Introduction: Esophageal adenocarcinoma (EAC) has become the predominant type of esophageal cancer in the United States, and prognosis of EAC remains poor despite modern combination therapies. Therefore, new therapeutic approaches are urgently needed. Hypoxia or insufficient tissue oxygenation contributes to EAC aggressiveness and poor clinical prognosis. Typically, EAC cell lines are used in two-dimensional (2D) cultures for screening of anticancer agents, and they do not represent the hypoxic tumor microenvironment. In this study, we established a novel three dimensional (3D) spheroid co-culture model of EAC. We then observed treatment response of chemo and hypoxia-targeting therapies in 2D, 3D and in vivo models of experimental EAC.

Methods: EAC 3D spheroids were generated from co-culturing human EAC and fibroblast cell lines. NanoCulture® plates and dishes were used for 3D spheroid cultures. Hypoxic status was detected by adding hypoxia probe LOX-1 and fluorescent microscopy. Paclitaxel (PT), carboplatin (CP) and nanoparticle albumin-bound paclitaxel (NPT) were used as chemotherapeutic agents, whereas acriflavin was used as hypoxia-targeting agent. In vitro cell growth was detected by WST-1 assay, in vivo tumor growth was detected by measuring subcutaneous xenograft using OE19 EAC cells, apoptosis was detected by analysis of cleaved caspase 3 and PARP expressions, hypoxia-targeting was detected by analysis of HIF-1α expression and stem cell phenotype was detected by flow cytometric analysis of aldehyde dehydrogenase (ALDH) activity.

Results: The results demonstrated that the 3D culture was more resistant to antiproliferative/proapoptotic effects of chemotherapeutic agents PT, CP, NPT and their combinations over 2D monolayer culture. Contrary to that, hypoxia-targeting agent acriflavin showed stronger antiproliferative/proapoptotic effects in 3D culture than in 2D culture. We observed strong expression of hypoxia inducible factor-1α (HIF-1α) in 3D culture with no expression of HIF-1α in 2D culture, and acriflavin treatment completely abolished the HIF-1α expression in 3D culture. We also observed hypoxia inside the 3D culture spheroids, but not in cells grown in 2D culture. Interestingly, we observed a reduced number of ALDH positive cells in 3D culture after acriflavin treatment compared to that after NPT treatment, indicating the preferential antiproliferative effect of acriflavin over NPT on cancer stem cells. In addition, acriflavin showed significant antitumor efficacy both as monotherapy and in combination with NPT. The xenograft tumor growth inhibition rate after a 2-week treatment with acriflavin, NPT and their combination was 44.97, 58.35 and 67.29 percent respectively (p<0.05).

Conclusion: Thus 3D cultures may be better than 2D cultures in simulating the important in vivo tumor characteristic of hypoxia, and HIF-targeting therapy acriflavin could be a novel treatment strategy for EAC.

Citation Format: Md Sazzad Hassan, Marisa Lenga, Lucia Petrova, Urs von Holzen. Therapeutic targeting of hypoxia in experimental esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1230.