Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

Abstract

Intercellular transfer of organelles via tunneling nanotubes (TNTs) is a novel means of cell-to-cell communication. Here we demonstrate the existence of TNTs between co-cultured RT4 and T24 bladder cancer cells using light microscopy, fluorescence imaging, and scanning electron microscopy (SEM). Spontaneous unidirectional transfer of mitochondria from T24 to RT4 cells was detected using fluorescence imaging and flow cytometry. The distribution of mitochondria migrated from T24 cells was in good agreement with the original mitochondria in RT4 cells, which may imply mitochondrial fusion. We detected cytoskeleton reconstruction in RT4-Mito-T24 cells by observing F-actin redistribution. Akt, mTOR, and their downstream mediators were activated and increased. The resultant increase in the invasiveness of bladder cancer cells was detected in vitro and in vivo. These data indicate that TNTs promote intercellular mitochondrial transfer between heterogeneous cells, followed by an increase in the invasiveness of bladder cancer cells.

Keywords: bladder cancer; cell connection; cell invasion; mitochondria; tunneling nanotubes.

Figure 1. Identification of TNT structure between T24 and RT4 cells by light and fluorescence microscopy

A. TNT structure. T24 and RT4 cells were co-cultured at a 1:1 ratio for 24 h, and images of TNTs, thin microtubular connections, were captured under white-light visual (a) and fluorescence (b) microscopy. (c) is the merged of visual image (a) and fluorescence image (b). Bar = 50 μm. B. F-actin based structure. T24 and RT4 cells were co-cultured for 24 h, and stained with Actin-Tracker Green and DAPI. (a) Actin-Tracker (Green); (b) nuclei-DAPI (Blue); (c) the merged image of (a) and (b). TNTs were observed and marked by Actin-Tracker Green (White arrows), which indicated TNTs had an F-actin based structure. Images were captured under fluorescence microscopy. Bar = 50 μm.

Figure 2. Identification of TNTs micro-structure between T24 and RT4 cells by scanning electron microscopy

A. Open-ended filopodia-like cell protrusions of TNTs between T24 and RT4 cells. TNTs (a, black arrows) were observed between T24 cells and RT4 cells (b, c, d). Continuous, membranous, micro-tubular connection between T24 (b) and RT4 (d) cells were featured. The caliber of the membranous tubes ranged from 100-200 nm, and the lengths of TNTs between T24 and RT4 cells spanned a large range from 20 μm to 1 mm. B. Blindly ending filopodia-like cell protrusions of TNTs between T24 and RT4 cells. TNTs (a, black arrows) were extended from T24 cells (b), indicating TNTs were originally formed by T24 cells.

Figure 3. Intercellular transfer of mitochondria via TNTs between T24 and RT4 cells

A. MitoTracker Deep Red labeled T24 (a, red) and CFSE Green labeled RT4 cells (b, green) were co-cultured for 24 h, and nuclei were marked by DAPI (c, blue); (d) is the merged images of (a), (b), and (c). Spontaneous mitochondria trafficking (white arrows) from T24 cells to RT4 cells were obtained by capturing “double positive” (red and green) RT4 cells under fluorescence microscopy (d). Bar = 25 μm. B. Mitochondria in T24 cells were labeled by MitoTracker Deep Red, and could be observed as a thin red line in the tube-like structures between T24 and RT4 cells (white arrows) (a). Then F-actin was labeled by Actin-Tracker Green (b), and nuclei were labeled by DAPI (c). Mitochondria could be observed migrating from T24 cells to RT4 cells via F-actin based TNTs (d, merged images of a, b, and c). Bar = 10 μm. C. Latrunculin B was used to inhibit TNT formation and mitochondria transfer between T24 cells and RT4 cells. (a) T24 cells were labeled by MitoTracker Deep Red and co-cultured with RT4 cells and Latrunculin B (1.25 μmol/L) for 24 h. No mitochondria transportation from T24 cells to RT4 cells can be observed. (b) T24 and RT4 cells were labeled by Actin-Tracker Green. (c) The nuclei in T24 and RT4 cells was marked by DAPI. (d) is the merged images of a, b, and c. Bar = 10 μm. D. MitoTracker Deep Red labeled T24 cells were co-cultured with MitoTracker Green labeled RT4 cells. Double labeled RT4-Mito-T24 cells were sorted out and obtained by FACS. (a) In RT4-Mito-T24 cells, mitochondria originally migrated from T24 cells, and was labeled by MitoTracker Deep Red. Image was captured under a red fluorescent filter. (b) In RT4-Mito-T24 cells, mitochondria originally in RT4 cells were labeled by MitoTracker Green. Image was captured under a green fluorescent filter. After nuclei were marked by DAPI (c), mitochondria distribution in RT4-Mito-T24 cells was observed by LCM. The immigrated mitochondria (a) from T24 cells and original mitochondria (b) from RT4 cells had a high concordance in sub-cellular distribution (d, white arrows, d is the merged images of a, b, and c), which implied mitochondria fusion. Bar = 10 μm.

Figure 4. Transwell assay shows RT4 cells’ invasive ability is lower than RT4-Mito-T24 cells

A. The invasive ability of RT4, T24, and RT4-Mito-T24 cells were detected by Transwell assay. After the incubation, images of cells migrating through the Matrigel-coated filter were captured respectively. Bar = 50 μm. B. Cells invading the Matrigel and reaching the lower surface of the filter were counted. The invasive ability in RT4-Mito-T24 cells was up-regulated compared to parental RT4 cells.

Figure 5. Wound healing assay shows RT4 cells’ invasive ability lower than RT4-Mito-T24 cells

A. The invasive ability of RT4, T24, and RT4-Mito-T24 cells were detected by wound healing assays. The images of the cells along the wound were captured at 0 h and 24 h, and marked by lines under an inverted microscope. Bar = 50 μm. B. Then the healing area was analyzed. The closure of the wounded area was accelerated in RT4-Mito-T24 cells relative to parental RT4 cells.

Figure 6. Xenograft tumor growth in athymic mice inhibited in RT4 cells

A. RT4, T24, and RT4-Mito-T24 cells were inoculated in the forelimb of athymic mice (A Left) respectively. After 30 days, mice were sacrificed, and the volume of the xenografts were measured (A Right). B. Tumor growth curves indicated that the average size of the tumors in the RT4 group was smaller than that in the other two groups.

Figure 7. Increase of relative vascular index was not identical in RT4-Mito-T24 cell xenografts

A. After tumor formation, blood flow circumference around xenografts (green square area, shown in A lower panel) were detected and measured by ultrasound in vivo. B. T24 group obtained a higher relative vascular index than the RT4 group. RT4-Mito-T24 group had a higher relative vascular index than parental RT4 cells, but the difference was not statistically significant.

Figure 8. F-actin was redistributed in RT4-Mito-T24 cells

RT4, T24, and RT4-Mito-T24 cells were cultured separately for 24 h and labeled by MitoTracker Deep Red, F-actin (green), and DAPI (blue). The images of F-actin and mitochondria distribution (white arrows) were captured by LCM. F-actin staining was restricted to the inner membrane of RT4 cells, and diffused along the filopodia-like cell protrusions. The mitochondria were restricted around the R24 nuclei, but diffusely distributed in the cytoplasm of T24 and RT4-Mito-T24 cells. Bar = 10 μm.

Figure 9. Akt and mTOR signaling were upregulated in RT4-Mito-T24 cells

A, B. Akt expression was increased in RT4-Mito-T24 cells compared to parental RT4 cells. However, the p-Akt levels between the three types of cells had no significant difference. Both mTOR and p-mTOR were increased in RT4-Mito-T24 cells compared to RT4 cells. As two main downstream regulators of mTOR, the expression of 4EBP1 was higher in RT4-Mito-T24 cells, while no difference was noted in p70S6K levels.