Kenneth Gyabaah

Novel 3D Co-Culture Model for Epithelial-Stromal Cells Interaction in Prostate Cancer

Paracrine function is a major mechanism of cell-cell communication within tissue microenvironment in normal development and disease. In vitro cell culture models simulating tissue or tumor microenvironment are necessary tools to delineate epithelial-stromal interactions including paracrine function, yet an ideal three-dimensional (3D) tumor model specifically studying paracrine function is currently lacking. In order to fill this void we developed a novel 3D co-culture model in double-layered alginate hydrogel microspheres, incorporating prostate cancer epithelial and stromal cells in separate compartments of the microspheres. The cells remained confined and viable within their respective spheres for over 30 days. As a proof of principle regarding paracrine function of the model, we measured shedded component of E-cadherin (sE-cad) in the conditioned media, a major membrane bound cell adhesive molecule that is highly dysregulated in cancers including prostate cancer. In addition to demonstrating that sE-cad can be reliably quantified in the conditioned media, the time course experiments also demonstrated that the amount of sE-cad is influenced by epithelial-stromal interaction. In conclusion, the study establishes a novel 3D in vitro co-culture model that can be used to study cell-cell paracrine interaction.

Cell Therapy with Human Renal Cell Cultures Containing Erythropoietin-Positive Cells Improves Chronic Kidney Injury

New therapeutic strategies for chronic kidney disease (CKD) are necessary to offset the rising incidence of CKD and donor shortage. Erythropoietin (EPO), a cytokine produced by fibroblast‐like cells in the kidney, has recently emerged as a renoprotective factor with anti‐inflammatory, antioxidant properties. This study (a) determined whether human renal cultures (human primary kidney cells [hPKC]) can be enriched in EPO‐positive cells (hPKC(F+)) by using magnetic‐bead sorting; (b) characterized hPKC(F+) following cell separation; and (c) established that intrarenal delivery of enriched hPKC(F+) cells would be more beneficial in treatment of renal injury, inflammation, and oxidative stress than unsorted hPKC cultures in a chronic kidney injury model. Fluorescence‐activated cell sorting analysis revealed higher expression of EPO (36%) and CD73 (27%) in hPKC(F+) as compared with hPKC. After induction of renal injury, intrarenal delivery of hPKC(F+) or hPKC significantly reduced serum creatinine, interstitial fibrosis in the medulla, and abundance of CD68‐positive cells in the cortex and medulla (p < .05). However, only hPKC(F+) attenuated interstitial fibrosis in the renal cortex and decreased urinary albumin (3.5‐fold) and urinary tubular injury marker kidney injury molecule 1 (16‐fold). hPKC(F+) also significantly reduced levels of renal cortical monocyte chemotactic protein 1 (1.8‐fold) and oxidative DNA marker 8‐hydroxy‐deoxyguanosine (8‐OHdG) (2.4‐fold). After 12 weeks, we detected few injected cells, which were localized mostly to the cortical interstitium. Although cell therapy with either hPKC(F+) or hPKC improved renal function, the hPKC(F+) subpopulation provides greater renoprotection, perhaps through attenuation of inflammation and oxidative stress. We conclude that hPKC(F+) may be used as components of cell‐based therapies for degenerative kidney diseases.

TRAIL and Interferon-α Act Synergistically to Induce Renal Cell Carcinoma Apoptosis

PURPOSE Despite modern targeted therapy metastatic renal cell carcinoma remains a deadly disease. Interferon-alpha (Calbiochem(R)) is currently used to treat this condition, mainly combined with the targeted anti-vascular endothelial growth factor antibody bevacizumab. TRAIL (Apo2 ligand/tumor necrosis factor related apoptosis inducing ligand) (Calbiochem) is a novel antineoplastic agent now in early phase clinical trials. Interferon-alpha and TRAIL can act synergistically to kill cancer cells but to our knowledge this has never been tested in the context of renal cell carcinoma. We hypothesized that TRAIL and interferon-alpha could synergistically induce apoptosis in renal cell carcinoma cells. MATERIALS AND METHODS We treated renal cell carcinoma cell lines with recombinant TRAIL and/or interferon-alpha. Viability and apoptosis were assessed by MTS assay, flow cytometry and Western blot. Synergy was confirmed by isobologram. Interferon-alpha induced changes in renal cell carcinoma cell signaling were assessed by Western blot, flow cytometry and enzyme-linked immunosorbent assay. RESULTS TRAIL and interferon-alpha acted synergistically to increase apoptotic cell death in renal cell carcinoma cells. Interferon-alpha treatment altered the ability of cells to activate extracellular signal-regulated kinase while inhibiting extracellular signal-regulated kinase with UO126 abrogated TRAIL and interferon-alpha apoptotic synergy. Interferon-alpha did not induce changes in TRAIL or death receptor expression, or change other known mediators of the intrinsic and extrinsic apoptotic cascade in the cells. CONCLUSIONS TRAIL plus interferon-alpha synergistically induces apoptosis in renal cell carcinoma cells, which is due at least in part to interferon-alpha mediated changes in extracellular signal-regulated kinase activation. TRAIL and interferon-alpha combination therapy may be a novel approach to advanced renal cell carcinoma that warrants further testing in vivo.

Novel In Vivo Model for Combinatorial Fluorescence Labeling in Mouse Prostate

The epithelial layer of prostate glands contains several types of cells, including luminal and basal cells. Yet there is paucity of animal models to study the cellular origin of normal or neoplastic development in the prostate to facilitate the treatment of heterogenous prostate diseases by targeting individual cell lineages.

Cell line modeling to study biomarker panel in prostate cancer.

African–American men with prostate cancer (PCa) present with higher‐grade and ‐stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well‐characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose.

Controlled Regulation of Erythropoietin by Primary Cultured Renal Cells for Renal Failure Induced Anemia

PURPOSE Renal failure induced anemia develops as a result of inadequate production of erythropoietin, which is the primary regulator of red blood cell production. We previously noted that culture expanded primary renal cells stably express erythropoietin and suggested that these cells may be used as a potential treatment for renal failure induced anemia. We investigated whether these cells are able to regulate erythropoietin expression in a controlled manner under different oxygen and environmental conditions. MATERIALS AND METHODS Primary rat renal cells were exposed to different hypoxic (0.1% to 1% O(2)) and normoxic environments. Erythropoietin expression was assessed using reverse transcriptase-polymerase chain reaction. Erythropoietin production was measured in culture medium using Meso Scale Discovery® assays. Results were plotted to compare different levels of production to the control. RESULTS Cultured renal cells expressed high levels of erythropoietin under hypoxia for up to 24 hours with a gradual decrease thereafter. However, erythropoietin expression was decreased when cells were switched from a hypoxic to a normoxic environment within the initial 24 hours. This indicated that cultured renal cells have the capacity to sense environmental oxygen tension and regulate erythropoietin expression accordingly. In addition, erythropoietin release in medium followed a pattern similar to that of gene expression under normoxic and hypoxic conditions. CONCLUSIONS These findings indicate that primary renal cells have the ability to regulate erythropoietin gene expression and release through environment dependent mechanisms. This also suggests that with further study the possibility exists of developing these cells as a potential method to treat renal failure induced anemia.

Abstract LB-190: Soluble E-Cadherin promotes cell invasion but not proliferation in prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Metastasis is responsible for the majority of prostate cancer-related deaths. Despite progress in early detection and management, there remains no effective, long-term cure for metastatic prostate cancer, partially due to the unclear molecular mechanism of cancer cell invasion and metastasis. Clinical reports discovered that soluble E-cadherin (sE-Cad, the cleaved ectodomain of human E-Cadherin protein) was elevated in body fluids and serum of patients with a variety of cancers, and its serum levels have been shown to correlate positively with metastatic cancers and disease invasiveness. However, little is known about the role of sE-Cad in metastasis, or the molecular mechanism of its abnormal accumulation in cancerous microenvironment. Our lab generated C4-2 prostate cancer cell lines which would secret synthetic sE-Cad by Doxycycline induction. We found that increased sE-Cad in the conditioned medium promotes cell invasion, but not cell proliferation. We analyzed the protein subcellular localizations in different cell compartments (membrane, cytoskeleton, nucleus and cytoplasm) and observed increased binding of synthetic sE-Cad to membrane E-Cadherin, indicating that sE-Cad could promote cell motility by interacting with membrane proteins and possibly disrupting the cell-cell adhesion. This work is supported by Wake Forest University institutional funds to KC Balaji and by NIH grant CA079448 to X Fang. Citation Format: Xiaolan Fang, K. C. Balaji, Bita NickKholgh, Kenneth Gyabaah. Soluble E-Cadherin promotes cell invasion but not proliferation in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-190. doi:10.1158/1538-7445.AM2014-LB-190

Abstract 4000: Biological basis of aggressive phenotype in African-American prostate cancer cell line

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: It is well established that African American men (AAM) with prostate cancer (PC) present more frequently with higher grade tumors compared to Caucasians. While the disparity is likely multifactorial, the biological basis is perhaps the strongest contender. Protein kinase D1 (PKD1) is a tumor and metastasis suppressor in PC. Down regulation of PKD1 was shown to increased cell proliferation, migration and metastasis in PC. Aim and experimental procedure: In order to address the fundamental question whether PC in AAM is inherently aggressive, we compared the proliferation, invasion and the expression of PKD1-centered signaling based 13-biomarker panel in primary and metastatic AAM cell lines (E006AA and MDApca2b) to benign (RWPE) and metastatic Caucasian cell lines (LNCaP and C4-2) using Western blot within different cell compartments. The results were further validated by RT-PCR and immunofluoroscent staining. Results: The proliferation, viability and invasion assays showed that E006AA cells proliferate and invade matrigel significantly more rapidly than metastatic LNCaP and MDAPca2b cells. The 13-biomarker panel analysis showed PKD1-centered dysregulaiton in AAM prostate cancer cell lines. Although E006AA is a primary PC cell line, PKD1 was down regulated similar to more aggressive C4-2 cells. Nuclear androgen receptor (AR), which shows active AR, was higher in LNCaP and MD2Pca2b; Vimentin showed higher expression in C4-2 and E006AA. Localization of E-Cadherin, N-Cadherin, T120 phosphorylated s- Catenin, MMP-2 and S11 phosphorylated snail in MD2Pca2b was similar to LNCaP cells but different to C4-2 and E006AA. Conclusion: Our Data demonstrates that the dysregulaiton of PKD1-centered signaling occurs in primary AAM prostate cancer cell line E006AA is akin to other metastatic PC cell lines suggesting an inherently aggressive nature of PC in AAM. Acknowledgment: Authors would like to thank Dr. Shahriar Koochekpour (Roswell park institute, University at Buffalo,State University of NewYork) for his kind gift E006AA cell line. Citation Format: Bita Nickkholgh, Xiaolan Fang, Kenneth Gyabaah, K.C. Balaji. Biological basis of aggressive phenotype in African-American prostate cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4000. doi:10.1158/1538-7445.AM2014-4000

Abstract 4078: Novel 3D co-cultural model for epithelial-stromal cells interaction in prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Paracrine function is a major mechanism of cell-cell communication within tissue microenvironment in both normal development and disease. In vitro cell culture models simulating tissue or tumor microenvironment are necessary tools to delineate epithelial stromal interactions including paracrine function. Yet an ideal 3-dimensional (3D) tumor model specifically studying paracrine function is currently lacking. In order to fill this void we developed a novel 3D co-culture model, using double layered alginate hydrogel microspheres to incorporate prostate cancer epithelial and stromal cells. The cells remained confined and viable within their respective spheres for over 30 days. As a proof of principle regarding paracrine function of the model, we measured shedded component of E-cadherin (sE-cad) in the conditioned media, a major membrane bound cell adhesive molecule that is highly dysregulated in cancers including prostate. In addition to demonstrating that sE-cad can be reliably quantified in the conditioned media, the time course experiments also proved that the amount of sE-cad is influenced by epithelial stromal interaction. In conclusion, the study establishes a novel 3D in vitro co-culture model that can be used to study cell-cell paracrine interaction. Citation Format: Xiaolan Fang, Sittadjody Sivanandane, Kenneth Gyabaah, K.C. Balaji. Novel 3D co-cultural model for epithelial-stromal cells interaction in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4078. doi:10.1158/1538-7445.AM2013-4078