Spheroids to Investigate the Role of Obesity in The Development of Endometrial Cancer

Introduction:: The incidence of endometrial cancer is increasing year after year and is mainly attributed to advanced age, estrogen exposure, and recently, the obesity epidemic, whose profile has modified the advanced age range, affecting women at an earlier age. A meta-analysis has revealed that each 5 units increase in body mass index (BMI) is associated with a 50% increased risk of developing endometrial cancer. In US, endometrial cancer accounts for 40% of all cancer diagnoses, so it is expected to surpass lung cancer and colorectal cancer by 2030 and become the third most common cancer in women. Recent data has shown that adipose stem cells (ASCs) infiltrate the endometrial microenvironment of obese, but not non-obese, patients, with elevated numbers of ASCs correlating with increased obesity. The reorganization of the extracellular matrix (ECM) by ASCs within the microenvironment and their interactions with the ECM via integrins (ITGs) may influence oncogenic transformation of the epithelial compartment. Understanding this ASC-ECM interactome will provide information critical to the design of new therapy options for endometrial cancer and target ASC actions within the tumor microenvironment.


Materials and Methods:: In this study, we used 2D models and spheroids of the ASC-ECM interaction. First, to evaluate how oncogenic endometrial cells affect ASCs ITGs expression, we exposed human ASCs to conditioned media (CM) derived from an endometrial cancer cell line (KLE) and non-oncogenic endometrial epithelial cells (EECs, EME6/7). Changes in integrin expression were evaluated using RT-PCR and cyTOF analysis. Then, to understand the effect of ASC in the endometrial microenvironment, spheroids were formed with co-cultures of human ASCs and EECs. Different ratios of ASCs (1:1, 1:4 and 1:10) were tested in co-culture formation with 0.25% w/w methylcellulose diluted in the culture media in a low-adherent round-bottom 96-well plate. Samples were incubated at 37 °C and 5% CO2 for 10 days. Spheroid formation and size was monitored through time and at day 10, changes in ITGs expression were quantified by RT-PCR.


Results, Conclusions, and Discussions:: Exposure of ASC to CM in 2D studies showed that expression of ITGA5 and ITGA7 was significantly increased in ASCs exposed to KLE CM compared to non-oncogenic EME6/7 CM (p< 0.05) or control media (p< 0.0001). On the other hand, expression levels of ITGAV and ITGB1 did not change. cyTOF analysis also showed an increase expression of ITGA5 when ASCs were exposed to KLE CM and EME6/7 CM. Co-culture spheroids were successfully formed and there was not difference in size or form among different ratios of ASC. Gene analysis of the spheroids showed that as the proportion of ASCs increased, there was a significant increase in ITGA5 compared to spheroids forms with just ASCs or just EME6/7 (p< 0.05). ITGA7 was increased in the 1:1 ratio and 1:4 ratio compared to ASCs or EME6/7 spheroids alone. These results suggest that specific ITG changes (ITGA5 & ITG7A) occur in ASCs in response to the endometrial tumor microenvironment. Upregulation of integrins that play a key role in cell adhesion and communication in the endometrium, might lead to alterations in ECM composition that can change preneoplastic lesion development. The induction of specific ITGs in ASCs, provides an intriguing possibility to target endometrial-infiltrating ASCs for delivery of therapeutic drugs.

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