Disulfiram reduces metastatic osteosarcoma tumor burden in an immunocompetent Balb/c or-thotopic mouse model.

INTRODUCTION: The overall survival rate of patients with osteosarcoma (OS) and pulmonary metastases has remained stagnant at 15-30% for several decades. Disulfiram (DSF) is an FDA-approved aldehyde dehydrogenase inhibitor that reduces the metastatic phenotype of OS cells in vitro. Here we evaluate its in vivo efficacy, as compared to doxorubicin chemotherapy, in a previously-validated orthotopic model of metastatic OS. RESULTS: All treatment groups displayed a significantly reduced quantitative OS metastatic burden compared with controls. The metastatic burden of Lo DSF-treated animals was equivalent to the DXR group. Ninety-five percent of control animals displayed evidence of metastatic disease, which was significantly greater than all treatment groups. DISCUSSION: Disulfiram treatment resulted in a reduced burden of OS metastatic disease compared with controls. This was statistically-equivalent to doxorubicin. No additive effect was observed between these two therapies. MATERIALS AND METHODS: One-hundred twenty immunocompetent Balb/c mice received proximal tibia paraphyseal injections of 5 × 105 K7M2 murine OS cells. Therapy began three weeks after injection: saline (control), low-dose disulfiram (Lo DSF), high-dose disulfiram (Hi DSF), doxorubicin (DXR), Lo DSF + DXR, and Hi DSF + DXR. Transfemoral amputations were performed at 4 weeks. Quantitative metastatic tumor burden was measured using near-infrared indocyanine green (ICG) angiography.

Quantitative Primary Tumor Indocyanine Green Measurements Predict Osteosarcoma Metastatic Lung Burden in a Mouse Model.

BACKGROUND: Current preclinical osteosarcoma (OS) models largely focus on quantifying primary tumor burden. However, most fatalities from OS are caused by metastatic disease. The quantification of metastatic OS currently relies on CT, which is limited by motion artifact, requires intravenous contrast, and can be technically demanding in the preclinical setting. We describe the ability for indocyanine green (ICG) fluorescence angiography to quantify primary and metastatic OS in a previously validated orthotopic, immunocompetent mouse model. QUESTIONS/PURPOSES: (1) Can near-infrared ICG fluorescence be used to attach a comparable, quantitative value to the primary OS tumor in our experimental mouse model? (2) Will primary tumor fluorescence differ in mice that go on to develop metastatic lung disease? (3) Does primary tumor fluorescence correlate with tumor volume measured with CT? METHODS: Six groups of 4- to 6-week-old immunocompetent Balb/c mice (n = 6 per group) received paraphyseal injections into their left hindlimb proximal tibia consisting of variable numbers of K7M2 mouse OS cells. A hindlimb transfemoral amputation was performed 4 weeks after injection with euthanasia and lung extraction performed 10 weeks after injection. Histologic examination of lung and primary tumor specimens confirmed ICG localization only within the tumor bed. RESULTS: Mice with visible or palpable tumor growth had greater hindlimb fluorescence (3.5 ± 2.3 arbitrary perfusion units [APU], defined as the fluorescence pixel return normalized by the detector) compared with those with a negative examination (0.71 ± 0.38 APU, -2.7 ± 0.5 mean difference, 95% confidence interval -3.7 to -1.8, p < 0.001). A strong linear trend (r = 0.81, p < 0.01) was observed between primary tumor and lung fluorescence, suggesting that quantitative ICG tumor fluorescence is directly related to eventual metastatic burden. We did not find a correlation (r = 0.04, p = 0.45) between normalized primary tumor fluorescence and CT volumetric measurements. CONCLUSIONS: We demonstrate a novel methodology for quantifying primary and metastatic OS in a previously validated, immunocompetent, orthotopic mouse model. Quantitative fluorescence of the primary tumor with ICG angiography is linearly related to metastatic burden, a relationship that does not exist with respect to clinical tumor size. This highlights the potential utility of ICG near-infrared fluorescence imaging as a valuable preclinical proof-of-concept modality. Future experimental work will use this model to evaluate the efficacy of novel OS small molecule inhibitors. CLINICAL RELEVANCE: Given the histologic localization of ICG to only the tumor bed, we envision the clinical use of ICG angiography as an intraoperative margin and tumor detector. Such a tool may be used as an alternative to intraoperative histology to confirm negative primary tumor margins or as a valuable tool for debulking surgeries in vulnerable anatomic locations. Because we have demonstrated the successful preservation of ICG in frozen tumor samples, future work will focus on blinded validation of this modality in observational human trials, comparing the ICG fluorescence of harvested tissue samples with fresh frozen pathology.

Notch Signaling Mediates Skeletal Muscle Atrophy in Cancer Cachexia Caused by Osteosarcoma.

Skeletal muscle atrophy in cancer cachexia is mediated by the interaction between muscle stem cells and various tumor factors. Although Notch signaling has been known as a key regulator of both cancer development and muscle stem cell activity, the potential involvement of Notch signaling in cancer cachexia and concomitant muscle atrophy has yet to be elucidated. The murine K7M2 osteosarcoma cell line was used to generate an orthotopic model of sarcoma-associated cachexia, and the role of Notch signaling was evaluated. Skeletal muscle atrophy was observed in the sarcoma-bearing mice, and Notch signaling was highly active in both tumor tissues and the atrophic skeletal muscles. Systemic inhibition of Notch signaling reduced muscle atrophy. In vitro coculture of osteosarcoma cells with muscle-derived stem cells (MDSCs) isolated from normal mice resulted in decreased myogenic potential of MDSCs, while the application of Notch inhibitor was able to rescue this repressed myogenic potential. We further observed that Notch-activating factors reside in the exosomes of osteosarcoma cells, which activate Notch signaling in MDSCs and subsequently repress myogenesis. Our results revealed that signaling between tumor and muscle via the Notch pathway may play an important role in mediating the skeletal muscle atrophy seen in cancer cachexia.

BMP2 is superior to BMP4 for promoting human muscle-derived stem cell-mediated bone regeneration in a critical-sized calvarial defect model.

Muscle-derived cells have been successfully isolated using a variety of different methods and have been shown to possess multilineage differentiation capacities, including an ability to differentiate into articular cartilage and bone in vivo; however, the characterization of human muscle-derived stem cells (hMDSCs) and their bone regenerative capacities have not been fully investigated. Genetic modification of these cells may enhance their osteogenic capacity, which could potentially be applied to bone regenerative therapies. We found that hMDSCs, isolated by the preplate technique, consistently expressed the myogenic marker CD56, the pericyte/endothelial cell marker CD146, and the mesenchymal stem cell markers CD73, CD90, CD105, and CD44 but did not express the hematopoietic stem cell marker CD45, and they could undergo osteogenic, chondrogenic, adipogenic, and myogenic differentiation in vitro. In order to investigate the osteoinductive potential of hMDSCs, we constructed a retroviral vector expressing BMP4 and GFP and a lentiviral vector expressing BMP2. The BMP4-expressing hMDSCs were able to undergo osteogenic differentiation in vitro and exhibited enhanced mineralization compared to nontransduced cells; however, when transplanted into a calvarial defect, they failed to regenerate bone. Local administration of BMP4 protein and cell pretreatment with N-acetylcysteine (NAC), which improves cell survival, did not enhance the osteogenic capacity of the retro-BMP4-transduced cells. In contrast, lenti-BMP2-transduced hMDSCs not only exhibited enhanced in vitro osteogenic differentiation but also induced robust bone formation and nearly completely healed a critical-sized calvarial defect in CD-1 nude mice 6 weeks following transplantation. Herovici's staining of the regenerated bone demonstrated that the bone matrix contained a large amount of type I collagen. Our findings indicated that the hMDSCs are likely mesenchymal stem cells of muscle origin and that BMP2 is more efficient than BMP4 in promoting the bone regenerative capacity of the hMDSCs in vivo.

Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.

This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process, whereas a slowly adhering cell (SAC) fraction containing muscle-derived stem cells is obtained from the later stages of the process. This protocol outlines the methods and materials needed to isolate RAC and SAC populations from murine skeletal muscle. The procedure involves mechanical and enzymatic digestion of skeletal muscle tissue with collagenase XI, dispase and trypsin followed by plating the resultant muscle slurry on collagen type I-coated flasks where the cells adhere at different rates. The entire preplate technique requires 5 d to obtain the final preplate SAC population. Two to three additional days are usually required before this population is properly established. We also detail additional methodologies designed to further enrich the resultant cell population by continuing the modified preplating process on the SAC population. This process is known as replating and requires further time.

A role for cell sex in stem cell-mediated skeletal muscle regeneration: female cells have higher muscle regeneration efficiency.

We have shown that muscle-derived stem cells (MDSCs) transplanted into dystrophic (mdx) mice efficiently regenerate skeletal muscle. However, MDSC populations exhibit heterogeneity in marker profiles and variability in regeneration abilities. We show here that cell sex is a variable that considerably influences MDSCs' regeneration abilities. We found that the female MDSCs (F-MDSCs) regenerated skeletal muscle more efficiently. Despite using additional isolation techniques and cell cloning, we could not obtain a male subfraction with a regeneration capacity similar to that of their female counterparts. Rather than being directly hormonal or caused by host immune response, this difference in MDSCs' regeneration potential may arise from innate sex-related differences in the cells' stress responses. In comparison with F-MDSCs, male MDSCs have increased differentiation after exposure to oxidative stress induced by hydrogen peroxide, which may lead to in vivo donor cell depletion, and a proliferative advantage for F-MDSCs that eventually increases muscle regeneration. These findings should persuade researchers to report cell sex, which is a largely unexplored variable, and consider the implications of relying on cells of one sex.

Murine bone marrow stromal progenitor cells elicit an in vivo cellular and humoral alloimmune response.

Stromal progenitor cells (SPC) exhibit immunosuppressive effects in vitro that have led to speculation regarding their capacity to evade host immune recognition and to treat autoimmune diseases and gravt-versus-host disease. However, there is little in vivo experimental data to support these immunologic claims. To assess immune recognition of SPC in vivo, we evaluated the immune response of animals transplanted with SPC. C57BL/6 (B6) or Balb/c adult, murine, bone marrow-derived SPC (AmSPC) were administered by intraperitoneal injection into B6 recipients. T cell proliferation and alloantibody response was assessed from spleens and peripheral blood harvested from transplanted animals and analyzed by cell proliferation assay and flow cytometry. To assess tolerance induction, transplanted animals also received allogeneic skin grafts. Animals injected with allogeneic AmSPC mounted an accelerated CD4 response to alloantigen compared to syngeneic AmSPC injected and uninjected controls. Allogeneic AmSPC-injected animals also demonstrated high titers (> or =1:1000) of antibody directed against allogeneic AmSPC targets. Animals primed with donor or host-matched AmSPC also failed to induce tolerance, and all animals exhibited rejection of allogeneic skin grafts (n = 7, P < .0001). In contrast to their in vitro behavior, our data demonstrate that AmSPC are recognized by the host immune system in vivo, elicit a cellular and humoral immune response, and fail to induce tolerance. These findings have significant implications for all allogeneic SPC-based therapeutic strategies.

Optimized large-scale production of high titer lentivirus vector pseudotypes.

The goal of the present study was to develop an efficient transient transfection method for large-scale production of high titer lentivirus vector stocks of eight different pseudotypes. The envelope genes used for this purpose were those from VSV-G, Mokola, Rabies, MLV-Ampho, MLV-10A1, LCMV-WE, and LCMV-Arm53b. All envelopes were cloned into phCMV, which yielded lentivirus vector titers one, two, or three orders of magnitude higher than the original plasmids for the Rabies, MLV-10A1, and MLV-Ampho envelopes, respectively. When these newly constructed envelope expression plasmids were used for packaging, treatment with sodium butyrate resulted in almost five-fold increase in titers for some of the pseudotypes, had no effect for others (VSV-G and Rabies), and negatively impacted titers for the LCMV-derived pseudotypes. Production of vectors in serum-free media yielded titers only slightly lower than those obtained in the presence of serum. The efficiency of concentrating vector supernatants by ultracentrifugation or ultrafiltration was compared, with higher recovery efficiencies for the latter method, but the highest titers for most pseudotypes were obtained by ultracentrifugation. The best conditions for each individual pseudotype yielded lentivirus vector stocks with titers above 1 x 10(9) tu/mL for most pseudotypes, and higher than 1 x 10(10) tu/mL for VSV-G.

Transduction of human glial and neuronal tumor cells with different lentivirus vector pseudotypes.

Lentiviral vectors have proven to be valuable tools for in vitro and in vivo gene delivery because they can transduce dividing and non-dividing cells efficiently, and mediate long-term gene expression. Pseudotyping of lentiviral vectors with envelope proteins other than VSV-G has resulted in enhanced transduction of certain cell types and tissues. In order to improve lentiviral vector-based gene therapy for peripheral neuroectodermal and brain tumors, we compared the efficiency of eight different lentivirus pseudotypes in transducing neuronal and glial tumor cell lines. Here, lentiviral vectors pseudotyped with the envelopes from human foamy virus, rabies, Mokola or amphotropic murine leukemia virus displayed the highest transduction efficiency in neuroblastomas, whereas pseudotyping with the lymphocytic choriomeningitis virus glycoprotein from strain Armstrong 53b resulted in the highest transduction efficiency in gliomas.