Head and neck squamous cell carcinoma (HNSCC) has a poor prognosis with less than 50% mortality rate with the multimodal therapies. Although surgery, chemotherapy and radiation therapy are most important treatment modalities used, there are associated long term side effects with poor life expectancy which bids for new emerging treatment strategies. In this study, MN mediated delivery of iron oxide nanoparticles & oxaliplatin with immune checkpoint inhibitors (anti PD-L1, anti-CTLA-4) was evaluated for its induction of ferroptosis and immunomodulation in Head and neck squamous cell carcinoma.

HNSCC cell line MOC-1 was treated with IONPs, Oxaliplatin, and ICI which were assessed in both In-vitro & In-vivo systems. For In-vitro studies, Cytotoxicity (CCK-8), Ferroptosis induction ROS (DCF-DA) and LPO (BODIPY C11), Apoptosis (Annexin-PI), Cell Cycle (PI) analyzed through spectrophotometry & Flow Cytometry and Ferroptosis/Apoptosis markers (GPX4, COX2, c-Caspase 3) through Western blot to compare treated/untreated groups. For In-vivo studies, MOC1-lucitag cells injected in C57BL/6 mice subcutaneously and tumor sizes assessed in dissolvable MN NP treated groups by bioluminescence signals. IHC-P was performed with treated/untreated tissue samples. Differential Expressed Genes (DEGs) and PCA deduced through RNA-Seq and data was analyzed through EBSeq.

IONP DLIN showed the most effective cytotoxicity with least IC50 (0.20 mM) compared to unconjugated IONP (0.90 mM) and Oxaliplatin (0.45 mM) in 24h MOC-1 cells In-vitro. Significant ferroptosis induction seen in DLIN (0.30 mM) treated cells with high LPO+ (52%) and high ROS+ (64%) compared to untreated cells. Oxaliplatin (0.30 mM) showed higher ROS/LPO induction among all treatment groups In-vitro. Apoptosis induction was higher in Oxaliplatin with Annexin V + cells (30%) than DLIN (14%) or Erastin (9%) as compared to untreated (5%). Significant cell death with high sub G1 % in DLIN (86%) than apoptotic cells and changes in protein markers such reduced GPX4 and increased COX2 expression in Erastin, DLIN and mix treated cells is implicative of ferroptosis induction. In-vivo MN-treatment showed decreased tumor with highest reduction in mixed (IONP/Oxali/ICI) as compared to untreated group. Immunomodulation was corroborated with high CD8 than CD4 and lower PD-L1, Ki67 expression in DLIN, Oxaliplatin & Mix IHC-P groups. RNA-Seq data revealed ~ 4600-7500 differential expressed genes (DEGs)(FDR < 0.05) in DLIN samples as compared to untreated both in-vitro/in-vivo groups.

IONP NPs has shown significant cytotoxicity with ferroptosis induction in cancer cells which was comparable to Oxaliplatin. The synergistic effect of the MN-mediated delivery of IONP/Oxali with the immune checkpoint inhibitors had enhanced cytotoxicity in tumor cells which could be targeted in cancer therapeutics strategies.