Engineering an adipose tissue model for studying ovarian cancer metastasis

Introduction: Ovarian cancer (OC) has the highest incidence-to-mortality ratio of all cancers. Considered a silent killer because of its nonspecific symptoms that present late in the disease course, OC is most often diagnosed after metastasis has already occurred. OC preferentially metastasizes to a vascularized layer of white, visceral adipose tissue that covers the abdominal organs (Fig. A), and patient 5-year survival drops 50% once OC has spread to this location. Limited physiologically relevant preclinical models exist to study the diverse tumor microenvironment (TME) of OC metastasis to abdominal adipose tissue. It is also thought that adiposity, or the presence of excess body fat, may act as a catalyst for OC metastasis, especially to this adipose tissue layer. As such, accurate modeling of this fat layer can help us understand if and how adiposity contributes to increased metastatic tumor burden in OC patients.
Therefore, our overall objective is to accurately recapitulate the TME of OC metastasis by creating a model that includes relevant biological components, with a focus on adipocytes, in a 3D construction. We demonstrate the novel cell types and cell culture conditions needed to create this model, which can be used to study the physical and biological transformations that occur in adipose tissue due to OC metastasis. In the absence of representative models of OC metastasis to this adipose layer, elucidating the mechanisms behind metastasis, investigating effective anti-cancer therapies, and improving patient prognosis post-metastasis will remain difficult.

Materials and

Methods: Three relevant preadipocyte cell lines from different origins were evaluated for their differentiation capabilities into mature adipocytes: StemPro subcutaneous human adipose-derived stem cells (ADSCs), immortalized patient-derived subcutaneous preadipocytes (SPADs), and immortalized patient-derived visceral preadipocytes (VPADs). Several variations of adipocyte induction media were also tested amongst the cell lines for their ability to produce effective and timely differentiation while minimizing adipocyte browning. A patient-derived physiologically relevant 3D model based on crosslinking of human plasma fibrinogen in combination with collagen that generated a personalized 3D scaffold was used for culture of pre-adipocytes (Fig. B). Preadipocytes were differentiated in 3D and stained with BODIPY505/515 at 7 and 14 days to evaluate lipid drop formation. Z-Stack confocal images were taken, and maximum projection images were used for analysis, and lipid staining intensity was used as a measure of differentiation. Individual cell stiffness of differentiated adipocytes and undifferentiated preadipocytes was also measured using atomic force microscopy (AFM). In addition, RNAseq data of samples of OC metastasis to adipose tissue were obtained from the Gene Expression Omnibus (GEO). Transcriptomic analysis was conducted on these samples using DESeq2 in RStudio, and body mass index (BMI) greater than 30 was used as a measure of adiposity.

Results, Conclusions, and Discussions: ADSC differentiation was found to be limited by cell passage number, with cells above passage six failing to form lipid droplets, while SPADs and VPADs demonstrated reproducible differentiation across higher passage numbers. VPADs also showed significantly less differentiation and produced smaller lipid droplets compared to the SPADs and ADSCs, likely reflective of their unique physiological origin in visceral fat (Fig. C). Several variations of adipocyte induction media were tested in order to characterize lipid droplet size and distribution mimicking white fat-like behavior. White-adipose induction media produced larger lipid droplets more characteristic of white fat across all cell lines, with the greatest differentiation seen with increased dexamethasone concentrations (Fig. D). Differentiation not only produced physical changes in appearance but also in cell stiffness, with differentiated ADSCs having significantly decreased cytoplasmic stiffness (Fig. E). This data demonstrates how our model captures the unique physical and biological characteristics of this white adipose tissue, which is necessary to accurately study OC metastasis. In addition, transcriptomic analysis of OC metastasis to visceral fat showed significantly different gene expression when samples were sorted by adiposity, with upregulated IGHA1 and IGHA2 and downregulated CXCL14 in patients with higher BMIs (Fig. F). Gene ontology (GO) analysis of significantly different genes showed increased leukocyte, lymphocyte, T-cell, and B cell activation and proliferation in patients with higher BMIs (Fig. G). Critically, these results demonstrate that adiposity may play a role in promoting OC metastasis, potentially by causing changes in immune function within host tissues.
In conclusion, we have recapitulated the TME of the visceral, white adipose tissue of preferential OC metastasis. This model will have a significant positive impact by allowing for further study of the cellular and extracellular processes that characterize OC metastasis, the effects of adiposity on patient prognosis, and the investigation of novel targeted therapies for metastatic OC.

Acknowledgements (Optional): This project is supported by Sanford Research start-up funds. We thank Dr. Klingelhutz for providing some of the lines used in this research. We also thank Mrs. Isaiah from the Bioinformatics Core for help with the transcriptomics analsyis