The effect of natural
protease inhibitors and a
chemoattractant on
tumor cell invasion were studied with the use of a new in vitro quantitative assay of
tumor cell penetration of native connective tissue. Human amnion membrane denuded of its epithelium is composed of a continuous basement membrane (BM) attached to a dense avascular collagenous stroma. M5076 reticulum
sarcoma cells, known to be highly invasive in vivo, were placed on the BM side of the amnion connective tissue.
Tumor cells penetrating the full thickness of the connective tissue barrier were collected on the stromal side with a Millipore filter.
N-Formylmethionyl-leucyl-phenylalanine (FMLP) at an optimal concentration of 10(-7) M stimulated the penetration of up to 600% more
tumor cells into the connective tissue after 20 hours in comparison to the number of
tumor cells spontaneously penetrating in
serum-free media. Natural
protease inhibitors blocked both FMLP-stimulated and spontaneous invasion. A
bovine cartilage extract containing inhibitors of both
serine proteinases and
metalloproteinases caused a 500% decrease in invasion. Furthermore, a 500% inhibition of invasion was produced by a purified
collagenase (
metalloproteinase) inhibitor. In contrast, soybean
trypsin inhibitor and
bovine serum albumin did not significantly alter the invasion rate. The
protease inhibitors were nontoxic and did not reduce
tumor cell proliferation, attachment to the amnion, and the rate of
tumor cell migration through Nuclepore filters. These data support the hypothesis that collagenolytic
metalloproteinases play a necessary role in
tumor cell invasion of native connective tissue.