Effect of Simulated Microgravity on Murine Melanoma Cells : Altered Tumor Cell and Tumorigenicity
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Spheroid cells which can grow as nonattached spheroids in vitro culture condition are considered as tumor-initiating cells that have properties similar to those of stem cells. However, the existence of spheroid cells in WM-266-4, a human malignant metastatic melanoma cell line, has not yet been reported.Accordingly, we investigated whether WM-266-4 can form spheroids, and characterized these spheroids using qRT-PCR, histology, immunohistochemistry and xenograft.WM-266-4 contains a small subpopulation of cells that grow as spheroids and express genes strongly related to tumor malignancy and stem-like factors. Second, histological analysis of the spheres revealed that they consist of 300-400 round cells per sphere with a high karyoplasmic ratio. They have a basophilic cytoplasm and are highly pleomorphic in size, and sometimes multinucleated and giant. Third, although there were differences between the spheroid and bulk cells, they both have high tumorigenic potential, as both cell types formed a tumor mass upon injection of only 100 cells in nude mice.We characterized the spheroid cells in an established melanoma cell line. We suggest that enriched spheroid cells might contain more dedifferentiated progenitor cells, but we could not conclude spheroid cells are cancer stem cells.
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The effects of adipose tissue-derived mesenchymal stem cells (AT-MSCs) on the growth of human malignancies, including melanoma, are controversial and the underlying mechanisms are not yet-well understood. The aim of the present study was to investigate the in vitro and in vivo anti-tumor effects of human AT-MSCs on human melanoma.The inhibitory effect of AT-MSC-conditioned medium (AT-MSC-CM) on the growth of A375SM and A375P (human melanoma) cells was evaluated using a cell viability assay. Cell-cycle arrest and apoptosis in melanoma cells were investigated by flow cytometry and western blot analysis. To evaluate the in vivo anti-tumor effect of AT-MSCs, CM-DiI-labeled AT-MSCs were circumtumorally injected in tumor-bearing athymic mice and tumor size was measured.AT-MSC-CM inhibited melanoma growth by altering cell-cycle distribution and inducing apoptosis in vitro. AT-MSCs suppressed tumor growth in tumor-bearing athymic mice and fluorescence analysis showed that AT-MSCs migrated efficiently to tumor tissues.AT-MSCs inhibit the growth of melanoma suggesting promise as a novel therapeutic agent for melanoma.
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This report describes the modulation of malignancy through coinoculation in vivo of a malignant mouse (A9) cell line, and a phenotypically stable, nontumorigenic hybrid clone derived from it by fusion with a transformed mutant human cell. Thus, the tumorigenicity of the A9 cells was suppressed by the hybrid cells, but not by unrelated normal cells. The hybrid cells also inhibited tumor development of unrelated human cancer cells.
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Tumorigenicity refers to the ability of cultured cells to develop viable tumors in immune-deficient animals. The goal of this protocol is to illustrate tumorigenicity assay by subcutaneous tumor-cell-transplantation in nude mice. Target cells are transplanted to 6-week-old nude mice subcutaneously and the tumor growth is monitored over a period of observation or treatment. When tumor grows to a pre-determined size or by the end of the limited period, the nude mice will be euthanatized and the xenograft will be removed for further examination.
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Clonal isolates of mouse 3T3 cells and primary rat embryo cells, recovered nonselectively after infection by simian virus 40 (SV40), have been tested for tumorigenicity in the immune-deficient nude mice in order to determine the cellular growth properties in vitro specifically correlated with neoplastic growth in vivo. In addition, mouse 3T3 cells transformed by murine sarcoma virus (MuSV, Kirsten strain), and revertants isolated from cells fully transformed by either SV40 or MuSV were also studied. Results suggest that the single cellular property consistently associated with tumorigenicity in nude mice is the acquisition by virus-transformed cells of the ability to proliferate in vitro in the absence of anchorage. Other cellular parameters of virus-induced transformation, such as lack of sensitivity to high cell density and the capacity to grow in low serum concentration, are dissociable from cellular tumorigeneicity. This conclusion is supported further by the demonstration that specific selection in vivo for tumorigenic cells from anchorage-dependent cells results in the isolation of anchorage-independent cells. Conversely, a single-step selection in vitro for anchorage-independent cells from nontumorigenic cells results in a simultaneous selection of highly tumorigenic subclones.
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Spaceflight involves numerous biological stressors that could affect long-term cancer incidence and tumor behavior. Ground-based models of microgravity can be used to investigate in vitro and in vivo tumor growth as a preparation for later work in space. The incidence of tumor growth and carcinogenesis in microgravity is as yet unknown. Hence, we investigated the effects of modeled microgravity on tumor growth and tumorigenicity using ground-based in vitro and in vivo models.Murine B16-F10 melanoma cells were cultured in a tissue culture flask (FL) and in a rotating-wall vessel bioreactor (BIO) designed by NASA to simulate some aspects of microgravity. We then measured cell growth, melanin production, and apoptosis. After 48 h of cultures in FL and BIO, cells were inoculated subcutaneously in C57BL/6 mice, syngeneic hosts for B16-F10 tumor cells. Tumor sizes were then measured every other day.BIO cultures had 50% decreases in growth when compared with FL cultures while demonstrating an inversely proportional increase in doubling time. Melanin production (a marker of differentiation) increased at 24 and 48 h in BIO. Flow cytometry analysis demonstrated that there was an increase in the percentage of apoptotic cells in the BIO when compared with that in the FL. When BIO-cultured melanoma cells were inoculated subcutaneously in mice, there was a significant increase in tumorigenicity as compared with FL-cultured cells.Our results indicate that simulated microgravity may have altered the tumor cell characteristics and enhanced the invasive property. It is possible that the microgravity analogue culture environment may have selected highly tumorigenic cells for survival despite the decreased overall growth in the microgravity analogue.
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Previously we've described the obtainment of a subpopulation of cancer stem cells from a human colorec- tal carcinoma cell line MIP101. These cells possess elevated clonogenic and tumorigenic capacities. According to our data, depletion of stem compartment in a cancer cell population blocks its tumorigenicity. The current work is dedicated to the comparison of tumorigenic potential between cell populations with enriched or depleted stem compartment. We show that tumor growth following xenografting of enriched stem cell population can be suppressed by intramuscular injections of ganciclovir. Thus, we report a method to obtain a cell population with high Oct4 promoter expression within the MIP101 colorectal carcinoma cell line and to eliminate these cells from the population in vitro as well as in vivo.
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To examine the effect of bone microenvironmental factors on the growth of metastatic cells, the in vivo proliferative features of three murine cell lines were determined at skeletal metastatic sites and correlated with their ability to grow in vitro in the presence of bone-derived factors. Bones, ovaries, adrenals and the brain were most affected by metastasis, following an intraarterial injection of B16/F1 and B16/F10 melanoma and FS/L10 fibrosarcoma cells into C57BL/6 mice. Melanoma cells showed a marked metastatic preference for bone, while fibrosarcoma cells developed brain metastasis in all animals. Tumor burden in bones was highest (19+/-2%) for B16/F10 cells, compared to B16/F1 (10+/-2%) or FS/L10 (3+/-1%) cells. Autoradiographic studies demonstrated organ- and cell type-specific differences in tumor cell proliferation, with B16/F10 cells displaying the lowest labelling indexes in bone (12+/-2% for B16/F10 vs 28+/-2% and 27+/-4% for B16/F1 and FS/L10 cells, respectively). To test if bone-derived factors differentially affected tumor cell growth in these three cell lines H-3-thymidine uptake by these tumor cells was assessed after in vitro incubation with bone-derived conditioned medium. Under these conditions, we observed stimulation of B16/F10 cell proliferation, but inhibition of uptake in the other two cell lines. Thus, these results demonstrate that, in this in vivo experimental model, growth properties of metastatic cells are organ- and cell type-specific. Additionally, we show that the in vitro proliferative behavior of tumor cells in the presence of bone-derived factors correlates and may predict skeletal tumor growth properties in vivo.
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