(22g) Invited: Tissue Engineered Cancer Models for Recapitulating the Tumor Microenvironment | AIChE

(22g) Invited: Tissue Engineered Cancer Models for Recapitulating the Tumor Microenvironment

Authors 

Lipke, E. - Presenter, Auburn University
Hassani, I., Auburn University
Habbit, N., Auburn University
Ahmed, B., Auburn University
Anbiah, B., Auburn University
Greene, M. W., Auburn University

Elizabeth Lipke Normal Elizabeth Lipke 1 1 2019-04-13T03:45:00Z 2019-04-13T03:46:00Z 2 622 3552 29 8 4166 16.00

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justify;line-height:normal"> Calibri;mso-fareast-font-family:Calibri;mso-hansi-font-family:Calibri;
mso-bidi-font-family:Arial">Consistent evidence from epidemiological studies
reveals that obesity is linked with up to 70%
increased risk of colorectal cancer (CRC). However, the mechanisms of
obesity-promoted CRC are still anonymous at the molecular levels, in part, due
to the lack of relevant experimental models. Here, we have established an in
vitro model of the obese tumor microenvironment by engineering cancer tissues
utilizing patient-derived xenograft (PDX) CRC cells and investigated the
ability of our model to recapitulate an in vivo obese model of orthotopically
implanted PDX CRC.

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justify;line-height:normal"> Calibri;mso-fareast-font-family:Calibri;mso-hansi-font-family:Calibri;
mso-bidi-font-family:Arial">Briefly, PDXs were established
through subcutaneous propagation for 3-4 passages in NOD-SCID mice. The PDXs were then excised, and the cells were isolated and
encapsulated within a biomimetic material, PEG-fibrinogen (PEG-Fb), to generate
3D engineered PDX CRC tumors (3DePCCTs). To determine if the PDX CRC cells
could maintain their viability in the 3DePCCTs, long-term cell viability assays
were performed. To determine how closely the 3DePCCTs
mimic the in vivo PDX tumor, cell colony growth, cell subpopulations by flow
cytometry, immunostaining, H&E staining, and mechanical stiffness by
parallel plate compression testing of within the 3DePCCTs were
carried out and compared to those of in vivo PDX tumor. To demonstrate
model responsiveness to the growth-promoting effects of obesity, the 3DePCCTs
were co-cultured with insulin sensitive (IS) or resistant (IR) (treated with 20
ng/mL TNF Calibri;mso-bidi-font-family:Arial;mso-bidi-font-style:italic">a Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:Arial"> and 1%
hypoxia) adipocytes (model of leanness or obesity, respectively),
differentiated from 3T3-L1 cells. Tumor cell colony area within the co-cultures
was quantified over 29 days and compared to our in vivo model in which Rag1tm1Mom
mice were fed either a high-fat Western diet + 4% sugar water (HFWD+S) or chow
diet for 12-weeks; PDX CRC tumor fragments were then orthotopically implanted;
diets were continued, and tumor growth assessed after seven weeks.

normal"> Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:Arial">

normal"> Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:Arial">Since PDXs are maintained through in vivo subculture, we first assessed
the ability of our 3DePCCTs to maintain PDX CRC cells in culture. Based on
viability assays, PDX CRC cells within the 3ePCCTs remained viable over 29 days
of culture (n=3 3DePCCTs, p < 0.05). Flow cytometry
cell quantification revealed that human ( font-family:Symbol;mso-ascii-font-family:Calibri;mso-fareast-font-family:Calibri;
mso-hansi-font-family:Calibri;mso-bidi-font-family:Arial;mso-char-type:symbol;
mso-symbol-font-family:Symbol"> Symbol">b Calibri;mso-fareast-font-family:Calibri;mso-hansi-font-family:Calibri;
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mso-bidi-font-family:Arial">2 microglobulin+ (B2M+)) cells
and the cells expressing a CRC marker, Cytokeratin 20+ (CK20+), remained fairly
constant over time within the 3DePCCTs and percentages of each cell
subpopulation were similar to the original tumor, whereas supporting mouse
stromal cells overtook 2D PDX CRC cultures (n=3 batches, p < 0.05). Based on
H&E staining, the cell colonies within the 3DePCCTs were similar to those
of the in vivo PDX tumor. Immunostaining showed that the human cells formed
colonies, while mouse stromal cells were elongated. Mechanical stiffness of the
3DePCCTs increased over time and the range of the 3DePCCTs stiffness was within
that of the in vivo PDX tumor (n=3 in vivo tumor
tissues or 3DePCCTs, p < 0.05). To examine the ability to recapitulate
differences in tumor growth rate, 3DePCCTs were formed
using PDX tumors from patients with stage II, III-B, and IV CRC adenocarcinomas.
In vivo, the stage II tumor had the fastest growth rate, followed by the stage
IV and then the stage III-B. These relative growth rates were
recapitulated within the 3DePCCTs for all stages. To study
obesity-promoted tumor growth in vitro and in vivo, the slower growing stage IV
CRC tumor was employed. Most notably, co-culture of the
3DePCCTs with IR adipocytes resulted in a higher density of PDX CRC cell
colonies after 8 days of co-culture as compared to co-culture with IS
adipocytes and this difference increased through day 29 (n= at least 3 3DePCCTs,
p < 0.05). Similarly, the weight of orthotopic PDX CRC tumors in obese
(HFWD+S diet) mice was over 2-fold higher than that of tumors in lean (chow
diet) mice (n=6 PDX tumors, p < 0.05).

normal"> Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:Arial">

normal"> Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:Arial">We have
established novel in vitro obesity-mimetic colorectal cancer model based on PDX
CRC engineered tumor tissue which is responsive to the
growth-promoting effects of obesity that can potentially be used to study the
mechanistic link between obesity and colorectal cancer.

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