Mai Yamagishi ^1,2* Yutaka Hori ³ Nobutake Suzuki ^1,2 Yu Peng ⁴ Yoshie Harada ⁴ Takashi Funatsu ² Osamu Ohara ⁵ Yoshitaka Shirasaki ^2,6*

• ¹ Live Cell Diagnosis Co., Ltd, Saitama, Kanagawa, Japan
• ² Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo, Tōkyō, Japan
• ³ Department of Applied Physics and Physico-informatics, Faculty of Science and Technology, Keio University, Hiyoshi Campus, Yokohama, Kanagawa, Japan
• ⁴ Institute for Protein Research, Osaka University, Suita, Ōsaka, Japan
• ⁵ Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan
• ⁶ Research Center for Advanced Science and Technology, The University of Tokyo, Komaba, Japan

Heterogeneity in the cellular microenvironment in vivo affects the variability of reactivity among immune cells. Individual-specific microenvironmental differences play a crucial role in determining macroscopic outcomes, such as the efficacy of immunotherapy and disease progression. The microenvironment is also featured by cytokines released from cells, significantly regulating immune cell function. However, the overall understanding, at single-cell resolution, of how cytokines shape the microenvironment and promote paracrine signaling remains unclear. In this manuscript, we propose a methodology that addresses both the microenvironment itself and the response to the microenvironment to comprehend microenvironment behavior at the single-cell level. Our objective is to contribute to basic understanding of the interplay between immune cells and their microenvironment, with particular relevance to implications for immune therapy and disease progression.