Abstract 5571: Collagen stabilization directs immune suppression in the head and neck squamous cell carcinoma hypoxic subtype
Authors: Srivastava Sonal , Powers John , Chaudhary Ritu , Marzban Sadegh , Slebos Robbert J. , West Jeffrey , Song Xiaofei , Guevara-Patino Jose A. , Chung Christine H. , Amelio Antonio L. 
Published: March 2024

Abstract
Head & neck squamous cell carcinoma (HNSCC) show limited clinical benefit to immune checkpoint inhibitors (ICI). Interactions between extracellular matrix (ECM), tumor, and immune cells underly the mechanisms leading to immunosuppression and resistance to ICIs. Collagen matrix remodeling within tumor stroma can influence accessibility and function of immune cells. Specifically, telopeptidyl lysine residues in fibrillar collagen can be hydroxylated by Lysyl hydroxylase 2 (LH2) prior to cross-linking which leads to stiffened, stable collagen. We previously showed that this LH2-modified tumor microenvironment (TME) drives HNSCC invasion and metastasis. Here, we elucidate the interplay between LH2-modified collagen and the immune landscape within TME of hypoxic and immunosuppressive HNSCC.
Bioinformatics analyses of RNAseq datasets were performed to assess pathways regulating collagen deposition and stabilization. Tissue microarray having 81 primary, 28 recurrent primary, and 3 lymph node metastases tumor cores was analyzed. Cores were classified according to our published Hypoxia-Immune signature (Chaudhary R. et al. 2023, Can Res Comm). We performed Second Harmonic Generation imaging of H&E stained TMA section to analyze the orientation and length of collagen fibrils. Picrosirius red (PSR) staining allowed quantification of total collagen and collagen maturation was assessed by polarized light imaging of birefringence. Further, we performed multiplex immunofluorescence staining for CD3+ (T-cell); CD14+ (Monocyte & Macrophage); CD11c+ (Dendritic); Pax5+ (B-cell); Pan-Cytokeratin+ (tumor), and LH2.
Low (0-1) LH2 expression was seen in 29.5% of analyzed cores whereas 70.5% of cores scored high (2-3) for LH2 expression. Cores classified under the hypoxia subtype exhibit high LH2 and enrichment of collagen stability signature. Late-stage tumors have highly oriented collagen fibers compared to normal tissue or early-stage tumors which have randomly distributed fibers. CT-Fire-based measurements revealed that LH2hi cores have significantly increased fiber length and lower coefficient of variance for fiber angles. The area of collagen measured by PSR staining had no correlation with tumor stage or subtype indicating that collagen deposition is similarly high in HNSCCs. However, birefringence measurements showed that a higher fraction of mature fibrillar collagen is associated with late-stage LH2hi tumors. Interestingly, LH2 expression positively correlated with increased CD3+ T cells in stroma suggesting that anti-tumor T cells are physically obstructed by the presence of LH2-stabilized and stiffened collagen fibers.
LH2 expression influences the quality of collagen but not the quantity. Understanding the effect of LH2-mediated collagen modifications on the TIME may have implications for therapeutic strategies to enhance anti-tumor immune response in HNSCC.
Citation Format: Sonal Srivastava, John Powers, Ritu Chaudhary, Sadegh Marzban, Robbert J. Slebos, Jeffrey West, Xiaofei Song, Jose A. Guevara-Patino, Christine H. Chung, Antonio L. Amelio. Collagen stabilization directs immune suppression in the head and neck squamous cell carcinoma hypoxic subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5571.