It has been suggested that chemoresistance of
chondrosarcoma (CHS), the cartilage
tumor, is caused by the phenotypic microenvironmental features of the
tumor tissue, mainly the chondrogenic extracellular matrix (ECM), and
hypoxia. We developed and characterized a multicellular
tumor spheroid (MCTS) of human
chondrosarcoma HEMC-SS cells to gain insight into
tumor cell biology and drug response. At Day 7, HEMC-SS spheroids exhibited a homogeneous distribution of proliferative Ki-67 positive cells, whereas in larger spheroids (Day 14 and Day 20), proliferation was mainly localized in the periphery. In the core of larger spheroids, apoptotic cells were evidenced by TUNEL assay, and
hypoxia by
pimonidazole staining. Interestingly,
VEGF excretion, evidenced by ELISA on
culture media, was detectable from Day 14 spheroids, and increased as the spheroids grew in size. HEMC-SS spheroids synthesized a chondrogenic extracellular matrix rich in
glycosaminoglycans and type-2
collagen. Finally, we investigated the sensitivity of Day 7 and Day 14
chondrosarcoma MCTS to
hypoxia-activated
prodrug TH-302 and
doxorubicin compared with their 2D counterparts. As expected,
TH-302 exhibited higher cytotoxic activity on larger hypoxic spheroids (Day 14) than on non-hypoxic spheroids (Day 7), with multicellular resistance index (MCRI) values of 7.7 and 9.1 respectively. For
doxorubicin, the larger-sized spheroids exhibited higher drug resistance (MCRI of 5.0 for Day 7 and 18.3 for Day 14 spheroids), possibly due to impeded drug penetration into the deep layer of spheroids, evidenced by its auto-fluorescence property. We have developed a model of human
chondrosarcoma MCTS that combines an ECM rich in
glycosaminoglycans with a high hypoxic core associated with
VEGF excretion. This model could offer a more predictive in vitro
chondrosarcoma system for screening drugs targeting
tumor cells and their microenvironment.