seminiferous tubule staging shows that stages X to XII, where late meiotic prophase and meiotic divisions take place, are delayed and disrupted in TR4(-/-) mice. Histological examination of testis sections from TR4(-/-) mice shows degenerated primary spermatocytes and some necrotic tubules. Testis-specific gene analyses show that the expression of sperm 1 and cyclin A1, which are genes expressed at the end of meiotic prophase, was delayed and decreased in TR4(-/-) mouse testes. Taken together, results from TR4(+/+) and TR4(-/-) mice indicate that TR4 is essential for normal spermatogenesis in mice.

Spermatogenesis and testis development are normal in mice lacking testicular orphan nuclear receptor 2.

Early in vitro cell culture studies suggested that testicular orphan nuclear receptor 2 (TR2), a member of the nuclear receptor superfamily, may play important roles in the control of several pathways including retinoic acids, vitamin D, thyroid hormones, and ciliary neurotrophic factor. Here we report the surprising results showing that mice lacking TR2 are viable and have no serious developmental defects. Male mice lacking TR2 have functional testes, including normal sperm number and motility, and both male and female mice lacking TR2 are fertile. In heterozygous TR2(+/-) male mice we found that beta-galactosidase, the indicator of TR2 protein expression, was first detected at the age of 3 weeks and its expression pattern was restricted mainly in the spermatocytes and round spermatids. These protein expression patterns were further confirmed with Northern blot analysis of TR2 mRNA expression. Together, results from TR2-knockout mice suggest that TR2 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. Alternatively, the roles of TR2 may be redundant and could be played by other close members of the nuclear receptor superfamily such as testicular orphan receptor 4 (TR4) or unidentified orphan receptors that share many similar functions with TR2. Further studies with double knockouts of both orphan nuclear receptors, TR2 and TR4, may reveal their real physiological roles.