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

## TL;DR

A new lab method called the Chamber Gap Assay helps study how pancreatic cancer cells attract and change macrophages, offering a better way to understand and target this process.

## Contribution

The Chamber Gap Assay is introduced as a novel in vitro method for real-time visualization and quantification of macrophage migration and morphological changes induced by pancreatic cancer cells.

## Key findings

- The Chamber Gap Assay enables accurate detection of directional macrophage migration toward cancer cells.
- The method reveals distinct migration patterns and improved sensitivity compared to conventional transwell assays.
- The assay allows visualization of morphological changes in macrophages influenced by pancreatic cancer cells.

## Abstract

Pancreatic cancer ranks among the most lethal malignancies. A key contributor to its poor prognosis is the capacity of tumor cells to recruit macrophages and reprogram them into a tumor-promoting rather than tumor-eliminating phenotype. Therefore, elucidating how and why macrophages are recruited to the tumor is essential for designing new therapeutic strategies. However, widely used laboratory methods do not permit direct visualization of cell movement or accurate quantification of migration dynamics. Here, we adapted and evaluated a novel in vitro method, the Chamber Gap Assay (CGA). By culturing cancer cells and macrophages in separate but connected compartments, this system permits real-time visualization of macrophage migration toward cancer cells and the resulting morphological changes. This approach enables more accurate quantification of migration compared with conventional methods. Using this approach, we showed that pancreatic cancer cells strongly attract macrophages and demonstrated that the CGA method is highly sensitive in detecting tumor-induced immune recruitment and morphological alteration in macrophages. The assay offers an accessible platform to study how tumors manipulate immune cells and may facilitate the development of therapies that target pathological immune cell recruitment.

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.