Breast cancer exosome-like microvesicles and salivary gland cells interplay alters salivary gland cell-derived exosome-like microvesicles in vitro

Abstract

Saliva is a useful biofluid for the early detection of disease, but how distal tumors communicate with the oral cavity and create disease-specific salivary biomarkers remains unclear. Using an in vitro breast cancer model, we demonstrated that breast cancer-derived exosome-like microvesicles are capable of interacting with salivary gland cells, altering the composition of their secreted exosome-like microvesicles. We found that the salivary gland cells secreted exosome-like microvesicles encapsulating both protein and mRNA. We also showed that the interaction with breast cancer-derived exosome-like microvesicles communicated and activated the transcriptional machinery of the salivary gland cells. Thus, the interaction altered the composition of the salivary gland cell-derived exosome-like microvesicles on both the transcriptomically and proteomically.

Figure 1. PKH-labeled 231-derived exosome-like microvesicles can label human salivary gland cells in the presence of serum.

(A) Microscopy (scale bar = 25 µm) and (B) FACS results showed that HSG cells were labeled after treatment for 1 hour with PKH-labeled 231-derived exosome-like microvesicles, and minimally labeled when treated only with PKH dye or lysed microvesicles. * P<0.05, ** P<0.01, and *** P<0.001; n = 3. All experiments were independently performed a minimum of three times.

Figure 2. Up-regulation of HSG RNA induced by 231-derived exosome-like microvesicles.

A) Nano Ladder. (B) Basal RNA level in serum-starved HSG cells, along with 18 s and 28 s ribosomal RNA peaks. Increased total RNA levels were observed after a 12-hour treatment with 231-derived exosome-like microvesicles (Exo) compared to lysed 231-derived exosome-like microvesicles (control, Lys Exo). Transcription inhibition by actinomycin D (ActD) diminished the increase in RNA levels induced by 231-derived exosome-like microvesicles, suggesting that transcription is activated by 231-derived exosome-like microvesicles. (C) Cell count and (D) cell viability were not affected by the treatments. * P<0.05, ** P<0.01, and *** P<0.001; n = 6. All experiments were independently performed a minimum of three times.

Figure 3. HSG-derived exosomal protein content was altered by 231-derived exosome-like microvesicles.

2D-DIGE identified 88 spots (circled) differing by 1.5-fold or more were observed compared to HSG-derived exosomal proteins treated with lysed 231-derived exosome-like microvesicles after treatment with intact 231-derived exosome-like microvesicles (n = 1).

Figure 4. Interplay between 231-derived exosome-like microvesicles altered the HSG exosomal mRNA composition.

(A) Heat map of microarray analysis results for mRNA transcripts from exosome-like microvesicles isolated from HSG cells treated with 231-derived exosome-like microvesicles or lysed 231-derived exosome-like microvesicles (control). (B) Ontological analysis of the 66 mRNA transcripts distinct to exosome-like microvesicles derived from HSG cells that interacted with 231-derived exosome-like microvesicles implicated in various molecular functions and (C) biological processes. (D, E) Array analysis using R 2.7.0 revealed the top 10 up- or down-regulated HSG-derived exosomal mRNA transcripts after treatment with 231-derived exosome-like microvesicles with respect to control. The results were generated via three independent trials.