Long-distance communication between laryngeal carcinoma cells

Abstract

Tunneling nanotubes and epithelial bridges are recently discovered new forms of intercellular communication between remote cells allowing their electrical synchronization, transfer of second messengers and even membrane vesicles and organelles. In the present study, we demonstrate for the first time in primary cell cultures prepared from human laryngeal squamous cell carcinoma (LSCC) samples that these cells communicate with each other over long distances (up to 1 mm) through membranous tunneling tubes (TTs), which can be open-ended or contain functional gap junctions formed of connexin 43. We found two types of TTs, containing F-actin alone or F-actin and α-tubulin. In the LSCC cell culture, we identified 5 modes of TT formation and performed quantitative assessment of their electrical properties and permeability to fluorescent dyes of different molecular weight and charge. We show that TTs, containing F-actin and α-tubulin, transport mitochondria and accommodate small DAPI-positive vesicles suggesting possible transfer of genetic material through TTs. We confirmed this possibility by demonstrating that even TTs, containing gap junctions, were capable of transmitting double-stranded small interfering RNA. To support the idea that the phenomenon of TTs is not only typical of cell cultures, we have examined microsections of samples obtained from human LSCC tissues and identified intercellular structures similar to those found in the primary LSCC cell culture.

Figure 1. Characterization of LSCC cells.

(A and a) Moderately differentiated squamous cell carcinoma of the larynx. The parenchyma (P) of the tumor consists of atypical epithelial cells with numerous hyperchromatic giant nuclei and atypical mitoses (white arrows). Several keratinizing cells are shown by yellow arrows. The surrounding stroma (S) is composed of the connective tissue with lymphocyte infiltration (hematoxylin and eosin staining). (B) LSCC cells after 3 days in vitro (TTs are indicated by yellow arrows). (C) Division rate of LSCC cells at the eighth passage (n = 5). No major differences in the division rate were observed between passages 5 to 15. (D) Preincubation of LSCC cells with colchicine (10 µM), an inhibitor of microtubule polymerization, for 24 h resulted in a complete loss of TTs. (E–G) TTs are not attached to the substratum as confirmed by applying negative or positive pressure through a broken patch pipette. The TT2 was formed by the rear lamellipodium of the cell-1 attached to the rear lamellipodium of cell-2 (encircled).

Figure 2. Formation of TT1s between LSCC cells.

(A–D) TT1s form in the process of cell division and successive dislodgment. (E–G) TT1s contain both F-actin and α-tubulin.

Figure 3. Formation of TT2s between LSCC cells.

(A) The leading lamellipodia of cell-1 and cell-2 indicated by white arrows are involved in cell movement. Rear lamellipodia are indicated by red arrows, and one of them outgrowing from the cell-1 forms the TT2 with the cell-2. (B–D) TT2s contain both F-actin and α-tubulin. (E) Crawling endings (“paws”, indicated by yellow arrows in A and B) of the rear and secondary lamellipodia, in addition to F-actin and α-tubulin, contain Cx43 hemichannel clusters. (F–G) Top view (DIC image) and Z-X reconstruction showing the TT2 raised above the substratum.

Figure 4. Formation of TT3s between LSCC cells.

(A) The secondary lamellipodium (red arrow) outgrowing from the rear lamellipodium of the cell-2 forms TT3 with the cell-1. (B–D) TT3s contain both F-actin and α-tubulin. (E–F) The TT3 between the cell-1 and the cell-2 was found raised 11 µm above the substratum (DIC image). (G) TT4s form between the intersecting rear or secondary lamellipodia establishing functional Cx43 GJs (the arrow in the inset) as confirmed by patch-clamp measurements.

Figure 5. Formation of TT5s between LSCC cells.

(A–C) TT5s contain F-actin but not α-tubulin. Cx43 hemichannel clusters, which can be seen inside and/or on the TT5 surface, form functional GJs at the cell border. (D) The multiple TT5s are formed between the cell-1 and the rear lamellipodium of the cell-2 (DIC image). (E) TT5s can be formed by protrusions from apposing cells, which come into contact and form functional Cx43 GJs (green). (F and G) Several TT5s between the cell-1 and the cell-2 were found raised ∼6 µm above the substratum (DIC image).

Figure 6. Permeability and electrical properties of TTs.

(A) AF350 dye introduced into the cell-1 through the patch pipette-1 diffuses via the TT to the cell-2. (B) Kinetics of dye accumulation in the cell-1 and the cell-2 after opening of patch-1 (indicated by arrow). (C–D) Electrical properties of TT were measured by opening patch-2 at the end of the experiment, applying the voltage ramp of negative polarity from 0 to −120 mV (upper panels), and measuring junctional current in the cell-1 (lower panels). I_T responses in (C) and (D) are typical of TTs without and with GJs, respectively. (E) g_T/V_T dependence is calculated from I_T response to V_T ramp (shown in C and D) and presented with its symmetric counterpart. The absence or presence of V_T gating indicates that the TT does not contain or contains GJs (upper and lower panel, respectively). (F) Correlation between TT1 conductance and geometry. (G) Correlation between TT(2–5)s conductance and geometry. TT5s thinner than 200 nm, the diameter of which cannot be measured by conventional microscopy, were not included into statistical analysis. (H) Correlation between TT permeability and electrical conductance. (I) Recording of single-channel current i_j (lower trace) in response to 2-min −80 mV voltage pulse V_j (upper panel). (J) Single channel substate (S) and open state (O) conductance γ_j was estimated by an all-point histogram fitted by the Gaussian function (pClamp 10 software). Two functioning channels can be recognized with respective substates and open states typical of Cx43 channels (more details are provided in the main text).

Figure 7. TTs between live LSCC cells in the culture contain mitochondria.

(A) A phase-contrast image shows the TT1 connecting the cell-1 and the cell-2, and the TT2 connecting the cell-3 and the cell-4 (arrows). (B) A dense network of mitochondria stained with MitoTracker Green is present in all cell bodies and also in the TT1 and the TT2 (insets b1 and b2). (C and c) TTs can be involved in cargo transport (DIC image). (D) The TT2 contains small DAPI-positive vesicles. (E) Cargoes can be transported along an outer surface of TTs (red arrow) (DIC image).

Figure 8. Permeability of TTs between LSCC cells to siRNA/AF488 in the culture.

(A) A phase-contrast image shows the TT2 connecting the cell-1 and the cell-2. SiRNA/AF488 (2 µm) introduced into the patch pipette enters the cell-1 after patch opening (B), rapidly diffuses along the TT2 to the “paw,” and then slowly accumulates through the GJ in the cell-2 (C). (E and inset) Kinetics of siRNA/AF488 accumulation in the cell-1, “paw,” and cell-2. (F and inset) SiRNA transfer through the TT2 containing GJs was confirmed by application of octanol (0.5 mM), which reversibly arrested siRNA/AF488 accumulation in the cell-2. A red arrow indicates the patch opening time in the cell-1.

Figure 9. TT5-like tunneling tubes in the LSCC tissue samples.

(A–D) TTs containing F-actin but not α-tubulin.

Figure 10. TT(1–4)-like tunneling tubes in the LSCC tissue samples.

(A–D) TTs containing F-actin and α-tubulin. (E) TTs containing mitochondria co-localizing with F-actin.