Background: Triple-negative (MDA-MB-231) and hormone-responsive (MCF-7) breast cancers exhibit complex immune-evasive mechanisms within the three-dimensional (3D) tumour microenvironment. While vitamin C (Vit-C) has shown immunomodulatory potential in two-dimensional systems, its efficacy and synergistic potential with chemotherapy in 3D architectures remain poorly defined. Drug resistance and toxicity remain major challenges in breast cancer (BC) therapy. We investigated a novel strategy combining high-dose Vit-C with chemotherapy to enhance anti-tumour immunity in 3D BC models. Methods: MDA-MB-231 and MCF-7 spheroids were established and co-cultured with human natural killer (NK) cells and CD8⁺ T cells. To isolate functional enhancement from pro-oxidant damage, immune cells were pre-treated with catalase (CAT) and subsequently primed with 1 mM Vit-C for 6 h. These primed effectors were then co-cultured for 24 h (at a 2:1 effector-to-target ratio for single cell types or 1:1:1 for mixed NK+CD8⁺ T cells) with spheroids that had been pre-conditioned for 24 h with sub-lethal doses of doxorubicin (DOX, 0.5 μM), docetaxel (DOCE, 0.5 μM), Vit-C, or their triple combination. Spheroid viability was assessed via ATP bioluminescence (CellTiter-Glo®), and cytotoxicity was quantified by lactate dehydrogenase (LDH) release. Secreted effector molecules—interferon-gamma (IFN-γ) and granzyme B—were measured in co-culture supernatants by ELISA. Results: Vit-C priming significantly enhanced both NK and CD8⁺ T cell cytotoxicity against BC spheroids (p < 0.001). A potent synergistic effect was observed when Vit-C-primed immune cells were co-cultured with chemotherapy-conditioned spheroids. This combination drove a massive escalation in IFN-γ and granzyme B production. The triple combination (Vit-C + DOX + DOCE) elicited the most robust response (p < 0.0001), yielding IFN-γ concentrations of 18.17 ± 1.476 ng/mL and granzyme B concentrations of 67.20 ± 5.211 ng/mL. This represents a significant 26-fold increase in IFN-γ and a 54-fold increase in granzyme B over untreated controls. Conclusion: This study demonstrates that Vit-C, when combined with sub-lethal chemotherapeutic conditioning, acts as a powerful adjuvant to overcome 3D tumor resistance. These findings provide a strong preclinical foundation for integrating pharmacological Vit-C into current chemo-immunotherapy regimens for BC.
Hamid et al. (Mon,) studied this question.