Abstract 3960: Therapeutic targeting of HIF-1 alpha induced CD73 expression in experimental esophageal adenocarcinoma
Authors: Hassan Md Sazzad , Ali Aktar , Ponna Saisantosh , Scofield Dimitri , Awasthi Niranjan , Jantz Mark , von Holzen Urs 
Published: April 2023

Introduction: Esophageal adenocarcinoma (EAC) is one of the most aggressive human cancers with poor prognosis, and the overall 5-year survival rate is less than 20 percent. Prognosis for EAC remains poor even with modern combination therapies due to high resistance to chemotherapy. Therefore, new therapeutic approaches for EAC treatment improvements are urgently needed. Hypoxia or insufficient tissue oxygenation contributes to cancer aggressiveness and poor clinical prognosis. Overexpression of hypoxia-inducible factor 1-alpha (HIF-1 alpha) and immunosuppressive CD73, an ecto-5’-nucleotidase enzyme in cancer can give rise to tumor progression with drug resistance. CD73 has never been proposed as a therapeutic target in EAC and its relationship with hypoxia or HIF-1 alpha has not also been investigated in EAC. In this study, we therefore investigated the therapeutic targeting of HIF-1 alpha and CD73 by acriflavine in experimental EAC.

Methods: Hypoxia in EAC cells were induced by 3D culture and hypoxic exposure. NanoCulture® plates and dishes were used for 3D cultures. For hypoxic exposure, cells were placed in a sealed modular incubator chamber flushed with a gas mixture containing 1% O2, 5% CO2 and 94% N2. Hypoxic status was detected by adding hypoxia probe LOX-1 and fluorescent microscopy. Nanoparticle albumin-bound paclitaxel (NPT) was used as chemotherapeutic agent, whereas acriflavine was used as hypoxia-targeting agent. In vitro cell growth was detected by WST-1 and Cell Titer-Glo (CTG) luminescent assays, in vivo tumor growth was detected by measuring subcutaneous xenografts, apoptosis was detected by cleaved caspase 3/PARP expressions and hypoxia-targeting was detected by HIF-1 alpha/CD73 expressions.

Results: We observed overexpression of both HIF-1 alpha and CD73 in 3D culture and hypoxic exposure of EAC cells. Interestingly acriflavine treatment drastically inhibited both HIF-1 alpha and CD73 expression in EAC 3D culture and hypoxic exposure. 3D culture was more resistant to antiproliferative effect of chemotherapeutic agent NPT over 2D monolayer culture. Contrary to that, hypoxia-targeting agent acriflavine showed stronger antiproliferative effects in 3D culture than in 2D culture. We also observed hypoxia inside the 3D culture spheroids. In addition, acriflavine showed significant in vivo antitumor efficacy both as monotherapy and in combination with NPT. In subcutaneous xenografts using OE19 EAC cells, acriflavine monotherapy exhibited a significant decrease in relative tumor volume to 55.02% compared to control (p=0.04) and addition of NPT with acriflavine also showed a significant enhancement effect of tumor regression as tumor size decreased to 32.70% compared to control (p=0.002).

Conclusion: These results support the potential of acriflavine as HIF-1 alpha and CD73 targeting and its combination with chemotherapy NPT as an effective option for EAC therapy.

Citation Format: Md Sazzad Hassan, Aktar Ali, Saisantosh Ponna, Dimitri Scofield, Niranjan Awasthi, Mark Jantz, Urs von Holzen. Therapeutic targeting of HIF-1 alpha induced CD73 expression in experimental esophageal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3960.