Melike F. Demir ^1,2 Lin Yuhsien ² Pedro H. Cruz ² Masaki Tajima ^2* Tasuku Honjo ¹ Elisabeth Müller ^1,3,4*

• ¹ Department of Immunology and Genomic Medicine, Faculty of Medicine, Kyoto University, Kyoto, Kyōto, Japan
• ² Division of Integrated High-Order Regulatory Systems, Center for Cancer Immunotherapy and Immunobiology, Kyoto, Japan
• ³ Tumor Immunology Group, Institute of Pathology, Oslo University Hospital, Oslo, Norway
• ⁴ Therapy Prediction in Lung Cancer, Department of Cancer Genetics, Institute of Cancer Research, Oslo University Hospital, Oslo, Norway

S100A9, a multifunctional protein mainly expressed by neutrophils and monocytes, poses an immunological paradox. In virus infections or sterile inflammation, it functions as an alarmin attracting innate immune cells, as well as mediating proinflammatory effects through TLR4 signaling. However, in cancer, S100A9 levels have been shown to associate with poor prognosis and lack of response to immunotherapy. Its expression by myeloid cells has been related to an immune suppressive phenotype, the so-called myeloid derived suppressor cells (MDSCs). Targeting S100A9 in cancer has therefore been proposed as a potential way to relieve myeloid-mediated immune suppression. Surprisingly, we found that blocking the extracellular TLR4 signaling from S100A9 using the inhibitor Paquinimod, resulted in increased tumor growth and a detrimental effect on anti-PD-L1 efficacy in the CT26 tumor model. This effect was caused by a reduction in the tumor immune infiltration to about half of untreated controls, and the reduction was made up of a 5-fold decrease in Ly6Chigh monocytic cells. The suppressive Ly6G+ myeloid cells compartment was not reduced by Paquinimod treatment, suggesting alternative mechanisms by which S100A9 contributes to myeloid-mediated suppression. Intratumoral injection of recombinant S100A9 early after mice inoculation with CT26 cells had an anti-tumor effect. These findings indicate an important yet understudied role of S100A9 as an alarmin and immune stimulatory signal in cancer settings, and highlight the potential to exploit such signals to promote beneficial anti-tumor responses.