In vitro cross-talk between metastasis-competent circulating tumor cells and platelets in colon cancer: a malicious association during the harsh journey in the blood

Abstract

Background: Platelets are active players in hemostasis, coagulation and also tumorigenesis. The cross-talk between platelets and circulating tumor cells (CTCs) may have various pro-cancer effects, including promoting tumor growth, epithelial-mesenchymal transition (EMT), metastatic cell survival, adhesion, arrest and also pre-metastatic niche and metastasis formation. Interaction with CTCs might alter the platelet transcriptome. However, as CTCs are rare events, the cross-talk between CTCs and platelets is poorly understood. Here, we used our established colon CTC lines to investigate the colon CTC-platelet cross-talk in vitro and its impact on the behavior/phenotype of both cell types. Methods: We exposed platelets isolated from healthy donors to thrombin (positive control) or to conditioned medium from three CTC lines from one patient with colon cancer and then we monitored the morphological and protein expression changes by microscopy and flow cytometry. We then analyzed the transcriptome by RNA-sequencing of platelets indirectly (presence of a Transwell insert) co-cultured with the three CTC lines. We also quantified by reverse transcription-quantitative PCR the expression of genes related to EMT and cancer development in CTCs after direct co-culture (no Transwell insert) with platelets. Results: We observed morphological and transcriptomic changes in platelets upon exposure to CTC conditioned medium and indirect co-culture (secretome). Moreover, the expression levels of genes involved in EMT (p < 0.05) were decreased in CTCs co-cultured with platelets, but not of genes encoding mesenchymal markers (FN1 and SNAI2). The expression levels of genes involved in cancer invasiveness (MYC, VEGFB, IL33, PTGS2, and PTGER2) were increased. Conclusion: For the first time, we studied the CTC-platelet cross-talk using our unique colon CTC lines. Incubation with CTC conditioned medium led to platelet aggregation and activation, supporting the hypothesis that their interaction may contribute to preserve CTC integrity during their journey in the bloodstream. Moreover, co-culture with platelets influenced the expression of several genes involved in invasiveness and EMT maintenance in CTCs.

Keywords: cancer; circulating tumor cells (CTCs); epithelial-to-mesenchymal transition (EMT); metastasis; platelets; tumor-educated platelets (TEPs).

FIGURE 1

Platelet morphological changes after exposure to conditioned medium from three CTC lines. (A) Representative images (×20 magnification—zoom ×40 magnification) of the morphological changes and aggregation of platelets (PLT) incubated with conditioned medium from three colon CTC lines (CTC41, CTC41.4, and CTC41.5G), with control medium, and with thrombin (positive control). (B) Quantification of CD41/61⁺, CD62P⁺, and CD41/61⁺CD62P⁺ platelets in the different culture conditions described in (A) by flow cytometry. Values are the mean ± SEM, n = 3/ group; *p < 0.05, **p < 0.005 vs. control (PLT) (unpaired t-test).

FIGURE 2

Platelet transcriptomic changes upon indirect co-culture with three CTC lines. (A) Volcano plots showing the differential gene expression analysis of RNA-seq data. The log-transformed p-values are plotted on the y-axis and the log2 fold change values of platelets after indirect co-culture with CTC41, CTC41.4, and CTC41.5G cells vs. control (platelets alone) are plotted on the x-axis. The criteria were relative expression fold change ≤ −2.0 or ≥2.0 and p ≤ 0.05. Red dots and blue dots represent increased and decreased RNAs, respectively. (B) Venn diagram showing the number of shared differentially expressed mRNAs in platelets after indirect co-culture with CTC41, CTC41.4, and CTC41.5G cells vs. control. (C–F) Gene Set Enrichment Analysis (GSEA) was performed using the RNA-seq data to compare the transcriptome profiles of platelets after indirect co-culture with CTC41, CTC41.4, and CTC41.5G cells vs. control. (C,D) GSEA results showing significant enrichment of the EMT and Hypoxia signaling pathways among mRNAs increased in platelets after indirect co-culture with CTC41, CTC41.4, and CTC41.5G cells vs. control. (E,F) GSEA results showing that the Inflammatory response and Apoptosis pathways are significantly enriched in platelets after indirect co-culture with CTC41, CTC41.4, and CTC41.5G cells vs. control. Venn diagrams show the shared enriched transcripts in each experimental condition. The heatmaps (C–F) of the common transcripts among the three sample groups illustrate their expression level (red, high; blue, low) compared with control. Abbreviations: PLT = Platelets, NES = Normalized enrichment score, FDR = False discovery rate.

FIGURE 3

RT-qPCR analysis of (A) FN1 and SNAL2, (B) CK19, EPCAM and CDH1, (C) MYC, (D) VEGFB, (E) IL33, (F) TGFß2, (G) PTGS2, and (H) PTGER2 in CTC lines co-incubated with platelets (striped bars) compared with control (CTCs alone; without stripes); expression in control was set to 1; values are the mean ± SEM, n = 3/group; *p < 0.05, **p < 0.005, ***p < 0.0005 (unpaired t-test). Abbreviations: PLT = platelets, FN1 = fibronectin, CK19 = cytokeratin 19, EPCAM = epithelial cell adhesion molecule, CDH1 = E-cadherin, MYC = MYC proto-oncogene, VEGFB = vascular endothelial growth factor B, IL33 = interleukin 33, TGFß2 = transforming growth factor beta 2, PTGS2 = prostaglandin-endoperoxide synthase 2, PTGER2 = prostaglandin E receptor 2.

FIGURE 4

Schematic representation of the CTC-platelet cross-talk. (A) CTC-platelet interactions cause phenotypic changes, activation, and transcriptome alternation in platelets, and also (B) increase the invasive properties of CTCs by upregulating MYC, VEGFB, IL33, PTGS2, and PTGER2 as well as TGFß2 in CTCs. Co-culturing CTCs with platelets induces or maintains the EMT state of CTCs. These cells have a major epithelial phenotype with some features of invasive cells. Upon EMT induction by interacting with platelets, the expression of epithelial markers was decreased, without upregulation of mesenchymal markers, in the three colon CTC lines. This might be explained by a specific biological lock to maintain CTCs in a partial EMT, allowing them to undergo MET immediately. This plasticity, called epithelial-to-mesenchymal plasticity, gives to CTCs a more aggressive phenotype and facilitates their quick adaptation in their new tumor environment to initiate a new tumor at a distant site. Abbreviations: EMT = epithelial-to-mesenchymal transition, MET = mesenchymal-to-epithelial transition, EMP = epithelial-to-mesenchymal plasticity; TGFß2 = transforming growth factor beta 2, MYC = MYC proto-oncogene, VEGFB = vascular endothelial growth factor B, IL33 = interleukin33, PTGS2 = prostaglandin-endoperoxide synthase 2, PTGER2 = prostaglandin E receptor 2.