Ascorbate in Pharmacological Concentrations Potentiates the Anti-Tumor Activity of NK and CD8+ T Cells and Synergizes with Chemotherapy for Enhanced Anti-Tumor Immune Response against 3D Breast Cancer Spheroid Models

Ali Mussa; Mahasin Hamid; Ahmad Hafiz Murtadha; Mustafa Talib; Khalid Hajisa; Noor Fatmawati Mokhtar; Rohimah Mohamud; Mohammad AI Al-Hatamleh; Rosline Hassan

Abstract

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.

References

1.
Li, J.J.; Tsang, J.Y.; Tse, G.M. Tumor Microenvironment in Breast Cancer Updates on Therapeutic Implications and Pathologic Assessment. Cancers 2021, 13, 4233. https://doi.org/10.3390/cancers13164233.
2.
Kundu, M.; Butti, R.; Panda, V.K.; et al. Modulation of the Tumor Microenvironment and Mechanism of Immunotherapy Based Drug Resistance in Breast Cancer. Mol. Cancer 2024, 23, 92. https://doi.org/10.1186/s12943-024-01990-4.
3.
Rodríguez Bejarano, O.H.; Parra López, C.; Patarroyo, M.A. A Review Concerning the Breast Cancer Related Tumour Microenvironment. Crit. Rev. Oncol. Hematol. 2024, 199, 104389. https://doi.org/10.1016/j.critrevonc.2024.104389.
4.
Guo, Z.; Zhu, Z.; Lin, X.; et al. Tumor Microenvironment and Immunotherapy for Triple Negative Breast Cancer. Biomark. Res. 2024, 12, 166. https://doi.org/10.1186/s40364-024-00714-6.
5.
Luchtel, R.A.; Bhagat, T.; Pradhan, K.; et al. High Dose Ascorbic Acid Synergizes with Anti PD1 in a Lymphoma Mouse Model. Proc. Natl. Acad. Sci. USA 2020, 117, 1666–1677. https://doi.org/10.1073/pnas.1908158117.
6.
Ma, J.; Zhang, C.; Shi, G.; et al. High Dose VitC plus Oncolytic Adenoviruses Enhance Immunogenic Tumor Cell Death and Reprogram Tumor Immune Microenvironment. Mol. Ther. 2022, 30, 644–661. https://doi.org/10.1016/j.ymthe.2021.09.015.
7.
Magrì, A.; Germano, G.; Lorenzato, A.; et al. High Dose Vitamin C Enhances Cancer Immunotherapy. Sci. Transl. Med. 2020, 12, eaay8707. https://doi.org/10.1126/scitranslmed.aay8707.
8.
Mussa, A.; Mohd Idris, R.A.; Ahmed, N.; et al. High Dose Vitamin C for Cancer Therapy. Pharmaceuticals 2022, 15, 711. https://doi.org/10.3390/ph15060711.
9.
Mussa, A.; Afolabi, H.A.; Syed, N.H.; et al. The NF κB Transcriptional Network Is a High Dose Vitamin C Targetable Vulnerability in Breast Cancer. Biomedicines 2023, 11, 1060. https://doi.org/10.3390/biomedicines11041060.
10.
Mussa, A.; Hamid, M.; Hajissa, K.; et al. Pharmacological Vitamin C Induced High H2O2 Generation Mediates Apoptotic Cell Death by Caspase 3/7 Activation in Breast Cancer Tumor Spheroids. J. Transl. Med. 2025, 23, 31. https://doi.org/10.1186/s12967-024-06016-7.
11.
Mitrakas, A.G.; Tsolou, A.; Didaskalou, S.; et al. Applications and Advances of Multicellular Tumor Spheroids: Challenges in Their Development and Analysis. Int. J. Mol. Sci. 2023, 24, 6949. https://doi.org/10.3390/ijms24086949.
12.
Kciuk, M.; Gielecińska, A.; Mujwar, S.; et al. Doxorubicin—An Agent with Multiple Mechanisms of Anticancer Activity. Cells 2023, 12, 659. https://doi.org/10.3390/cells12040659.
13.
Imran, M.; Saleem, S.; Chaudhuri, A.; et al. Docetaxel: An Update on Its Molecular Mechanisms, Therapeutic Trajectory and Nanotechnology in the Treatment of Breast, Lung and Prostate Cancer. J. Drug Deliv. Sci. Technol. 2020, 60, 101959. https://doi.org/10.1016/j.jddst.2020.101959.
14.
Ma, Z.; Zhang, W.; Dong, B.; et al. Docetaxel Remodels Prostate Cancer Immune Microenvironment and Enhances Checkpoint Inhibitor Based Immunotherapy. Theranostics 2022, 12, 4965–4979. https://doi.org/10.7150/thno.73152.
15.
Chen, W.; Wong, C.; Vosburgh, E.; et al. High Throughput Image Analysis of Tumor Spheroids: A User Friendly Software Application to Measure the Size of Spheroids Automatically and Accurately. J. Vis. Exp. 2014, 91, 51639. https://doi.org/10.3791/51639.
16.
Lee, S.J.; Jeong, J.H.; Lee, I.H.; et al. Effect of High Dose Vitamin C Combined with Anti Cancer Treatment on Breast Cancer Cells. Anticancer Res. 2019, 39, 751–758. https://doi.org/10.21873/anticanres.13172.
17.
Pilco Ferreto, N.; Calaf, G.M. Influence of Doxorubicin on Apoptosis and Oxidative Stress in Breast Cancer Cell Lines. Int. J. Oncol. 2016, 49, 753–762. https://doi.org/10.3892/ijo.2016.3558.
18.
Wang, S.; Konorev, E.A.; Kotamraju, S.; et al. Doxorubicin Induces Apoptosis in Normal and Tumor Cells via Distinctly Different Mechanisms: Intermediacy of H2O2- and p53-Dependent Pathways. J. Biol. Chem. 2004, 279, 25535–25543. https://doi.org/10.1074/jbc.M400944200.
19.
Saueressig, S.; Tessmann, J.; Mastelari, R.; et al. Synergistic Effect of Pyrazoles Derivatives and Doxorubicin in Claudin Low Breast Cancer Subtype. Biomed. Pharmacother. 2018, 98, 390–398. https://doi.org/10.1016/j.biopha.2017.12.062.
20.
Cabeza, L.; Ortiz, R.; Arias, J.L.; et al. Enhanced Antitumor Activity of Doxorubicin in Breast Cancer through the Use of Poly(butylcyanoacrylate) Nanoparticles. Int. J. Nanomed. 2015, 10, 1291–1306. https://doi.org/10.2147/ijn.S74378.
21.
Turan, T.; Kannan, D.; Patel, M.; et al. Immune Oncology, Immune Responsiveness and the Theory of Everything. J. Immunother. Cancer 2018, 6, 50. https://doi.org/10.1186/s40425-018-0355-5.
22.
Huijskens, M.J.A.J.; Walczak, M.; Sarkar, S.; et al. Ascorbic Acid Promotes Proliferation of Natural Killer Cell Populations in Culture Systems Applicable for Natural Killer Cell Therapy. Cytotherapy 2015, 17, 613–620. https://doi.org/10.1016/j.jcyt.2015.01.004.
23.
Xu, Y.-p.; Lv, L.; Liu, Y.; et al. Tumor Suppressor TET2 Promotes Cancer Immunity and Immunotherapy Efficacy. J. Clin. Invest. 2019, 129, 4316–4331. https://doi.org/10.1172/JCI129317.
24.
Toliopoulos, I.; Simos, Y.; Verginadis, I.; et al. NK Cell Stimulation by Administration of Vitamin C and Aloe vera Juice in Vitro and in Vivo: A Pilot Study. J. Herb. Med. 2012, 2, 29–33. https://doi.org/10.1016/j.hermed.2012.04.002.
25.
Raskov, H.; Orhan, A.; Christensen, J.P.; et al. Cytotoxic CD8⁺ T Cells in Cancer and Cancer Immunotherapy. Br. J. Cancer 2021, 124, 359–367. https://doi.org/10.1038/s41416-020-01048-4.
26.
Sant, D.W.; Mustafi, S.; Gustafson, C.B.; et al. Vitamin C Promotes Apoptosis in Breast Cancer Cells by Increasing TRAIL Expression. Sci. Rep. 2018, 8, 5306. https://doi.org/10.1038/s41598-018-23714-7.
27.
Zhao, X.; Liu, M.; Li, C.; et al. High Dose Vitamin C Inhibits PD L1 by ROS pSTAT3 Signal Pathway and Enhances T Cell Function in TNBC. Int. Immunopharmacol. 2024, 126, 111321. https://doi.org/10.1016/j.intimp.2023.111321.
28.
Ghebeh, H.; Lehe, C.; Barhoush, E.; et al. Doxorubicin Downregulates Cell Surface B7 H1 Expression and Upregulates Its Nuclear Expression in Breast Cancer Cells: Role of B7 H1 as an Anti Apoptotic Molecule. Breast Cancer Res. 2010, 12, R48. https://doi.org/10.1186/bcr2605.
29.
Zhou, S.; Wang, B.; Wei, Y.; et al. PD 1 Inhibitor Combined with Docetaxel Exerts Synergistic Anti Prostate Cancer Effect in Mice by Down Regulating the Expression of PI3K/AKT/NFKB P65/PD L1 Signaling Pathway. Cancer Biomark. 2024, 40, 47–59. https://doi.org/10.3233/cbm-230090.
30.
Symes, J.C.; Kurin, M.; Fleshner, N.E.; et al. Fas Mediated Killing of Primary Prostate Cancer Cells Is Increased by Mitoxantrone and Docetaxel. Mol. Cancer Ther. 2008, 7, 3018–3028. https://doi.org/10.1158/1535-7163.MCT-08-0335.
31.
Sawasdee, N.; Wattanapanitch, M.; Thongsin, N.; et al. Doxorubicin Sensitizes Breast Cancer Cells to Natural Killer Cells in Connection with Increased Fas Receptors. Int. J. Mol. Med. 2022, 49, 64. https://doi.org/10.3892/ijmm.2022.5095.
32.
Wennerberg, E.; Sarhan, D.; Carlsten, M.; et al. Doxorubicin Sensitizes Human Tumor Cells to NK Cell and T Cell Mediated Killing by Augmented TRAIL Receptor Signaling. Int. J. Cancer 2013, 133, 1643–1652. https://doi.org/10.1002/ijc.28163.
33.
Twomey, J.D.; Zhao, L.; Luo, S.; et al. Tubulin Couples Death Receptor 5 to Regulate Apoptosis. Oncotarget 2018, 9, 36804–36815. https://doi.org/10.18632/oncotarget.26407.
34.
Lee, W.H.; Kim, S.C.; Kim, S.H.; et al. Docetaxel Enhances Tumor Necrosis Factor Related Apoptosis Inducing Ligand Mediated Apoptosis in Prostate Cancer Cells via Epigenetic Gene Regulation by Enhancer of Zeste Homolog 2. World J. Mens Health 2023, 41, 649–658. https://doi.org/10.5534/wjmh.220073.
35.
Xiong, Y.; Xu, S.; Fu, B.; et al. Vitamin C Induced Competitive Binding of HIF 1α and p53 to Ubiquitin E3 Ligase CBL Contributes to Anti Breast Cancer Progression through p53 Deacetylation. Food Chem. Toxicol. 2022, 168, 113321. https://doi.org/10.1016/j.fct.2022.113321.
36.
Riffle, S.; Hegde, R.S. Modeling Tumor Cell Adaptations to Hypoxia in Multicellular Tumor Spheroids. J. Exp. Clin. Cancer Res. 2017, 36, 102. https://doi.org/10.1186/s13046-017-0570-9.
37.
Fourie, C.; du Plessis, M.; Mills, J.; et al. The Effect of HIF 1α Inhibition in Breast Cancer Cells Prior to Doxorubicin Treatment under Conditions of Normoxia and Hypoxia. Exp. Cell Res. 2022, 419, 113334. https://doi.org/10.1016/j.yexcr.2022.113334.
38.
Li, H.; Sun, X.; Li, J.; et al. Hypoxia Induces Docetaxel Resistance in Triple Negative Breast Cancer via the HIF 1α/miR 494/Survivin Signaling Pathway. Neoplasia 2022, 32, 100821. https://doi.org/10.1016/j.neo.2022.100821.
39.
Ramasubramanian, T.S.; Adstamongkonkul, P.; Scribano, C.M.; et al. A Live Tumor Fragment Platform to Assess Immunotherapy Response in Core Needle Biopsies While Addressing Challenges of Tumor Heterogeneity. J. Transl. Med. 2026, 24, 18. https://doi.org/10.1186/s12967-025-07378-2.

Scilight Press

Author Information

Abstract

Keywords

References

About Scilight

Journals

Publishing Policies

Contact Us