Contact-dependent carcinoma aggregate dispersion by M2a macrophages via ICAM-1 and β2 integrin interactions

Abstract

Tumor-associated macrophages (TAMs) can constitute up to 50% of the tumor mass and have strong implications in tumor progression and metastasis. Macrophages are plastic and can polarize to various subtypes that differ in terms of surface receptor expression as well as cytokine and chemokine production and effector function. Conventionally, macrophages are grouped into two major subtypes: the classically activated M1 macrophages and the alternatively activated M2 macrophages. M1 macrophages are pro-inflammatory, promote T helper (Th) 1 responses, and show tumoricidal activity, whereas M2 macrophages contribute to tissue repair and promote Th2 responses. Herein, we present a microfluidic system integrating tumor cell aggregates and subtypes of human monocyte-derived macrophages in a three-dimensional hydrogel scaffold, in close co-culture with an endothelial monolayer to create an in vitro tumor microenvironment. This platform was utilized to study the role of individual subtypes of macrophages (M0, M1, M2a, M2b and M2c) in human lung adenocarcinoma (A549) aggregate dispersion, as a representation of epithelial-mesenchymal transition (EMT). A significant difference was observed when M2a macrophages were in direct contact with or separated from A549 aggregates, suggesting a possible mechanism for proximity-induced, contact-dependent dissemination via ICAM-1 and integrin β2 interactions. Indeed, M2a macrophages tended to infiltrate and release cells from carcinoma cell aggregates. These findings may help in the development of immunotherapies based on enhancing the tumor-suppressive properties of TAMs.

Keywords: cancer microenvironment; epithelial-mesenchymal transition; macrophage phenotypes; macrophage polarization; microfluidics.

Figure 1. Microfluidic co-culture platform to study the interactions between carcinoma aggregates and macrophages

A. Photograph of the polydimethyl siloxane (PDMS) device. B. Schematic images of an enlarged, isometric view of the channel layout showing the orientation of co-culturing carcinoma cell aggregates and endothelial cells (HUVECs), with macrophages either physically contacting (left panel; 1: media channel; 2: A549 aggregates and macrophages gel channel; 3. supporting gel channel; 4: HUVEC monolayer) or cultured under separated conditions (right panel; 1: media channel; 2: A549 aggregates gel channel; 3. macrophages gel channel; 4: HUVEC monolayer). C. HUVEC monolayers formed in the microfluidic channel. Green: GFP-HUVECs. D–F. E-cadherin immunocytochemical staining. E-cadherin expression of A549 aggregates at 0 h (D), E-cadherin expression of A549 aggregates in the absence (E) or presence (F) of macrophages at 36 h. Green: E-cadherin staining, red: mCherry A549 nuclei.

Figure 2. Characterization of polarized macrophages

M0, M1 and M2a macrophages were immunostained for markers at either 0 h or after culturing for 36 h in the microfluidic device in co-culture with A549 carcinoma aggregates: A. CD80 (M1 marker); B. CD209 (M2a marker). Scale bars 50 μm.

Figure 3. A549 aggregates dispersion induced by various subtypes of macrophages

A. Quantitative measurement of aggregate dispersion at 36 h for macrophages under either “contact” or “separated” conditions. * indicates statistical calculations compared to no macrophage conditions, where *P < 0.05 and ****P < 0.0001. # indicates a statistical calculation between “contact” versus “separated” culture conditions, where #P < 0.01. Data are shown as a box plot with Tukey outliers. Ctrl_0 represents the control without HUVECs and without macrophages. Ctrl represents the control with HUVECs but without macrophages. B. Images of M2a inducing A549 aggregate dispersion under “contact” and “separated” conditions at 0 h or after culture for 36 h. Red: mCherry A549 nuclei. Scale bars 100 μm.

Figure 4. Migration of macrophage subtypes

A. Migration direction (white arrows) of M0, M1, M2b, and M2c subtypes of macrophages under “contact” conditions with the carcinoma aggregates imaged at 6 h. B. Time-lapsed images of the M2a subtype under “contact” condition at specific times (top left). The migration direction (white arrows) of the M2a subtype that were ≤50 μm (M2a-proximity; top right) or ≥50 μm (M2a-peripheral; bottom left) from the carcinoma aggregate under “contact” conditions or M2a cells under “separated” conditions (bottom right). Cells were imaged for 6 h. C. Migration speed of macrophages situated ≤50 μm from the carcinoma aggregates. D. Migration speed of M2a cells situated either ≤50 μm (M2a-proximity) or ≥50 μm (M2a-peripheral) from the carcinoma aggregates and grown under “separated” conditions. E. Radial velocity of M0, M1 and M2a subtypes under “contact” conditions. All data shown are mean ± SEM of at least three experiments. *P < 0.05, **P < 0.01. Green: DiO-labeled macrophages, red: mCherry A549 nuclei.

Figure 5. Involvement of integrins in carcinoma aggregate dispersion by macrophages

A. Surface expression of CD11a, CD11b, and CD11c integrins on macrophage subtypes, as determined by FACS and expressed as mean fluorescence intensity (MFI). B. Normalized aggregate dispersion induced by M2a macrophages in the absence (−) or presence of various blocking antibodies at 12 h. *P <0.05 and **P < 0.01. C. Fluorescent image showing an M2a macrophage extending a projection (white arrow) as it attempts to dissociate a cancer cell from the aggregate. Green: DiO-labeled macrophages, red: mCherry A549 nuclei. D. Migration speed of M2a macrophages in the absence or presence of the indicated blocking antibodies. Data shown are mean ± SEM of three experiments.

Figure 6. Transwell migration assay of A549 carcinoma cells

A. Migration of A549 cells in the absence (No Mac) or presence of M2a macrophages without (−) or with the indicated blocking antibodies, measured after 12 h. Data are the mean ± SEM. *P < 0.05. B. Diagram showing the possible adhesion mechanism governing M2a-induced A549 aggregate dispersion via the interaction of ICAM-1 and β₂ integrin.