Limited penetration of methotrexate into human osteosarcoma spheroids as a proposed model for solid tumor resistance to adjuvant chemotherapy

Abstract

It is generally accepted that oxygen has a limited ability to diffuse into solid tumor masses. However, the question of the ability of chemotherapy agents to penetrate solid tumor masses has not been evaluated. This clearly would have an impact on the ability of chemotherapy to control microscopic disease during the "avascular" phase of growth. An attempt was made to evaluate the ability of methotrexate to penetrate solid tumor masses when grown in three dimensions (spheroids). Since methotrexate is used in the clinical management of human osteosarcoma, we chose this drug-tumor combination for our studies. This was done by growing human osteosarcoma cells into spheroids and exposing spheroids of various sizes to tritiated methotrexate. Audioradiographs were obtained from sections through the center of spheroids of various sizes. Our findings suggest that methotrexate has a limited ability to penetrate into avascular tumor masses when grown in three dimensions. This is most evident when the tumor masses are approximately 250 micron and larger in diameter. In addition, we compared the degree of penetration of methotrexate to the growth fraction of the tumor, as measured by tritiated thymidine, and found that the growth fraction was much greater than the fraction of cells reached by methotrexate. We conclude that the limited ability of methotrexate to penetrate solid tumor masses offers an alternative explanation for the limited effectiveness of methotrexate when used as an adjuvant for osteosarcoma. We question whether the established biochemical mechanisms for methotrexate resistance are comprehensive explanations for its limited clinical effectiveness.