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.

The ability of a cancer cell to migrate through the dense extracellular matrix (ECM) within and surrounding the solid tumor is a critical determinant of metastasis. Macrophages enhance invasion and metastasis in the tumor microenvironment but the basis for their effects are not fully understood. Using a microfluidic 3D cell migration assay, we found that the presence of macrophages enhanced the speed and persistence of cancer cell migration through a 3D extracellular matrix in a matrix metalloproteinases (MMP)-dependent fashion. Mechanistic investigations revealed that macrophage-released TNFα and TGFβ1 mediated the observed behaviors by two distinct pathways. These factors synergistically enhanced migration persistence through a synergistic induction of NF-κB-dependent MMP1 expression in cancer cells. In contrast, macrophage-released TGFβ1 enhanced migration speed primarily by inducing MT1-MMP expression. Taken together, our results reveal new insights into how macrophages enhance cancer cell metastasis, and they identify TNFα and TGFβ1 dual blockade as an anti-metastatic strategy in solid tumors.

<div>Abstract<p>The ability of a cancer cell to migrate through the dense extracellular matrix within and surrounding the solid tumor is a critical determinant of metastasis. Macrophages enhance invasion and metastasis in the tumor microenvironment, but the basis for their effects is not fully understood. Using a microfluidic 3D cell migration assay, we found that the presence of macrophages enhanced the speed and persistence of cancer cell migration through a 3D extracellular matrix in a matrix metalloproteinases (MMP)-dependent fashion. Mechanistic investigations revealed that macrophage-released TNFα and TGFβ1 mediated the observed behaviors by two distinct pathways. These factors synergistically enhanced migration persistence through a synergistic induction of NF-κB–dependent MMP1 expression in cancer cells. In contrast, macrophage-released TGFβ1 enhanced migration speed primarily by inducing MT1-MMP expression. Taken together, our results reveal new insights into how macrophages enhance cancer cell metastasis, and they identify TNFα and TGFβ1 dual blockade as an antimetastatic strategy in solid tumors. <i>Cancer Res; 77(2); 279–90. ©2016 AACR</i>.</p></div>

<p>This file contains the figure captions for the supplementary information figures. Again, Fig. S1 and S2 is related to Fig. 1; Fig. S3 and S4 is related to Fig. 2; Fig. S5, S6, S7 is related to Fig. 3; Fig. S8-11 is related to Fig. 4; Fig. S12 is related to Fig. 6. Each figure caption describes its corresponding figure, summarize the results, and describes the statistics of the data.</p>

<p>This file contains figures in the supplementary information. These data complements data in the main figures. It contains: Figure S1: Microfluidic Assay for cell migration in 3D ECM; Figure S2: Macrophages increase cancer cell invasion rate in 3D ECM; macrophages increase cancer cell migration total speed but decrease directedness on 2D substrates; Figure S3: MMP activity is crucial for cancer cell migration, and macrophages enhance cancer cell MMP expression; Figure S4: The abilities of macrophages to enhance cancer cell migration dynamics and the expression of MMPs are due primarily to the paracrine factors secreted by macrophages; Figure S5: Neutralizing antibody blocking is specific to factors released by macrophages; Figure S6: Blocking TNFalpha and TGFbeta1 secreted by macrophages significantly reduced macrophage-induced MMP expression and macrophage-enhanced cancer cell invasion rate; Figure S7: TNFalpha and TGFbeta1 secreted by macrophages mostly accounts for macrophage-enhanced cancer cell migration total speed and directedness; Figure S8: TGFbeta1 has a larger effect on cancer cell migration total speed than TNFalpha. TNFalpha and TGFbeta1 synergistically increase cancer cell migration directedness; Figure S9: Cancer cell migration total speed is correlated with cancer cell expression of MT1-MMP, while cancer cell migration directedness is correlated with cancer cell expression of MMP1; Figure S10: Inhibiting both TNFalpha and TGFbeta1 in a mice model of breast tumor effectively reduces lung metastasis; Figure S11: TNFalpha and TGFbeta1 co-treatment synergistically up-regulate cancer cell MMP1 protein production and secretion. TGFbeta1 is mainly responsible for the increase in MT1-MMP protein production; Figure S12: TNFalpha and TGFbeta1 co-treatment synergistically enhance NF-κB nuclear localization in MDA-MB-435S cells. Fig. S1 and S2 is related to Fig. 1; Fig. S3 and S4 is related to Fig. 2; Fig. S5, S6, S7 is related to Fig. 3; Fig. S8-11 is related to Fig. 4; Fig. S12 is related to Fig. 6</p>

<p>This files contains detailed methods on procedures used in this study, which complements the method described in the main paper. It also expands on the methods described in the main paper. Specifically, it contains method on Microfluidic 3D cell migration assay; 2D Cell Migration Assay; Immunofluorescent staining, confocal microscopy, and DQ-Collagen I Release Assay; ELISA; Cancer cell-macrophage co-culture for western blot analysis; Treatment of cancer cells for western blot analysis; Protein isolation and western blot analysis; Total RNA extraction and mRNA detection; In vivo metastasis assay</p>

<p>This file contains figures in the supplementary information. These data complements data in the main figures. It contains: Figure S1: Microfluidic Assay for cell migration in 3D ECM; Figure S2: Macrophages increase cancer cell invasion rate in 3D ECM; macrophages increase cancer cell migration total speed but decrease directedness on 2D substrates; Figure S3: MMP activity is crucial for cancer cell migration, and macrophages enhance cancer cell MMP expression; Figure S4: The abilities of macrophages to enhance cancer cell migration dynamics and the expression of MMPs are due primarily to the paracrine factors secreted by macrophages; Figure S5: Neutralizing antibody blocking is specific to factors released by macrophages; Figure S6: Blocking TNFalpha and TGFbeta1 secreted by macrophages significantly reduced macrophage-induced MMP expression and macrophage-enhanced cancer cell invasion rate; Figure S7: TNFalpha and TGFbeta1 secreted by macrophages mostly accounts for macrophage-enhanced cancer cell migration total speed and directedness; Figure S8: TGFbeta1 has a larger effect on cancer cell migration total speed than TNFalpha. TNFalpha and TGFbeta1 synergistically increase cancer cell migration directedness; Figure S9: Cancer cell migration total speed is correlated with cancer cell expression of MT1-MMP, while cancer cell migration directedness is correlated with cancer cell expression of MMP1; Figure S10: Inhibiting both TNFalpha and TGFbeta1 in a mice model of breast tumor effectively reduces lung metastasis; Figure S11: TNFalpha and TGFbeta1 co-treatment synergistically up-regulate cancer cell MMP1 protein production and secretion. TGFbeta1 is mainly responsible for the increase in MT1-MMP protein production; Figure S12: TNFalpha and TGFbeta1 co-treatment synergistically enhance NF-κB nuclear localization in MDA-MB-435S cells. Fig. S1 and S2 is related to Fig. 1; Fig. S3 and S4 is related to Fig. 2; Fig. S5, S6, S7 is related to Fig. 3; Fig. S8-11 is related to Fig. 4; Fig. S12 is related to Fig. 6</p>

<div>Abstract<p>The ability of a cancer cell to migrate through the dense extracellular matrix within and surrounding the solid tumor is a critical determinant of metastasis. Macrophages enhance invasion and metastasis in the tumor microenvironment, but the basis for their effects is not fully understood. Using a microfluidic 3D cell migration assay, we found that the presence of macrophages enhanced the speed and persistence of cancer cell migration through a 3D extracellular matrix in a matrix metalloproteinases (MMP)-dependent fashion. Mechanistic investigations revealed that macrophage-released TNFα and TGFβ1 mediated the observed behaviors by two distinct pathways. These factors synergistically enhanced migration persistence through a synergistic induction of NF-κB–dependent MMP1 expression in cancer cells. In contrast, macrophage-released TGFβ1 enhanced migration speed primarily by inducing MT1-MMP expression. Taken together, our results reveal new insights into how macrophages enhance cancer cell metastasis, and they identify TNFα and TGFβ1 dual blockade as an antimetastatic strategy in solid tumors. <i>Cancer Res; 77(2); 279–90. ©2016 AACR</i>.</p></div>