Interstitial flow promotes macrophage polarization toward an M2 phenotype

Abstract

Tumor tissues are characterized by an elevated interstitial fluid flow from the tumor to the surrounding stroma. Macrophages in the tumor microenvironment are key contributors to tumor progression. While it is well established that chemical stimuli within the tumor tissues can alter macrophage behaviors, the effects of mechanical stimuli, especially the flow of interstitial fluid in the tumor microenvironment, on macrophage phenotypes have not been explored. Here, we used three-dimensional biomimetic models to reveal that macrophages can sense and respond to pathophysiological levels of interstitial fluid flow reported in tumors (∼3 µm/s). Specifically, interstitial flow (IF) polarizes macrophages toward an M2-like phenotype via integrin/Src-mediated mechanotransduction pathways involving STAT3/6. Consistent with this flow-induced M2 polarization, macrophages treated with IF migrate faster and have an enhanced ability to promote cancer cell migration. Moreover, IF directs macrophages to migrate against the flow. Since IF emanates from the tumor to the surrounding stromal tissues, our results suggest that IF could not only induce M2 polarization of macrophages but also recruit these M2 macrophages toward the tumor masses, contributing to cancer cell invasion and tumor progression. Collectively, our study reveals that IF could be a critical regulator of tumor immune environment.

Biophysical regulation of macrophages in health and disease

“…( F ) Immunofluorescence image of F4/80 (red) and Ki67 (green) cell proliferation marker of murine peritoneal macrophages undergoing cyclic mechanical stretch (4%, biaxial, 0.67 Hz) compared to static controls (left) and quantification of cell number and Ki67 (right), adapted from Sager Hendrik et al . ( H ) Immunofluorescence images of nuclei (Dapi, blue) and CD206 (M2 marker, red) in BMDM exposed to interstitial flow (3 µm/s) compared to static controls (left), and quantification of CD206 levels (right), adapted from Li et al …”

“…Li et al. developed a microfluidic system to expose macrophages within collagen gels to interstitial flow, or flow through tissues, which was aimed at recapitulating flow emanating from a tumor to the surrounding stromal tissues . Using this system, the group showed that interstitial fluid flow increased the expression of phosphorylated STAT3/6 and CD206 expression, markers associated with macrophage pro‐healing activation (Fig.…”

“…(B) Image of IgG-coated latex beads (cyan) uptaken by RAW264.7 macrophages on stiff compared to soft surfaces (left), adapted from Patel et al, 38 and TNF secretion by primary murine macrophages on surfaces different stiffness (right), adapted from Previtera and Sengupta. 40 (D) Immunofluorescence image of Arg1 (M2 marker, red) and nuclei (blue) of macrophages cultured on flat and 1D wrinkled surfaces (left), and quantification of Arg1 expression as measured by Western blot (right), adapted from Wang et al 59 (F) Immunofluorescence image of F4/80 (red) and Ki67 (green) cell proliferation marker of murine peritoneal macrophages undergoing cyclic mechanical stretch (4%, biaxial, 0.67 Hz) compared to static controls (left) and quantification of cell number and Ki67 (right), adapted from Sager Hendrik et al71 (H) Immunofluorescence images of nuclei (Dapi, blue) and CD206 (M2 marker, red) in BMDM exposed to interstitial flow (3 µm/s) compared to static controls (left), and quantification of CD206 levels (right), adapted from Li et al83…”

“…Together, these studies suggest that healthy shear stress maintains endothelial function with minimal leukocyte recruitment, and pathological shear conditions contribute to the adhesion and recruitment of monocyte/macrophages to tissues.Fluid flow has also recently been shown to directly influence macrophage functional activation and migration. Li et al developed a microfluidic system to expose macrophages within collagen gels to interstitial flow, or flow through tissues, which was aimed at recapitulating flow emanating from a tumor to the surrounding stromal tissues 83. Using this system, the group showed that interstitial fluid flow increased the expression of phosphorylated STAT3/6 and CD206 expression, markers associated with macrophage pro-healing activation (Fig.…”

Integratedin silicoand 3Din vitromodel of macrophage migration in response to physical and chemical factors in the tumor microenvironment

“…Thus, we hypothesized that a subset of G proteins may represent the integration point and the source of the synergy between these signaling pathways. In addition, IF-modulated signaling, as mediated by integrin-β2 and FAK, is known to drive the activation and downstream migratory activity associated with Rho GTPases [51,77]. Finally, because our in vitro experimental data show that IF and TSF induced a similar increase in directedness and speed, we hypothesized that IL-8, CCL2 and IF regulate one or more of the relevant Rho GTPases, namely CDC42, Rac1 and/or RhoA [38][39][40].…”