Background 3D pMicrotumor with Cell Lines 3D DRP

A Complex 3D Model of Glioblastoma for Patient-Specific Drug Response
Profiling
#611
Lauren O’Donnell1, Lillia Holmes1,, Qi Guo1, David Schammel2, Jeff Edenfield3, Charles C. Kanos3, Howland Crosswell1, Teresa M. DesRochers1
1KIYATEC Inc.; Greenville, South Carolina 29605 USA | 2 Pathology Consultants; Greenville, South Carolina 29605 USA | 3 Greenville Health Systems; Greenville, South Carolina 29605 USA
3D pMicrotumor with Cell Lines
Neurospheres
Endothelial Cells
ECM
Resected
Tumor
•
•
•
•
Neurospheres
Generated
3D
Spheroids
•
•
•
•
•
•
Non-Lytic Analysis
Cell Metabolism
Biomarker Release
Flow Cytometry
Multiphoton
Microscopy
Lytic Analysis
Cell Viability
DNA Content
Gene Expression
Phosphoproteome
Tissue Morphology
Epigenetic
Signatures
A.
B.
B.
2 Weeks
Static
1 Week
Perfusion
Co-Culture Model Development
B.
Static
Methods
1 Week
Day 0
Day 7
C.
With Special Thanks to the
Biorepository
Day 14
Day
Day 21
Day
%CD56+
0
0.60
7
11.34
14
0.11
21
15.57
28
20.69
P ic o G re e n
6
D N A c o n t e n t (u g /m L )
40000
30000
20000
10000
0
4
2
E.
n
k
S
B
la
N
5
1
B
B
N
N
A
A
1
B
5
la
N
n
k
S
0
Drugs
Cisplatin
Carboplatin
LY294002
Erlotinib
Temozolomide
Irinotecan
Afatinib
Axitinib
Topotecan
Everolimus
BEZ235
IC50 (uM)
19.23
>100
82.96
>100
>100
>100
7.04
>100
92.27
19.44
53.25
Figure 5: EV3D DRP of primary GBM
predicts EGFR inhibitor response when
EGFR is amplified. Resected tumor from
2 different GBMs were processed and run
in KIYATEC’s EV3D DRP Assay against
afatanib (Golotrif®), which kills by
inhibiting EGFR. The tumor tissue was
very similar, except one was EGFR
amplified (blue line) and one was not (red
line). As predicted, afatanib was 30x more
active in the EGFR amplified tumor tissue
(50% cell kill (IC50) at 0.2 v 6uM), n = 6
150
E G F R A m p lifie d
100
E G F R N o n A m p lifie d
50
0
-4
-2
0
2
4
L o g D o s e (u M )
Day 21
%CD133+
*Alive
(UL)
*Dead
(UR)
0
0.38
0.00
7
0.00
0.00
14
0.02
0.02
21
0.00
0.00
28
0.16
0.00
Day 28
Day 28
P r e s t o B lu e
50000
Conclusions
Day 7
Day 14
Tumors were removed and enzymatically digested into single cells. The single
cells were either cultured as 3D spheroids in KIYATEC’s EV3D™ DRP platform or
allowed to develop into neurospheres. 3D spheroids were treated with 11
clinically relevant chemotherapy and targeted agents. Viability was assessed
and dose response curves were generated using non-linear regression.
Perfused 3D heterotypic Microtumors (pMT) were made of neurospheres (NS)
and endothelial cells in ECM and cultured for up to 2 weeks in the 3DKUBE™
perfusion microbioreactor, which supports non-lytic analysis. Additionally, the
impact of normoxic environmental (20% O2) conditions and hypoxic
environmental conditions (5% O2) on the cellular microenvironment was
examined by culturing 3D heterotypic Microtumors made of NS for up to 2
weeks in the 3DKUBE perfusion microbioreactor.
D.
C.
Figure 4: 3D DRP and pMT with cells isolated from primary GBM. Cells were isolated
from BNA15 and used for EV3D drug response (E). Cells were also used for NS expansion
and subsequently cultured for 7 days in the 3D pMT platform. BNA15 primary tumor (A)
and NS (B), fixed with formalin and stained with H&E, show similar morphology. H&E
staining (C) and DNA analysis (D) confirmed presence of NS in 3D pMT, although Presto
Blue (D) showed no cell metabolism in 3D perfusion. Error bars = standard deviation, n = 3.
B.
Day 0
B.
2 Weeks
Figure 2: H&E of 3D GBM pMT. (A) NS 3D pMT visualized in 3D scaffold (B) NS/HBEC coculture promotes tighter appearing cell clusters in 3D pMT environment. Scale bars = 100
µm
A.
A.
M ean R FU
Figure 1: Perfusion culture affects
3D pMT viability. (A) 3D pMT
containing U87 NS cells with and
without HBECs were grown in either
static culture conditions or under
perfusion flow. (B) 3D pMT
containing U87 NS cells were grown
in static culture conditions and
perfusion culture conditions in
hypoxic environmental conditions
and
normoxic
environmental
conditions. Cellular metabolism and
DNA content were assayed over 14
days.
A.
Perfusion
Approximately 70,000 new cases of brain tumors will be diagnosed this
year with glioblastoma (GBM) accounting for about 17% of those
cases. Unfortunately, the median survival for patients with GBM is only
14.6 months due to the complexity of the tumor, the pattern of diffuse
spread within the brain, and the lack of effective therapeutic options.
Complex in vitro tumor models developed in 3D better mimic in vivo
biology, and, when utilizing patient-derived cells, may offer robust
platforms for new drug development and patient-specific drug
response profiling. We have previously shown this to be true with both
breast and ovarian cancer and we have adopted this approach to
modelling GBM ex vivo. We hypothesize that primary derived GBM
tissues and stem cells cultured in complex 3D microenvironments can
recapitulate the intra-tumor heterogeneity and drug resistance similar
to that found in the clinical. To this end, we have developed 3D
models of GBM that incorporate tumor cells and endothelial cells
within a complex extracellular matrix and cultured them for up to 2
weeks under perfusion flow. We have created these models using both
cell lines and primary patient cells.
3D DRP & pMicrotumor with Primary Cells
% V ia b ility
Background
•Cell viability is affected by perfusion
•NS cultured with HBECs show a general increase in cellular metabolism
•NS grown under normal oxygen tension show general increase in cellular
metabolism compared to hypoxia
•U87 NS grown in suspension culture lose CD133 expression, but show
increased CD56 expression over time
•Drug response profiling is consistent with Temozolomide resistance and NGS
sequencing results
This work is supported by NCI SBIR Contract #: HHSN261201300043C
Acknowledgements: Chris Corless, MD, PhD at Knight Cancer Inst.
Figure 3: CD133 & CD56 expression in U87 NS
over time. (A) Day 0 FSC vs SSC U87 NS culture
(B) U87 NS CD133 expression over 28 days. (C)
U87 NS CD56 (NCAM) expression increases over
28 days. Black peaks = isotype control, blue peaks
= markers.
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