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. 2016 Jul 12;7(28):43150-43161.
doi: 10.18632/oncotarget.9504.

Tunneling nanotube formation is stimulated by hypoxia in ovarian cancer cells

Snider Desir  1   2 , Elizabeth L Dickson  3 , Rachel I Vogel  4 , Venugopal Thayanithy  2 , Phillip Wong  2 , Deanna Teoh  3 , Melissa A Geller  3 , Clifford J Steer  5 , Subbaya Subramanian  6 , Emil Lou  1   2
Affiliations

Tunneling nanotube formation is stimulated by hypoxia in ovarian cancer cells

Snider Desir et al. Oncotarget. .

Abstract

In this study, we demonstrated that hypoxic conditions stimulated an increase in tunneling nanotube (TNT) formation in chemoresistant ovarian cancer cells (SKOV3, C200).We found that suppressing the mTOR pathway using either everolimus or metformin led to suppression of TNT formation in vitro, verifying TNTs as a potential target for cancer-directed therapy. Additionally, TNT formation was detected in co-cultures including between platinum-resistant SKOV3 cells, between SKOV3 cells and platinum-chemosensitive A2780 cells, and between SKOV3 cells cultured with benign ovarian epithelial (IOSE) cells; these findings indicate that TNTs are novel conduits for malignant cell interactions and tumor cell interactions with other cells in the microenvironment. When chemoresistant C200 and parent chemosensitive A2780 cells were co-cultured, chemoresistant cells displayed a higher likelihood of TNT formation to each other than to chemosensitive malignant or benign epithelial cells. Hypoxia-induced TNT formation represents a potential mechanism for intercellular communication in ovarian cancer and other forms of invasive refractory cancers.

Keywords: chemoresistance; hypoxia; intercellular communication; mTOR; tunneling nanotubes.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1. Differing patterns of TNT formation among malignant (chemoresistant and chemosensitive) and also benign ovarian cells
(A) Representative confocal microscopy image of a TNT within an intact human malignant ovarian tumor (adenocarcinoma). Arrowheads indicate mitochondria within a TNT stained with MitoTracker orange-fluorescent dye. (B) Representative phase contrast microscopy images of TNTs connecting the cisplatin- and doxorubicin-resistant SKOV3 ovarian cancer cells; platinum-resistant C200 cells, and their parent chemosensitive cell line A2780; and a benign ovarian epithelial cell line (IOSE). (C) Quantification of TNTs/cell per field in cultures of chemoresistant, chemosensitive, and benign ovarian epithelial cell lines across replicates over four days plotted and summarized using the median (line). An Olympus IX70 inverted microscope with 20× objective lens was used to visualize and count the number of TNTs and cells in 10 randomly chosen fields. This experiment was performed in duplicate.
Figure 2
Figure 2. Hypoxia induces HIF-1α expression and TNT formation in ovarian cancer cell lines
(A) Quantitative Western blot analysis shows increased expression of HIF-1α in SKOV3, C200, and A2780 cells cultured under hypoxic conditions. (B–D) Number of TNTs (mean ± standard deviation) in chemoresistant SKOV3 (B) and C200 (C) cells and chemosensitive A2780 (D) cells cultured in standard and TNT media under normoxic (left panel) or hypoxic (right panel) conditions. For all experiments, the number of TNTs in 10 high-power fields (hpf) were counted and averaged. This experiment was performed in duplicate.
Figure 3
Figure 3. TNT formation between chemoresistant and chemosensitive ovarian cancer cell lines and between chemoresistant ovarian cancer and benign epithelial ovarian cells
(A) The number of TNTs in co-cultures of chemoresistant SKOV3 (R) and chemosensitive A2780 (S) cells across replicates and summarized using the median (line). (B) Representative Zeiss Axio widefield fluorescence microscopy images of TNTs forming among DiI red-labeled A2780 (chemosensitive) and GFP (green)-labeled SKOV3 (cisplatin, adriamycin resistant) cell lines. (C) The number of TNTs in co-cultures of chemoresistant C200 (R) cells and chemosensitive A2780 (S) cells across replicates and summarized using the median (line). (D) Representative microscopy image of TNTs forming among DiI red-labeled A2780 (chemosensitive) and DiO green-labeled C200 cell lines. (E) The number of TNTs in co-cultures of chemoresistant SKOV3 (R) cells and normal ovarian epithelial IOSE (S) cells across replicates and summarized using the median (line). (F) Representative microscopy image of TNTs forming among GFP green-labeled SKOV3 cells and DiI red-labeled IOSE cells. For all images in this figure, cells were counted per 20× high power field during a 24-hour period at 15 minute intervals.
Figure 4
Figure 4. Effects of metformin and everolimus on TNT formation
For all experiments, the number of TNTs in 10 high-power fields (hpf) were counted and averaged and each experiment was performed in duplicate. (A) Number of TNTs (mean ± standard deviation) in A2780 cells with or without addition of metformin or everolimus under normoxia. (B) Number of TNTs (mean ± standard deviation) in C200 cells with or without addition of metformin or everolimus under normoxia. (C) Number of TNTs (mean ± standard deviation) in SKOV3 cells with or without addition of metformin or everolimus under normoxia. (D) Number of TNTs (mean ± standard deviation) in A2780 cells with or without addition of metformin or everolimus under hypoxia. (E) Number of TNTs (mean ± standard deviation) in C200 cells with or without addition of metformin or everolimus under hypoxia. (F) Number of TNTs (mean ± standard deviation) in SKOV3 cells with or without addition of metformin or everolimus under hypoxic conditions.
Figure 5
Figure 5. Summary diagram
Physiologic stress induced under hypoxic conditions leads to an increase in HIF-1α expression and subsequent increase in TNTs. This increase in TNTs allows cells to form cellular networks that facilitate sharing of cellular signals. Inhibition of the mTOR pathway using clinically available drugs (everolimus, metformin) can suppress TNT-mediated intercellular communication.
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References

    1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9–29. - PubMed
    1. Rauh-Hain JA, Nitschmann CC, Worley MJ, Bradford LS, Berkowitz RS, Schorge JO, Campos SM, del Carmen MG, Horowitz NS. Platinum resistance after neoadjuvant chemotherapy compared to primary surgery in patients with advanced epithelial ovarian carcinoma. Gynecol Oncol. 2013;129:63–68. - PubMed
    1. Frankfurt OS, Seckinger D, Sugarbaker EV. Intercellular transfer of drug resistance. Cancer Res. 1991;51:1190–1195. - PubMed
    1. Zhang FF, Zhu YF, Zhao QN, Yang DT, Dong YP, Jiang L, Xing WX, Li XY, Xing H, Shi M, Chen Y, Bruce IC, Jin J, et al. Microvesicles mediate transfer of P-glycoprotein to paclitaxel-sensitive A2780 human ovarian cancer cells, conferring paclitaxel-resistance. European Journal of Pharmacology. 2014;738:83–90. - PubMed
    1. Tang K, Zhang Y, Zhang HF, Xu PW, Liu J, Ma JW, Lv M, Li DP, Katirai F, Shen GX, Zhang GM, Feng ZH, Ye DY, et al. Delivery of chemotherapeutic drugs in tumour cell-derived microparticles. Nat Commun. 2012:3. - PubMed

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