Role of Hypoxia in RetinoblastomaY79 cell line

Abstract

Purpose : Low oxygen tensions (hypoxic microenvironment) is common in many advanced tumors, and are believed to play a role in tumor progression and response to treatment, more so in tumors like Retinoblastoma, a malignant intraocular tumor. In this study, we aimed to evaluate the effect of the hypoxic microenvironment on growth, proliferation, metastasis and stemness potential in Y79 Retinoblastoma cell lines through in-vitro and in-vivo assays.

Methods : Y79 Rb cells were maintained at 37^oC in RPMI-1640 with 10% FBS. Hypoxic treatment was carried out either in hypoxic chamber (Don Whitley Scientific, UK) with1% O₂ v/v and 5% CO₂ v/v or by chemical induction using 100uM CoCl₂(hypoxia mimic chemical). The expression of hydroxy-Hypoxia Inducible Fator-1α (hyHIF-1α) was measured using Western blot analysis. Proliferation of hypoxic and normoxic cells were determined by EdU proliferation assay; nodule growth and metastases were analyzed by Chick Embryo-CAM assay by transplanted 2*10⁶ normoxic and hypoxic primed Y79 Rb cells. Genetic markers for angiogenesis (VEGF), metastasis (MACC1) and stemness (Oct-4, CD44) were evaluated in vitro Y79 cells cultured in hypoxic and normoxic conditions.

Results : Western blot analysis showed the highest level of hyHIF-1α protein after 16hours of hypoxic conditions and was similar in both CoCl₂-induced and chamber-induced hypoxic cells. Y79 Rb cells cultured for 96 hours in hypoxia showed reduced proliferation and tumor nodule formation on the CAM (n=4), and increased metastasis in abdominal region of Chick Embryo compared to normoxic cells (Fig 1).Gene expression analyses for a panel of markers for hypoxia, angiogenesis, metastasis and stemness are ongoing, with initial screening indicating low level CD44 expression, previously notable for its lack in Y79 cells.

Conclusions : This study provides evidence of dynamic hyHIF-1αprotein expression and cell proliferation in Y79 Rb cells during hypoxic preconditioning over 96 hours. Despite the reduction in proliferation, hypoxia-primed Y79 Rb cells promoted metastasis in the chick embryo model compared to the normoxic cells, supporting the potential role of hypoxia in the dissemination of tumor cells to metastatic sites. Of particular interest are preliminary reports of CD44 expression in Y79 Rb cells, which require further validation. It is possible that the promotion of stem-like characteristics under hypoxia promote the spread of cancer cells.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.