Exploring Mechanisms of Early Acquired Resistance to Doxorubicin in Melanoma in 3D Model

Chemoresistance continues to remain a significant barrier to the effective treatment of various cancers, including melanoma. Previous studies, including our own, have demonstrated the limited efficacy of doxorubicin (DOX) against melanoma cells and tumors, both in vitro and in vivo, primarily due to acquired chemoresistance. Furthermore, the lack of favorable outcomes for patients with metastatic melanoma treated with liposomal DOX underscores the urgent need for identifying early biomarkers of DOX-induced chemoresistance and develop combination strategies to improve its therapeutic efficacy and overcome drug resistance. This study examines early adaptive responses in B16.F10 melanoma cells grown as spheroids, after two exposures to DOX used at sub-inhibitory concentration (≈IC ₃₀ ). The investigated processes associated with early establishment of chemoresistance involved cycle regulation, DNA damage and repair mechanisms, apoptosis, angiogenesis, and dissemination potential. Melanoma spheroids were cultured in ultra-low attachment plates and treated with DOX in two 48-hour phases separated by 48-hour recovery periods, followed by viability, morphology, gene/protein expression, and enzyme activity assessments using RNA-seq, RT-qPCR, Western blot, and gelatinase assay, respectively. Functional analysis confirmed downregulation of cell cycle pathways, suppression of stress response and survival pathways (e.g., TNFA_SIGNALING_VIA_NFKB and MTORC1_SIGNALING). Despite mRNA data showing cell cycle arrest (downregulated CDK1, Ticrr, Cdc20 ) and major DNA damage and impaired repair (downregulated BRIP1, BARD1, Rad51, Rmi2 ), after first exposure to DOX, cells probably bypassed cell cycle checkpoints by upregulating transcripts and proteins linked to pro-survival ( Tnfrsf19 , cIAP-2) and angiogenesis (VEGF, eotaxin-1), and by upregulating transcripts related to cell adhesion ( Pcdh beta family members), and ECM remodeling ( Ecm1 , Jam2 ), suggesting early adaptation mechanisms to DOX. With a second DOX exposure, melanoma spheroids showed stronger chemoresistance, evidenced by enhanced angiogenesis (upregulated Aqp1, VEGF, and Ackr3 ), increased invasiveness (upregulated MMP-2 , favored MMP-9 activity), and a 40% increase in pNF-kB levels, reinforcing drug tolerance and aggressiveness. This study provides valuable insights into the molecular drivers of chemoresistance, revealing that early DOX resistance in melanoma arises from adaptive mechanisms enhancing survival, angiogenesis, and invasiveness. Repeated exposure to DOX reinforces these traits, sustaining drug tolerance.