Abstract 1978: Sarcoma invasion associated with heparan sulfate degradation

Abstract INTRODUCTION: Sarcomas are locally aggressive, angiogenic and spread hematogenously. The purpose of this study was to quantify the effect of heparan sulfate degrading activity on invasion of K-ras-transformed sarcoma cells. METHODS: Heparan degrading enzyme assays were performed in triplicate on lysate from Balb/c 3T3 (fibroblasts) and K234 cells (K-ras-transformed 3T3) cells (sarcoma) using an enzyme assay kit (cat# MK412;Takara). Invasion assays were performed using Corning Matrigel Invasion Chamber (cat#354480; ThermoFisher ). 7.5 x 104 cells were placed in serum-free DMEM in the upper chamber (insert), and DMEM with 10% FBS as attractant in the lower chamber. After incubation for 24 h at 37 °C, the insert was removed and cells on the undersurface of the insert were fixed with 5% glutaraldehyde for 10 min, then stained with 1% crystal violet in 2% ethanol for 20 min. After the insert was dry, the cells on the filter were counted by microscope over multiple fields. For experiments with inhibitors, 10 ug/ml λ-carageenan (cat# 22049; Sigma), anti-NAP-2 antibody (cat# sc-19224; Santa Cruz Biotech), or anti-HPA1 antibody were used. RESULTS: Heparan sulfate-degrading activity was 0.0625 U/ug for K234 cell lysate compared to 0.0125 U/ug for Balb/c3T3 cells. λ-carageenan inhibited the activity completely confirming glycanase inhibition. Anti-NAP2 and anti-HPA1 antibody inhibited heparan sulfate degrading activity in 3T3 lysate. Invasion was two-fold greater by K234 cells than for 3T3 cells. When anti-NAP2 antibody was added to the invasion assay, both K234 and 3T3 cell invasion was inhibited by 55% and 54%, respectively. Less than 1% inhibition of invasion occurred with anti-HPA1 antibody. CONCLUSION: The invasion of heparan sulfate-containing Matrigel by K234 cells is partly influenced by heparan sulfate-degrading activity. The inhibition of the degrading activity by an anti-chemokine antibody decreased invasion, and invokes other processes for the malignant sarcoma phenotype. Citation Format: Donghong Ju, Leonard Lipovich, Mary A. Kosir. Sarcoma invasion associated with heparan sulfate degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1978.