(T1430-03-19) Selenium-Functionalized Sulfated Hyaluronic Acid (sHASe): A Novel Multi-Targeted Therapeutic Strategy for Overcoming Therapy Resistance and Metastasis in Head and Neck Squamous Cell Carcinoma

Purpose: Head and neck squamous cell carcinoma (HNSCC) remains a significant global health burden, with an estimated 890,000 new cases and 450,000 deaths annually¹. Early-stage HNSCC patients achieve 5-year survival rates of 70–90% with curative treatment, but outcomes for advanced-stage cases—comprising two-thirds of patients—have remained poor, with survival stagnating around 50% for decades². This stagnation is largely due to restricted treatment options, disease recurrence, and/or treatment failure caused by therapy resistance^2,3. Despite advancements in chemotherapy, radiotherapy, and immunotherapy, recurrence and resistance remain a critical challenge^2,3. With the current standard of care, 10–20% of early-stage HNSCC and 50% of locally advanced HNSCC cases experience recurrence over time^4,5, emphasizing the urgent need for novel therapeutic strategies to improve treatment efficacy, overcome resistance mechanisms, and enhance long-term survival outcomes for patients with advanced-stage HNSCC. A key driver of treatment failure is the activation of multiple tumor-promoting pathways. Key pathways include the C-X-C Motif Chemokine (CXC) Ligand 12 (CXCL12)/CXC Receptor 4 (CXCR4) axis, which regulates cancer cell migration and invasion, Vascular Endothelial Growth Factor (VEGF) C/VEGF Receptor 3 (VEGFR3) signaling pathway, which promotes tumor-associated lymphangiogenesis and metastatic dissemination, and Nuclear Factor Erythroid 2-Related Factor 2 (NRF2)-mediated antioxidant defenses, which enable cancer cells to adapt to oxidative stress and resist therapy-induced cell death^6–9. Although these pathways are critically implicated in HNSCC progression and therapy resistance, no therapies specifically targeting CXCL12/CXCR4, VEGFC/VEGFR3, or NRF2 are currently approved for the treatment of HNSCC. To address this gap, we have developed selenium-functionalized sulfated hyaluronic acid (sHASe), a first-in-class, tunable glycosaminoglycan-based therapeutic designed to simultaneously inhibit CXCL12/CXCR4 and VEGFC/VEGFR3-mediated tumor invasion, metastasis and lymphangiogenesis through the sulfated glycosaminoglycan backbone, while selectively disrupting NRF2-driven redox homeostasis in cancer cells through selenium functionalization. By targeting these key resistance and metastasis pathways in parallel, sHASe offers a novel strategy to sensitize tumors to standard therapies, inhibit metastatic progression, and improve clinical outcomes for patients with advanced-stage HNSCC.

Methods: sHASe was synthesized by chemical sulfation of low molecular weight hyaluronic acid, followed by conjugation to methyl selenocysteine (MSC) (Figure 1). To optimize biological activity, three lead candidates—sHASe 011, 111, and 112—were generated by systematically varying the degree of sulfation and selenium incorporation. Structural integrity and chemical modifications were confirmed using Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (¹H-NMR), while molecular weight distribution and selenium content were quantified by gel permeation chromatography (GPC) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. The anticoagulant profile of sHASe was evaluated through prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays to ensure safety and minimize bleeding risk. Cellular cytocompatibility was assessed using the CCK-8 assay in human umbilical vein endothelial cells (HUVECs) as a healthy control and Human Tongue Squamous Carcinoma Cell (HSC-3) to establish baseline tolerability and anti-tumor cytotoxicity.

Results: All three sHASe candidates were successfully synthesized through controlled sulfation and covalent amide bond formation with MSC. FTIR and ¹H-NMR confirmed successful sulfation and conjugation (Figures 2a and 2b), while GPC analysis demonstrated narrow molecular weight distributions (Figure 2c). Elemental analysis by ICP-MS verified tunable selenium and sulfur incorporation, with selenium and sulfur content ranging from 0-7.4μg/g and 0-59μg/g respectively across the lead candidates (Figure 2d). In coagulation assays, sHASe 011 (35μM) and sHASe 111 (28μM) exhibited no anticoagulant activity, with PT and aPTT values comparable to untreated controls. sHASe 112 demonstrated no effect on PT but induced a two-fold increase in aPTT at 26μM, which was significantly reduced at 13μM (p < 0.0001). In contrast, unfractionated heparin at 1.3μM increased anticoagulant activity by over ten-fold, highlighting the superior safety profile of sHASe (Figures 3a and 3b). In cytocompatibility assays, MSC significantly increased HUVEC viability up to 2.5mM, with cytotoxicity (~60% reduction) observed at 5mM. sHASe 102 and sHASe 112 similarly enhanced HUVEC viability across concentrations up to 155μM and 105μM respectively. sHASe 011 had no significant effect, while sHASe 111 increased viability at 28μM and 57μM (Figures 3d–3h). The enhanced viability is likely due to improved cellular antioxidant defenses and survival signaling driven by selenium and sulfated glycosaminoglycan components. In contrast, all sHASe candidates significantly reduced HSC-3 tumor cell viability, with sHASe 111 (p < 0.0085) and sHASe 102 (p < 0046) demonstrating the strongest tumor-killing effects at 17μM and 23μM respectively (Figure 3c). These findings collectively demonstrate that sHASe possess tunable chemical properties, favorable safety profile in healthy cells, and strong selective cytotoxicity against HNSCC cells.

Conclusion: This study presents the successful development and initial validation of sHASe as a safe and promising multi-functional therapeutic candidate for resistant HNSCC. The structural and functional properties of sHASe, combined with selective cytotoxicity in cancer cells, support its potential for further mechanistic studies targeting NRF2, VEGFC/VEGFR3, and CXCL12/CXCR4 pathways. Future work will focus on evaluating receptor interactions, therapy sensitization, and mechanistic validation in advanced in vitro and ex vivo HNSCC models.

References: 1. Barsouk A, Aluru JS, Rawla P, Saginala K, Barsouk A. Epidemiology, Risk Factors, and Prevention of Head and Neck Squamous Cell Carcinoma. Medical Sciences. 2023;11(2):42. doi:10.3390/MEDSCI11020042
2. Khera N, Rajkumar AS, Abdulkader M Alkurdi K, et al. Identification of multidrug chemoresistant genes in head and neck squamous cell carcinoma cells. Mol Cancer. 2023;22(1):1-8. doi:10.1186/S12943-023-01846-3/FIGURES/2
3. Nissi L, Suilamo S, Kytö E, Vaittinen S, Irjala H, Minn H. Recurrence of head and neck squamous cell carcinoma in relation to high-risk treatment volume. Clin Transl Radiat Oncol. 2021;27:139-146. doi:10.1016/J.CTRO.2021.01.013
4. Meci A, Goyal N, Slonimsky G. Mechanisms of Resistance and Therapeutic Perspectives in Immunotherapy for Advanced Head and Neck Cancers. Cancers (Basel). 2024;16(4):703. doi:10.3390/CANCERS16040703
5. Argiris A, Harrington KJ, Tahara M, et al. Evidence-Based Treatment Options in Recurrent and/or Metastatic Squamous Cell Carcinoma of the Head and Neck. Front Oncol. 2017;7(MAY):72. doi:10.3389/FONC.2017.00072
6. Jin X, Lou X, Qi H, et al. NRF2 signaling plays an essential role in cancer progression through the NRF2-GPX2-NOTCH3 axis in head and neck squamous cell carcinoma. Oncogenesis 2024 13:1. 2024;13(1):1-13. doi:10.1038/s41389-024-00536-z
7. Dumitru CS, Raica M. Vascular Endothelial Growth Factor Family and Head and Neck Squamous Cell Carcinoma. Anticancer Res. 2023;43(10):4315-4326. doi:10.21873/ANTICANRES.16626
8. Samara GJ, Lawrence DM, Chiarelli CJ, et al. CXCR4-mediated adhesion and MMP-9 secretion in head and neck squamous cell carcinoma. Cancer Lett. 2004;214(2):231-241. doi:10.1016/J.CANLET.2004.04.035
9. Albert S, Riveiro ME, Halimi C, et al. Focus on the role of the CXCL12/CXCR4 chemokine axis in head and neck squamous cell carcinoma. Head Neck. 2013;35(12):1819-1828. doi:10.1002/HED.23217

Acknowledgements: The authors declare no conflicts of interest or financial disclosures relevant to this work.


Figure 1. Synthetic strategy for the generation of selenium-functionalized sulfated hyaluronic acid (sHASe) via controlled sulfation and covalent amide bond conjugation with methylselenocysteine.


Figure 2. Structural and elemental characterization of selenium-functionalized sulfated hyaluronic acid (sHASe) by FTIR, ¹H-NMR, GPC, and ICP-MS. (a) FTIR and (b) ¹H-NMR confirmed successful sulfation and amide bond formation with MSC. (c) GPC analysis showed narrow molecular weight distributions. (d) ICP-MS revealed tunable selenium and sulfur incorporation across candidates, ranging from 0–7.4 μg/g and 0–59 μg/g, respectively.


Figure 3. Anticoagulant profiling and differential cytocompatibility of sHASe candidates assessed by PT, aPTT, and cell viability in non-malignant HUVEC and HNSCC-derived HSC-3 cells. (a–b) PT and aPTT assays demonstrated minimal anticoagulant activity for sHASe 011 and 111, and modest, dose-dependent effects for sHASe 112, compared to strong anticoagulation by heparin. (c) All sHASe candidates significantly reduced HSC-3 cell viability, with sHASe 111 (p < 0.0085) and sHASe 102 (p <0046) demonstrating the strongest tumor-killing effects. (d–h) HUVEC viability remained stable or increased in response to sHASe treatment, indicating favorable cytocompatibility.