(22b) “Extracellular Vesicles Derived Supported Bilayer As a Platform for Cell Culture to Understand the Interactions between Adipose Stem Cells and Extracellular Vesicles"

normal"> " tahoma>“ONCOGENIC
EXTRACELLULAR VESICLES DERIVED SUPPORT BILAYER AS A PLATFORM FOR CELL CULTURE
TO STUDY INTERACTIONS BETWEEN ADIPOSE STEM CELLS AND EXTRACELLULAR VESICLES”

normal"> " tahoma>Johana Uribe ^{mso-bidi-font-size:10.0pt;font-family:" tahoma>1}, Han-Yuan Liu ^{mso-bidi-font-size:10.0pt;font-family:" tahoma>2}, Claudia Fischbach ^{mso-bidi-font-size:10.0pt;font-family:" tahoma>1}, and Susan Daniel ^{mso-bidi-font-size:10.0pt;font-family:" tahoma>2}*

1 font-family:" tahoma>Meinig
School of Biomedical Engineering, Cornell University, Ithaca, NY.

2 font-family:" tahoma>Robert
Frederick Smith School of Chemical and Biomolecular Engineering, Cornell
University, Ithaca, NY.

normal"> " tahoma> 

10.0pt;font-family:" tahoma color:black>Introduction: 10.0pt;font-family:" tahoma color:black>Extracellular vesicles (EVs) are encapsulated particles secreted
by eukaryotic cells. EVs are known to work as information “exchangers” between
cells facilitating intercellular communication and leading to diverse
biological outcomes ^{10.0pt;font-family:" tahoma color:black>1} 10.0pt;font-family:" tahoma color:black>. In the specific hallmark of cancer, EVs are known to play an
important role in cancer aggressiveness, angiogenesis, and metastasis^{1, 2.} For instance, Adipose derived stem
cells (ADSCs) have shown signs of myofibroblast transformation and
proangiogenic activity behavior upon interactions with oncogenic EVs². However, there is a lack of
understanding on the mechanisms behind such interactions and an absence of
techniques to study those in vivo ^{mso-bidi-font-size:10.0pt;font-family:" tahoma>3.} mso-bidi-font-size:10.0pt;font-family:" tahoma> Therefore, using SLB (supporting lipid bilayers)
techniques developed by our group ^{mso-bidi-font-size:10.0pt;font-family:" tahoma>4} 10.0pt;font-family:" tahoma color:black>, we originated a tunable in
vitro model with a planar geometry as a cell culture substrate to study the
interactions between EVs and cells at the single particle level, extracellular
vesicles derived supported lipid bilayer (ESLB). Our results displayed
successful culture of ADSCs and healthy development of the same on ESLBs, as
well as biological outcomes caused by their interactions with ESLBs. As a
result, we can propose the use of SLBs derived from EVs as a cell culture
substrate/platform to understand the interactions at the surface level between
stromal cells and EVs.

mso-bidi-font-size:10.0pt;font-family:" tahoma> 

mso-bidi-font-size:10.0pt;font-family:" tahoma> 

10.0pt;font-family:" tahoma color:black>Materials and Methods: mso-bidi-font-size:10.0pt;font-family:" tahoma> EVs from breast cancer cells, MDA-MB-231, were isolated
using vacuum filtering and differential centrifugation techniques giving origin
to two EVs populations, microvesicles and exosomes⁵. Using techniques previously developed
in our group ^{font-family:" tahoma>4,} we incubated EVs on glass slides
and add fusogenic liposomes (POPC-PEG) to aid EVs rupture and subsequent
formation of an ESLB. Using Fluorescence Recovery after Photobleaching (FRAP),
formation of SLBs was confirmed and their fluidity and mobile fraction were
calculated. Total Internal Reflection Fluorescence Microscopy (TIRFM) was
utilized to detect the expression of EVs surface markers, EGFR for
microvesicles and HSP70 and CD63 for exosomes, on the ESLBs. ADSCs were seeded
on ESLBs and incubated at 37 ֯C and 5% CO₂ for optimal growth. Cell adhesion
and surface area were assessed by vinculin and F-actin staining 24 hours after
seeding. Cell viability was assessed on day 1, 3, and 5 using a LIVE/DEAD kit.
ADSCs angiogenic activity was analyzed utilizing an ELISA Duo Set to measure
VEGF secreted on day 1 and 4. All images were captured by epifluorescence
microscopy.

mso-bidi-font-size:10.0pt;font-family:" tahoma> 

mso-bidi-font-size:10.0pt;font-family:" tahoma> 

10.0pt;font-family:" tahoma color:black>Results and Discussion: mso-bidi-font-size:10.0pt;font-family:" tahoma> Using isolated cancer derived EVs, we generated ESLBs able
to recover after photobleaching, showing proper formation and fluidity of a
SLB. Its diffusivity coefficients were significantly lower compared to their
value in synthetic SLBs, making ESLBs more attractive as a cell culture
substrate. Treatment of ESLBs with EVs markers showed that exosomes SLB highly
expressed HSP70 and CD63, as well as microvesicles SLB highly expressed EGFR.
These observations suggest that native components of EVs are conserved in our
generated ESLBs. ADSCs seeded on ESLBs showed a healthier morphology, higher
cell surface area and spreading, and stronger adhesion to the substrate
compared to cells on synthetic SLBs. Cell viability was significantly higher on
ADSCs on ESLBs compared to those seeded on synthetic SLBs over all examination
days. Overall, cells on ESLBs showed a cell behavior comparable the one
presented by cells grown cell culture glass. Finally, ADSCs on ESLBs secreted
higher concentration of VEGF compared to cells on synthetic SLBs and glass on
day 1 and 4. These results show that ADSCs in contact with EVs surface mimic by
ESLBs have higher proangiogenic activity compared to cells without contact with
EVs surface (SLBs and glass). It supports the observations by Song et al. in
which ADSCs treated with EVs have a higher angiogenic activity compared to their
non-treated counterpart and it shows that just surface interactions between EVs
and ADSCs are enough to cause a change in cell angiogenic behavior. 

mso-bidi-font-size:10.0pt;font-family:" tahoma> 

mso-bidi-font-size:10.0pt;font-family:" tahoma> 

normal">Figure 1. normal">Cell surface area and focal adhesions of ADSCs cultured on ESB, POPC
SLB, and EZ-slides. (a-d) ASDCs seeded on MVs- ESB, exosomes-ESB, POPC-SLB,
and EZ-slides. Vinculin generates a red signal and DAPI, in cell nuclei, a blue
signal. a) Cells on show uniform cell spreading and strong vinculin expression.
b) Cells display prolongated lengthwise spreading and high expression of
vinculin. c) Cells show unhealthy and rounded morphology and low spreading. d)
Cells are uniformly spread, have healthy cell morphology, and high expression
of vinculin. (e-h) Individual focal adhesions from images on the top panel
(a-d) visualized utilizing ImageJ [48] for quantitative analysis of focal
adhesions. i) Quantitative analysis of cell surface
area for ADSCs cultured on different substrates performed using ImageJ. i) Quantitative analysis of number of focal adhesions per
cell. n = 43, mean ± SD, *p ≤ 0.05, **p ≤ 0.01, and
***p≤0.001.

normal"> " tahoma>Conclusions: 
" tahoma>We are
presenting the first developed extracellular vesicle derived supported lipid
bilayer (ESLB), containing the native composition of EVs membranes, as a cell
culture platform to study the interactions between extracellular vesicles and
stromal cells. Our method allows the production of different EVs populations
(microvesicles and exosomes) derived ESLBs to study individually the
interactions of EVs and stromal cells and to decouple the biological outcomes
produced by each of the EVs population. It could be utilized as a method to
investigate cell to cell interactions, and
extracellular particles to cell interactions in different types of cells and
for several disease scenarios. Lately, it will facilitate the study cell to
cell recognition understanding ligand- receptor interactions.  Future work
includes the study of interactions between stem cells and ELSBs at the single
particle level using single particle tracking (SPT) techniques. Preliminary
results suggest that most of the interactions between EVs and stem cells take
place at the plasma membrane level rather than by intake of EVs cargo.

mso-bidi-font-size:10.0pt;font-family:" tahoma> 

mso-bidi-font-size:10.0pt;font-family:" tahoma> 

normal"> " tahoma>References:

mso-bidi-font-size:10.0pt;font-family:" tahoma> 

1. 7.0pt;mso-bidi-font-size:10.0pt;font-family:" times new roman>    Xu, Rong, Alin Rai, Maoshan Chen, Wittaya Suwakulsiri, David W. Greening, and Richard J. Simpson
(2018), “Extracellular vesicles in cancer-implications for future improvements
in cancer care,” font-family:" tahoma background:white>Nature Reviews Clinical Oncology normal">, volume 15, pp 617–638.

mso-bidi-font-size:10.0pt;font-family:" tahoma>  " tahoma>

2.    Song, Young Hye, white">Christine Warncke, Sung Jin Choi, white">Siyoung Choi, Aaron E. Chiou, Lu Ling, Han-Yuan Liu, Susan
Daniel, Marc A. Antonyak, Richard
A. Cerione, and Claudia
Fischbach (2017), “Breast Cancer-Derived Extracellular Vesicles
Stimulate Myofibroblast Differentiation and Pro-Angiogenic Behavior of Adipose
Stem Cells,”  Journal of the International Society for
Matrix Biology (July) 60-61: 190–205.

3.  Van Dommelen, Susan M., Margaret Fish, Arjan
D. Barendrecht, Raymond M. Schiffelers,
Omolola Eniola-Adefeso, and Pieter Vader (2017),
“Interaction of Extracellular Vesicles with Endothelial Cells Under
Physiological Flow Conditions,” In Exosomes and Microvesicles: Methods and Protocols,
Hill, A. F., Ed. Springer New York, NY; pp 205-213.

4.  Richards, Mark J., Chih-Yun Hsia, Rohit R. Singh, Huma
Haider, Julia Kumpf, Toshimitsu
Kawate, and Susan Daniel (2016), “Membrane Protein Mobility and
Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma
Membrane Blebs," Langmuir 2016, 32
(12), 2963-2974.

5.  Antonyak, Marc A., Bo Li, Lindsey K.
Boroughs, Jared L. Johnson, Joseph E. Druso, Kirsten
L. Bryant, David A. Holowka, and Richard A.
Cerione (2011), " 12.0pt;font-family:" tahoma> Cancer
cell-derived microvesicles induce transformation by transferring tissue
transglutaminase and fibronectin to recipient cells," 2011, 108 (12), 4852-4857.

font-family:" tahoma>