Development and characterization of paired in vivo and in vitro models to examine obesity-linked colorectal cancer

Abstract

Currently, obesity has become a global issue and is referred to as an epidemic. Contemporarily, colorectal cancer (CRC) is the fourth most common cause of cancer-associated death worldwide. Several epidemiological studies demonstrated that obesity is linked to CRC. Obesity is characterized as a chronic low-grade inflammation which is assumed to be a crucial risk factor for CRC. However, the mechanism behind obesity-associated CRC is elusive. To investigate the link between obesity and CRC, and to develop a platform to screen and test therapeutic agents targeting CRC in the obese state, our objectives were to develop an innovative experimental model of obese insulin resistance in vitro which can facilitate the in vivo obese insulin resistance adipose tissues and also to develop a new obesity-linked engineered 3D cancer co-culture system that may serve as a platform to screen therapeutic agents against obesity-linked CRC. To develop an obese insulin resistance model in vitro, differentiated 3T3-L1 adipocytes were treated with Tumor Necrosis Factor alpha (TNF-α) and hypoxia for 24 hours, and then, extra glucose and Fetal Bovine Serum‎ (FBS) were added for 72 hours. TNF-α and hypoxia significantly reduced expression of insulin-sensitive genes and induced expression of insulin resistant genes. Adipocytes lost lipid content over time and the insulin-stimulated AKT phosphorylation significantly decreased. This result indicates that combined TNF-α and hypoxia have the potential to induce long-term an obese insulin resistance phenotype in vitro. To investigate CRC in vivo, we developed a standardized platform for obesity-linked CRC study in vivo using PDX CRC tumors implanted orthotopically. PDX CRC tumors were implanted in the diet-induced obese Rag1 (B6.129S7-Rag1tm1Mom/J) mice. Diet-induced obese Rag1 mice developed insulin resistance, which promoted tumor growth. To establish a unique in vitro platform, we fabricated 3D engineered CRC tissues. Three lines of tumors were obtained from three different patients, and tumors were propagated in SCID (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice subcutaneously for 29 days. Cells from the PDX tumors were also encapsulated and propagated in Poly (ethylene glycol)-fibrinogen (PEG-Fb) hydrogel to form a 3D tissue, which we define 3D engineered CRC tissue. The results showed that the phenotype of the growth of 3D engineered CRC tissues followed the growth of the in vivo tumors. We also examined the 3D engineered CRC tissues grown by co-culturing with the long-term insulin resistant adipocytes. The in vitro 3D tissues were enabled to recapitulate the in vivo microenvironment, and most prominently the obese insulin resistant adipocytes were able to enhance of the growth of the 3D engineered CRC tissues. Finally, we performed mRNA sequencing of PDX CRC tumors propagated in SCID mice and 3D engineered CRC tissues. Interestingly, the sequencing data demonstrated that the gene expression characteristics of PDX CRC tumors propagated in SCID mice clustered with the 3D engineered CRC tissues, indicating that each PDX line has a unique gene expression profile. Overall, our study observed that the 3D engineered CRC tissue scaffold facilitates the in vivo tumor microenvironment similar to the tumor propagated in SCID. The co-culture study has demonstrated that the insulin resistant adipocyte model can lead to enhanced 3D engineered CRC tissue growth, which may serve as a platform for further research on obesity-linked CRC study.