Gene transfer in a newly established spontaneous feline fibrosarcoma cell line

Fibrosarcoma is a deadly disease in cats and is most often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene therapy in NVS fibrosarcoma. The NVS fibrosarcoma cell line (Cocca-6A) was isolated from a spontaneous fibrosarcoma that occurred in a domestic calico cat. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Giemsa block staining of metaphase spreads of the Cocca-6A cells demonstrated deletion of one of the E1 chromosomes, where feline p53 maps. Adenoviral gene therapy demonstrated a remarkable effect on the viability and growth of the Cocca-6A cells following transduction with adenoviruses carrying RB/p105, Ras-DN, Mda-7/IL-24 or their combination. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. The use of Ad.Ras-DN, Ad.Rb/p105 and Ad.Mda7/IL-24 has been identified as a gene therapy for a possible future adjuvant treatment in spontaneous feline fibrosarcoma. More gene therapy studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas

Investigation Of Ultrasound Targeted Microbubbles As A Therapeutic Gene Delivery System For Prostate Cancer

A major challenge for effective gene therapy is systemic delivery of viruses carrying therapeutic genes into affected tissue. The immunogenic nature of human adenoviruses (Ads) limits their use for intratumoral (IT) injection in gene therapy. Ads transfection is further hampered by the fluctuating presence of Coxsackie and Adenovirus Receptor (CAR) and integrins on the cells’ surface. To circumvent these limitations we developed a novel approach wherein Ads are encapsulated inside the shell of lyophilized, lipid-encapsulated, perfluorocarbon microbubbles (MBs)/ultrasound (US) contrast agents, which act as delivery vehicles for a sitespecific gene transfer system. We performed infection studies with Ad.GFP (Green Fluorescent Protein), Ad.mda-7 (melanoma differentiation associated gene 7) and CTV.mda-7 on human DU145 and mouse prostate cancer cells as well as observed enhanced GFP expression when Ad.GFP was delivered by MBs and US. Our results show that US breaks open the MB/Ads complexes by undergoing cavitation at the sonoporated site, which allows Ads to transfer their transgene only to the sonoporated region. Cavitation collapse of the MBs creates small shockwaves that increase cell permeability by forming temporary micropores on the cell surface bypassing the receptormediated dependence of Ads for transfection. Fetal bovine serum (FBS) containing complement did not allow the unprotected Ads to infect the cells; however, MBs complexed with Ad.GFP did infect DU145 and TRAMP-C2 cells in a FBS rich media. We studied MB assisted gene delivery of reporter (GFP) and therapeutic genes (p53, Rb, Rb2 (p130) and Mda-7/IL-24) into prostate cancer (PC) xenografted in immune-compromised athymic mice. The results demonstrated that MBs protect the host from unspecific viral immune response thus protecting the viral payload and allowing for intravenous (IV) injection rather than IT injection. Additionally, Ad gene transfer was enhanced at the targeted/sonoporated mice tumor xenografts. This research demonstrated mda-7’s efficacy in reducing primary (treated) and untreated tumors that simulated the presence of metastasis in athymic mice xenograft models bearing human PC cells. Bystander anti-tumor activity of mda-7, a secreted cytokine was noted for non-targeted tumors. Earlier in vitro studies on the combination of radiation and gene therapy (Ad.p53, Ad.Rb, and Ad.p130) demonstrated an increase in the percentage of cell death for DU145 cells. We also studied UTMD (ultrasound targeted microbubble destruction) gene therapy in combination with external beam radiation for radiation resistant PC. The results demonstrated an enhanced therapeutic benefit of tumor suppressor genes in radiation resistant PC. We also demonstrated an increase in the expression of tumor suppressor genes at the tumor site due to MBs and US. These findings highlight the potential therapeutic benefit of this novel image guided gene transfer technology alone or in combination with external beam radiation for prostate cancer patients with therapy resistant disease

Microbubble-mediated delivery of human adenoviruses does not elicit innate and adaptive immunity response in an immunocompetent mouse model of prostate cancer

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Lack of standardization of clinically compliant culture protocols of mesenchymal stem cells for re-implantation in humans have hindered clinical progress in the field of tissue regeneration to repair maxillofacial and orthopedic defects. The goal of this study was to establish a clinically relevant osteogenic protocol for collection and expansion of autologous stem cells to be used at Marshall University for re-implantation and repair of maxillofacial and orthopedic conditions. Human bone marrow (hBM) samples were collected from patients undergoing intramedullary nail fixation for closed femoral fractures. hBM mesenchymal cells were expanded by growing them first in Petri dishes for two weeks, followed by a week of culture using Perfecta 3D Hanging Drop Plates®. Various scaffold materials were tested and analyzed for cellular integration, vitality, and differentiation capacity of harvested hBM-MSCs including: 60/40 blend of hydroxyapatite biomatrix; Acellular bone composite discs; Allowash®, cancellous bone cubes; PLGA (poly lactic-co-glycolic acid); and Woven chitin derived fiber. We found that the 3D spheroid culture allowed production of hBM mesenchymal cells that retained osteoblast differentiation capacity over a monolayer culture of hBM-MSCs without the need to use chemical or hormonal modulation. We also observed that hydroxyapatite and Allowash cancellous bone scaffolds allowed better cell integration and viability properties as compared to other materials tested in this study. In conclusion, the multimodal culture methodology we developed creates actively differentiating stem-cell spheroids that can then be readily utilized in clinical practices to improve the regeneration of tissues of the head and the body

Targeting a Newly Established Spontaneous Feline Fibrosarcoma Cell Line by Gene Transfer

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas

Microbubble-mediated delivery of human adenoviruses does not elicit innate and adaptive immunity response in an immunocompetent mouse model of prostate cancer

Background Gene transfer to malignant sites using human adenoviruses (hAds) has been limited because of their immunogenic nature and host specificity. Murine cells often lack some of the receptors needed for hAds attachment, thus murine cells are generally non-permissive for human adenoviral infection and replication, which limits translational studies. Methods We have developed a gene transfer method that uses a combination of lipid-encapsulated perfluorocarbon microbubbles and ultrasound to protect and deliver hAds to a target tissue, bypassing the requirement of specific receptors. Results In an in vitro model, we showed that murine TRAMP-C2 and human DU145 prostate cancer cells display a comparable expression pattern of receptors involved in hAds adhesion and internalization. We also demonstrated that murine and human cells showed a dose-dependent increase in the percentage of cells transduced by hAd-GFP (green fluorescent protein) after 24 h and that GFP transgene was efficiently expressed at 48 and 72 h post-transduction. To assess if our image-guided delivery system could effectively protect the hAds from the immune system in vivo, we injected healthy immunocompetent mice (C57BL/6) or mice bearing a syngeneic prostate tumor (TRAMP-C2) with hAd-GFP/MB complexes. Notably, we did not observe activation of innate (TNF-α and IL-6 cytokines), or adaptive immune response (neutralizing antibodies, INF-γ+ CD8+ T cells). Conclusions This study brings us a step closer to demonstrating the feasibility of murine cancer models to investigate the clinical translation of image guided site-specific adenoviral gene therapy mediated by ultrasound-targeted microbubble destruction

Microbubble-mediated delivery of human adenoviruses does not elicit innate and adaptive immunity response in an immunocompetent mouse model of prostate cancer

Abstract Background Gene transfer to malignant sites using human adenoviruses (hAds) has been limited because of their immunogenic nature and host specificity. Murine cells often lack some of the receptors needed for hAds attachment, thus murine cells are generally non-permissive for human adenoviral infection and replication, which limits translational studies. Methods We have developed a gene transfer method that uses a combination of lipid-encapsulated perfluorocarbon microbubbles and ultrasound to protect and deliver hAds to a target tissue, bypassing the requirement of specific receptors. Results In an in vitro model, we showed that murine TRAMP-C2 and human DU145 prostate cancer cells display a comparable expression pattern of receptors involved in hAds adhesion and internalization. We also demonstrated that murine and human cells showed a dose-dependent increase in the percentage of cells transduced by hAd-GFP (green fluorescent protein) after 24 h and that GFP transgene was efficiently expressed at 48 and 72 h post-transduction. To assess if our image-guided delivery system could effectively protect the hAds from the immune system in vivo, we injected healthy immunocompetent mice (C57BL/6) or mice bearing a syngeneic prostate tumor (TRAMP-C2) with hAd-GFP/MB complexes. Notably, we did not observe activation of innate (TNF-α and IL-6 cytokines), or adaptive immune response (neutralizing antibodies, INF-γ+ CD8+ T cells). Conclusions This study brings us a step closer to demonstrating the feasibility of murine cancer models to investigate the clinical translation of image guided site-specific adenoviral gene therapy mediated by ultrasound-targeted microbubble destruction

Chemo-predictive assay for targeting cancer stem-like cells in patients affected by brain tumors.

Administration of ineffective anticancer therapy is associated with unnecessary toxicity and development of resistant clones. Cancer stem-like cells (CSLCs) resist chemotherapy, thereby causing relapse of the disease. Thus, development of a test that identifies the most effective chemotherapy management offers great promise for individualized anticancer treatments. We have developed an ex vivo chemotherapy sensitivity assay (ChemoID), which measures the sensitivity of CSLCs as well as the bulk of tumor cells to a variety of chemotherapy agents. Two patients, a 21-year old male (patient 1) and a 5-month female (patient 2), affected by anaplastic WHO grade-III ependymoma were screened using the ChemoID assay. Patient 1 was found sensitive to the combination of irinotecan and bevacizumab, which resulted in a prolonged disease progression free period of 18 months. Following recurrence, the combination of various chemotherapy drugs was tested again with the ChemoID assay. We found that benzyl isothiocyanate (BITC) greatly increased the chemosensitivity of the ependymoma cells to the combination of irinotecan and bevacizumab. After patient 1 was treated for two months with irinotecan, bevacizumab and supplements of cruciferous vegetable extracts containing BITC, we observed over 50% tumoral regression in comparison with pre-ChemoID scan as evidenced by MRI. Patient 2 was found resistant to all treatments tested and following 6 cycles of vincristine, carboplatin, cyclophosphamide, etoposide, and cisplatin in various combinations, the tumor of this patient rapidly progressed and proton beam therapy was recommended. As expected animal studies conducted with patient derived xenografts treated with ChemoID screened drugs recapitulated the clinical observation. This assay demonstrates that patients with the same histological stage and grade of cancer may vary considerably in their clinical response, suggesting that ChemoID testing which measures the sensitivity of CSLCs as well as the bulk of tumor cells to a variety of chemotherapy agents could lead to more effective and personalized anticancer treatments in the future

The Effects of the combination of Adenoviral gene transfer on the feline fibrosarcoma cells Cocca-6A.

<p>Every panel shows the phase contrast imaging (200× magnification, 24-hours post transduction) and the propidium iodide flow cytometry analysis of Cocca-6A cells transduced with 50 MOIs of various combinations of double or triple transductions using Ads carrying Ad.<i>RB/p105</i>, Ad.<i>Ras</i>-DN, Ad.<i>mda-7/IL24</i>, CTV.<i>mda-7/IL24</i> or CTV.<i>IFNγ.</i> (a–g) Single adenoviral transduction and controls: (a) Control untransduced Cocca-6A cells. (b) Ad.GFP, (c) Ad.Rb/p105, (d) Ad.Ras-DN, (e) Ad.Mda7/IL-24, (f) CTV.Mda7/IL-24, (g) CTV.IFNγ transduced Cocca-6A cells. (h–q) Combinations of double or triple adenoviral transductions: (h) Ad.Rb/p105+ Ad.Ras-DN, (i) Ad.Rb/p105+ Ad.Mda7/IL-24, (j) Ad.Rb/p105+ CTV.Mda7/IL-24, (k) Ad.Rb/p105+ CTV.<i>IFNγ,</i> (l) Ad.Ras-DN + Ad.Mda7/IL-24, (m) Ad.Ras-DN + CTV.Mda7/IL-24, (n) Ad.Ras-DN + CTV.<i>IFNγ,</i> (o) Ad.Rb/p105+ Ad.Ras-DN + Ad.mda7/IL-24, <i>(p)</i> Ad.Rb/p105+ Ad.Ras-DN + CTV.mda7/IL-24, (q) Ad.Rb/p105+ Ad.Ras-DN + Ad.CTV.<i>IFNγ</i> transduced Cocca-6A cells.</p

Establishment of a feline fibrosarcoma cell line.

<p>(A) Hematoxylin & Eosin staining of the pathologic specimen. Optical microscope 100× magnification of the neoplastic tissue shows cells that appear to have indistinct cell borders, round to oval nuclei with finely stippled chromatin, and small single or multiple nucleoli. (B–F) Phase contrast imaging of the Cocca-1A, -2A, -3A, -6A, and -3B clones after 48 hours of culture (200× magnification). (G) Phase contrast, 200× magnification of the clone 6A after 3 months of continuous culture. (H–J) Phase contrast, 200× magnification of the clone 6A after 6-, 12-, and 24 months of continuous culture.</p