Abstract Purpose: Brain tumors develop within complex neural microenvironments shaped by dynamic interactions between tumor cells and surrounding neural cells. Recapitulating key features of these environments in vitro remains challenging. We build on a previously established 3D microfluidic platform that enables spatial organization and gradient formation characteristic of tumor microenvironments, while supporting non-destructive, longitudinal analysis. Within this context, astrocytes represent abundant and highly responsive components of neural microenvironments and actively engage inflammatory signaling pathways. Such interactions elicit context-dependent inflammatory responses in astrocytes. Among these, NF-κB–dependent transcriptional programs represent a major axis of astrocyte responsiveness to inflammatory cues. Here, we establish an adeno-associated virus (AAV) -based NF-κB reporter as a real-time readout of astrocyte inflammatory responses and demonstrate its compatibility with a previously established 3D microfluidic platform that enables spatially controlled microenvironments. Experimental Procedures: Human astrocytes derived from neural progenitors from previously approved sources were transiently transduced with adeno-associated viral (AAV) vectors to express an NF-κB–responsive NanoLuc reporter. Multiple AAV serotypes were evaluated to optimize astrocyte transduction efficiency. Astrocyte viability, morphology, and marker expression were assessed following viral infection. Endogenous NF-κB signaling was evaluated by quantifying total and phosphorylated p65 levels after TNF-α stimulation. AAV-infected astrocytes were incorporated into 3D collagen hydrogels within the previously described microfluidic platform, which supports spatial organization and non-destructive, longitudinal monitoring of cellular responses in complex microenvironments. Results: AAV-mediated transduction produced efficient reporter expression in human astrocytes without altering cell number or morphology. Expression of established astrocytic markers, including GFAP and ALDH1L1, was preserved following viral infection. TNF-α induced dose-dependent NF-κB activation, with comparable magnitude and kinetics of p65 phosphorylation in AAV-transduced and non-transduced astrocytes, indicating preserved endogenous inflammatory signaling. Among the tested vectors, AAV3 produced the most consistent reporter signal. Reporter astrocytes remained responsive within the microfluidic system, enabling real-time detection of inflammatory activation in a 3D environment. Conclusions: This work establishes an AAV-based NF-κB reporter in human astrocytes and integrates it into a 3D microfluidic platform, enabling non-destructive, real-time monitoring of astrocyte inflammatory signaling within spatially controlled microenvironments. AI-based tools were used to support text drafting and editing; all authors are responsible for the accuracy and integrity of the content. Citation Format: Anderson F. Cunha, Trish Hoang, John Van Herwynen, Rithvik V. Turaga, Jose M. Ayuso, Stevens Rehen, Helena. Borges. Development of a human astrocyte NF-κB reporter for real-time inflammatory signaling in 3D microfluidic platforms abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (6Suppl): Abstract nr B013.
Cunha et al. (Mon,) studied this question.