Huff Janice L.

Patel, Zarana S.

NASA Technical Reports Server

 Radiation Effects on Tissue Differentiation in a 3-D Organotypic 
Aerodigestive Tract Model 
1 Universities Space Research Association, Houston, TX 77058 
Zarana S. Patel1 and Janice L. Huff1 
INTRODUCTION 
CONCLUSIONS 
RESULTS 
ACKNOWLEDGMENTS 
This work was supported by NASA grant NNJ06HG25A to JLH.  The esophageal epithelial and 
fibroblast cell lines were kindly provided by Dr. Anil Rustgi (University of Pennsylvania). The 
authors gratefully acknowledge Carol Johnston (MD Anderson Cancer Center) for assistance 
with histology on organotypic cultures. We also thank Dr. Eleanor Blakely for helpful advice and 
our colleagues at JSC and NSRL for their support. Track structure diagrams were created 
using RITRACKS software created by Ianik Plante (USRA). 
REFERENCES 
[1] Okawa et al., Genes and Development 2007, 21: 2788-2803. 
[2] Fenech. Nature Protocols 2007, 2: 1084 - 1104. 
Clonogenic Survival Assay 
EPC2-hTERT cells in sister flasks were irradiated and frozen. Upon 
thawing, cells were plated at increasing densities (n=3 plates per density). 
At 12 days, cells were fixed and stained with 0.1% crystal violet in 10% 
NBF and colonies containing more than 50 cells were counted.   
METHODS 
2-D Cell Culture 
Normal immortalized human esophageal epithelial cells (EPC2-hTERT [1]) 
were cultured in Keratinocyte Serum Free Media (KSFM) for growth and in 
Epidermalization media (EPI, with DMEM, Ham’s F12 and supplements) for 
micronucleus assays (unless otherwise indicated). FEF3 cells (normal human 
esophageal fibroblasts) were cultured in DMEM (10% FBS+ pen/strep).  
Cytokinesis-Block Micronucleus Assay 
 Following irradiation, cells were fed with media containing 
cytochalasin B to inhibit cytoplasmic division and allow for  
accumulation of binucleated cells and micronuclei (MN) [2]. For 
the 3-D assay, cultures were irradiated one day prior to airlift. 
Cells were harvested 65-70 hours post-irradiation. 
Irradiation 
Low-LET irradiation was conducted with a 137cesium gamma source. High-
LET particle was conducted at the NASA Space Radiation Laboratory at 
Brookhaven National Laboratory during campaigns NSRL ’11C and ‘12A. 
Experimental set-up and beam line at NSRL. Cells were irradiated in “track-segment mode” 
with the beam (arrow) perpendicular to the culture with constant LET across sample.  
3-D Organotypic Culture 
3-D cultures consisted of FEF3 
fibroblasts in a layer of collagen I 
and Matrigel. EPC2-hTERT cells 
were seeded on top of this matrix 
and cultured in EPI media. Creation 
of an air-liquid interface triggered 
differentiation and stratification of 
the epithelial layer. Total culture time 
was 14-16 days.  
Stratified layers of 
epithelial cells
Collagen/Matrigel matrix 
with fibroblasts
Acellular layer of collagen
Medium
H&E section of normal stratified 
squamous esophageal epithelium. 
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3-D organotypic culture of human esophageal 
cells grown at Johnson Space Center.   
Epithelium
Basal cells
Stroma
The mucosa of the human aerodigestive tract is a typical stratified squamous 
epithelium where the outermost, luminal region consists of an epithelial cell 
compartment overlying a supportive stromal matrix. As the basal cells at the stromal 
interface divide and move upward, they become increasingly committed to terminal 
differentiation, lose the capacity to replicate, and are ultimately shed from the luminal 
surface. This process is fed by a population of basal cells with stem-like characteristics 
that serve as a reservoir for maintenance of the normal tissue structure. In the 
esophagus, cell fate determination controlling normal tissue homeostasis is dependent 
on intercellular Notch signaling (Ohashi 2010). This evolutionarily conserved pathway 
is important in embryonic development and differentiation as well as in angiogenesis, 
apoptosis, proliferation, stem cell renewal, and epithelial-mesenchymal transition 
(EMT). The outcome of Notch activity is dependent on cellular framework and is 
contingent on the timing and levels of pathway activity. In the esophagus, studies 
suggest a tumor suppressive function of Notch via promotion of differentiation and 
suppression of cancer-promoting EMT. In this study, we investigated the effects of 
radiation exposure (100 cGy 137Cs gamma-rays) on differentiation in a 3-D 
organotypic esophageal model using immunohistochemistry for differentiation markers 
(CDH1, IVL, KRT13 and 14) combined with focused gene expression analysis using 
qRT-PCR for Notch-specific pathway effectors and downstream targets. Qualitative 
changes in tissue morphology and distribution of differentiation markers were not 
observed in the irradiated compared to unirradiated samples at 3, 5 and 7 days 
following exposure. However, qRT-PCR analysis revealed significant upregulation of 
Notch pathway genes and downstream effectors that were consistent with an 
enhanced terminal differentiation in irradiated cultures. Although the exact role of these 
genes in the radiation response in this tissue remains to be elucidated, one possible 
explanation is that activation of a differentiation program following radiation exposure 
functions as a protective mechanism to eliminate genetically damaged cells from the 
replicating cell population.  
Ohashi et al., 2010. Gastroenterology 139(6): 2113–23. 
Total RNA samples was isolated using Qiagen RNeasy spin columns from 
EPC2-hTERT cells cultured in epidermalization (EPI) media or keratinocyte 
serum free media (KSFM) at 72 hours post-irradiation. Fold changes in 
relative gene expression from triplicate samples were quantified using the 
comparative CT method with data normalized to GAPDH . 
RT-qPCR 
Immunocytochemistry 
Monolayer cultures were fixed in 4% paraformaldehyde and immunostained 
with rabbit antibody to p21 (BD Biosciences) and Alexa594 labeled 
secondary antibody (Invitrogen). Nuclei were counterstained with DAPI. 
p21 Waf/CIP       K5 
KSFM 
EPI or Calcium 
Growth Curves
0.E+00
5.E+05
1.E+06
2.E+06
2.E+06
3.E+06
0 1 2 3 4 5 6
Day
C
e
ll
 N
u
m
b
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r
KSFM
Epiderm
2-D Differentiation of EPC2-hTERT Cells  
•Clustered cell growth 
in EPI with evidence 
of stratification, vs. 
dispersed monolayer 
growth in KSFM 
•Slower population 
growth in EPI media 
•KSFM cultures 
represent 
proliferating basal 
population 
2-D Media Comparison: Differentiation Markers 
•Markers of epithelial cell late differentiation (Involucrin, 
K13) are observed in cells grown in EPI media but not 
KSFM 
•Markers of basal-like cells (K5, TP63) are expressed in 
cells grown in both EPI and KSFM with higher levels in 
KSFM cultures 
Differentiation Marker Expression in 2-D EPC2-
hTERT +/- Ca at 72 hours 
Calcium treatment enhanced 
expression of the differentiation 
markers CDH1, Notch 3, and p21 
while p63 expression was 
suppressed.  
 
IR exposure enhanced 
expression of CDH1, Notch 1, and 
KRT13 while p63 expression was 
suppressed and no change 
observed for IVL.  
IR Effects on Differentiation Markers in 2-D EPC2-hTERT 
Proliferative basal 
population fuels tissue 
stratification. Cells 
become increasingly 
committed to terminal 
differentiation as they 
move upwards through 
epithelial layer and are 
ultimately shed from the 
luminal surface. 
 
 
A B
C D
Day 7Day 5Day 3 Day 9
Day 3 Day 5
Day 7 Day 9
Differentiation in 3-D Organotypic Culture 
Tissue differentiation modeled in 3-D organotypic culture of EPC2-hTERT esophageal epithelial cells. Cultures were 
harvested at day 3 (panel A), day 5 (panel B), day 7 (panel C) and day 9 (panel D) post epithelial seeding and fixed 
in formalin and processed for histology. Top panel shows immunostaining of  sections (6 µm) with antibodies against 
p63 (red) and involucrin (green). Bottom panel: Real time qPCR analysis of differentiation genes in RNA harvested 
from sister cultures. Data are ddCt values normalized to the housekeeping genes GAPDH and RPL13.   
 
Day 3 
Day 5 
Day 7 
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Target Genes 
Notch Signaling Pathway RT PCR Array (SA Biosciences) 
Small perturbations observed in mRNA expression of 
Notch receptor and ligands, as well as target genes that 
implement Notch signals.  Coincide with alterations in 
Wnt receptor expression.  
 
Day 3 Day 5 Day 7 
Dynamic role of Notch signaling 
pathway 
10
100
0 100 200 300 400 500
Su
rv
iv
al
 (%
)
Radiation Dose (rad)
Ti 350 (158 keV/µm)
Cs 137
Equitoxic Dose Calculation – 2D Clonogenic Survival 
70 
Radiation Effects on Markers of Differentiation in Organotypic Culture 
A 
B 
• Gamma-rays and 
HZE particles 
irradiation both 
result in increased 
expression of 
differentiation 
markers at 72 hours 
post-exposure  
 
•Upregulation of 
Notch genes 
provides a clue to 
mechanism of 
radiation impact on 
cellular 
differentiation in this 
model 
0 vs.100 rad Gamma 
0 vs.30 rad Titanium (350 MeV/amu) 
0 rad 100 rad 
30 rad 0 rad 
Notch signaling pathway perturbed following radiation 
exposure with both γ-rays and heavy ions; in 
both 2-D and 3-D cultures of EPC2-hTERT 
cells.  
Functional significance of Notch alterations unknown. 
Possible considerations are enhanced 
differentiation, or cell survival functions of this 
pleiotropic pathway.  
Future studies to determine activation state of notch proteins 
following radiation, combined with pathway 
inhibitors will refine these preliminary results. 
Investigate role of p53.  
3D EPC2-hTERT 100r γ-ray Day 3 Day 5 Day 7
Notch Signaling
Notch Receptors Notch1 1.72 1.21 1.23
Notch3 1.12 1.06 1.18
Notch Ligands DLL1 1.23 1.26 2-
DLL4 1.23 1.26 2-
Jag1 1.49 1.03 1.21
DTX1 1.37 1.46 1.06
Transcription 
Cofactor
MAML2 1.38 1.27 1.14-
Notch Target Genes
Cell Cycle CDKN1A 1.74 1.07 1.35
Transcription 
Factors
Hes5 1.77 1.91 1.1-
FOS 1 1.66 1.13-
NR4A2 1.42 1.13 1.08
RUNX1 1.48 1.14 1.43
Immune CHUK1 1.43 1.68 1.05
Pathways that Crosstalk with Notch
Wnt Signaling Fzd4 1.87 1.64 1.06
FzD7 1.37 1.61 1.48
https://ntrs.nasa.gov/search.jsp?R=20140002495 2019-08-29T14:43:14+00:00Z


Radiation Effects on Tissue Differentiation in a 3-D Organotypic Aerodigestive Tract Model

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