The Chamber Gap Assay Is a Simple and Sensitive In Vitro Method for Studying Pancreatic Cancer-Induced Macrophage Recruitment and Morphological Alteration.

Pancreatic cancer is characterized by an immunosuppressive tumor environment in which macrophages are recruited and reprogrammed to support tumor growth. Studying macrophage migration and polarization is crucial for understanding disease progression and identifying therapeutic targets. However, existing in vitro methods such as the transwell assay provide limited spatial resolution and do not allow visualization of cell movement or morphological changes. Here, we established and evaluated the Chamber Gap Assay, a modified two-compartment culture system that enables direct, time-resolved observation and quantification of macrophage migration toward pancreatic cancer cells as well as phenotypic alterations. Using murine and human macrophage-cancer cell models, we compared the performance of the Chamber Gap Assay with the transwell assay. We found that macrophage monocultures displayed substantial spontaneous migration in the transwell system, while cancer cells induced only modest additional macrophage recruitment. In contrast, the Chamber Gap Assay demonstrated clear and highly significant directional macrophage movement toward cancer cells, with distinct migration patterns and improved sensitivity for detecting group differences. The method also enabled visualization of cancer cell movement within the same system. Furthermore, CGA offers observations of morphological changes in immune cells under the influence of pancreatic cancer cells. Our findings indicate that the Chamber Gap Assay provides a robust and physiologically relevant method for studying tumor-induced immune cell recruitment and associated morphological changes.