Abstract 7625: Development of a blood-based tumor hypoxia detection assay

Abstract We have developed a method designed for detection of hypoxia-based markers in the vesicles shed from tumor cells in vitro and in vivo to non-invasively assess the degree of hypoxia present in a patient (patent pending). Our initial studies indicated that vesicle proteins can be labeled by various nitroimidazoles and detected by western blotting of exosome lysates from blood samples or tissue culture. In addition, vesicles secreted from 4T1 murine breast tumor cells incubated with pimonidazole in hypoxia (0.5% O2) could be detected using flow cytometry or plate-reader fluorescence quantification of purified exosome samples assayed with a fluorescent anti-pimonidazole primary antibody. There was a 2.4-fold elevation in binding signal in exosomes sampled from hypoxic vs oxic culture. Recently, we have also studied the production of pimonidazole- labeled exosomes in the U118 human brain tumor cell line and found a marked difference in signal between exosomes isolated from aerobic culture vs. exosomes isolated in hypoxic culture. In 4T1 tumor tissue lysates, hypoxia induced a maximum over 8-fold increase in pimonidazole adducts by day 11 of tumor growth. However, at day 4 of tumor growth there was a maximum of 3.95-fold increase in adducts from exosomes isolated from a blood sample, and the signal decreased, yet remained elevated compared to samples from non-tumor bearing mice, in blood samples taken at day 7, 11 or 14 or tumor growth. We theorized that the more advanced the tumor becomes, the less blood circulation and more necrosis are present which prevents hypoxic exosomes from entering the circulation. In U118 brain tumor cell exosomes from in vitro culture, there were similar distinct signal enhancements found, where there was a 3.8 fold enhancement of pimonidazole adduct bands in exosomes harvested from U118 cells cultured in 0.5% O2 overnight compared to exosomes from cells cultured in normoxia. We have most recently investigated a click-chemistry based detection of nitroimidazoles in biological samples, thereby avoiding the need for antibody detection and thus improving the sensitivity and specificity. We have observed marked hypoxia-specific binding of the click-compatible dye in cells cultured in hypoxia and found a distinct reduced azido-aza associated peak using Raman spectroscopy, which could be a more efficient and rapid test for clinically obtained samples. Finally, a dot blot approach we developed has demonstrated that vesicles obtained from the blood of tumor bearing mice indicate an average signal increase of 40% in tumor-bearing mouse exosomes vs. animals without tumor. We surmise that the detection of vesicles being shed from a hypoxic niche could lead to a non-invasive blood test for tumor or ischemia presence, as well as a surrogate marker for treatment success or tumor recurrence. Efforts to correlate individual tumor hypoxic fraction and the exosome signal are ongoing to verify the specificity of the approach. Citation Format: Azemat Jamshidi-Parsian, Ruud Petrus Dings, Rajshekar A. Kore, Narasimhan Rajaram, Varsha Karunakaran, Robert J. Griffin. Development of a blood-based tumor hypoxia detection assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7625.