ABSTRACT
A parental line of mouse B16 melanoma cells (B16) and two derived cloned lines, either pigmented (B16P) or non pigmented (B16NP), were cultured in vitro as spheroids. After 48 hrs, the pigmented cells (B16, B16P) formed smaller and looser aggregates, with higher rates of cell proliferation and lower amounts of extracellular matrix as compared to B16NP spheroids. The three lines were more tumorigenic when inoculated subcutaneously as spheroids than as isolated cells. Furthermore, B16P or B16 spheroids developed richly vascularized subcutaneous tumors and metastases more rapidly than B16NP aggregates. After intravenous injection of spheroids, the measurement with an image analyzer of the area of sections in lung colonies indicated that B16P colonies were larger and more numerous than those induced by B16NP cells.
Subject(s)
Melanoma, Experimental/pathology , Animals , Cell Adhesion/physiology , Cell Aggregation/physiology , Glycoproteins/analysis , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Melanoma, Experimental/chemistry , Melanoma, Experimental/secondary , Mice , Mice, Inbred C57BL , Neoplasm Invasiveness , Neoplasm Transplantation , Pigmentation , Tumor Cells, CulturedABSTRACT
Multicellular spheroids which promote cell-cell and cell-matrix interactions were prepared in culture with mouse B16 melanoma cells (pigmented or non pigmented) alone or mixed with mouse 3T3 fibroblasts. Their volume and proliferation or necrosis rate were evaluated. As measured by dot blot immunoassay, laminin was mainly produced by fibroblasts rather than by melanoma cells. High levels of laminin B1 chain mRNA were detected only in spheroids composed of 3T3 fibroblasts. The levels of 67 kD laminin binding protein mRNA were high in all cell populations studied here.
Subject(s)
Laminin/analysis , Melanoma, Experimental/pathology , Receptors, Laminin/analysis , 3T3 Cells , Animals , Fibroblasts/cytology , Fibroblasts/metabolism , Immunoenzyme Techniques , Immunohistochemistry , Kinetics , Laminin/genetics , Melanoma, Experimental/metabolism , Mice , Mitotic Index , Molecular Weight , RNA, Messenger/analysis , Receptors, Laminin/genetics , Tumor Cells, CulturedABSTRACT
Cytotoxic and mitogenic soluble factors are released into media conditioned by pure or mixed populations of mouse 3T3 fibroblasts and B16 melanoma cells cultivated in vitro. These activities are demonstrated by the use of MTT cell survival test and 3HTDR incorporation. Mitogenic (M.W. greater than 10,000) and cytotoxic factors (M.W. less than 1,000) are present and are generally more active on B16 cells than on fibroblasts. Their release into conditioned media is related to the rate of pigmentation in B16 cells and to the mode of cultivation (monolayers or cell aggregates).
Subject(s)
Cell Survival , Growth Inhibitors/isolation & purification , Melanoma, Experimental/metabolism , Mitogens/isolation & purification , Animals , Cell Line , Cell Survival/drug effects , Culture Media , Culture Techniques/methods , DNA Replication , Fibroblasts/cytology , Growth Inhibitors/pharmacology , Mice , Mitogens/pharmacology , UltrafiltrationABSTRACT
Culture media conditioned (CM) by mixed populations of mouse B16 melanoma cells and 3T3 fibroblasts cultivated as monolayers exert cytological effects on B16 or 3T3 cells when treated separately in culture. By ultrafiltration of these CM, we show that a stimulatory activity on B16 melanoma cells proliferation is present in fractions with M.W. greater than 10,000 daltons. A strong cytotoxic activity for B16 melanoma cells and, to a lower degree, for 3T3 fibroblasts is detected in fractions with M.W. less than 1,000 daltons. The ultrastructural analysis of cells (B16 or 3T3) treated with cytotoxic fractions reveals in them mitochondrial swelling, blebs, broken membranes and dead cells.
