Human mast cells promote colon cancer growth via bidirectional crosstalk: studies in 2D and 3D coculture models

Abstract

Chronic inflammation drives the development of colorectal cancer (CRC), where tumor-infiltrating immune cells interact with cancer cells in a dynamic crosstalk. Mast cells (MC), one of earliest recruited immune cells, accumulate in CRC tissues and their density is correlated with cancer progression. However, the exact contribution of MC in CRC and their interaction with colon cancer cells is poorly understood. Here, we investigated the impact of primary human MC and their mediators on colon cancer growth using 2D and 3D coculture models. Primary human MC were generated from peripheral CD34⁺ stem cells. Transwell chambers were used to analyze MC chemotaxis to colon cancer. Colon cancer cells HT29 and Caco2 differentially recruited MC by releasing CCL15 or SCF, respectively. Using BrdU proliferation assays, we demonstrated that MC can directly support colon cancer proliferation and this effect was mediated by their cellular crosstalk. 3D coculture models with cancer spheroids further confirmed the pro-tumor effect of MC on colon cancer growth, where direct cell-cell contact is dispensable and increased production of multiple soluble mediators was detected. Moreover, TLR2 stimulation of MC promoted stronger growth of colon cancer spheroids. By examining the transcriptome profile of colon cancer-cocultured MC versus control MC, we identified several MC marker genes, which were deregulated in expression. Our study provides an advanced in vitro model to investigate the role of human MC in cancer. Our data support the detrimental role of MC in CRC development and provide a molecular insight into the cellular crosstalk between MC and colon cancer cells.

Keywords: 3D coculture; CCL15; Human mast cells; TLR2; cellular crosstalk; colorectal cancer; gene expression; stem cell factor.

Figure 1.

Generation of primary human MC. (a) Granule development in CD34⁺ derived human MC during the course of culture (scale bar, 20μm). (b) Viability and purity of human MC followed by dead cell removal and CD117⁺ positive selection. (c) β-hexosaminidase (β-hex) release by human MC in response to FcɛRI-mediated activation. Bars represent mean value ± SEM. Data shown are a representative of three independent experiments.

Figure 2.

Colon cancer cells recruit human MC via the release of soluble mediators. (a) MC chemotaxis to conditioned medium (CM) of human colon cancer cell lines HT29 and Caco2. (b) MC migration induced by HT29 CM and Caco2 CM in the presence of SCF blocking antibody. (c) MC migration induced by HT29 CM and Caco2 CM in the presence of CCL15 blocking antibody. Bars represent mean value ± SEM. Data shown are a representative of three independent experiments. **P ˂ 0.005; ***P ˂ 0.001; ****P < 0.0001, assessed by unpaired Student’s t test or one-way ANOVA.

Figure 3.

Human MC directly promote colon cancer proliferation. Proliferation of colon cancer cells (HT29 and Caco2), as measured by BrdU uptake, cultured with different ratios of human MC (1:0, 1:0.5, 1:1, 1:2, 1:4) in a serum-free medium for 72 hr. Data are displayed as mean ± SEM of three independent experiments (n=3). *P ˂ 0.05; **P ˂ 0.005; ***P ˂ 0.001, assessed by one-way ANOVA.

Figure 4.

Crosstalk between human MC and colon cancer cells is necessary for cancer proliferation. Proliferation percentage (% proliferation) of colon cancer cells (HT29 and Caco2) cultured with MC directly or MC supernatant alone in a serum-free medium for 72 hr. The proliferation of cancer cells cultured in serum-free medium alone serves as a negative control (NEG) and in FCS-containing medium serves as a positive control (POS). Proliferation percentage was calculated as follows: BrdU uptake [(treated group - NEG)/(POS – NEG)] x 100%. Data are displayed as mean ± SEM of three independent experiments (n=3). *P ˂ 0.05; **P ˂ 0.005; ***P ˂ 0.001, assessed by one-way ANOVA.

Figure 5.

TLR2-stimulated MC induce stronger growth of colon cancer in a 3D spheroid model. (a) Representative confocal images of HT29 cancer spheroids cultured in 10% FCS medium, 1% BSA medium or 1% BSA medium in presence of non-stimulated human MC (MC NS) or FSL-1 stimulated human MC (MC S) for 6 days. GFP transfected HT29 cells are shown in green, cell nuclei are shown in blue and CMTPX-labeled MC in red. (Scale bar, 200μm). (b) Spheroid area of HT29 as an indicator of cancer growth. (c) Border regularity of HT29 spheroid as an indicator of cancer invasiveness. Border regularity was calculated in a formula: 4π (spheroid area/spheroid perimeter^2). Data are displayed as mean ± SEM of two independent experiments (n=2) .**P ˂ 0.005; **** P < 0.0001, assessed by one-way ANOVA, Tukey’s multiple comparisons.

Figure 6.

Coculture of colon cancer spheroids with MC induces production of various cytokines. (a) Human antibody cytokine arrays to assess mediators secreted from HT29 spheroids cultured in 1% BSA medium with or without non-stimulated human MC (MC NS) or FSL-1 stimulated human MC (MC S) for 6 days. Increased levels of angiogenin, IL-8, MIF, TIMP-1, TIMP-2 and uPAR were detected in human MC-HT29 coculture compared with HT29 spheroids alone (BSA). MIF: macrophage migration inhibitory factor; TIMP-1/2: tissue inhibitor of metalloproteinases-1/2; uPAR: urokinase receptor. (b) ELISA assays to quantify the concentration of angiogenin, IL-8, MIF, TIMP-1, TIMP-2 and uPAR secreted from HT29 spheroids alone, human MC alone and human MC-HT29 coculture. Data are displayed as mean ± SEM of two independent experiments (n=2). *P ˂ 0.05; **P ˂ 0.005; ***P ˂ 0.001, ****P < 0.0001, assessed by one-way ANOVA.

Figure 7.

Deregulated genes in MC cocultured with colon cancer spheroids. (a) Differentially expressed genes in MC cocultured with HT29 spheroids (CCS) versus MC alone (CTR). Genes labeled in red were significantly differentially up or down regulated, respectively. MMP2: matrix metalloproteinase 2; PDGFA: platelet derived growth factor subunit a; PTGS2: prostaglandin-endoperoxide synthase 2; RELA: NF-κB subunit P65; PTGER4: prostaglandin E receptor 4; VEGFA: vascular endothelial growth factor A; ITGA2/3: integrin alpha-2/3; CIT: citron; RHOB: ras-related homology B. The P values were computed by Wald test and corrected by the Benjamini-Hochberg procedure. The horizontal dash-line denotes the significance level of α = 0.05 (n=2). The vertical dash-line denotes the value of log₂-transformed gene expression fold change (log₂FC) = 0.2. (b) Gene expression (Reads Per Kilobase of transcript, per Million mapped reads, RPKM) of MC associated genes were compared between CTR and CCS. The P values were computed by Wald test and corrected by the Benjamini-Hochberg procedure (n=2).

Figure 8.

Schematic overview of deregulated genes and associated pathways in colon cancer-cocultured MC. Differentially upregulated genes are shown in red and downregulated genes in blue. Predicted pathways and effects are presented.