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Transcriptomic Characterization of Doxorubicin Resistance in Bone Sarcoma Organoids

ABSTRACT: Tumor organoids have emerged as the most promising avatar to model cancer in vitro and to be used as drug testing platform in personalized medicine protocols. The derivation of organoids from epithelial tumors is well established, and their use has proven useful for biomarker discovery, drug screening, and clinical decision support. However, the generation of organoids from sarcomas is much less developed and current protocols require further optimization to fully harness their research and clinical potential. In this study, we explored the challenges of generating basement membrane extract (BME)-based organoids from patient-derived sarcoma samples. Of the 19 samples processed, 3D structures formed in 7 (37%), yet none of these organoids could be propagated beyond the first passage. As an alternative approach, we successfully generated 3D organoids with long-term growth potential from sarcoma xenografts. Of the 14 xenograft samples tested, 7 (50%) could be maintained through at least three passages and were successfully biobanked. Specifically, organoids derived from xenografts grown from parental and doxorubicinresistant 143B osteosarcoma cells could be maintained as long-term cultures and used as a 3D platform to investigate drug resistance in osteosarcoma. Transcriptomic profiling and functional assays performed in these models identified the nonreceptor protein tyrosine kinase SRC as a key mediator of resistance to doxorubicin in osteosarcoma. Importantly, we demonstrated that inhibition of SRC signaling using dasatinib restores doxorubicin sensitivity in osteosarcoma cells by suppressing c-JUN–driven ABCB1 expression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE303294 | GEO | 2026/06/03

REPOSITORIES: GEO

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Project description:Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chem-otherapy during the past decades the survival rates of osteosarcoma patients remains usatisfacto-ry. Drug resistance is one of the main reasons leading to treatment failure and poor progno-sis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug re-sistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofi-bromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interac-tions in this process. Moreover, high throughput sequencing analysis identified differential regula-tion of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, in-cluding p53 apoptosis effector related to PMP-22 (Perp). Deletion of CD44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.

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Transcriptomic Characterization of Doxorubicin Resistance in Bone Sarcoma Organoids

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