Inflamed neutrophils sequestered at entrapped tumor cells via chemotactic confinement promote tumor cell extravasation

Abstract

Systemic inflammation occurring around the course of tumor progression and treatment are often correlated with adverse oncological outcomes. As such, it is suspected that neutrophils, the first line of defense against infection, may play important roles in linking inflammation and metastatic seeding. To decipher the dynamic roles of inflamed neutrophils during hematogenous dissemination, we employ a multiplexed microfluidic model of the human microvasculature enabling physiologically relevant transport of circulating cells combined with real-time, high spatial resolution observation of heterotypic cell-cell interactions. LPS-stimulated neutrophils (PMNs) and tumor cells (TCs) form heterotypic aggregates under flow, and arrest due to both mechanical trapping and neutrophil-endothelial adhesions. Surprisingly, PMNs are not static following aggregation, but exhibit a confined migration pattern near TC-PMN clusters. We discover that PMNs are chemotactically confined by self-secreted IL-8 and tumor-derived CXCL-1, which are immobilized by the endothelial glycocalyx. This results in significant neutrophil sequestration with arrested tumor cells, leading to the spatial localization of neutrophil-derived IL-8, which also contributes to increasing the extravasation potential of adjacent tumor cells through modulation of the endothelial barrier. Strikingly similar migration patterns and extravasation behaviors were also observed in an in vivo zebrafish model upon PMN-tumor cell coinjection into the embryo vasculature. These insights into the temporal dynamics of intravascular tumor-PMN interactions elucidate the mechanisms through which inflamed neutrophils can exert proextravasation effects at the distant metastatic site.

Keywords: cell migration; extravasation; inflammation; metastasis; neutrophils.

Fig. 1.

Multiplexed microvascular network assay allows quantification of TC–PMN arrest and extravasation dynamics. (A) Eight independent hydrogel regions where microvascular networks, connected by branching channels, are formed per chip. Fluorescence Inset depicts a confocal projection of one perfused vascular network. (Scale bar: 200 μm.) (B) A reservoir sustaining a hydrostatic pressure drop of ∼5 mm water is secured on top of the chip to generate continuous perfusion. (C) TC–PMN clusters in microvessels. Higher magnification examples of extravasating and nonextravasated MA2 cells in TC–PMN clusters. (D) Degree of tumor cell–PMN aggregate formation during intravascular arrest. Aggregation index is defined as the fraction of TCs clustered with neutrophils multiplied by the average number of neutrophils per cluster (n = 10–13 devices per condition). **P < 0.01, ***P < 0.001, and error bars indicate SD.

Fig. 2.

Cluster-associated PMNs exhibit migratory confinement that is dependent on autologous chemotaxis to secreted factors. (A) Cluster-associated and free PMNs arrested intraluminally. (B) Migration speeds and end-to-end distance from original position of cluster-associated vs. free PMNs (n = 43–53 PMNs per condition over five devices). (C) Tracks of free (blue) and cluster-associated (red) PMNs over 90 min (40-s time step). Dotted circle delineates the 150-μm radius. (D) Cytokine array showing relative magnitudes of secreted factors from MA2 alone, PMN alone, LPS-activated PMNs, or MA2 + PMN coincubation, after 4 h of culture (two replicates). (E) Migration speed and end-to-end distance from original position of cluster-associated PMNs with or without anti–CXCL-1 + anti–IL-8 (n = 30–34 PMNs per condition, over five devices). Error bars indicate SD, **P < 0.01, ***P < 0.001. (F) Migration tracks of cluster-associated PMNs incubated with anti–CXCL-1 + anti–IL-8 or control (Ctrl) IgG.

Fig. 3.

Confined PMN migration sequesters PMNs near TCs for >6 h, and is enhanced by the endothelial glycocalyx. (A) Cluster-associated PMNs and their dispersion from TCs over 2 h, in the absence and presence of anti–CXCL-1 + anti–IL-8. (B) Fraction of PMNs remaining in individual clusters over 3 h (every 10 min) when the system is treated with IL-8 and/or CXCL-1 neutralizing antibodies, or when tumor cells are replaced with 15 μm polystyrene beads (n = 15 clusters per group over three independent devices, at each time point). (C) Fraction of PMN remaining per TC–PMN cluster at 6 h. Devices are treated with either IgG or anti–IL-8 and/or anti–CXCL-1. In some cases, only tumor-derived IL-8 and/or CXCL-1 is ablated via siRNA. Each point represents one device with at least five TC–PMN clusters averaged per device (n > 5 devices per condition). **P < 0.01, ***P < 0.001, n.s. = not significant, error = SEM.

Fig. 4.

IL-8 released by LPS-stimulated PMNs increases TC extravasation rates through the action of IL-8 on local EC barrier function. (A) Percentage of extravasated cells at 6 h for A375, A375-MA2, and MDA-MB-231 when coperfused with quiescent PMNs or LPS-stimulated PMNs at a 1:5 ratio (n = 12 devices). (B) Percentage of MA2 extravasated in the PMN-associated or nonassociated subpopulations within the same device, with or without anti–IL-8 (n = 6 devices). (C) Extravasation rates of MA2 at 6 h when coincubated with conditioned media (CM) generated from various conditions (n > 9 devices per condition). (D) Extravasation rates of MA2 in the presence of stimulated PMNs and neutralizing antibodies at 6 h postinjection (n = 5 devices). (E) Permeability of microvessels to 70 kDa dextran after 6-h treatment with stimulated PMN conditioned media. Extravasation rates (at 6 h) of MA2 tumor cells in PMN-conditioned media pretreated vessels (for 6 h) (n = 3 devices). (F) Percentage of original (at t = 0) PMNs remaining in microvessel beds after 6 h of flow in the absence or presence of anti–IL-8 + anti–CXCL-1 (n = 10 devices). (G) Percentage of PMNs remaining in cluster-associated or free subpopulations after 6 h of flow (with MA2 coperfusion). Each point represents the average of at least five clusters across five regions of interest (ROIs), or at least 20 free PMNs over five ROIs. In A–G, error bars indicate SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Inflamed PMNs are sequestered at TC–PMN clusters and enhance extravasation rates of A375 and MA2 in vivo. (A) Intravascular cluster-associated and free PMNs (pink) when coinjected with MA2 cells (green) in flk:dsRed (red) zebrafish embryos. (B) Migration tracks of cluster-associated or free PMNs in vessels of zebrafish embryos. (C) Extravasation rates of A375 and MA2 cells with or without coperfusion with inflamed PMNs at 6 h and 24 h (n = 7–12 embryos for A375, n = 18–23 embryos for MA2). Error bars indicate SEM, **P < 0.01, ***P < 0.001.