Highly skewed distribution of miRNAs and proteins between colorectal cancer cells and their exosomes following Cetuximab treatment: biomolecular, genetic and translational implications

Abstract

Exchange of molecules via exosomes is a means of eukaryotic intercellular communication, especially within tumour microenvironments. However, no data are available on alterations of exosomal molecular cargo by environmental cues (eg, pharmacological treatments). To approach this issue, we compared the abundance of 754 miRNAs and 741 cancer-related proteins in exosomes secreted by Caco-2 (Cetuximab-responsive) and HCT- 116 (Cetuximab-resistant) CRC cells, before and after Cetuximab treatment, with that in their source cells. Cetuximab significantly altered the cargo of Caco-2 exosomes: it increased abundance of miRNAs and proteins activating proliferation and inflammation and reduced miRNAs and proteins related to immune suppression. These alterations did not precisely mirror those in source cells, suggesting a Cetuximab-linked effect. Analogous alterations were detected in HCT-116. Transfection of exosomes from Cetuximab-treated Caco-2 into HCT-116 significantly increased HCT-116 viability; conversely, no viability alteration was detected in Caco-2 transfected with exosomes from Cetuximab-treated HCT-116. Analysis of networks, comprising targets of differentially expressed (DE) exosomal miRNAs and DE exosomal proteins, demonstrates a significant involvement of processes related to proliferation, inflammation, immune response, apoptosis. Our data extend existing knowledge on molecular mechanisms of eukaryotic intercellular communication, especially in oncological processes. Their translation to clinical settings may add new weapons to existing therapeutic repertoires against cancer.

Keywords: Cetuximab; Colon Cancer; Exosomes; Proteins; miRNAs.

Figure 1. Characterization of Caco-2 and HCT-116 exosomes

(A) Average particle sizes in exosome samples were determined by dynamic light scattering. Y-axes: signal intensity (%); X-axes: size of particles (nm). (B) FACS analysis was performed based on exosome markers CD9, CD63 and CD81 on nanoparticles isolated from Caco-2 and HCT-116 cells.

Figure 2. Comparison of miRNA sets found in exosomes and their source cells

Venn diagrams show the overlap between miRNA sets in exosomes and their matching source cells: (A) Caco-2; (B) HCT-116. A third diagram (C) shows a comparison of miRNAs content in exosomes from Caco-2 and HCT-116 cells, respectively; (D) same data are shown for cellular miRNAs in Caco-2 and HCT-116 cells (for details see Materials and Methods).

Figure 3. Quantitative asymmetric distribution of miRNAs in CRC cells and their exosomes

Relative quantities (RQ) of exosomal miRNAs were compared to those from source cells: Caco-2 (A); HCT-116 (B). Values are shown as log10 of RQ.

Figure 4. Biological functions of exosomal miRNAs in CRC

(A) Prediction of the biological functions of the most abundant exosomal miRNAs. Based on their validated or putative targets, the related function was predicted for both cell lines at steady-state; GO annotations are shown that exhibited a significant overrepresentation in upregulated miRNAs with respect to those downregulated in exosomes from (B) Caco-2 and (C) HCT-116 cells after Cetuximab treatment.

Figure 5. Comparison of biological functions attributed to cellular DE miRNAs in Caco-2 and HCT-116 cells after Cetuximab treatment

A comparison is shown of biological terms from Biocarta, Gene Ontology, KEGG and Reactome that are associated with DE miRNAs in Caco-2 and HCT-116 cells after Cetuximab treatment. Values plotted in the histogram are show as –log10 of the adjusted p-value.

Figure 6. Antibody array data from CRC exosomes after Cetuximab treatment

(A) Detrended correspondence analysis of array data from Caco-2 and HCT-116 exosomes before (Ctrl) and after treatment (Cetuximab). (B) Hierarchical clustering of the exosome samples.

Figure 7. Biological functions of DE exosomal proteins in HCT-116

Analysis of biological functions associated with DE exosomal proteins from Cetuximab-treated HCT-116 cells based on an Ingenuity System analysis: (A) canonical pathways; (B) network involvement; (C) functional annotation categories.

Figure 8. Biological functions of DE exosomal proteins in Caco-2

Analysis of biological functions associated with DE exosomal proteins from Cetuximab-treated Caco-2 cells based on an Ingenuity System analysis: (A) canonical pathways; (B) network involvement; (C) functional annotation categories.

Figure 9. CRC cells viability after incubation with exosomes from untreated and Cetuximab-treated cells

(A) MTT analysis of HCT-116 cells incubated for 24 h and 48 h with exosomes. Left and centre: untreated cells (CTRL) were compared to cells to which 2 μg or 5 μg, respectively, of exosomes from Caco-2 cells at steady-state had been added. Right: differences of incubations with exosomes from Caco-2 cells with or without Cetuximab treatment. (B) Results of an MTT assay on Caco-2 cells incubated for 24 h and 48 h. Left and centre: untreated cells (CTRL) were compared to cells to which 2 μg or 5 μg of exosomes from HCT-116 cells at steady-state had been added. Right: differences of incubations with exosomes from HCT-116 cells with or without Cetuximab treatment. CTRL: solvent used for exosome resuspension (PBS). Unpaired t-test, * P ≤ 0.05; ** P ≤ 0.01.

Figure 10. Viability of CRC cells simultaneously treated with Cetuximab and exosomes from untreated or treated cells

(A) Results of MTT assays on HCT-116 cells treated with Cetuximab for 24 h and co-incubated with 5 μg of exosomes from Caco-2 cells that had been treated or not with Cetuximab. (B) Results of MTT assays on Caco-2 cells treated with Cetuximab for 24 h and co-incubated with 5 μg of exosomes from HCT-116 cells that had been treated or not with Cetuximab. CTRL: solvent used for Cetuximab resuspension (PBS). Unpaired t-test, * P ≤ 0.05; ** P ≤ 0.01.