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.

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.

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.

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.

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.

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.

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.

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.

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 Genomic Alterations of 5α-Reductases and Their Inhibitor Finasteride's Effect in Bladder Cancer.

BACKGROUND/AIM: Since androgens affect urothelial bladder cancer (UBC), we examined whether 5α-reductases (5-AR) have genomic alterations in UBC and whether 5α-reductase inhibitors (5-ARI) affect UBC. MATERIALS AND METHODS: The cBioPortal was used to analyze genomic alternations of 5-ARs in UBC cancer genomic datasets. Next, we used the Taiwan National Health Insurance Research database to conduct a population-based case-control study to investigate the effect of a 5-ARI, finasteride on UBC incidence. We also performed an XTT assay to examine the direct effect of finasteride on UBC cells. RESULTS: We found that 5-AR genomic alternations were observed in 29% of UBC patients and patients with alternations had shorter disease-free survival. Also, the use of finasteride with >180 cDDDs reduced the risk of UBC. Finasteride could directly inhibit UBC cell growth. CONCLUSION: Based on our findings, we concluded that 5-AR could be explored as a therapeutic target for UBC with 5-ARIs.

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.

Androgen receptor facilitates the recruitment of macrophages in tumor microenvironment to promote upper urinary tract urothelial cell carcinoma progression.

Interactions between infiltrating macrophages in the tumor microenvironment (TME) and tumor cells contribute to tumor progression. The potential impacts of recruited macrophages to the upper urinary tract urothelial cell carcinomas (UUTUCs) progression remain unclear. Here we found human UUTUCs might recruit more macrophages than surrounding normal urothelial cells in human clinical specimens and in in vitro co-culture experiments with UUTUC cells and macrophages. The consequences of recruiting more macrophages to UUTUCs might then enhance UUTUC cell growth, migration and invasion. Further investigation found that the androgen receptor (AR) not only enhanced UUTUC cells capacity to recruit more macrophages, it could also promote the macrophages-enhanced UUTUC cells growth, migration and invasion. Downstream AR target cytokine search found AR might function through modulating CCL5 expression to influence UTTUC progression. Interruption of CCL5 partially reversed the AR-regulated macrophage-enhanced UUTUC progression. AR in UUTUC cells also increased tumor formation in vivo. Taken together, these results suggest that macrophages recruitment may enhance UUTUC progression, modulated by AR-CCL5 signal through alterations in chromatin state to establish a tumor microenvironment with recruited macrophages and cytokines to facilitate cell growth, migration and invasion.

Androgen receptor expands the population of cancer stem cells in upper urinary tract urothelial cell carcinoma cells.

Androgen receptor (AR) affects the development and progression of upper urinary tract urothelial cell carcinoma (UUTUC). However, the regulatory mechanism exerted by AR to affect UUTUC cells remains unclear. Here we investigated whether AR promotes UUTUC development and progression, possibly by expanding the population of cancer stem cells (CSCs), which are a particular population of cells within cancer cells responsible for tumor initiation, drug resistance and metastasis. We compared UUTUC cells with or without the addition of AR on their CSC population with flow cytometry, colony formation and sphere formation assay to determine the effect of AR on CSC activity, and real-time PCR was used to detect the expression stemness genes and miRNAs. In vivo tumor formation was evaluated with the implantation of cancer cells in nude mice. We found that the addition of AR in UUTUC cells, significantly increased the population of CSC, clonogenicity, sphere formation and the expression of stemness genes (Oct4, Bmi1 and Nanog), altered CSC-related miRNA profile, as well as promoted epithelial mesenchymal transition (EMT). And AR inhibitor, enzalutamide was shown to suppress AR's effect on tumorsphere formation. Furthermore, in an immune-deficient mouse model, the addition of AR in UUTUC cells also increased the tumor formation capacity. This study will help us better understand the extent to which AR contributes to UUTUC progression by expanding their CSC population and capacity. Our findings could explain high incidence of UUTUC observed in males. And targeting AR may lead to novel therapeutic approaches for genetically diversified urothelial carcinomas in precision medicine era.

Antiandrogen Therapy with Hydroxyflutamide or Androgen Receptor Degradation Enhancer ASC-J9 Enhances BCG Efficacy to Better Suppress Bladder Cancer Progression.

Recent studies suggest that the androgen receptor (AR) might play important roles in influencing bladder cancer progression, yet its clinical application remains unclear. Here, we developed a new combined therapy with Bacillus Calmette-Guérin (BCG) and the AR degradation enhancer ASC-J9 or antiandrogen hydroxyflutamide (HF) to better suppress bladder cancer progression. Mechanism dissection revealed that ASC-J9 treatment enhanced BCG efficacy to suppress bladder cancer cell proliferation via increasing the recruitment of monocytes/macrophages that involved the promotion of BCG attachment/internalization to the bladder cancer cells through increased integrin-α5ß1 expression and IL6 release. Such consequences might then enhance BCG-induced bladder cancer cell death via increased TNFα release. Interestingly, we also found that ASC-J9 treatment could directly promote BCG-induced HMGB1 release to enhance the BCG cytotoxic effects for suppression of bladder cancer cell growth. In vivo approaches also concluded that ASC-J9 could enhance the efficacy of BCG to better suppress bladder cancer progression in BBN-induced bladder cancer mouse models. Together, these results suggest that the newly developed therapy combining BCG plus ASC-J9 may become a novel therapy to better suppress bladder cancer progress.

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.

Androgen receptor increases CD133 expression and progenitor-like population that associate with cisplatin resistance in endometrial cancer cell line.

Endometrial cancer (EMC) is a sex steroid hormone-related female malignancy. Androgen and androgen receptor (androgen/AR) signals have been implicated in EMC progression. Cancer stem/progenitor cells (CSPCs) are suspected to link to chemoresistance in patients with EMC. In this study, we examined the androgen/AR roles in cisplatin resistance and CSPC population. We found AR expression increased naive EMC side population, CSPC population, cell migration, and epithelial-mesenchymal transition. Meanwhile, it decreased cisplatin cytotoxic effect on EMC cells. Collaterally, endogenous AR expressions in EMC cells were upregulated in the cisplatin-resisting state. Moreover, AR expression could further enhance CD133 expression, CSPC-related markers, and drug-resistance gene messenger RNA expression in EMC cells. Finally, the AR-associated gene expression might go through indirect regulation. This is the first report revealing AR function on EMC cells' CSPC and cisplatin resistance.

Epidermal growth factor enhances androgen receptor­mediated bladder cancer progression and invasion via potentiation of AR transactivation.

Androgen receptor (AR) plays a critical role in bladder cancer (BCa) development. Our early studies found AR knock-out mice (with few androgens and deleted AR) failed to develop BCa, yet 50% of castrated mice (with few androgens and existing AR) still developed BCa in an N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) carcinogen-induced BCa mouse model, suggesting the existing AR in BCa of castrated mice may still play important roles in promoting BCa development at the castration level of androgens. The mechanism underlying this and/or which factors potentiate AR function at the castration level of androgen remains unclear. Epidermal growth factor (EGF), a key player in BCa progression, has been demonstrated to be able to potentiate AR transactivation in prostate cancer. In the present study, we found that EGF could increase BCa cell growth, migration and invasion in the presence of AR under the low amount of androgen and EGF was able to potentiate AR transactivation through EGFR by activating PI3K/AKT and MAPK pathway at castration androgen level. The increased suppression effects by EGFR inhibitor of PD168393 on AR function after addition of anti-androgen, Casodex, further suggested AR might play a key role in the effects of EGF on BCa progression and metastasis. Collectively, our results indicate that EGF may be able to potentiate AR transactivation that leads to enhancing BCa progression, which may help us to develop a better therapeutic approach to treat BCa via targeting both EGF and AR signaling.

MicroRNA-21 promotes the ovarian teratocarcinoma PA1 cell line by sustaining cancer stem/progenitor populations in vitro.

INTRODUCTION: Resistance of cancer stem/progenitor cells (CSPCs) to chemotherapy can lead to cancer relapse. Ovarian teratocarcinoma (OVTC) arises from germ cells and comprises pluripotent cells that can be used to study cancer cell stemness. In this study, we evaluated whether microRNA-21 (miR-21) promotes ovarian teratocarcinoma by maintaining cancer stem/progenitor populations. METHODS: The lentiviral delivery system was used to upregulate or to suppress the expression of miR-21 in the human ovarian teratocarcinoma cell line PA1 and cell growth assays were used to monitor the expression of miR-21 at different time points. Antibodies directed toward CD133, a stem cell marker, were used to identify CSPCs in the PA1 cell population, and the level of miR-21 expression was determined in enriched CSPCs. Stem cell functional assays (sphere assay and assays for CD133 expression) were used to assess the effects of miR-21 on progression of the CD133+ population. RESULTS: Knockdown of miR-21 in PA1 cells attenuated growth of PA1 cells whereas overexpression of miR-21 promoted cell growth. Moreover, knockdown of miR-21 resulted in a marked reduction in the CD133+ population and sphere formation of CSPCs. In contrast, overexpression of miR-21 resulted in a marked increase in the population of CD133+ cells as well as sphere formation of CSPCs. CONCLUSIONS: MicroRNA-21 plays a significant role in cancer growth by regulating stemness in cancer cells.

Androgen receptor promotes the migration and invasion of upper urinary tract urothelial carcinoma cells through the upregulation of MMP-9 and COX-2.

Dysregulated androgen receptor (AR) signaling is implicated in several types of tumor, including carcinomas of the prostate, breast, liver and bladder. However, the contribution of AR to the progression of upper urinary tract urothelial carcinomas (UUTUC) has not been fully investigated. In the present study, we demonstrated that the AR is involved in the metastasis and invasiveness of UUTUC cells. We investigated the role of the AR in UUTUC by using UUTUC-derived BFTC 909 cells. The overexpression of AR promotes the migration and invasion of BFTC 909 cells. Expression of migration/invasion-related genes was increased in BFTC 909 cells overexpressing AR determined by qPCR and western blot analyses. The results showed that AR-enhanced migration and invasion of UUTUC cells are linked to the upregulation of the matrix-degrading enzyme MMP-9 and cyclooxygenase (COX)-2. Subsequently, the blocking of MMP-9 and COX-2 signaling by inhibitors suppressed AR-enhanced cell migration and invasion. The results of the present study provide evidence for the first time of the role of AR in the motility and invasion of UUT cancer cells and support the hypothesis that the AR may play a critical role in the establishment of the invasive phenotype in urothelial neoplasia of UUT. Thus, the AR may also serve as a novel biomarker and potential therapeutic target for UUT cancer.