miR-200-containing extracellular vesicles promote breast cancer cell metastasis

Abstract

Metastasis is associated with poor prognosis in breast cancer patients. Not all cancer cells within a tumor are capable of metastasizing. The microRNA-200 (miR-200) family, which regulates the mesenchymal-to-epithelial transition, is enriched in the serum of patients with metastatic cancers. Ectopic expression of miR-200 can confer metastatic ability to poorly metastatic tumor cells in some settings. Here, we investigated whether metastatic capability could be transferred between metastatic and nonmetastatic cancer cells via extracellular vesicles. miR-200 was secreted in extracellular vesicles from metastatic murine and human breast cancer cell lines, and miR-200 levels were increased in sera of mice bearing metastatic tumors. In culture, murine and human metastatic breast cancer cell extracellular vesicles transferred miR-200 microRNAs to nonmetastatic cells, altering gene expression and promoting mesenchymal-to-epithelial transition. In murine cancer and human xenograft models, miR-200-expressing tumors and extracellular vesicles from these tumors promoted metastasis of otherwise weakly metastatic cells either nearby or at distant sites and conferred to these cells the ability to colonize distant tissues in a miR-200-dependent manner. Together, our results demonstrate that metastatic capability can be transferred by the uptake of extracellular vesicles.

Figures

Figure 15. Circulating MB-231 cells acquire colonization capability from primary tumor–expressing miR-200.

(A) Schema for imaging lung colonization. Two weeks after CA1a cells, untreated or transduced with anti–miR-200–TuD vector, were injected into the flanks of nude mice, MB-231–Fluc–mCherry cells were injected into the tail vein. Luminescence was imaged every week until the mice were sacrificed when tumor diameter approached 20 mm (week 3 or 4). (B) Levels of miR-200 miRNAs and (C) their targets in CA1a-control and CA1a-200-TuD cells, as determined by TaqMan or Ssofast qPCR, respectively (3 experiments). (D) Representative luminescent images of the mice at indicated times after i.v. injection. The type of primary flank tumor is indicated at left for each group of mice. (E) log2 luminescent photon flux (photons/s) of the whole body. The number of mice in each group with detectable metastases is indicated. (F) Representative lung luminescent images (left) and the average lung luminescent photon flux at time of sacrifice (right). *P < 0.05; **P < 0.01, Student’s t test.

Figure 14. EVs from human metastatic CA1a and BPLER cells promote colonization of MB-231 cells in the lung.

(A) Schema for imaging lung colonization. MB-231–Fluc–mCherry cells treated with EVs purified from the culture supernatants of MB-231, CA1a, or BPLER cells for 3 days were injected into the tail vein of nude mice. Luminescence was imaged every week until sacrifice at 5 weeks. (B) Representative luminescent images of mice at indicated times after tail-vein injection. (C) log2 luminescent photon flux (photons/s) of the whole body captured at the indicated times after the tail-vein injection, including 5 mice in the MB-231 EV group, 6 mice in the CA1a EV group, and 6 mice in the BPLER EV group. (D) Representative lung luminescent images (top) and the average lung luminescent photon flux 5 weeks after i.v. injection (bottom). *P < 0.05; **P < 0.01, Mann-Whitney U test.

Figure 13. miR-200 miRNAs are secreted by human metastatic breast cancer cells and transferred to poorly metastatic cells.

(A) Expression of miR-200s in human poorly metastatic MB-231 cells and highly metastatic CA1a and BPLER cells. (B) miRNA copy number in EVs purified from the supernatants of MB-231, CA1a, and BPLER cells. (C) miRNA and mRNA levels in MB-231 cells, cocultured for 3 days separated by a Transwell with MB-231, CA1a, or BPLER cells in which miR-200 was inhibited or not by TuD. (D) miRNA and mRNA levels in MB-231 cells treated for 3 days with EVs purified from the supernatants of MB-231, CA1a, or BPLER cells. In A–D, miRNAs and mRNAs were quantified by TaqMan and Ssofast Evagreen qRT-PCR, respectively (3 experiments). (E) miRNA levels in the sera of mice uninjected (n = 6) or injected with CA1a (n = 6) or BPLER tumors (n = 8) 12 weeks after implantation, determined using the Firefly Circulating miRNA Assay. *P < 0.05; **P < 0.01, Student’s t test.

Figure 12. Poorly metastatic 4TO7 orthotopic tumors acquire metastatic capability from distal metastatic tumors expressing miR-200s.

(A) BALB/c SCID mice were injected with 4TO7-Fluc-mCherry cells in the right fourth mammary fat pads and an equal number of GFP+ 4TO7V, 4TO7OE, or 4T1E cells in the left fourth mammary fat pads. Luminescent images were taken every 5 days, and the mice were sacrificed on day 20. (B) Representative luminescent images. The type of GFP+ cells implanted in the right mammary fad pads is indicated at left. (C) log2 luminescent photon flux of metastases in the anterior halves of the mice measured after implantation. Number of mice with metastasis versus the total is indicated. (D) Volume of the mCherry tumors in the right mammary fat pad and GFP+ tumors in the left mammary fat pad measured by calipers in mice that received different types of GFP+ cells, as indicated. Asterisks indicate differences at day 20. (E) Representative lung luminescent images (left) and the average lung luminescent photon flux (right). (F) Representative lung mCherry images (left) and the average mCherry photon flux (right). (G) Representative lung GFP images (left) and the average GFP photon flux (right). *P < 0.05; **P < 0.01, Mann-Whitney U test.

Figure 11. Mammary fat-pad tumor 4T1E cells promote spontaneous metastasis of neighboring tumor cells in a miR-200–dependent manner.

(A) BALB/c SCID mice were injected into the fourth right mammary fat pad with an equal mixture of 4T1E cells and 4TO7-Fluc-mCherry expressing a control or anti–miR-200 TuD (Cont-TuD or 200-TuD). Luminescent images were taken every 5 days, and mice were sacrificed on day 23. (B) Representative luminescent images. The type of coimplanted 4TO7 cells is indicated at left. (C) log2 luminescent photon flux of metastases in the anterior halves of the mice after tumor cell implantation. The number of mice with metastases is shown. (D) Tumor volume on day 23 as measured by calipers. (E) Representative lung luminescent images (left) and the average luminescent photon flux at time of sacrifice (right). (F) Representative lung mCherry images (left) and the average mCherry photon flux (right). **P < 0.01 by Mann-Whitney U test.

Figure 10. miR-200 expression in mammary fat-pad tumor cells promotes spontaneous metastasis of neighboring tumor cells.

(A) BALB/c SCID mice were injected into the fourth right mammary fat pad with an equal mixture of 4TO7-Fluc-mCherry cells and 4T1E-GFP, 4TO7OE-GFP, or 4TO7V-GFP cells. Luminescent images were taken every 5 days, and mice were sacrificed on day 23. (B) Representative luminescent images. The type of coimplanted GFP cell is indicated at left. (C) log2 luminescent photon flux of metastases in the anterior halves of the mice after tumor cell implantation. The number of mice with metastases is shown in each condition. (D) Tumor volume as measured by calipers. Asterisks indicate differences at day 23. (E) Representative lung luminescent images (left) and the average lung luminescent photon flux on day 23 (right). (F) Representative lung mCherry images (left) and the average mCherry photon flux (right). (G) Representative lung GFP images (left) and the average GFP photon flux (right). *P < 0.05; **P < 0.01, Mann-Whitney U test.

Figure 9. Circulating 4TO7 cells acquire colonization capability from primary tumors expressing miR-200.

(A) Schema for imaging of lung colonization. When mammary fat-pad tumors of 4TO7V-GFP or 4TO7OE-GFP cells in BALB/c SCID mice approached 9 mm in diameter, 4TO7-Fluc-mCherry cells were injected into the tail vein and luminescence was imaged every 2 days until sacrifice on day 8. (B) Representative luminescent images of mice after tail-vein injection. The type of mammary fat-pad tumor is indicated at left. (C) log2 luminescent photon flux (photons/s) of the whole body captured at the indicated times after the tail-vein injection, including 6 mice bearing 4TO7V tumors and 7 mice bearing 4TO7OE tumors. (D) Orthotopic tumor volume measured by calipers. (E) Representative lung luminescent images (left) and the average lung luminescent photon flux, 8 days after the i.v. injection (right). (F) Representative lung mCherry images (left) and the average mCherry photon flux (right). (G) Representative GFP images (left) and the average GFP photon flux (right). *P < 0.05; **P < 0.01, Mann-Whitney U test.

Figure 8. miR-200 overexpression in mammary fat-pad tumors promotes lung colonization by circulating 4TO7 cells.

(A) Schema of in vivo metastasis assay. 4TO7V or 4TO7OE cells were injected into the mammary fat pad of BALB/c mice, and 10 days later, when the tumor diameter approached 9 mm, 4TO7 cells were injected into the tail vein. Macroscopic lung colonies were counted 8 days later. (B) Representative photographs of the lungs. (C) Colony number per lung. Seven mice in each control group did not receive tail-vein injection; 8 mice bearing 4TO7V tumors and 11 mice bearing 4TO7OE tumors were injected with 4TO7 cells by tail vein. *P < 0.05, Student’s t test. (D) Mammary tumor volume as measured by calipers.

Figure 7. Endogenous miR-200s secreted by metastatic cells mediate the prometastatic effect of EVs.

(A) 4TO7 cells were incubated with EVs purified from 4T1E stably expressing a control (Cont-TuD) or anti–miR-200–TuD (200-TuD). (B) Levels of miR-200s and their targets in 4T1E cells expressing TuD (3 experiments). (C) Representative photographs of the lungs treated as in A. (D) Number of tumor colonies in the lungs (n = 11 mice). *P < 0.05; **P < 0.01, Student’s t test.

Figure 6. Inhibition of miR-200s in recipient cells reduces the prometastatic effect of EVs derived from metastatic cells.

(A) 4TO7 cells were transfected with control anti-miR (AM) or a combination of anti–miR-200c and anti–miR-141 (200c/141 anti-miR) and then incubated with 4TO7V or 4TO7OE EVs. Treated 4TO7 cells were injected into the tail vein of BALB/c mice, and the lungs were analyzed 8 days later. (B) Levels of miR-200 miRNAs and their targets in 4TO7 cells transfected with anti-miRs and treated with EVs (3 experiments). (C) Representative photographs of the lungs treated as in A. (D) Number of tumor colonies in the lungs (n = 9 mice). *P < 0.05; **P < 0.01, Student’s t test.

Figure 5. EVs containing miR-200 miRNAs promote colonization of 4TO7 cells in the lung.

(A) Schema of in vivo colony assay. 4TO7 cells were incubated for 3 days with 500 μg EVs purified from culture supernatants of 4TO7 or 4T1E cells and then injected into the tail vein of BALB/c mice. Lungs were fixed 8 days later. (B) Representative photographs of the lungs. 4TO7 cells overexpressing miR-200c and miR-141 (4TO7OE cells) were used as a positive control for metastasis. (C) Average number of tumor colonies in the lungs (n = 9 mice). (D) Expression of miR-200c and miR-141 in 4T1E cells, 4TO7OE cells, and empty vector–control 4TO7 cells (4TO7V) relative to U6 snRNA (3 experiments), determined by TaqMan assay. (E) miR-200c copy number in EVs released from 4TO7 cells, 4T1E cells, and 4TO7OE cells (3 experiments), determined by TaqMan assay. **P < 0.01, Student’s t test (C–E).

Figure 4. Transferred miR-200c associates with donor CD63 and AGO2.

(A) Schema of 4T1E cells transfected with CD63-RFP plasmid and Cy5–miR-200c and cocultured with 4TO7-GFP cells. (B) A representative image of donor 4T1E cells expressing CD63-RFP (red) and Cy5–miR-200c (green) 24 hours after transfection. (C) A representative image of 4TO7-GFP cells (blue) that internalized CD63-RFP (red) and Cy5–miR-200c (green) after coculture with 4T1E cells. Green and blue channels were swapped for easy visualization. (D) Schema of 4T1E cells, cotransfected with eGFP-AGO2 plasmid and Cy5–miR-200c and cocultured with 4TO7 cells. After 48 hours, recipient 4TO7 cells were stained with CellMask Membrane Dye and imaged. (E) A representative image of 4TO7 cells with blue membrane and internalized eGFP-AGO2 (green) and Cy5–miR-200c (red) after coculture. Arrows and insets (×2.4) highlight colocalization. Scale bars: 10 μm. Each experiment was repeated twice.

Figure 3. miR-200 miRNAs are transferred across a Transwell or by EVs.

(A) miRNA and mRNA levels in 4TO7 cells, untreated or cocultured for 6 days with 4TO7 or 4T1E cells at a 1:4 ratio separated by Transwell. (B) Luciferase reporter assay of 4TO7 cells, transfected with an empty vector (control) or a miRNA reporter for miR-200c or miR-141, and cocultured with 4TO7 or 4T1E cells at a 1:4 ratio in a Transwell plate for 2 days. Renilla luciferase activity, normalized to firefly luciferase, is presented as percentage relative to the control 4TO7 coculture. (C) miRNA and mRNA levels in 4TO7 cells untreated or treated for 3 days with EVs from the culture supernatants of 4TO7 or 4T1E cells. (D) Protein expression in 4TO7 cells untreated or treated as in C. In all panels, 3 experiments are represented. *P < 0.05; **P < 0.01, Student’s t test.

Figure 2. miR-200 miRNAs are transferred from metastatic 4T1E cells to poorly metastatic 4TO7 cells.

(A and B) miRNA and mRNA levels in 4T1E cells, untreated 4TO7 cells, and 4TO7-GFP cells sorted after 48-hour coculture with 4T1E cells in a 1:1 or 1:4 (4TO7/4T1E) ratio. miRNAs and mRNAs were quantified by TaqMan assay and SsoFast EvaGreen qRT-PCR, respectively (3 experiments). *P < 0.05; **P < 0.01, Student’s t test. (C) Western blot analysis in 4T1E and 4TO7-GFP cells that were untreated or cocultured with 4T1E cells for 1 week. (D) Flow cytometry analysis of E-cadherin (Ecad) staining in 4TO7-GFP cells that were untreated (U) or cocultured (C) with 4T1E cells at a 1:4 ratio for 1 week, gated on GFP+ single viable cells (3 experiments). *P < 0.05, Student’s t test. (E) E-cadherin staining (red) of 4T1E cells, 4TO7-GFP cells (green), and after 1 week, coculture of the 2 cell lines at a 1:4 ratio. DAPI (blue) marks nuclei. Scale bars: 10 μm.

Figure 1. miR-200 microRNAs are secreted in EVs from 4T1E cells.

(A) Size distribution of EVs released by 4TO7 and 4T1E cells in 48-hour culture supernatants (3 experiments). (B) Immunoblot of proteins in 4T1E cells and their EVs (repeated twice). (C) Relative expression (log2) of miRNAs quantified by Firefly Cellular miRNA Assay and normalized using miR-16. Each value is an average of 4 independent experiments with values presented from low (green) to high (red). Undetected miRNAs (ND) are gray. (D) miR-200 copy number in 4T1E or 4TO7 EVs from cells, untreated or treated with RNase A and/or Triton X-100, quantified by TaqMan assay (3 experiments). (E) Representative images of lung sections, stained with H&E, from mice bearing 4TO7 or 4T1E mammary tumors (n = 6 mice). The number of mice with lung metastases is indicated below the images. Original magnification, ×10. (F) miRNA copy number in EVs in the circulation of tumor-free mice or mice bearing 4TO7 or 4T1E tumors. Blood was collected when primary tumors reached 15 mm in diameter. EVs were purified from the serum, and miRNAs were quantified by TaqMan assay (3 experiments). **P < 0.01, Student’s t test.