An immunohistochemical and ultrastructural study of human melanoma colonies grown in soft agar for up to 50 days was performed. Three morphological variants of developing tumor colonies are reported: (1) large light colonies, (2) small dark colonies, and (3) smooth‐edged colonies. The large light colony variant is the most frequently observed in the soft agar assay (≈70%), followed by the dark colony variant (≈27%), and the smooth‐edged colony variant (≈3%). Major morphological characteristics are associated with each variant, as shown with light microscopy (LM) and transmission electron microscopy (TEM). Both LM and TEM analyses demonstrated that the large light colony variant was hypomelanotic and contained a microfibrillar extracellular matrix (ECM). The small dark colony variant was found to be hypermelanotic and contained a less demonstrable ECM. The smooth‐edged variant has an encapsulated periphery, no demonstrable ECM, and tightly packed cells with desmosome‐like junctions. In order to characterize further the ECM in the most commonly observed variant, the large light colony, specific antibodies to fibronectin (FN) and collagen types IV and V (COLs IV and V) were applied and observed with immunofluorescence microscopy and immu‐noperoxidase. In paraffin sections of melanoma colonies, FN was observed associated with both the cell surface and the ECM. However, no specific staining was seen for COLs IV and V. In addition, ruthenium red was used to preserve and selectively bind to glycosaminoglycans (GAGs) and proteoglycans (PGs). TEM studies reveal GAG‐like granules stained with ruthenium red in the fibrillar ECM and a dotted, punctate staining of the cell surface. Understanding the biological and architectural composition of developing melanoma tumor colonies in soft agar could contribute to the development of more efficient chemo‐therapeutic strategies.
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