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Papers by Igor Moskalev

Urologic Oncology: Seminars and Original Investigations

Urologic Oncology-seminars and Original Investigations, 2017

The Journal of Urology

INTRODUCTION AND OBJECTIVES: We previously reported that high MET and matriptase expression in RC... more INTRODUCTION AND OBJECTIVES: We previously reported that high MET and matriptase expression in RCC cells in bone metastasis indicates their importance in bone metastasis (Mukai et al. Hum Cell, 2015). MET is a high-affinity receptor tyrosine kinase of hepatocyte growth factor (HGF). HGF is secreted as an inactive singlechain precursor, which requires proteolytic activation for conversion to an active form. Matriptase is the most efficient known cellular activator of pro-HGF. Furthermore, activation of matriptase is regulated by HGF activator inhibitor (HAI). In this study, we employed a previously reported mouse model of bone metastasis (Strube et al. Clin Exp Metastasis, 2010) to clarify the significance of the matriptase-induced HGF/MET signaling axis in RCC bone metastasis. METHODS: Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of female nude mice (5 weeks old). After 6 weeks, we confirmed the formation of bone metastasis by whole-body bioluminescent imaging, and extracted specimens. Expression of matriptase, MET and HAI was analyzed by PCR, immunohistochemistry (IHC) and immunoblots. Phosphorylation of MET was also investigated. Based on the result, we produced HAI-2 (specific inhibitor of matriptase) stable knock down (KD) 786-O cells, and analyzed the difference of expression in each molecule, cell-migration assay and invasion assay. RESULTS: Expression of matriptase was increased significantly in bone metastasis compared with parent cell line, and we confirmed increased phosphorylation of MET in bone metastasis. On the other hand, decreased expression of HAI-2 was observed in bone metastasis. Interestingly, increased matriptase expression was observed by HAI-2 KD in 786-O cells. In addition, invasive activity was increased significantly by knock down of HAI-2. CONCLUSIONS: These results have suggested that matriptase contributes to the HGF-dependent MET activation in the pericellular microenvironment of bone metastasis in RCC. In addition, upregulation of matriptase and downregulation of HAI-2 may have important roles in their progression.

Cancer Research, 2016

Purpose of study To develop a reproducible and reliable orthopic xenograft mouse model for Suniti... more Purpose of study To develop a reproducible and reliable orthopic xenograft mouse model for Sunitinib resistant renal cell carcinoma (RCC). Introduction Clear-cell renal cell carcinoma (ccRCC) is the most common malignancy of the kidneys and has been steadily increasing at a rate of 3% yearly over the last decade. Furthermore, 30% of patients with ccRCC have de novo metastatic disease. Until the last decade, immunotherapy was the only option for metastatic ccRCC treatment. Current first-line systemic therapies for metastatic ccRCC all primarily target the angiogenesis pathway via tyrosine kinase inhibition (TKI) and have increased progression free survival dramatically. However, durable responses are rare, drug resistance invariably arises after short-term disease stabilization and metastatic ccRCC remains almost uniformly lethal. There is currently no animal model for TKI resistance in RCC. We developed a reliable and reproducible orthotopic xenograft mouse model of Suntinib resistance in RCC. Methods Luciferin expressing-CAKI-1 human renal cancer cells were injected under the renal capsule of athymic nude mice using ultrasound guidance. After initial tumor growth mice were treated with Sunitinib malate at an initial dose of 40mg/kg which was increased sequentially (60 and 80 mg/kg) at each tumor passage (Fig.1). Tumors were monitored twice weekly by ultrasound and bioluminescence (IVIS system). Tumors exceeding 200mm3 by ultrasound or 10⁁10 photons/sec by IVIS were considered end points. Before passaging, tumors were harvested and immediately dissociated with a Dispase/Collagenase mix. 5×105 cells were injected into the following batch of mice, considering all these steps as one cycle or passage. Tumors were passaged with increasing doses of Sunitinib 5 times. Results Tumor take rates were above 95% in each cycle. Initial treatment of Sunitinib at 40mg/kg resulted in a decrease in tumor size via ultrasound and IVIS of about 30%. At around 14 days of treatment tumors started to regrow despite further treatment. In the second cycle tumors did not show a response to a subsequent treatment of 40mg/kg, proving resistance. Even after increasing the dose of Suntinib in the following cycles to 60 and 80 mg/kg respectively, there was no response observed. The fifth cycle (100mg/kg) had to be terminated due to severe side effects in the animals (skin coloring, weight loss), however no decrease in tumor size was observed. We therefore created a Sunitinib resistance model in the range of the maximal tolerated long-term dose in vivo. Conclusions This study describes the first orthotopic xenograft renal cell carcinoma Sunitinib resistant mouse model created in situ. The development of Sunitinib resistance in an angiogenic environment establishes a more realistic and translational model, which opens the possibility to study not only the mechanism of Sunitinib resistance but also to find alternative therapies to overcome Sunitinib resistance. Citation Format: Sebastian K. Frees, Igor Moskalev, Betty Zhou, Peter Raven, Claudia Chavez-Munoz, Peter Black, Alan I. So. An orthotopic xenograft renal cell carcinoma Sunitinib resistant murine model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5191.

The Journal of Urology, 2016

BCG treatment. A total of 13 patients (7 BCG responders and 6 non responders) were included. BCG ... more BCG treatment. A total of 13 patients (7 BCG responders and 6 non responders) were included. BCG response was defined by no recurrence neither progression for at least 24 month after therapy. Hematoxilin-eosin (HE) and immunohistochemical stains were performed at the pathology department, using monoclonal antibodies specific either for the Th2 associated transcription factor GATA-3(L50-823) or the Th1-associated transcription factor T-bet (H-210). Number of peritumoral GATA-3+ and T-bet + were determined. The ratio Th2/ Th1(GATA-3+/T-bet +) was calculated. Results obtained from these metric were correlated with response to treatment. Statistical analysis for comparisions between groups was performed. The median values were compared using a nonparametric Mann Whitney test. RESULTS: Variable lymphocytic response was found in peritumoral stroma in all cases. Patients who responded to BCG (R) had a higher Th2 infiltration (GATA 3+ cells) than patients who did not respond (NR) (median149.5AE34 and 91.70AE18 cells, respectively) and a minor presence of Th1 cells (T-bet +) (median 30.33 AE8.9 and 48.60AE8.7 cells, respectively). The GATA 3+/T-bet + (Th2/Th1) ratio was higher in responders than in non responders (4.9 and 2.3, respectively). CONCLUSIONS: The presence of an infiltrating Th-1 type immune response in peritumoral tissue before BCG therapy may be associated with lack of response to treatment. Determining the immune polarization (Th1 vs Th2) could be useful as a prognostic marker to BCG therapy in these patients.

European Urology Supplements, 2015

Purpose: Unilateral ureteral obstruction halts ureteral peristalsis, and may cause pain and lead ... more Purpose: Unilateral ureteral obstruction halts ureteral peristalsis, and may cause pain and lead to infection. Ureteral ability to recover after obstruction removal remains unclear. Erythropoietin has protective effects in nonhematopoietic organs and restores peristalsis in hypocontractile intestinal smooth muscle cells. We investigated the role of erythropoietin in ureteral smooth muscle function and its therapeutic value for unilateral ureteral obstruction. Materials and Methods: Unilateral ureteral obstruction was created for 24, 48 and 72 hours in 22 mice per group using a nontraumatic microclip via laparotomy. We determined erythropoietin, erythropoietin receptor and b-common receptor expression in obstructed and unobstructed ureters by reverse transcriptasepolymerase chain reaction and immunohistochemistry. Ten mice per group received 20 IU erythropoietin for 4 days and controls received saline. Hydronephrosis regression after obstruction removal was assessed by ultrasound. Peristalsis was determined microscopically before and after obstruction removal. Results: Erythropoietin, erythropoietin receptor and b-common receptor were expressed in the unobstructed and obstructed ureters of untreated mice. Erythropoietin mRNA was up-regulated in response to obstruction and erythropoietin expression was identified in ureteral smooth muscle. After obstruction removal hydronephrosis and ureteral dysfunction correlated with obstruction duration. Hydronephrosis resolution and ureteral peristalsis restoration were significantly accelerated in erythropoietin treated mice compared to controls. Conclusions: Erythropoietin treatment significantly promoted functional recovery of the ureter after obstruction removal. Erythropoietin may be a helpful strategy for ureteral motility recovery and hydronephrosis resolution in ureteral obstruction.

As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolve... more As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

The Journal of Urology, 2014

Urologic Oncology: Seminars and Original Investigations

Prostate Cancer and Prostatic Diseases

Molecular cancer therapeutics, Jan 27, 2018

The significance of lactate transporters has been recognized in various cancer types, but their r... more The significance of lactate transporters has been recognized in various cancer types, but their role in urothelial carcinoma (UC) remains mostly unknown. The aim of the present study was to investigate the functional importance of the monocarboxylate transporter (MCT) 4 in preclinical models of UC and to assess its relevance in patient tumors. The association of MCT4 expression with molecular subtypes and outcome was determined in The Cancer Genome Atlas (TCGA) cohort and two independent cohorts of patients with UC. Silencing of MCT4 was performed using small-interfering RNAs in UC cell lines. Effects of MCT4 inhibition on cell growth, apoptosis and production of reactive oxygen species were assessed. Moreover, effects on lactate efflux were determined. The in vivo effects of MCT4 silencing were assessed in an orthotopic xenograft model. MCT4 expression was higher in the basal subtype. Decreased MCT4 methylation, and increased RNA and protein expression were associated with worse ov...

Oncotarget, Jan 20, 2018

Bone metastasis is an important prognostic factor in renal cell carcinoma (RCC). The calcium-sens... more Bone metastasis is an important prognostic factor in renal cell carcinoma (RCC). The calcium-sensing receptor (CaSR) has been associated with bone metastasis in several different malignancies. We analyzed the impact of CaSR in bone metastasis in RCC and . The RCC cell line 786-O was stably transfected with the gene and treated with calcium alone or in combination with the CaSR antagonist NPS2143. Afterwards migration, adhesion, proliferation and prominent signaling molecules were analyzed. Calcium treated -transfected 768-O cells showed an increased adhesion to endothelial cells and the extracellular matrix components fibronectin and collagen I, but not to collagen IV. The chemotactic cell migration and proliferation was also induced by calcium. The activity of SHC, AKT, ERK, P90RSK and JNK were enhanced after calcium treatment of -transfected cells. These effects were abolished by NPS2143. Development of bone metastasis was evaluated in a mouse model. Intracardiac injection of CaSR...

Oncotarget, Jan 27, 2018

Clear-cell renal cell carcinoma (ccRCC) is the sixth most common malignancy in men in North Ameri... more Clear-cell renal cell carcinoma (ccRCC) is the sixth most common malignancy in men in North America. Since ccRCC is a malignancy dependent on neovascularization, current first line systemic therapies like sunitinib, target the formation of new vessels allowing nutrient deprivation and cell death. However, recent studies have shown that patients develop resistance after approximately 1 year of treatment and show disease progression while on therapy. Therefore, we propose to identify the protein(s) responsible for increased migration with the aim of developing a new therapy that will target the identified protein and potentially slow down the progression of the disease. Human renal cancer cell lines (Caki-1, Caki-2, ACHN) were treated with increasing doses of sunitinib to develop a sunitinib-conditioned renal cell carcinoma cell line. mRNA microarray and qPCR were performed to compare the differences in gene expression between Caki-1 sunitinib-conditioned and non-conditioned cells. NT...

Human gene therapy, Jan 19, 2018

Muscle-invasive bladder cancer represents approximately 25% of patients diagnosed with bladder ca... more Muscle-invasive bladder cancer represents approximately 25% of patients diagnosed with bladder cancer and carries a significant risk of death. Oncolytic viruses (OV) are novel antitumor agents with the ability to selectively replicate and lyse tumor cells while sparing healthy tissue. Thus, we explored the efficiency of the oncolytic, YB-1 selective adenovirus XVir-N-31 in vitro and in an orthotopic mouse model for bladder cancer by intramural injection under ultrasound guidance. We demonstrated that XVir-N-31 replicates in bladder cancer cells and induced a stronger immunogenic cell death than adenovirus wildtype by facilitating enhanced release of HMGB1 and exosomal Hsp70. The intratumoral delivery of XVir-N-31 by ultrasound guidance delayed tumor growth in an immunodeficient model, demonstrating the feasibility of this approach to deliver oncolytic viruses directly into the tumor.

Methods in molecular biology (Clifton, N.J.), 2018

Orthotopic mouse models of urothelial cancer are essential for testing novel therapies and molecu... more Orthotopic mouse models of urothelial cancer are essential for testing novel therapies and molecular manipulations of cell lines in vivo. These models are either established by orthotopic inoculation of human (xenograft models) or murine tumor cells (syngeneic models) in immunocompromised or immune competent mice. Current techniques rely on inoculation by intravesical instillation or direct injection into the bladder wall. Alternative models include the induction of murine bladder tumors by chemical carcinogens (BBN) or genetic engineering (GEM).

Scientific reports, Jan 13, 2017

Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in canc... more Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in cancer-related deaths. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted signalling proteins whose normal roles reside in embryogenesis and neuronal development. In this context, semaphorins help establish chemotactic gradients and direct cell movement. Various semaphorin family members have been found to be up- and down-regulated in a number of cancers. One family member, Semaphorin 3 C (SEMA3C), has been implicated in prostate, breast, ovarian, gastric, lung, and pancreatic cancer as well as glioblastoma. Given SEMA3C's roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes epithelial-to-mesenchymal transition...

The Prostate, 2017

The vast majority of prostate cancer presents clinically localized to the prostate without eviden... more The vast majority of prostate cancer presents clinically localized to the prostate without evidence of metastasis. Currently, there are several modalities available to treat this particular disease. Despite radical prostatectomy demonstrating a modest prostate cancer specific mortality benefit in the PIVOT trial, several novel modalities have emerged to treat localized prostate cancer in patients that are either not eligible for surgery or that prefer an alternative approach. Athymic nude mice were subcutaneously inoculated with prostate cancer cells. The mice were divided into four cohorts, one cohort untreated, two cohorts received docetaxel (10 mg/kg) either subcutaneously (SC) or intravenously (IV) and the fourth cohort was treated using the magnetically-actuated docetaxel delivery device (MADDD), dispensing 1.5 μg of docetaxel per 30 min treatment session. Treatment in all three therapeutic arms (SC, IV, and MADDD) was administered once weekly for 6 weeks. Treatment efficacy wa...

European journal of immunology, Feb 1, 2017

Checkpoint blockade of CTLA-4 results in long-lasting survival benefits in metastatic cancer pati... more Checkpoint blockade of CTLA-4 results in long-lasting survival benefits in metastatic cancer patients. However, patients treated with CTLA-4 blockade have suffered from immune-related adverse events, most likely due to the breadth of the induced T-cell activation. Here, we investigated the efficacy of a local low-dose anti-CTLA-4 administration for treatment of subcutaneous or orthotopic murine bladder 49 (MB49) bladder carcinoma in C57BL/6 mice. When MB49 tumors were grown s.c., peritumoral (p.t.) injection of anti-CTLA-4 treatment was equally effective as intravenous or s.c. (nontumor bearing flank) administration. Notably, p.t. injection was associated with lower circulating antibody levels and decreased IL-6 serum levels as compared to systemic treatment. Ultrasound-guided intratumoral anti-CTLA-4 antibody treatment of orthotopically growing MB49 tumors resulted in tumor regression, with more than tenfold reduction in systemic antibody levels as compared to i.v. or s.c. administ...

European urology, Jul 10, 2017

Although cisplatin-based neoadjuvant chemotherapy (NAC) improves survival of unselected patients ... more Although cisplatin-based neoadjuvant chemotherapy (NAC) improves survival of unselected patients with muscle-invasive bladder cancer (MIBC), only a minority responds to therapy and chemoresistance remains a major challenge in this disease setting. To investigate the clinical significance of oncofetal chondroitin sulfate (ofCS) glycosaminoglycan chains in cisplatin-resistant MIBC and to evaluate these as targets for second-line therapy. An ofCS-binding recombinant VAR2CSA protein derived from the malaria parasite Plasmodium falciparum (rVAR2) was used as an in situ, in vitro, and in vivo ofCS-targeting reagent in cisplatin-resistant MIBC. The ofCS expression landscape was analyzed in two independent cohorts of matched pre- and post-NAC-treated MIBC patients. An rVAR2 protein armed with cytotoxic hemiasterlin compounds (rVAR2 drug conjugate [VDC] 886) was evaluated as a novel therapeutic strategy in a xenograft model of cisplatin-resistant MIBC. Antineoplastic effects of targeting ofC...

Urologic Oncology: Seminars and Original Investigations

Urologic Oncology-seminars and Original Investigations, 2017

The Journal of Urology

INTRODUCTION AND OBJECTIVES: We previously reported that high MET and matriptase expression in RC... more INTRODUCTION AND OBJECTIVES: We previously reported that high MET and matriptase expression in RCC cells in bone metastasis indicates their importance in bone metastasis (Mukai et al. Hum Cell, 2015). MET is a high-affinity receptor tyrosine kinase of hepatocyte growth factor (HGF). HGF is secreted as an inactive singlechain precursor, which requires proteolytic activation for conversion to an active form. Matriptase is the most efficient known cellular activator of pro-HGF. Furthermore, activation of matriptase is regulated by HGF activator inhibitor (HAI). In this study, we employed a previously reported mouse model of bone metastasis (Strube et al. Clin Exp Metastasis, 2010) to clarify the significance of the matriptase-induced HGF/MET signaling axis in RCC bone metastasis. METHODS: Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of female nude mice (5 weeks old). After 6 weeks, we confirmed the formation of bone metastasis by whole-body bioluminescent imaging, and extracted specimens. Expression of matriptase, MET and HAI was analyzed by PCR, immunohistochemistry (IHC) and immunoblots. Phosphorylation of MET was also investigated. Based on the result, we produced HAI-2 (specific inhibitor of matriptase) stable knock down (KD) 786-O cells, and analyzed the difference of expression in each molecule, cell-migration assay and invasion assay. RESULTS: Expression of matriptase was increased significantly in bone metastasis compared with parent cell line, and we confirmed increased phosphorylation of MET in bone metastasis. On the other hand, decreased expression of HAI-2 was observed in bone metastasis. Interestingly, increased matriptase expression was observed by HAI-2 KD in 786-O cells. In addition, invasive activity was increased significantly by knock down of HAI-2. CONCLUSIONS: These results have suggested that matriptase contributes to the HGF-dependent MET activation in the pericellular microenvironment of bone metastasis in RCC. In addition, upregulation of matriptase and downregulation of HAI-2 may have important roles in their progression.

Cancer Research, 2016

Purpose of study To develop a reproducible and reliable orthopic xenograft mouse model for Suniti... more Purpose of study To develop a reproducible and reliable orthopic xenograft mouse model for Sunitinib resistant renal cell carcinoma (RCC). Introduction Clear-cell renal cell carcinoma (ccRCC) is the most common malignancy of the kidneys and has been steadily increasing at a rate of 3% yearly over the last decade. Furthermore, 30% of patients with ccRCC have de novo metastatic disease. Until the last decade, immunotherapy was the only option for metastatic ccRCC treatment. Current first-line systemic therapies for metastatic ccRCC all primarily target the angiogenesis pathway via tyrosine kinase inhibition (TKI) and have increased progression free survival dramatically. However, durable responses are rare, drug resistance invariably arises after short-term disease stabilization and metastatic ccRCC remains almost uniformly lethal. There is currently no animal model for TKI resistance in RCC. We developed a reliable and reproducible orthotopic xenograft mouse model of Suntinib resistance in RCC. Methods Luciferin expressing-CAKI-1 human renal cancer cells were injected under the renal capsule of athymic nude mice using ultrasound guidance. After initial tumor growth mice were treated with Sunitinib malate at an initial dose of 40mg/kg which was increased sequentially (60 and 80 mg/kg) at each tumor passage (Fig.1). Tumors were monitored twice weekly by ultrasound and bioluminescence (IVIS system). Tumors exceeding 200mm3 by ultrasound or 10⁁10 photons/sec by IVIS were considered end points. Before passaging, tumors were harvested and immediately dissociated with a Dispase/Collagenase mix. 5×105 cells were injected into the following batch of mice, considering all these steps as one cycle or passage. Tumors were passaged with increasing doses of Sunitinib 5 times. Results Tumor take rates were above 95% in each cycle. Initial treatment of Sunitinib at 40mg/kg resulted in a decrease in tumor size via ultrasound and IVIS of about 30%. At around 14 days of treatment tumors started to regrow despite further treatment. In the second cycle tumors did not show a response to a subsequent treatment of 40mg/kg, proving resistance. Even after increasing the dose of Suntinib in the following cycles to 60 and 80 mg/kg respectively, there was no response observed. The fifth cycle (100mg/kg) had to be terminated due to severe side effects in the animals (skin coloring, weight loss), however no decrease in tumor size was observed. We therefore created a Sunitinib resistance model in the range of the maximal tolerated long-term dose in vivo. Conclusions This study describes the first orthotopic xenograft renal cell carcinoma Sunitinib resistant mouse model created in situ. The development of Sunitinib resistance in an angiogenic environment establishes a more realistic and translational model, which opens the possibility to study not only the mechanism of Sunitinib resistance but also to find alternative therapies to overcome Sunitinib resistance. Citation Format: Sebastian K. Frees, Igor Moskalev, Betty Zhou, Peter Raven, Claudia Chavez-Munoz, Peter Black, Alan I. So. An orthotopic xenograft renal cell carcinoma Sunitinib resistant murine model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5191.

The Journal of Urology, 2016

BCG treatment. A total of 13 patients (7 BCG responders and 6 non responders) were included. BCG ... more BCG treatment. A total of 13 patients (7 BCG responders and 6 non responders) were included. BCG response was defined by no recurrence neither progression for at least 24 month after therapy. Hematoxilin-eosin (HE) and immunohistochemical stains were performed at the pathology department, using monoclonal antibodies specific either for the Th2 associated transcription factor GATA-3(L50-823) or the Th1-associated transcription factor T-bet (H-210). Number of peritumoral GATA-3+ and T-bet + were determined. The ratio Th2/ Th1(GATA-3+/T-bet +) was calculated. Results obtained from these metric were correlated with response to treatment. Statistical analysis for comparisions between groups was performed. The median values were compared using a nonparametric Mann Whitney test. RESULTS: Variable lymphocytic response was found in peritumoral stroma in all cases. Patients who responded to BCG (R) had a higher Th2 infiltration (GATA 3+ cells) than patients who did not respond (NR) (median149.5AE34 and 91.70AE18 cells, respectively) and a minor presence of Th1 cells (T-bet +) (median 30.33 AE8.9 and 48.60AE8.7 cells, respectively). The GATA 3+/T-bet + (Th2/Th1) ratio was higher in responders than in non responders (4.9 and 2.3, respectively). CONCLUSIONS: The presence of an infiltrating Th-1 type immune response in peritumoral tissue before BCG therapy may be associated with lack of response to treatment. Determining the immune polarization (Th1 vs Th2) could be useful as a prognostic marker to BCG therapy in these patients.

European Urology Supplements, 2015

Purpose: Unilateral ureteral obstruction halts ureteral peristalsis, and may cause pain and lead ... more Purpose: Unilateral ureteral obstruction halts ureteral peristalsis, and may cause pain and lead to infection. Ureteral ability to recover after obstruction removal remains unclear. Erythropoietin has protective effects in nonhematopoietic organs and restores peristalsis in hypocontractile intestinal smooth muscle cells. We investigated the role of erythropoietin in ureteral smooth muscle function and its therapeutic value for unilateral ureteral obstruction. Materials and Methods: Unilateral ureteral obstruction was created for 24, 48 and 72 hours in 22 mice per group using a nontraumatic microclip via laparotomy. We determined erythropoietin, erythropoietin receptor and b-common receptor expression in obstructed and unobstructed ureters by reverse transcriptasepolymerase chain reaction and immunohistochemistry. Ten mice per group received 20 IU erythropoietin for 4 days and controls received saline. Hydronephrosis regression after obstruction removal was assessed by ultrasound. Peristalsis was determined microscopically before and after obstruction removal. Results: Erythropoietin, erythropoietin receptor and b-common receptor were expressed in the unobstructed and obstructed ureters of untreated mice. Erythropoietin mRNA was up-regulated in response to obstruction and erythropoietin expression was identified in ureteral smooth muscle. After obstruction removal hydronephrosis and ureteral dysfunction correlated with obstruction duration. Hydronephrosis resolution and ureteral peristalsis restoration were significantly accelerated in erythropoietin treated mice compared to controls. Conclusions: Erythropoietin treatment significantly promoted functional recovery of the ureter after obstruction removal. Erythropoietin may be a helpful strategy for ureteral motility recovery and hydronephrosis resolution in ureteral obstruction.

As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolve... more As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

The Journal of Urology, 2014

Urologic Oncology: Seminars and Original Investigations

Prostate Cancer and Prostatic Diseases

Molecular cancer therapeutics, Jan 27, 2018

The significance of lactate transporters has been recognized in various cancer types, but their r... more The significance of lactate transporters has been recognized in various cancer types, but their role in urothelial carcinoma (UC) remains mostly unknown. The aim of the present study was to investigate the functional importance of the monocarboxylate transporter (MCT) 4 in preclinical models of UC and to assess its relevance in patient tumors. The association of MCT4 expression with molecular subtypes and outcome was determined in The Cancer Genome Atlas (TCGA) cohort and two independent cohorts of patients with UC. Silencing of MCT4 was performed using small-interfering RNAs in UC cell lines. Effects of MCT4 inhibition on cell growth, apoptosis and production of reactive oxygen species were assessed. Moreover, effects on lactate efflux were determined. The in vivo effects of MCT4 silencing were assessed in an orthotopic xenograft model. MCT4 expression was higher in the basal subtype. Decreased MCT4 methylation, and increased RNA and protein expression were associated with worse ov...

Oncotarget, Jan 20, 2018

Bone metastasis is an important prognostic factor in renal cell carcinoma (RCC). The calcium-sens... more Bone metastasis is an important prognostic factor in renal cell carcinoma (RCC). The calcium-sensing receptor (CaSR) has been associated with bone metastasis in several different malignancies. We analyzed the impact of CaSR in bone metastasis in RCC and . The RCC cell line 786-O was stably transfected with the gene and treated with calcium alone or in combination with the CaSR antagonist NPS2143. Afterwards migration, adhesion, proliferation and prominent signaling molecules were analyzed. Calcium treated -transfected 768-O cells showed an increased adhesion to endothelial cells and the extracellular matrix components fibronectin and collagen I, but not to collagen IV. The chemotactic cell migration and proliferation was also induced by calcium. The activity of SHC, AKT, ERK, P90RSK and JNK were enhanced after calcium treatment of -transfected cells. These effects were abolished by NPS2143. Development of bone metastasis was evaluated in a mouse model. Intracardiac injection of CaSR...

Oncotarget, Jan 27, 2018

Clear-cell renal cell carcinoma (ccRCC) is the sixth most common malignancy in men in North Ameri... more Clear-cell renal cell carcinoma (ccRCC) is the sixth most common malignancy in men in North America. Since ccRCC is a malignancy dependent on neovascularization, current first line systemic therapies like sunitinib, target the formation of new vessels allowing nutrient deprivation and cell death. However, recent studies have shown that patients develop resistance after approximately 1 year of treatment and show disease progression while on therapy. Therefore, we propose to identify the protein(s) responsible for increased migration with the aim of developing a new therapy that will target the identified protein and potentially slow down the progression of the disease. Human renal cancer cell lines (Caki-1, Caki-2, ACHN) were treated with increasing doses of sunitinib to develop a sunitinib-conditioned renal cell carcinoma cell line. mRNA microarray and qPCR were performed to compare the differences in gene expression between Caki-1 sunitinib-conditioned and non-conditioned cells. NT...

Human gene therapy, Jan 19, 2018

Muscle-invasive bladder cancer represents approximately 25% of patients diagnosed with bladder ca... more Muscle-invasive bladder cancer represents approximately 25% of patients diagnosed with bladder cancer and carries a significant risk of death. Oncolytic viruses (OV) are novel antitumor agents with the ability to selectively replicate and lyse tumor cells while sparing healthy tissue. Thus, we explored the efficiency of the oncolytic, YB-1 selective adenovirus XVir-N-31 in vitro and in an orthotopic mouse model for bladder cancer by intramural injection under ultrasound guidance. We demonstrated that XVir-N-31 replicates in bladder cancer cells and induced a stronger immunogenic cell death than adenovirus wildtype by facilitating enhanced release of HMGB1 and exosomal Hsp70. The intratumoral delivery of XVir-N-31 by ultrasound guidance delayed tumor growth in an immunodeficient model, demonstrating the feasibility of this approach to deliver oncolytic viruses directly into the tumor.

Methods in molecular biology (Clifton, N.J.), 2018

Orthotopic mouse models of urothelial cancer are essential for testing novel therapies and molecu... more Orthotopic mouse models of urothelial cancer are essential for testing novel therapies and molecular manipulations of cell lines in vivo. These models are either established by orthotopic inoculation of human (xenograft models) or murine tumor cells (syngeneic models) in immunocompromised or immune competent mice. Current techniques rely on inoculation by intravesical instillation or direct injection into the bladder wall. Alternative models include the induction of murine bladder tumors by chemical carcinogens (BBN) or genetic engineering (GEM).

Scientific reports, Jan 13, 2017

Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in canc... more Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in cancer-related deaths. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted signalling proteins whose normal roles reside in embryogenesis and neuronal development. In this context, semaphorins help establish chemotactic gradients and direct cell movement. Various semaphorin family members have been found to be up- and down-regulated in a number of cancers. One family member, Semaphorin 3 C (SEMA3C), has been implicated in prostate, breast, ovarian, gastric, lung, and pancreatic cancer as well as glioblastoma. Given SEMA3C's roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes epithelial-to-mesenchymal transition...

The Prostate, 2017

The vast majority of prostate cancer presents clinically localized to the prostate without eviden... more The vast majority of prostate cancer presents clinically localized to the prostate without evidence of metastasis. Currently, there are several modalities available to treat this particular disease. Despite radical prostatectomy demonstrating a modest prostate cancer specific mortality benefit in the PIVOT trial, several novel modalities have emerged to treat localized prostate cancer in patients that are either not eligible for surgery or that prefer an alternative approach. Athymic nude mice were subcutaneously inoculated with prostate cancer cells. The mice were divided into four cohorts, one cohort untreated, two cohorts received docetaxel (10 mg/kg) either subcutaneously (SC) or intravenously (IV) and the fourth cohort was treated using the magnetically-actuated docetaxel delivery device (MADDD), dispensing 1.5 μg of docetaxel per 30 min treatment session. Treatment in all three therapeutic arms (SC, IV, and MADDD) was administered once weekly for 6 weeks. Treatment efficacy wa...

European journal of immunology, Feb 1, 2017

Checkpoint blockade of CTLA-4 results in long-lasting survival benefits in metastatic cancer pati... more Checkpoint blockade of CTLA-4 results in long-lasting survival benefits in metastatic cancer patients. However, patients treated with CTLA-4 blockade have suffered from immune-related adverse events, most likely due to the breadth of the induced T-cell activation. Here, we investigated the efficacy of a local low-dose anti-CTLA-4 administration for treatment of subcutaneous or orthotopic murine bladder 49 (MB49) bladder carcinoma in C57BL/6 mice. When MB49 tumors were grown s.c., peritumoral (p.t.) injection of anti-CTLA-4 treatment was equally effective as intravenous or s.c. (nontumor bearing flank) administration. Notably, p.t. injection was associated with lower circulating antibody levels and decreased IL-6 serum levels as compared to systemic treatment. Ultrasound-guided intratumoral anti-CTLA-4 antibody treatment of orthotopically growing MB49 tumors resulted in tumor regression, with more than tenfold reduction in systemic antibody levels as compared to i.v. or s.c. administ...

European urology, Jul 10, 2017

Although cisplatin-based neoadjuvant chemotherapy (NAC) improves survival of unselected patients ... more Although cisplatin-based neoadjuvant chemotherapy (NAC) improves survival of unselected patients with muscle-invasive bladder cancer (MIBC), only a minority responds to therapy and chemoresistance remains a major challenge in this disease setting. To investigate the clinical significance of oncofetal chondroitin sulfate (ofCS) glycosaminoglycan chains in cisplatin-resistant MIBC and to evaluate these as targets for second-line therapy. An ofCS-binding recombinant VAR2CSA protein derived from the malaria parasite Plasmodium falciparum (rVAR2) was used as an in situ, in vitro, and in vivo ofCS-targeting reagent in cisplatin-resistant MIBC. The ofCS expression landscape was analyzed in two independent cohorts of matched pre- and post-NAC-treated MIBC patients. An rVAR2 protein armed with cytotoxic hemiasterlin compounds (rVAR2 drug conjugate [VDC] 886) was evaluated as a novel therapeutic strategy in a xenograft model of cisplatin-resistant MIBC. Antineoplastic effects of targeting ofC...