Synergistic IL-6 and IL-8 paracrine signalling pathway infers a strategy to inhibit tumour cell migration

Abstract

Following uncontrolled proliferation, a subset of primary tumour cells acquires additional traits/mutations to trigger phenotypic changes that enhance migration and are hypothesized to be the initiators of metastasis. This study reveals an adaptive mechanism that harnesses synergistic paracrine signalling via IL-6/8, which is amplified by cell proliferation and cell density, to directly promote cell migration. This effect occurs in metastatic human sarcoma and carcinoma cells- but not in normal or non-metastatic cancer cells-, and likely involves the downstream signalling of WASF3 and Arp2/3. The transcriptional phenotype of high-density cells that emerges due to proliferation resembles that of low-density cells treated with a combination of IL-6/8. Simultaneous inhibition of IL-6/8 receptors decreases the expression of WASF3 and Arp2/3 in a mouse xenograft model and reduces metastasis. This study reveals a potential mechanism that promotes tumour cell migration and infers a strategy to decrease metastatic capacity of tumour cells.

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 (N₀) originate from the cell body, 1st generation protrusions (N₁) stem from N₀ and 2nd generation protrusions (N₂) stem from N₁. (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).

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).

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).

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).

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).