This presentation is from an AudiosSlides presentation on my lab's publication on microRNA transfer between cancer cells via tunneling nanotubes. The full presentation with audio can be found here: http://www.sciencedirect.com/science/article/pii/S1931524414001984 For more information on my lab's research work on intercellular communication in cancer, you can visit our webpage at the University of Minnesota: http://www.dom.umn.edu/hematology-oncology-and-transplantation/faculty/emil-lou/lou-lab/index.htm

1. Tumor-stromal cross-talk: Direct cell-to-
cell transfer of oncogenic microRNAs
via tunneling nanotubes
Venugopal Thayanithy, PhD1; Elizabeth L. Dickson, MD2; Clifford
Steer, MD3; Subbaya Subramanian, PhD4; Emil Lou, MD, PhD1
From the Departments of Medicine, Obstetrics & Gynecology,
Division of Gynecologic Oncology, and Department of Surgery
University of Minnesota
Translational Research, published online May 24, 2014
DOI: 10.1016/j.trsl.2014.05.011

2. Introduction
• Intercellular communication among distant and proximal
cells in the heterogeneous tumor microenvironment has
emerged as an important paradigm for understanding
tumor growth and invasion.
• Tunneling nanotubes (TnTs) are long, non-adherent, actin-
based cellular extensions that act as open conduits for
transport of cellular cargo between connected cells.
• TnTs represent a novel mechanism by which intercellular
components can be transferred from cell to cell in the
complex tumor microenvironment.
• In the present study, we examined intercellular transfer of
genetic material (microRNAs) between malignant cells –
and also between malignant and stromal cells – via TnTs.

3. Identification of nanotube structures in resected tumors

4. In vitro demonstration of intercellular tumor-tumor and tumor-stromal
interaction via TnTs in osteosarcoma. We also demonstrated the same occurs for
ovarian cancer.
TnTs formed between (A)
K7M2 murine osteosarcoma
cells; (B) K7M2 and MC3T3
murine osteoblast cells; and
(C) Mg63.2 human OS cells
and hFOB1.19 human OB
cells. Cells were labeled
using the lipophilic dyes DiI
(red) and DiO (green) per
the manufacturer’s protocol
and per our prior
demonstration using these
dyes to visualize TnTs with
fluorescence microscopy. In
cases of co-culture, cells
were cultured in a 1:1 ratio.

5. Identification of differential miRNA expression
A) miRNA overexpression of miR-19a in human osteosarcoma compared with normal
bone.
B) miRNA expression pattern of the miR-199 family in cisplatin-sensitive (A2780, on
left) and resistant (C200, on right) ovarian cancer cell lines.

6. Intercellular transfer of miRNA via TnTs, captured using
time-lapse imaging.
A) Alexa-Fluor-488-tagged miR-19a transfer between two K7M2 murine OS cells connected via a long TnT.
B) miR-19a-expressing K7M2 cell (green) extending a long TnT that connects with an MC3T3 OB cell marked by fluorescent DiI (red). The K7M2
forms a TnT connection which then allows transfer of the miRNA cargo to the OB cell.

7. Demonstration of miR-199a in multiple TnTs connecting SKOV3
human ovarian adenocarcinoma cells in vitro.
A) Multiple TnTs connecting SKOV3 cells expressing fluorescing miR-199a.
B) Demonstration of a long TnT connecting a miR-199a-transfected SKOV3 cell
(green) to a DiI-marked IOSE (red) ovarian epithelial cell, with intercellular
transport of miR-199a in progress.

8. FACS analysis of cells grown in open culture vs. separated by
membrane filter in modified invasion chambers

9. TnT-mediated intercellular transfer of miR-199a from an miR-199a-marked SKOV3 cell
(green) connected to DiI-stained IOSE cell (red).
For time-lapse video of this experiment, please see Supplemental Movie 3 online.

10. Highlights and Conclusions
• We demonstrate nanotube structures are present in
tumor explants from human patients with ovarian
cancer and also from an orthotopic murine model of
osteosarcoma.
• Here for the first time we demonstrate direct cell-to-cell
transfer of genetic material between tumor and stromal
cells via TnT conduits.
• Our findings of tumor-stromal cross-talk via TnTs, and
consequent intercellular transfer of microRNAs, has
potential relevance to the emerging paradigm that
intratumoral heterogeneity and tumor-stroma
interactions are important properties of tumor
formation, progression, and recurrence.

11. Other pertinent work on TnTs from our group
• E Lou, S Fujisawa, A Morozov, A Barlas, Y Romin, Y Dogan, AL Moreira, K Manova-Todorova, MAS
Moore. Tunneling Nanotubes Provide a Unique Conduit for Intercellular Transfer of Cellular
Contents in Human Malignant Pleural Mesothelioma. PLoS ONE 7(3):e33093. Epub 2012 Mar 9.
• E Lou, S Fujisawa, A Barlas, Y Romin, K Manova-Todorova, M.A.S. Moore, S Subramanian. Tunneling
Nanotubes: A new paradigm for studying intercellular communication and therapeutics in cancer.
Communicative & Integrative Biology 5(4): 399-403, July 2012. doi: 10.4161/cib.20569.
• E Lou, S Subramanian, CJ Steer. Pancreatic Cancer: Modulation of KRAS, MicroRNAs and
Intercellular Communication in the Setting of Tumor Heterogeneity. Pancreas 42(8):1218-26, 2013.
• V Thayanithy, V Babatunde, EL Dickson, P Wong, S Oh, X Ke, A Barlas, S Fujisawa, Y Romin, AL
Moreira, RJ Downey, CJ Steer, S Subramanian, K Manova-Todorova, MA Moore, E Lou. Tumor
exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells.
Experimental Cell Research 323 (1): 178-88, April 15 2014.
• Our Lab webpage:
http://www.dom.umn.edu/hematology-oncology-and-transplantation/faculty/emil-lou/lou-lab/index.htm