Selection of highly malignant tumour cells using reconstituted basement membrane matrix.

A new technique was developed to coat Nuclepore filters with basement membrane matrix components. Using the EHS tumour as a source, a proteoglycan and laminin-containing fraction was extracted with guanidine and collagen type IV was solubilized in a second fraction by limited digestion with pepsin. When the two fractions are combined and guanidine removed by dialysis, a gel is rapidly formed. A flat-bed dialysis apparatus was devised, allowing gels to form on filters that are soaked in the mixture, thus coating them with components of the basement membrane. Such filters were used for selection of B16 melanoma cells penetrating the gels. 10 clones were isolated after three selection passages, and assayed for spontaneous metastasis in C57Bl/6 mice after intracutaneous injection. The metastasis rates were strongly increased to the lungs and to inguinal and axillary lymph nodes. Less accessible sites such as mediastinal and maxillary lymph nodes were reached only by selected cells. There was no particular preference for the haematogenous or lymphatic routes, indicating that the ability to traverse the basement membrane leads to a general increase of metastasis. The described method provides a valuable tool for isolating those subpopulations able to traverse basement membranes and to assess this capacity in new tumour samples.