In-Vitro and In-Vivo Establishment and Characterization of Bioluminescent Orthotopic Chemotherapy-Resistant Human Osteosarcoma Models in NSG Mice

Abstract

Osteosarcoma, the most common bone malignancy with a peak incidence at adolescence, had no survival improvement since decades. Persistent problems are chemo-resistance and metastatic spread. We developed in-vitro osteosarcoma models resistant to chemotherapy and in-vivo bioluminescent orthotopic cell-derived-xenografts (CDX). Continuous increasing drug concentration cultures in-vitro resulted in five methotrexate (MTX)-resistant and one doxorubicin (DOXO)-resistant cell lines. Resistance persisted after drug removal except for MG-63. Different resistance mechanisms were identified, affecting drug transport and action mechanisms specific to methotrexate (RFC/SCL19A1 decrease, DHFR up-regulation) for MTX-resistant lines, or a multi-drug phenomenon (PgP up-regulation) for HOS-R/DOXO. Differential analysis of copy number abnormalities (aCGH) and gene expression (RNAseq) revealed changes of several chromosomic regions translated at transcriptomic level depending on drug and cell line, as well as different pathways implicated in invasive and metastatic potential (e.g., Fas, Metalloproteinases) and immunity (enrichment in HLA cluster genes in 6p21.3) in HOS-R/DOXO. Resistant-CDX models (HOS-R/MTX, HOS-R/DOXO and Saos-2-B-R/MTX) injected intratibially into NSG mice behaved as their parental counterpart at primary tumor site; however, they exhibited a slower growth rate and lower metastatic spread, although they retained resistance and CGH main characteristics without drug pressure. These models represent valuable tools to explore resistance mechanisms and new therapies in osteosarcoma.

Mechanisms of Resistance to Conventional Therapies for Osteosarcoma

“…MTX resistant OS cell lines exhibit high expression of DHFR. Indeed, a relationship between high expression of DHFR in xenografts and emergence of resistance has been shown [ 84 , 85 ]. High expression of DHFR is rare in initial biopsy samples (10% of samples) but is very frequently observed in metastases (62% of samples), suggesting that this resistance mechanism appears during treatment.…”

MT2A is an early predictive biomarker of response to chemotherapy and a potential therapeutic target in osteosarcoma

“…To evaluate if repression of MT2A can reverse chemo-resistance, we modified HOS cells that have acquired in vitro resistance to doxorubicin (Resistance Index = 88; see Methods section) 9 . This HOS-Doxo R cell line exhibited a cross-resistance to etoposide (RI = 140) but not to cisplatin or ifosfamide 9 . As expected, Hos-Doxo R cells modified with sh-MT2A sequences exhibited significantly lower MT2A mRNA level (−40% vs .…”

“…The human cell line OHS-4 was kindly provided by Drs Fournier and Price 24 . The human HOS-Doxo R cell lines was established by continual exposure to increasing concentration of Doxorubicin (from 0.01 µM up to 1.3 µM) 9 .…”

Targeting Molecular Mechanisms Underlying Treatment Efficacy and Resistance in Osteosarcoma: A Review of Current and Future Strategies

“…It primarily enters cells via the reduced folate carrier (RFC encoded by SLC19A1) at the cell membrane with folate receptor alpha only playing a minor role [284]. Decreased expression of RFC has been shown to mediate methotrexate resistance in osteosarcoma [285][286][287]. Overall, 65% of osteosarcoma biopsy samples were found to have decreased RFC expression, with low expression more commonly found in patients with poor histological response to chemotherapy [285].…”

“…Mutations in DHFR leading to reduced affinity for methotrexate are important in the acquisition of methotrexate resistance for some cancers, including acute leukaemia [320]. High levels of DHFR expression have been described in methotrexate-resistant osteosarcoma cell lines, and xenografts with high DHFR expression exhibit methotrexate resistance [287]. Gene duplications in DHFR are common mechanisms underlying methotrexate resistance in patients with acute lymphoblastic leukaemia, and these duplications are common in patients with concurrent p53 mutations [321].…”