Spheroids reveal hypoxia‑driven spatial restriction of adenoviral infection

www.nature.com/scientificreports OPEN Spheroids reveal hypoxia‑driven spatial restriction of adenoviral infection Tamara Büttner1, Xiaoyan Wang2, Brenda Krishnacoumar1, Athanasios Papadamakis2, Suna Cicek1, Yves Schild1, Sandra Winning1, Elisabeth Littwitz-Salomon2,4, Anja Ehrhardt3, Ulf Dittmer2, Wibke Bayer2, Joachim Fandrey1 & Anna Malyshkina1 Hypoxia is a hallmark of solid tumors and represents a major barrier for effective cancer therapies, including oncolytic virotherapy. While adenoviruses are widely studied as oncolytic agents, the impact of tumor-associated hypoxia on viral infection and spatial spread remains incompletely understood. Here, we investigated how oxygen availability influences adenovirus infection in two-dimensional (2D) cultures and three-dimensional (3D) tumor spheroids. We confirmed that cell lines commonly used in adenovirus research (HEK293A, A549), as well as KP4 pancreatic cancer cells, exhibited a physiological response to hypoxia. In KP4 monolayers, hypoxia strongly reduced adenoviral protein production. To model oxygen gradients found in solid tumors, we established stable KP4 spheroids and performed spatial analysis of HAdV5_GFP infection. When virus was added during spheroid formation and hypoxia development, infection was largely restricted to the well-oxygenated outer rim. In contrast, inoculation of virus under normoxia prior to spheroid formation resulted in a more uniform distribution of infected cells throughout the spheroid. Together, our findings demonstrate that hypoxia not only suppresses adenoviral replication in cell culture but also shapes the spatial pattern of infection in 3D tumor models, highlighting the importance of hypoxia-relevant 3D systems in preclinical evaluation of oncolytic adenoviruses. Oncolytic viruses (OV) represent a promising class of emerging anticancer immunotherapies, utilizing the natural ability of certain replication-competent viruses to selectively infect and destroy tumor cells while sparing normal, non-neoplastic tissues. Oncolytic human adenoviruses (HAdV), in particular, are a prominent focus in the development of cancer therapies. First, they have a HAdV type-specific natural tropism for a broad variety of cell types, allowing efficient tumor cell targeting. Second, their genome can accommodate large inserts, making them highly versatile for therapeutic engineering. Additionally, adenoviruses do not integrate into the host genome and stimulate strong immune response, amplifying antitumor activity1. Effective cancer treatment using oncolytic HAdV relies on the viral spread from infected to uninfected cells. To date, there has been limited success in clinical studies in the use of replicating adenoviruses. For instance, Tasadenoturev, HAdV type 5-based OV, showed a strong safety profile but achieved only modest efficacy in both pediatric2 and adult3 glioma patients. Along these lines, CG0070, a GM-CSF-armed HAdV type 5-based oncolytic virus developed for bladder cancer, demonstrated mixed results and did not become a widely adoptive option4. Similarly, Oncorine, another HAdV5-based therapy approved in China, elicited improved responses in certain settings, yet its broader clinical use remains limited5,6. While these examples do not achieve complete therapeutic efficacy, they highlight the potential of oncolytic adenoviruses as a promising approach that requires further investigation. The difference between laboratory findings and clinical results may arise from multiple factors, including preexisting immunity against the administered virus7, the dense extracellular matrix and aberrant tumor vasculature that generate interstitial hypertension8, and the negative impact of tumor hypoxia. Hypoxia, the condition characterized by low oxygen levels, is frequently observed in solid tumors9,10. It is difficult to determine the hypoxic state in tumors due to variations in oxygen content between tissues, as well as differences in tumor size and measurement methods, and tissue oxygenation is highly variable, also within the same organ. 1Institute of Physiology, University of Duisburg-Essen, University Hospital Essen, Essen, Germany. 2Institute for Virology, University of Duisburg-Essen, University Hospital Essen, Essen, Germany. 3Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Faculty of Health, Witten/Herdecke University, Witten, Germany. 4Institute for the Research on HIV and AIDS-associated Diseases, University of Duisburg-Essen, University Hospital Essen, Essen, Germany. email: Scientific Reports | (2026) 16:15864 | https://doi.org/10.1038/s41598-026-53319-4 1 However, the available results indicate that the measurement of tumor partial pressure of oxygen (pO2) in patients (polarographic technique) has demonstrated the presence of low values (< 10 mmHg) or equal 1,3% O2 in several different tumor types, including pancreatic cancer, head and neck tumors, breast cancer, cervical cancer, and melanoma. Specifically, the tumor microenvironment exhibits a median O₂ concentration of 1.3%11,12. In contrast, adenoviruses naturally infect tissues that are typically exposed to normal ambient oxygen concentrations (normoxia). Moreover, it was shown that hypoxia reduces adenoviral replication in 2D cell culture13,14. Sustained interest in the development of 3D culture models in recent years reflects the growing demand for physiologically relevant systems in cancer research. These models are particularly valuable in tumor biology, as they recapitulate key features of the tumor microenvironment, including oxygen gradients. Spheroids typically display proliferative, normoxic (or more precisely: physoxic) cells at the periphery, an intermediate hypoxic zone, and a central region that is often severely hypoxic, anoxic, and/or necrotic. Importantly, such gradients arise intrinsically during spheroid formation, avoiding the need for external hypoxic chambers. As a result, spheroids provide a more natural representation of tumor physiology, hypoxia-driven processes, and therapeutic responses. Thus, they are suitable to enhance the predictive value of preclinical studies for in vivo and clinical outcomes. In the present study, we examined how the intrinsic oxygen gradients that develop within spheroids influence HAdV infection. Our results show the hypoxic regions in the inner part of the spheroid which strongly correlate with limited infection rates. Thus, we indicate that such spheroids represent a valuable tool for cancer research and virotherapy. Specifically, we determined that 3D spheroid cultures recapitulate the inhibitory effects of hypoxia observed in 2D monolayers, thereby providing a more physiologically relevant model to study adenovirus replication in the context of tumor biology. Results Hypoxia stabilizes HIF-1α in A549, HEK293A, and KP4 cells First, we selected human cell lines as model systems: A549 lung carcinoma and HEK293A human embryonic kidney cells, wh (...truncated)