complex, 3d tissues for modeling the immune response in...
TRANSCRIPT
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The immune system plays an active role in both the prevention and
the promotion of cancer dependent upon its interaction with the
tumor cells. The roles of both macrophages and T-cells in cancer
progression have been heavily studied over the past few years.
Macrophages have been found to be either tumor promoting or
tumor preventing depending upon their differentiation status and the
tumor microenvironment while the homing and cell destroying
capabilities of T-cells have been manipulated to effect better, more
specific tumor cell cytotoxicity through the development of therapies
such as chimeric antigen receptor T-cells (CAR-T cells). Unfortunately
the majority of research in the area of immune-oncology has relied
upon either 2D cell culture or animal models. While a large amount of
information has been learned from these models, it has been well
established that 2D cell culture does not mimic in vivo biology and the
immune system of mouse models differs from that of humans in
numerous ways including T-cell subsets, cytokine receptors, and
costimulatory molecule expression. To overcome these limitations, we
have developed a number of 3D in vitro tissue models including multi-
cell type models of glioblastoma (GBM), breast, and ovarian cancer.
Complex, 3D Tissues for Modeling the Immune Response in Cancer and Predicting the Activity of ImmunotherapiesTeresa M. DesRochers1, Lillia Holmes1, Qi Guo1, Lauren O’Donnell1, Stephen Shuford1, Larry Puls2, Jeffrey Elder2, Jeff Edenfield2, Howland Crosswell1
1KIYATEC Inc.; Greenville, South Carolina 29605 USA | 2Greenville Health System; Greenville, South Carolina 29605 USA |
Background Ovarian – Immune Cells Affect Viability
Immune Tumor Models
Conclusions
Figure 4: The presence of neurospheres and huvecs affects the differentiation of
CD14+ PBMCs. Scaffolds containing NS/HUVEC or PBMCs were cultured together in
perfusion for 14 days. Both metabolism (A) and cell numbers (B) increased in the
NS/HUVEC scaffolds. However, while the PBMC scaffolds increased in metabolic activity,
they lost cell numbers. The PBMCs were labeled with PKH26. After 1 week in culture a very
small number of PKH26 cells were found in the NS/HUVEC scaffolds indicating a lack of
cellular migration by the PBMCs (C). At day 7 (D) and day 14 (E), the PBMCs were removed
from their scaffolds and examined for macrophage markers. At both days the cells were
positive for both CD163 and CD206 but the signal was reduced at day 14 compared to day
7. *p<0.05, **p,0.01, error bars = standard deviation, n=3.
Glioblastoma
• The presence of CD3+ cells negatively affects cell viability in ovarian cancer while CD14+
cells do not
•Ascitic fluid of ovarian cancer patients affects drug response with EpCAM+ cells from
ascitic fluid reflecting the response of the primary tumor.
• The different cell types of a tumor can affect the differentiation of CD14+ immune cells
•Future Directions: preclinical immuno-oncology agent testing and
predictive assay for checkpoint inhibitor responders
#5119
Resected
Tumor
Mince & Wash
ascitic fluid CD14+
CD3+
Ovari
an
BreastM1/M2 Macrophages
+ ECM
Neurospheres + ECM
GBM
Figure 1: Model Development. (Ovarian) Both tumors and matched ascitic fluid are
collected from patients. Tumor cells are then combined with immune cells in 3D spheroid
assays. (Breast) Tumors and matched ascitic fluid are collected from patients. Tumor cells,
endothelial cells, fibroblasts, and fat are then combined with immune cells in 3D
microtumors. (GBM) Tumors are collected from patients and neurospheres are established.
These are combined with differentiated macrophages from normal blood in 3D
microtumors.
0
500
1250
2500
5000
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
2 5 0 0 0
O V B 0 1 & M a tc h e d C D 3 +
C D 3 + c e lls
Bla
nk
ed
R
LU
T u m o r
T u m o r + C D 3 +
p < 0 .0 5
0
500
1250
2500
5000
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0O V B 0 3 & M a tc h e d C D 1 4 +
C D 1 4 + C e lls
T u m o r
T u m o r + C D 1 4 +
Bla
nk
ed
R
LU
Figure 2: Immune cells co-cultured
with patient-matched tumor cells
can affect proliferation in a cell-
type dependent manner. (A) When
ovarian tumors cells were co-cultured
with matched CD3+ cells for 96 hours,
there was a significant drop if the
viability of the tumor cell/CD3+
combo that was inconsistent with the
viability of the CD3+ cells alone. (B)
When ovarian tumor cells were co-
cultured with matched CD14+ cells for
96 hours, viability of the tumor
cell/CD14+ combos were consistent
with the viability of the CD14+ cells
alone.
Ovarian – Drug Response Profiling
NS
/HU
VE
C
PB
MC
0
1 0 0 0 0
2 0 0 0 0
3 0 0 0 0
4 0 0 0 0
5 0 0 0 0
P re s to B lu e
S c a ffo ld T y p e
Bla
nk
ed
RF
U
D a y 0
D a y 7
D a y 1 4
*
*
*
**
NS
/HU
VE
C
PB
MC
0
1 0
2 0
3 0
4 0
5 0
P ic o G re e n
S c a ffo ld T y p e
DN
A c
on
ten
t (u
g/m
L) D a y 0
D a y 7
D a y 1 4
*
*
-4 -2 0 2 4
0
5 0
1 0 0
1 5 0
A s c e t ic F lu id - P re -S u rg e ry
L o g D o s e (u M )
% V
iab
ilit
y
C is p la tin
T o p o te c a n
L Y 2 9 4 0 0 2
D o x o ru b ic in
G e m c ita b in e
C a rb o p la tin
T ra m e tin ib
A fa tin ib
P a c lita x e l
A. B.
C. D.
-4 -2 0 2 4
0
5 0
1 0 0
1 5 0
T u m o r
L o g D o s e (u M )
% V
iab
ilit
y
C is p la tin
T o p o te c a n
L Y 2 9 4 0 0 2
D o x o ru b ic in
G e m c ita b in e
C a rb o p la tin
T ra m e tin ib
A fa tin ib
P a c lita x e l
-4 -2 0 2 4
0
5 0
1 0 0
1 5 0
A s c e t ic F lu id - S u rg e ry
L o g D o s e (u M )
% V
iab
ilit
y
C is p la tin
T o p o te c a n
L Y 2 9 4 0 0 2
D o x o ru b ic in
G e m c ita b in e
C a rb o p la tin
T ra m e tin ib
A fa tin ib
P a c lita x e l-4 -2 0 2 4
0
5 0
1 0 0
1 5 0
A s c e tic F lu id - S u rg e ry
E p C A M + C e lls
L o g D o s e (u M )
% V
iab
ilit
y
C is p la tin
T o p o te c a n
L Y 2 9 4 0 0 2
D o x o ru b ic in
G e m c ita b in e
C a rb o p la tin
T ra m e tin ib
A fa tin ib
P a c lita x e l
IC50 (uM)
Drug
ascitic Fluid
Pre-SurgeryTumor
ascitic Fluid
Surgery
Unselected
ascitic Fluid
Surgery
EpCAM+
Cisplatin 22.45 13.91 30.34 6.716
Topotecan 3.493 3.964 3.097 3.976
LY 294002 20.47 60.35 23.14 34.82
Doxorubicin 0.3053 0.3359 0.2329 0.3238
Gemcitabine >100 >100 18.15 >100
Carboplatin 79.72 >100 >100 >100
Trametinib >100 >100 29 >100
Afatinib 0.654 1.901 1.664 0.2887
Paclitaxel 11.4 21.68 5.903 3.962
Figure 3: Drug response is affected by the source of the cells and the cell
types present. ascitic fluid (A, C, D) and tumor (B) from the same patient were
assessed for drug response in our EV3D DRP assay. The ascitic fluid was assessed
both before surgery and at the time of surgery. Prior to surgery it predicted
response to carboplatin however their was no response when the tumor and the
ascitic fluid at surgery was assessed. The ascitic fluid at surgery predicted response
to both Gemcitabine and Trametinib which was not predicted by the tumor.
When the surgical ascitic fluid was selected for EpCAM+ cells (D) which made 20%
of the cellular population, the drug response profile was more similar to the tumor
than either unselected ascitic fluid samples, n = 6.
A.
D.
C.B.
E.
0.49%
CD14 PKH26 CD163 CD206
CD14 PKH26 CD163 CD206
This work is supported in part by NCI SBIR Contract #: HHSN261201300043C
With Special Thanks to
the Biorepository