Background Stress keratin 17 (K17) is an intermediate filament keratin induced during would healing, inflammatory diseases, papillomavirus infection as well as in cancers. Our prior work described an immune-regulatory role of K17 in three distinct mouse cancer models.^1–3 We showed K17 expression decreased CXCL9 expression in the tumor microenvironment (TME), reduced T cell infiltration, and contributed to disease progression.^1–3 Additionally, K17 conferred resistance to immune checkpoint blockade (ICB) treatment in a HNC mouse model and in patients.² In current study, we identify tumor-intrinsic factors regulated by K17 that affects immune response in the TME to uncover novel mechanisms underlying K17-mediated immune regulation.

Methods We used MOC2 cells as a syngeneic mouse HNC model. K17 was knocked out from parental MOC2 cells by Crisper/Cas9 and a single cell clone of K17KO cells was generated. Bulk RNA sequencing of WT cells and K17KO cells were performed in triplicate. This K17KO clone and parental MOC2 cells were injected subcutaneously into C57BL/6 and NSG mice to confirm the rejection of K17KO cells was only observed in immunocompetent mice. MOC2 tumors and K17KO tumors growing in C57BL/6 mice were collected at day 7 post implantation, at the peak of K17KO tumor growth for single cell RNA seq analyses.

Results Our prior scRNA-seq analysis of sorted CD45+ cells showed that myeloid cells are the major producers of CXCL9 among immune cells in MOC2 tumors. And macrophages from WT tumors expressed less Nos2 (M1 marker) and more Arg1 (M2 marker) compared to those from K17KO tumors. Our new scRNA-seq data encompassing all cells in TME confirmed that CXCL9 expression is mainly detected in the myeloid compartment, and is low in fibroblasts and tumor cells. Additionally, single cell analysis of epithelial cells from tumors and bulk RNA seq of tumor cells in tissue culture identified 148 differentially expressed genes in common that were upregulated in WT tumor cells both in vitro and in vivo. Among these genes were CCL2 and galectin-3, known to contribute to M2-macrophage polarization.^{4 5} We therefore hypothesize that K17 expression in tumor cells upregulates CCL2 and galectin-3 expression, which in turn directs M2 polarization of macrophages to induce immune evasion in head and neck cancers.

Conclusions We identified CCL2 and galectin-3 as tumor factors regulated by K17 expression. Validation studies are needed to confirm their protein expressions and their direct role in tumor-macrophage communications.

References

1. Wang W, Uberoi A, Spurgeon ME, Gronski E, Majerciak V, Lobanov A, Hayes M, Loke A, Zheng ZM, Lambert PF. Stress keratin 17 enhances papillomavirus infection-induced disease by downregulating T cell recruitment. PLoS Pathog, 2020 Jan 22;16(1):e1008206.

2. Wang W, Lozar T, Golfinos AE, Lee D, Gronski E, Shaw EW, Hayes M, Bruce JY, Kimple RJ, Hu R, Harari PM, Xu J, Keske A, Sondel PM, Fitzpatrick MB, Dinh HQ, Lambert PF. Stress Keratin 17 Expression in Head and Neck Cancer Contributes to Immune Evasion and Resistance to Immune-Checkpoint Blockade. Clinical Cancer Research, 2022 May 27;OF1-OF16. doi: 10.1158/1078-0432. PMID: 35621713.

3. Wang W*, Spurgeon ME*, Pope A, McGregor S, Shaw EW, Gronski E, Lambert PF. Stress keratin 17 and estrogen support viral persistence and modulate the immune environment during cervicovaginal murine papillomavirus infection. Proceedings of the National Academy of Sciences. 2023 March 14; https://doi.org/10.1073/pnas.2214225120 (*Co-First Authors)

4. Alison C MacKinnon, Sarah L Farnworth, Philip S Hodkinson, Neil C Henderson, Kirsten M Atkinson, Hakon Leffler, Ulf J Nilsson, Christopher Haslett, Stuart J Forbes, Tariq Sethi. Regulation of Alternative Macrophage Activation by Galectin-3¹. J Immunol 15 February 2008;180(4):2650–2658. https://doi.org/10.4049/jimmunol.180.4.2650

5. Elena Sierra-Filardi, Concha Nieto, Ángeles Domínguez-Soto, Rubén Barroso, Paloma Sánchez-Mateos, Amaya Puig-Kroger, María López-Bravo, Jorge Joven, Carlos Ardavín, José L Rodríguez-Fernández, Carmen Sánchez-Torres, Mario Mellado, Ángel L Corbí. CCL2 Shapes Macrophage Polarization by GM-CSF and M-CSF: Identification of CCL2/CCR2-Dependent Gene Expression Profile. J Immunol 15 April 2014;192(8):3858–3867.

Ethics Approval Experiments were approved and performed in accordance with guidelines approved by the Association for Assessment of Laboratory Animal Care, at the University of Wisconsin Medical School. This study was approved by the University of Wisconsin School of Medicine and Public Health Institutional Animal Care and Use Committee under protocol number M005871.