Increase in tumour cell density enhances cell motility

To assess the potential effect of cell proliferation–and associated increase in local cell density–on cancer cell migration in vitro, human fibrosarcoma HT1080 cells, a cell line commonly used in studies of cell migration19,20,21,22, were embedded in 3D type I collagen matrices. Collagen I is not only the main extracellular matrix component of connective tissues, but is also enriched in the vicinity of carcinoma and sarcoma tumours23. Cell migratory patterns within the matrix were monitored for 16.5 h using live-cell phase-contrast microscopy at a rate of 30 frames/h every other day for 5 days. This analysis revealed that fibrosarcoma cells became progressively more motile as cells proliferated and increased local cell density (Fig. 1a–c). To investigate the role of increased cell density on cancer cell migration, we seeded increasing cell densities in 3D matrices and observed cell migration. The initial cell densities used in the experiments, ranging from 10 cells mm−3 to 120 cells mm−3, corresponded to average cell-to-cell distances from 470 to 130 μm in the 3D matrix, distances that were significantly larger than the average cell size (10–20 μm in diameter)24. This data revealed that cells became progressively more motile as cell density increased. This enhanced motility cannot be attributed to repulsive cell–cell interactions as the tracked cells did not come in contact with other cells. Cell speed eventually plateaued for cell densities higher than 100 cells mm−3 (Fig. 1d,e). Similar trends were observed for migration parameters such persistence of migration and invasive distance (Fig. 1f and Supplementary Fig. 1A and B)25.

Figure 1: Effect of cell density on cancer cell motility. (a) Phase contrast micrographs demonstrate confluence of human fibrosarcoma cells (HT1080WT) days after initial seeding. Scale bar, 100 μm. (b) Cell speed measured at a time lag of 2 min days after initial seeding. (c) Average distance to nearest cell (dR) relates density at different days to initial seeding density. (d) Randomly selected trajectories of human fibrosarcoma cells (HT1080WT) under different seeding densities of 10, 50, 120 cells mm−3 embedded in a 3D collagen matrix. Phase contrast micrographs demonstrate the confluence at each density. Scale bar, 100 μm. (e,f) Cell speed and persistence distance measured at a time lag of 2 min at different seeding densities. (g) Topology of protrusions for cells embedded in 3D collagen matrices: 0th generation protrusions (N 0 ) originate from the cell body, 1st generation protrusions (N 1 ) stem from N 0 and 2nd generation protrusions (N 2 ) stem from N 1 . (h) Cell speed and protrusion frequency are highly correlated. (i) Randomly selected trajectories of human carcinoma breast cancer cells (MDA-MB-231) under seeding densities of 10, 50, 120 cells mm−3. (j) Cell speed evaluated at a time lag of 2 min, at five different seeding densities. Cells at high seeding densities (ρ>50) show a significantly higher speed than cells seeded at low seeding density (ρ=10). (k) Average doubling time at increasing cell density demonstrates that proliferation is independent of cell density. In all panels, data is represented as mean±s.e.m. from three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (ANOVA) (n=3). Full size image

We have previously shown that cell motility in 3D matrices is predicted by the ability of cells to form dendritic pseudopodial protrusions19,26. Consistent with these observations, we found that the total number of main and dendritic protrusions generated per unit time by tumour cells steadily increased and then plateaued with cell density. The cell-density-dependent number of protrusions generated by the cells is strongly correlated with the cell-density-dependent cell speed (Fig. 1g,h and Supplementary Fig. 1C).

This remarkable relationship between tumour cell density and cell migration was also found in human metastatic carcinoma cells (MDA-MB-231) and human metastatic glioblastoma cells (U-87). Similar to fibrosarcoma cells, the migration of these two tumorigenic, metastatic/invasive cell lines increased with cell density (Fig. 1i,j and Supplementary Fig. 1D). In contrast, cell-density-dependent migration was not observed in tumorigenic, non-metastatic carcinoma cells (MCF7) and non-tumorigenic cell lines WI-38 human lung fibroblasts and MCF10A human epithelial cells (Supplementary Fig. 1E–G).

Interestingly, cell-density-dependent migration was not observed when cells were placed on two-dimensional (2D) collagen-coated substrates (Supplementary Fig. 1H–J). Moreover, in contrast to 3D cell migration, the proliferation of cells in 3D matrices was unaffected by cell density (Fig. 1k and Supplementary Fig. 1K), that is, cells continued to proliferate at a constant rate regardless of cell density. Together, these results indicate that cancer cell density enhances cancer cell migration, but not proliferation, and that cell-density-dependent migration could be unique to tumorigenic, metastatic cells in 3D microenvironments.

Cell migration enhancement is not caused by ECM remodelling

Cell-density-dependent migration could be mediated by the collagen matrix and cell-induced matrix remodelling. We investigated if cell density modulated the microstructural properties of the matrix, such as inter-fibre spacing (effective pore size) and local fibre alignment27. Using quantitative reflection confocal microscopy, we determined that local fibre alignment showed poor correlation with cell density and speed. Average inter-fibre spacing showed a poor correlation with cell speed as well.

As expected, a strong negative correlation was identified between average inter-fibre spacing and cell density. As the cell density increases, the forces exerted on the collagen fibrils by the cells increases, causing the space between the fibrils to decrease. (Fig. 2a–d and Supplementary Fig. 2A) Based on this result alone, we would have expected cell speed to decrease with increasing cell density but since the cells move faster as cell density increases this physical property cannot modulate cell-density-dependent migration. In sum, cell-density-dependent migration cannot be attributed to changes in the physical properties of the matrix.

Figure 2: Biochemical cues. (a) Reflection confocal micrograph. Singular of 3D collagen matrices. Scale bar, 10 μm. (b) Correlation plot of fibre alignment versus cell density. (c) Correlation plot of cell speed versus fibre alignment. (d) Correlation plot of average inter-fibre spacing versus cell density. (e) Method to prepare condition medium: medium is incubated for 24 h with a collagen matrix containing a high density of cells, 50 cells mm−3 (HD), which is then filtered using a 0.45-μm filter, and added to a matrix containing a low density of cells, 10 cells mm−3 (LD). (f) The addition of conditioned medium (CM) from a matrix containing a high cell density (HD) increases the speed of cells in a matrix containing a low cell density (LD). The HD cell speed in the presence of fresh medium (FM) is recapitulated in LD when using CM. (g) Secretomic analysis of CM harvested from human fibrosarcoma cells indicates that levels of interleukin 6 (IL-6) and interleukin 8 (IL-8) increase as a function of HT1080 cell density in the matrix, while levels of other major cytokines do not significantly change. (h) Secretomic analysis of conditioned medium from human breast carcinoma cells (MDA-MB-231) confirms our observations with HT1080 cells. (i,j) Increasing density of human fibrosarcoma cells in the matrix increases the concentrations of secreted IL-6 (A) and IL-8 (B), as analysed by ELISA. (k,l) Increasing cell density of human carcinoma cells in the matrix increases the concentrations of secreted IL-6 (A) and IL-8 (B), as analysed by ELISA. In all panels, data is represented as mean±s.e.m. from three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (ANOVA). Full size image

Secretomic profiles of matrix-embedded tumour cells

Based on these results, we hypothesized that cell-density-dependent migration was regulated by soluble molecules secreted by the cells in a cell-density-dependent manner. To test this hypothesis, we introduced conditioned medium collected from a matrix containing a high density of HT1080 cells (50 cells mm−3) into a matrix containing a low density of HT1080 cells (10 cells mm−3). We found that the enhanced cell velocity observed at high cell density could be recapitulated at a low cell density by adding condition medium collected from high cell density matrices (Fig. 2e,f). This result suggests that soluble molecules secreted by matrix embedded cancer cells are sufficient to promote enhanced cell migration.

To identify the soluble factor(s) driving enhanced motility, we measured and analysed the secretomic profiles of HT1080 and MDA-MB-231 cells embedded at low and high densities in 3D matrices, using a multiplex antibody microarray assay28. This assay simultaneously measured the concentration of 24 soluble molecules. We observed that the cytokines IL-6 and IL-8 were both secreted in relatively high concentrations and increased linearly with cell density for both cell lines. Remarkably, all other secreted proteins that were assayed including hepatocyte growth factor (HGF), which has been implicated in promoting tumour progression and tumour metastasis in several cancers29, were not elevated at higher cell densities during our experimental time window (Fig. 2g,h). Using ELISA, we confirmed our results and determined the precise concentrations of IL-6 and IL-8 at specific cell densities of matrix-embedded HT1080 and MDA-MB-231 cells (Fig. 2i–l). Together, this result suggests that IL-6 and IL-8 drive density-dependent cell migration in 3D matrices.

IL-6 and IL-8 together induce enhanced cell migration

Next, we systematically assessed whether IL-6 and IL-8 were required to drive cell-density-enhanced migration by conducting gain-of-function and loss-of-function experiments. We exposed matrix embedded HT1080 cells seeded at a low density to controlled concentrations of human recombinant IL-6 and IL-8. We found that IL-6 or IL-8 alone had no effect on cell migration, even at high concentrations (Fig. 3a,b and Supplementary Fig. 3A and B). In contrast, IL-6 and IL-8, when combined at the prescribed concentrations found at the high density of 50 cells mm−3 in the precise stoichiometric ratio of 5:2, induced cells at low density to move at the high velocity observed at high cell density and also detected for cells at low density exposed to conditioned medium (Fig. 3c). Strikingly, other stoichiometric ratios of IL-6 and IL-8 did not induce enhanced migration (Supplementary Fig. 3C). These results indicate that a mixture of IL-6 and IL-8 is sufficient to recapitulate the enhanced migration of cells embedded at high densities.

Figure 3: Functional influence of IL-6 and IL-8. (a,b) The addition of recombinant IL-6 alone or recombinant IL-8 alone do not increase cell speed. (c) The addition of recombinant IL-6 and IL-8 in combination at the precise concentrations found in a matrix containing a high density of 50 cells mm−3 (RM) recapitulates the high speed observed of human fibrosarcoma cells at high densities. (d) Decreased speed at LD (ρ=10) where cells are exposed to conditioned medium produced by IL-6 and IL-8 knockdown cells and conditioned medium obtained from a matrix containing a high cell density (HD) following exposure to specific IL-6 and IL-8 functional antibodies compared with control cells exposed to conditioned medium from wild-type cells at HD (ρ=50). (e) Decreased speed of the IL-6 and IL-8 knockdown cells at LD (ρ=10) and HD (ρ=50). (f) The addition of recombinant IL-6 and IL-8 in combination at the precise concentrations found in a matrix containing a high density of 100 cells mm−3 recapitulates the high speed observed of human carcinoma cells at high densities. (g) Cartoon depicts the fact that IL-6 and IL-8 are both required to influence cancer cell motility. (h) Decreased speed of the IL-6R and IL-8R knockdown cells at LD (ρ=10) and HD (ρ=50). (i) Cartoon depicts that Tocilizumab and Reparixin can be used to block the cognate receptors of IL-6 and IL-8. (j) Individually, Tocilizumab and Reparixin decreased cell speed of human fibrosarcoma cells embedded in a 3D matrix at LD (ρ=10) and HD (ρ=50) compared with cells exposed to fresh medium (0). (k,l) Tocilizumab and Reparixin in combination greatly decrease cell speed of cells embedded in a 3D matrix at LD (ρ=10) and HD (ρ=50) compared with cells exposed to fresh medium (0). In all panels, data is represented as mean±s.e.m. from three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (ANOVA). Full size image

To verify that both cytokines were required for cell-density-dependent migration, we conducted experiments with conditioned medium from HT1080 cells depleted of IL-6 or IL-8 via shRNA interference. Depleting either IL-6 or IL-8 prevented the conditioned medium from high density matrices to enhance cell migration of low density matrices (Fig. 3d). Similar results were obtained when we utilized specific neutralizing antibodies to block secreted IL-6 and IL-8. The loss-of-function assays conducted with matrix-embedded cells at low and high cell densities exposed to specific neutralizing antibodies and with matrix embedded HT1080 cells depleted of IL-6 and IL-8 demonstrated that the cell-density-dependent migration patterns observed previously were no longer detected (Fig. 3e and Supplementary Fig. 3G–I). These results were confirmed with matrix embedded MDA-MB-231 cells (Fig. 3f). Interestingly, as with HT1080 cells, the enhanced migration of MDA-MB-231 cells was observed when IL-6 and IL-8 were present in the stoichiometric ratio of 5:2 (Supplementary Fig. 3D). In marked contrast, tumorigenic, non-metastatic cells, MCF7, and non-tumorigenic cells, MCF10A, exposed to both IL-6 and IL-8 did not exhibit enhanced migration (Supplementary Fig. 3E and F). These results suggest that IL-6 and IL-8 are each individually required, but only sufficient in combination to induce enhanced migration in tumorigenic, metastatic cells (Fig. 3g).

Further, we hypothesized that enhanced migration through the synergistic signalling of IL-6 and IL-8 is sensed by the cells via a paracrine pathway through the receptors of IL-6 (IL-6R) and IL-8 (IL8R1/CXCR1 or IL8R2/CXCR2). Matrix-embedded cells are exposed to a gradient of secreted proteins that can readily build up around a cell and consequently paracrine signalling can occur as the inter-cellular distance is decreased with an increase in cell density. As a result, the paracrine signalling could trigger a response in cellular behaviour (for example, enhanced migration). Our results demonstrated that the expression of IL-6R and CXCR2 indeed increased as cell density increased, indicating that signalling pathway is paracrine (Supplementary Fig. 3J and K).

We next explored possible therapeutic targets to decrease metastatic capacity by inhibiting the cell-density-dependent paracrine signalling pathway. The receptors on tumour cell membranes are also important drivers in signalling pathways and thus could be targeted to inhibit a particular phenotype. HT1080 and MDA-MB-231 cells depleted of IL-6R and CXCR2 (Referred to from this point on as IL-8R) via shRNA interference were embedded in 3D collagen matrices. We found that the depletion of IL-6R had no effect on cell migration at low cell density. In contrast, this molecular intervention suppressed cell velocity at elevated cell densities. Interestingly, cells depleted of IL-8R displayed a reduction in cell velocity at both low and high cell densities (Fig. 3h and Supplementary Figs 3L and 6A–D).

To determine pharmacological agents for potential therapeutic interventions, inhibitors of IL-6R (Tocilizumab) and IL-8R (Reparixin) were added to matrix-embedded cells at low and high densities. Tocilizumab induced a small decrease in HT1080 cell velocity at low cell density, but induced a more visible decrease in cell velocity at high cell density. Reparixin decreased cell velocity at both low and high cell densities, with notable reduction in cell velocity at higher Reparixin concentrations (Fig. 3j). The combination of the two inhibitors showed a decrease in cell velocity at both low and high cell densities (Fig. 3k). We observed similar effects of the inhibitors on the velocity of MDA-MB-231 cells (Fig. 3l and Supplementary Fig. 3M and N).

Cell density induces a distinct transcriptional phenotype

To confirm the formation of a more invasive and migratory phenotype solely induced by cell proliferation and increase of cell density, we performed global transcriptional phenotype analysis by RNA sequencing (RNA-seq)30. The transcriptomes of HT1080 cells at low density (LD) and high density (HD) were sequenced and compared for differential gene expression. They were also compared with the transcriptomes of HT1080 cells at a low density exposed to recombinant IL-6 alone (IL-6), IL-8 alone (IL-8), and IL-6 and IL-8 found in the precise concentrations at the high density of 50 cells mm−3 (RM). To identify the sources of transcriptional variations caused by different conditions, we performed an ANOVA-like test to detect the genes most variable among multiple groups. To study the relationship of global transcriptomes, principle component analysis (PCA) of the top 930 most significant genes was performed. PCA demonstrated that the transcriptomes of LD, IL-6 and IL-8 cluster in close proximity in the third quadrant while RM shows a phenotypical shift toward HD in the second quadrant. RM and HD residing in the same quadrant indicate the phenotypic similarity between cell-proliferation-induced migratory phenotype and the phenotype generated by RM (Fig. 4a). The shift in transcriptional phenotype induced solely by cell density increase was confirmed with differential gene expression analysis.

Figure 4: Proposed mechanism. (a) Principle component analysis (PCA) of the top 930 most significant genes to determine the relationship of global transcriptomes. (b) Heat map demonstrating the difference between gene ontology categories. (c) Table demonstrating gene ontology categories. (d) Activity of STAT3 in 3D conditions at LD (ρ=10) and HD (ρ=50). (e) Decreased cell speed of human fibrosarcoma cells embedded in a 3D matrix exposed to JAK2 inhibitor, AG-490, STAT3 inhibitor, S3I-201, and Arp2/3 complex inhibitor, CK 666, at LD (ρ=10) and HD (ρ=50) compared with cells exposed to fresh medium (0). (f) Increased mRNA expression of ACTR2 at HD. (g) The addition of recombinant IL-6 and IL-8 alone does not increase protrusion frequency, however the addition of recombinant IL-6 and IL-8 in combination at the precise concentrations found in a matrix containing a high density of 50 cells mm−3 (RM) significantly increases the frequency of cellular protrusions. (h) Increased mRNA expression of WASF3 at HD and cells exposed to recombinant IL-6 and IL-8 in combination at the precise concentrations found in a matrix containing a high density of 50 cells mm−3. (i) Cartoon depiction of the IL-6 and the IL-8 signalling pathway leading to enhanced cell motility. In all panels, data is represented as mean±s.e.m. from three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (ANOVA). Full size image

Next, we performed ingenuity pathway analysis (IPA) to investigate the biological mechanisms underlying transcriptional phenotypes by analysing the functional annotation of differential expression gene clusters and pathway enrichment. The most enriched gene ontology category of the genes highly expressed in RM was ‘cell movement’. Another group of genes, which were highly expressed in IL-6, IL-8 and LD but downregulated in RM, was cellular metabolism and division-related pathways. This result suggests that RM induces a strong phenotype with enhanced cell movement. Other biological pathways contributing to the change of LD to HD cell phenotype include cell death and survival, cell metabolic activity, cell cycle and division. (Fig. 4b,c and Supplementary Fig. 4A and B).

Mechanism of cell-density-dependent migration

Signal transducer and activator of transcription 3, STAT3, is a transcription factor that is a common downstream effector in the individual pathways of IL-6 and IL-8 (refs 31, 32). Therefore, we hypothesized that STAT3 could regulate cell-density-dependent migration. We found that the activity of STAT3 in matrix embedded HT1080 cells at a high density was two fold higher than that of cells at a low density (Fig. 4d).

Based on our previous work, we further speculated that the Arp2/3 complex nucleates F-actin assembly and mediates dendritic protrusions required for cell-density-dependent migration19. Thus, we reasoned that enhanced migration may be regulated by the Arp2/3 complex through the (Janus kinase) JAK/STAT3 pathway. Through examinations of the migration of HT1080 cells at low and high cell densities exposed to the specific JAK2 inhibitor AG-490 (ref. 33), STAT3 inhibitor S3I-201 (ref. 34), or Arp 2/3 complex inhibitor CK666 (ref. 35), we determined that JAK2, STAT3 and the Arp2/3 complex were indeed required for cell-density-dependent migration. Treatment with any of the three inhibitors prevented cell-density-dependent migration by repressing protrusion activity (Fig. 4e and Supplementary Fig. 4C).

To further determine the role of the Arp2/3 complex in cell-density-dependent migration, we measured the mRNA expression of the ACTR2 and protein expression of ARP2 and ARP3 and determined they wereslightly upregulated at HD (Fig. 4f and Supplementary Fig. 6E–H). We also measured protrusions and branching frequency for LD, HD, IL-6, IL-8 and RM conditions and demonstrated that IL-6 and IL-8 did not increase protrusion frequency or branching frequency but RM significantly did. (Fig. 4g and Supplementary Fig. 4D).

Because WASF3 is involved in the regulation of actin cytoskeleton dynamics through the recruitment of the Arp2/3 complex36,37,38, we also hypothesized that WASF3 was an important intermediate between STAT3 and Arp2/3. Thus, we quantified the mRNA expression of WASF3 at LD, HD, IL-6, IL-8 and RM and found a relatively higher expression of WASF3 at HD and RM compared with LD, IL-6 and IL-8. Additionally, the protein level for WASF3 increased at HD (Supplementary Fig. 6I and J). RNAseq analysis also indicated that WASF3 was upregulated at RM but not when the interleukins were present individually (Fig. 4h and Supplementary Fig. 4E). Further we depleted WASF3 through shRNA interference and found that cell-density-dependent migration was not observed. (Supplementary Fig. 4F and G) These results suggest that WASF3 together with the Arp2/3 complex are important regulators in the pathway that controls cell-density-dependent migration (Fig. 4i).

Interestingly, when the expression of STAT3, WASF3 and ACTR2 were measured for differing stoichiometric ratios of IL-6 and IL-8, we observed that the expression of these three intermediates were maximally stimulated under the 5:2 conditions (Supplementary Fig. 4H–J).

Mouse xenograft model to test therapeutic strategies

Given that IL-6 and IL-8 cooperate to enhance migration in breast carcinoma cells, we sought to investigate their potential role in metastasis by inhibiting their cognate receptors using Tocilizumab and Reparixin. Tocilizumab is currently in clinical trials to study its efficacy against recurrent ovarian cancer20, while Reparixin is being evaluated for safety, tolerability, pharmacokinetics, and to detect early signs of antitumour activity in breast cancer patients22,23. The effect of these drugs on metastasis was examined by generating an animal model through the introduction of MDA-MB-231 carcinoma breast cancer cells into the mammary fat pad of NSG (NOD SCID Gamma) mice and injecting four sets of mice with saline, Tocilizumab alone (25 mg kg−1), Reparixin alone (30 mg kg−1), and Tocilizumab and Reparixin in combination every three days for 6 weeks.

As predicted from our in vitro results (Fig. 1k), we observed that the treatment had no effect on the rate of tumour growth (Supplementary Fig. 5A and B). We also determined that metastasis to the lungs, liver, and lymph nodes were suppressed in the treated group. Specifically, the combination of the two drugs was the most effective in repressing metastatic burden on the liver and the lymph nodes (Fig. 5a–f). Moreover, the expression of the key intermediates in the synergistic pathway, STAT3, WASF3 and ACTR2, were significantly decreased in the treated group suggesting that the cell-density-dependent paracrine signalling pathway was responsible for the decreased metastases observed. (Fig. 5g–i) Immuno-histochemical staining for ACTR2 confirmed expression in control tumours and markedly decreased expression in the tumours from mice treated with the combination of drugs (Fig. 5j,k).

Figure 5: In vivo validation. (a) Tumour volume measured over time. (b,c) Human genomic DNA content in mouse lungs and livers were quantified using qPCR to determine the metastatic burden. (d) Vimentin staining of lymph nodes quantified by image analysis. (e) Images of mice lungs that were stained with hematoxylin and eosin. Scale bar, 100 μm. (f) Images of lymph nodes that were stained with vimentin. Scale bar, 100 μm. (g–i) Decreased expression of STAT3, WASF3 and Arp2/3 in treated group compared with the control group. (j) Immunohistochemical staining of primary tumour sections for Arp2/3. Scale bar, 100 μm. (k) Arp 2/3 staining of primary tumour sections quantified by image analysis. In all panels, data is represented as mean±s.e.m. of five mice. *P<0.05; **P<0.01; ***P<0.001 (ANOVA). Full size image

Our in vitro and in vivo findings describe a novel synergistic paracrine signalling pathway between IL-6 and IL-8 that plays a critical role in metastasis through the regulation of cell-density-dependent tumour cell migration. Thus, this study infers a new therapeutic target to decrease the metastatic capacity of tumour cells and improve patient outcomes.