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. 2022 Mar 17;23(6):3262.
doi: 10.3390/ijms23063262.

Extracellular Vesicles as Signal Carriers in Malignant Thyroid Tumors?

Małgorzata Grzanka  1 , Anna Stachurska-Skrodzka  2 , Anna Adamiok-Ostrowska  1 , Ewa Gajda  1 , Barbara Czarnocka  1
Affiliations

Extracellular Vesicles as Signal Carriers in Malignant Thyroid Tumors?

Małgorzata Grzanka et al. Int J Mol Sci. .

Abstract

Extracellular vesicles (EVs) are small, membranous structures involved in intercellular communication. Here, we analyzed the effects of thyroid cancer-derived EVs on the properties of normal thyroid cells and cells contributing to the tumor microenvironment. EVs isolated from thyroid cancer cell lines (CGTH, FTC-133, 8505c, TPC-1 and BcPAP) were used for treatment of normal thyroid cells (NTHY), as well as monocytes and endothelial cells (HUVEC). EVs' size/number were analyzed by flow cytometry and confocal microscopy. Gene expression, protein level and localization were investigated by qRT-PCR, WB and ICC/IF, respectively. Proliferation, migration and tube formation were analyzed. When compared with NTHY, CGTH and BcPAP secreted significantly more EVs. Treatment of NTHY with cancer-derived EVs changed the expression of tetraspanin genes, but did not affect proliferation and migration. Cancer-derived EVs suppressed tube formation by endothelial cells and did not affect the phagocytic index of monocytes. The number of 6 μm size fraction of cancer-derived EVs correlated negatively with the CD63 and CD81 expression in NTHY cells, as well as positively with angiogenesis in vitro. Thyroid cancer-derived EVs can affect the expression of tetraspanins in normal thyroid cells. It is possible that 6 μm EVs contribute to the regulation of NTHY gene expression and angiogenesis.

Keywords: angiogenesis; extracellular vesicles; tetraspanins; thyroid cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) EVs from different cell lines labeled with Caveolin-1 and imaged by confocal microscopy: (a,b)—EVs isolated from CGTH; (c)—EVs isolated from TPC-1; (d)—EVs isolated from FTC-133, (green—Alexa Fluor 488); (B) Representative images of EVs’ release from the 8505c cell line (automatic sequential microscopic image registration (a–d) arrow—released EV).
Figure 2
Figure 2
Analyzing the sizes of extracellular vesicles (EVs) released by thyroid cancer cell lines. (a). Gating strategy for analyzing EV sizes. Visualization of size beads (with diameters of 2, 4, and 6 μm) shown in a forward scatter (FSC) and side scatter (SSC); (b). Size distribution of EVs released by the different thyroid cell lines. NTHY: cell line derived from normal human thyroid follicular cells; CGTG: Thyroid gland squamous cell carcinoma; FTC-133: follicular thyroid carcinoma; 8505c: anaplastic thyroid carcinoma; TPC-1: thyroid gland papillary carcinoma; BcPAP: thyroid gland papillary carcinoma. Full characteristics of the used cell lines are provided in Supplementary Table S1.
Figure 3
Figure 3
The average number of extracellular vesicles (EVs) released by thyroid cell lines per 100 cells. Data are shown as means with standard deviations (±SEM). ***: p < 0.001.
Figure 4
Figure 4
Relative expression of tetraspanins (a–e); Caveolin-1 (f); Ezrin (g); Moesin (h); Radixin (i); and Alix (j) in thyroid cell lines. Data are reported as means with standard deviation (±SEM). *: p < 0.05; ** p < 0.01; ***: p < 0.001.
Figure 5
Figure 5
The effect of EVs derived from thyroid cancer cells on the expression of tetraspanins in NTHY cells. The expression of tetraspanins is shown in separate panels: CD9 (a); CD81 (b); CD82 (c); and CD151 (d). Each panel shows: the level of gene expression (qRT-PCR) (the graphs placed on the left side of the figure); the level of protein expression (WB) (the images placed in the middle); the representative images of ICC/IF analysis (the microscopic photographs shown on the right side of the figure). Line 1: NTHY alone; 2: CGTH; 3: FTC-133; 4: 8505c; 5: TPC-1, 6: BcPAP. Data are shown as means with standard deviation (±SEM). *: p < 0.05, ***: p < 0.001.
Figure 6
Figure 6
The number of cancer-derived 6 μm EVs correlates with the expression of tetraspanins. The plots show the Pearson correlation between the expression levels of CD63 (a); and CD81 (b) in NTHY cells (after incubation with cancer-derived EVs) and the percentage of 6-µm extracellular vesicles (EVs) released by thyroid cancer cells.
Figure 7
Figure 7
The effect of thyroid cancer-derived extracellular vesicles (EVs) on NTHY (normal thyroid) cell proliferation and viability. (a): cell proliferation results by BrdU assay; (b): cell viability results by MTS assay.
Figure 8
Figure 8
Internalization of thyroid cancer-derived extracellular vesicles (EVs) by monocytes. (a–c): a 3D model of internalized EVs visualized by confocal microscopy (Zen software—ZEISS). Actin has been stained with Phalloidin-FITC (green), EV Caveoiln-1 with AF 594 (red), and nucleic DNA with DAPI (blue). (a,b): a monocyte with internalized EV in two different positions; (c): a monocyte with internalized EV without a channel for nucleus. (d): numbers of monocytes with absorbed EVs, released by different thyroid cell lines.
Figure 9
Figure 9
The effect of thyroid-derived EVs on angiogenesis. Upper panel: microscopic analysis of tube formation assay after 5 h: HUVEC cells incubated with: (a)—EVs from NTHY; (b)—EVs from CGTH; (c)—EVs from FTC-133; (d)—EVs from 8505c; (e)—EVs from TPC-1; (f)—EVs from BcPAP; Lower panel: (g)—total branching length (Data are reported as mean ± SEM, **: p < 0.01, ***: p < 0.001).
Figure 10
Figure 10
Pearson correlation between the percentage of 6-µm extracellular vesicles (EVs) released by cells and their pro-angiogenic potential.
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