Constructing a novel Dynamic Three Dimensional in Vitro Model for Investigation of Ovarian Carcinoma Progression at the Different Anatomic Sites of the Disease

Introduction: Ovarian carcinoma (OC) metastases reported to have a much higher malignancy and therapy resistance compare to the primary tumor. In addition, transition from a solid form of the tumor to a detached cellular spheroid form, in effusion presents a special challenge both in designing effective drug treatment and in understanding cellular processes during disease progression. There is a lack of an appropriate in vitro model of the tumor microenvironment mimicking three different sites of OC. Emerging evidence shows the various functions of sphingolipids in cellular trafficking and cell motility. Specifically, Sphingolipid-1-phosphate (S1P) has been implicated as a potent regulator of cancer progression. Herein, our main objective was to examine the main S1P-associated genes from different OC anatomical sites. Based on the acquired data, we aim to establish a three-dimensional (3D) in vitro culture system of OC cells, using alginate macroporous-based scaffolds under dynamic conditions to mimic the 3D tumor microenvironment.

Material and method: The basal mRNA levels of S1P receptors’ expression and relative proportion were established by RT-PCR analyzing of 250 OC samples. A multi chamber perfusion system was applied, designed for optimal flow conditions, homogeneity of the flow within all scaffolds and control over oxygen level, and thus, better mimicking in vivo conditions. 433 or ES2 OC cells were cultured in 4 different culture forms for 72 h: (a) monolayer (b) seeded into alginate porous scaffolds, under static conditions, (c) alginate scaffolds in perfusion bioreactor, (D) cell spheroids. RT-PCR analysis was conducted.

Results: Cultivation of 433 cells within alginate scaffolds, cultured under flow velocity of 50 mL/h resulted in S1P receptor mRNA expression levels similar to those of primary samples from OC patients. Moreover, the relative proportions of each receptor were also similar to those of the primary tumor samples. By contrast, no such similarity was detected for other culture methods: monolayer, spheroid cultures or static 3D alginate scaffolds. Similarly, ES2 culture in bioreactor lead to S1P receptor mRNA expression levels and relative proportions similar to those of effusion samples OC patients, compared to all other culture methods.

Conclusions: A novel in vitro model was designed and established for Primary OC, appropriated for experiments with a relatively large number of the samples.