Abstract 116: CD73+cancer-associated fibroblasts augment cancer stemness and drug resistance in hepatocellular carcinoma through adenosine accumulation

Abstract Background: Hepatocellular carcinoma (HCC) exhibits significant therapeutic resistance due to its high cancer stemness, which is strongly influenced by the tumor microenvironment (TME). Extracellular adenosine (eAdo), a critical metabolic regulator within the TME, is primarily generated from extracellular ATP via the enzymatic activity of CD73 (NT5E), which is highly expressed in cancer-associated fibroblasts (CAFs). In our pilot study, we found that the CAF populations marked by CD73 were drastically induced in a lenvatinib-resistant immune-competent mouse HCC model. Based on this data, we hypothesize that CD73+ CAFs confer cancer stemness-driven drug resistance via eAdo accumulation in the TME. Methodology: Liquid chromatography-mass spectrometry (LC-MS) was used to quantify adenosine levels, whereas flow cytometry was used to assess stemness-related surface markers. Tumor-initiating frequencies were evaluated using a limiting dilution assay, and CRISPR-Cas9-mediated CD73 knockdown in CAFs, the precursors of CAFs, was employed to validate CD73’s functional role. Western blot analysis was conducted to investigate the pathway activation.Results: Our results showed that CD73 is a functional marker of CAFs, and its overexpression is significantly correlated with poor prognosis of HCC patients. LC-MS detected a significant amount of adenosine in CAFs when compared with HCC cells, while a notable reduction in adenosine levels was observed in the conditioned medium (CM) of CD73-knockout CAFs. Interestingly, ATP-supplemented CM of CAF augments the cancer stemness of HCC cells, while its effect is offset upon CD73 knockout in CAFs. Echoed to this finding, adenosine treatment (10 µM) exerted similar effects, confirming the critical role of CD73 in CAF-driven cancer stemness via adenosine accumulation. Mechanistic studies have demonstrated that adenosine promotes stemness through activation of the A2AR/AKT/mTOR signaling pathway in HCC cells. Conclusion: This study establishes that CD73+ CAFs drive HCC stemness and drug resistance by modulating the TME through eAdo accumulation. Targeting CD73 and its downstream signaling pathways represents a promising therapeutic strategy to overcome drug resistance which potentially opens a new therapeutic avenue for HCC treatment. Citation Format: Hui Hai Yang, Carmen Oi Ning Leung, Terence Kin-Wah Lee. CD73+cancer-associated fibroblasts augment cancer stemness and drug resistance in hepatocellular carcinoma through adenosine accumulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 116.