Diptiman Chanda

Cancer gene therapy using mesenchymal stem cells expressing interferon-β in a mouse prostate cancer lung metastasis model

Cell-based therapy for cancer is a promising new field. Among cell types that can be used for this purpose, mesenchymal stem cells (MSCs) appear to hold great advantage for reasons including easier propagation in culture, possible genetic modification to express therapeutic proteins and preferential homing to sites of cancer growth upon in vivo transfer. The present study evaluated the potential of genetically modified MSC, constitutively expressing interferon (IFN)-β, in an immunocompetent mouse model of prostate cancer lung metastasis. A recombinant adeno-associated virus (rAAV) encoding mouse IFN-β was constructed and initially tested in vitro for high-level expression and bioactivity of the transgenic protein. MSCs were transduced by the rAAV-IFN-β or green fluorescent protein ex vivo and used as cellular vehicles to target lung metastasis of TRAMP-C2 prostate cancer cells in a therapy model. Cohorts of mice were killed on days 30 and 75 to determine the effect of therapy by measurement of tumor volume, histology, immunohistochemistry, enzyme-linked immunosorbent assay and flow cytometry. Results indicated a significant reduction in tumor volume in lungs following IFN-β-expressing MSC therapy. Immunohistochemistry of the lung demonstrated increased tumor cell apoptosis and decreased tumor cell proliferation and blood vessel counts. A significant increase in the natural kill cell activity was observed following IFN-β therapy correlating the antitumor effect. Systemic level of IFN-β was not significantly elevated from this targeted cell therapy. These data demonstrate the potential of MSC-based IFN-β therapy for prostate cancer lung metastasis.

Therapeutic Potential of Mesenchymal Stem Cells Producing Interferon‐α in a Mouse Melanoma Lung Metastasis Model

Adult stem cells represent a potential source for cell‐based therapy of cancer. The present study evaluated the potential of bone marrow‐derived mesenchymal stem cells (MSC), genetically modified to express interferon (IFN)‐α, for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A recombinant adeno‐associated virus (rAAV) 6 vector encoding IFN‐α was used to transduce mouse bone marrow‐derived MSC ex vivo. Expression and bioactivity of the transgenic protein from rAAV‐transduced MSC were confirmed prior to in vivo studies. A lung metastasis model of melanoma was developed by i.v. injection of B16F10 cells into 8‐week‐old C57BL/6 mice. Ten days later, MSC transduced with rAAV‐IFN‐α or green fluorescent protein were intravenously injected. One cohort of mice was sacrificed to determine the effects of the therapy at an earlier time point, and another cohort was observed for long‐term survival. Results indicated that systemic administration of MSC producing IFN‐α reduced the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis of the tumors from MSC‐IFN‐α‐treated animals indicated an increase in apoptosis and a decrease in proliferation and blood vasculature. These data demonstrate the potential of adult MSC constitutively producing IFN‐α to reduce the growth of lung metastasis in melanoma.

Therapeutic potential of genetically modified mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSC) are multipotent adult stem cells of mesodermal origin localized within the bone marrow compartment. MSC possess multilineage property making them useful for a number of potential therapeutic applications. MSC can be isolated from the bone marrow, expanded in culture and genetically modified to serve as cell carriers for local or systemic therapy. Despite their ability to differentiate into osteoblasts, chondrocytes, adipocytes, myocytes and neuronal cells under appropriate stimuli, distinct molecular signals that guide migration of MSC to specific targets largely remain unknown. The pluripotent nature of MSC makes them ideal resources for regenerative medicine, graft-versus-host disease and autoimmune diseases. Despite their therapeutic potential in a variety of diseases, certain issues need to be critically addressed both in in vitro expansion of these cells without losing their stem cell properties, and the long-term fate of the transplanted MSC in vivo following ex vivo modifications. Finally, understanding of complex, multistep and multifactorial differentiation pathways from pluripotent stem cells to functional tissues will allow us to manipulate MSC for the formation of competent composite tissues in situ. The present article will provide comprehensive account of the characteristics of MSC, their isolation and culturing, multilineage properties and potential therapeutic applications.

Therapeutic potential of adult bone marrow-derived mesenchymal stem cells in diseases of the skeleton

Mesenchymal stem cells (MSCs) are the most popular among the adult stem cells in tissue engineering and regenerative medicine. Since their discovery and functional characterization in the late 1960s and early 1970s, MSCs or MSC‐like cells have been obtained from various mesodermal and non‐mesodermal tissues, although majority of the therapeutic applications involved bone marrow‐derived MSCs. Based on its mesenchymal origin, it was predicted earlier that MSCs only can differentiate into mesengenic lineages like bone, cartilage, fat or muscle. However, varied isolation and cell culturing methods identified subsets of MSCs in the bone marrow which not only differentiated into mesenchymal lineages, but also into ectodermal and endodermal derivatives. Although, true pluripotent status is yet to be established, MSCs have been successfully used in bone and cartilage regeneration in osteoporotic fracture and arthritis, respectively, and in the repair of cardiac tissue following myocardial infarction. Immunosuppressive properties of MSCs extend utility of MSCs to reduce complications of graft versus host disease and rheumatoid arthritis. Homing of MSCs to sites of tissue injury, including tumor, is well established. In addition to their ability in tissue regeneration, MSCs can be genetically engineered ex vivo for delivery of therapeutic molecule(s) to the sites of injury or tumorigenesis as cell therapy vehicles. MSCs tend to lose surface receptors for trafficking and have been reported to develop sarcoma in long‐term culture. In this article, we reviewed the current status of MSCs with special emphasis to therapeutic application in bone‐related diseases. J. Cell. Biochem. 111: 249–257, 2010.

Depletion of Plasmacytoid Dendritic Cells Inhibits Tumor Growth and Prevents Bone Metastasis of Breast Cancer Cells

Elevated levels of plasmacytoid dendritic cells (pDC) have been reported in breast cancer patients, but the significance remains undefined. Using three immunocompetent mouse models of breast cancer bone metastasis, we identified a key role for pDC in facilitating tumor growth through immunosuppression and aggressive osteolysis. Following infiltration of macrophages upon breast cancer dissemination, there was a steady increase in pDC within the bone, which resulted in a sustained Th2 response along with elevated levels of regulatory T cells and myeloid-derived suppressor cells. Subsequently, pDC and CD4+ T cells, producing osteolytic cytokines, increased with tumor burden, causing severe bone damage. Microcomputed tomography and histology analyses of bone showed destruction of femur and tibia. The therapeutic significance of this finding was confirmed by depletion of pDC, which resulted in decreased tumor burden and bone loss by activating tumor-specific cytolytic CD8+ T cells and decreasing suppressor cell populations. Thus, pDC depletion may offer a novel adjuvant strategy to therapeutically influence breast cancer bone metastasis.

Breast cancer subtypes predispose the site of distant metastases.

OBJECTIVES The distant organs to which breast cancer preferentially metastasizes are of significant clinical importance. METHODS We explored the relationship between the clinicopathologic factors and the common sites of distant metastasis in 531 consecutive patients with advanced breast cancer. RESULTS Breast cancer subtype as a variable was significantly associated with all five common sites of relapse by multivariate analysis. The luminal tumors were remarkable for their significant bone-seeking phenotype and were less frequently observed in lung, brain, and pleural metastases and less likely to be associated with multiorgan relapse. The HER2 subtype demonstrated a significant liver-homing characteristic. African Americans were significantly less likely to have brain-only metastasis in patients with brain relapse. CONCLUSIONS These findings further articulate that breast cancer subtypes differ not only in tumor characteristics but also in their metastatic behavior, thus raising the possibility that this knowledge could potentially be used in determining the appropriate strategy for follow-up of patients with newly diagnosed breast cancer.

Effects of Sustained Antiangiogenic Therapy in Multistage Prostate Cancer in TRAMP Model

Antiangiogenic therapy is a promising alternative for prostate cancer growth and metastasis and holds great promise as an adjuvant therapy. The present study evaluated the potential of stable expression of angiostatin and endostatin before the onset of neoplasia and during the early and late stages of prostate cancer progression in transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Groups of 5-, 10-, and 18-week-old male TRAMP mice received recombinant adeno-associated virus-6 encoding mouse endostatin plus angiostatin (E+A) by i.m. injection. The effects of therapy were determined by sacrificing groups of treated mice at defined stages of tumor progression and following cohorts of similarly treated mice for long-term survival. Results indicated remarkable survival after recombinant adeno-associated virus-(E+A) therapy only when the treatment was given at an earlier time, before the onset of high-grade neoplasia, compared with treatment given for invasive cancer. Interestingly, early-stage antiangiogenic therapy arrested the progression of moderately differentiated carcinoma to poorly differentiated state and distant metastasis. Immunohistochemical analysis of the prostate from treated mice indicated significantly lower endothelial cell proliferation and increased tumor cell apoptosis. Vascular endothelial growth factor receptor (VEGFR)-2 expression was significantly down-regulated in tumor endothelium after treatment but not VEGFR-1. Analysis of the neuroendocrine marker synaptophysin expression indicated that antiangiogenic therapy given at an early-stage disease reduced neuroendocrine transition of the epithelial tumors. These studies indicate that stable endostatin and angiostatin gene therapy may be more effective for minimally invasive tumors rather than advanced-stage disease.

Therapeutic potential of adult bone marrow-derived mesenchymal stem cells in prostate cancer bone metastasis.

Purpose: Current evidence indicates that an osteoblast lesion in prostate cancer is preceded by osteolysis. Thus, prevention of osteolysis would reduce complications of bone metastasis. Bone marrow–derived mesenchymal stem cells have the ability to differentiate into osteoblast and produce osteoprotegerin, a decoy receptor for the receptor activator for nuclear factor κB ligand, naturally. The present study examined the potential of unmodified mesenchymal stem cells to prevent osteolytic bone lesions in a preclinical mouse model of prostate cancer. Experimental Design: The human prostate cancer cell line PC3 was implanted in tibiae of severe combined immunodeficient mice. After establishment of the tumor, either unmodified or genetically engineered mesenchymal stem cells overexpressing osteoprotegerin was injected at the site of tumor growth. The effects of therapy were monitored by bioluminescence imaging, micro–computed tomography, immunohistochemistry, and histomorphometry. Results: Data indicated significant (P < 0.001) inhibition of tumor growth and restoration of bone in mice treated with unmodified and modified mesenchymal stem cells. Detailed analysis suggested that the donor mesenchymal stem cell inhibited tumor progression by producing woven bone around the growing tumor cells in the tibiae and by preventing osteoclastogenesis. Conclusions: Overcoming the limitation of the number of mesenchymal stem cells available in the bone can provide significant amelioration for osteolytic damage without further modification. (Clin Cancer Res 2009;15(23):7175–85)

LL-37 as a therapeutic target for late stage prostate cancer

The antimicrobial peptide, leucine–leucine‐37 (LL‐37), stimulates proliferation, angiogenesis, and cellular migration, inhibits apoptosis and is associated with inflammation. Since these functional processes are often exaggerated in cancer, the aim of the present study was to investigate the expression and role of LL‐37 in prostate cancer (PCa) and establish its value as a therapeutic target.

Systemic osteoprotegerin gene therapy restores tumor-induced bone loss in a therapeutic model of breast cancer bone metastasis.

Enhanced production of receptor activator of nuclear factor-kappaB ligand (RANKL) and its binding to RANK on the osteoclasts have been associated with osteolysis in breast cancer bone metastasis. Osteoprotegerin (OPG) is a decoy receptor that prevents RANKL-RANK interaction. This study determined the effects of sustained expression of OPG using a recombinant adeno-associated viral (rAAV) vector in mouse model of osteolytic breast cancer. Bone metastasis was established by intracardiac injection of the human breast cancer cell line MDA-MB-435. Following this, mice were administered a one-time intramuscular injection of rAAV encoding either OPG.Fc (OPG) or green fluorescent protein (GFP). Mice were killed 1 month later and the effects of therapy on tumor growth and bone remodeling were evaluated. Bioluminescence imaging showed significant reduction of tumor growth in bone of OPG.Fc-treated mice. Micro-computed tomography (microCT) analysis and histomorphometry of the tibia indicated significant protection of trabecular and cortical bones after OPG.Fc therapy. Despite the prevention of bone loss and tumor growth in bone, OPG.Fc therapy failed to provide long-term survival. OPG.Fc-treated mice developed more bone than age-matched normal mice, indicating a requirement for regulated transgene expression. Results of this study indicate the potential of rAAV-OPG therapy for reducing morbidity and mortality in breast cancer patients with osteolytic bone damage.