R20 - A Microfluidic System for Real-Time Imaging of CAR T Cell Function in a 3D Hypoxic Tumor Microenvironment

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

JJ

Jiayi Jiang

Student
University of Southern California
Los Angeles, California, United States

Co-Author(s)

CK

Cem Kilic, MS

PhD Student
University of Southern California
Los Angeles, California, United States
Tianze Paul Guo

Research Assistant
USC
Los Angeles, California, United States

Last Author(s)

Keyue Shen, PhD

Associate Professor
University of Southern California, California, United States

Primary Investigator(s)

Keyue Shen, PhD

Associate Professor
University of Southern California, California, United States

Introduction: : CAR T cells have been effective in treating B cell malignancies, but their success has been limited in solid tumors due to the immunosuppressive tumor microenvironment (TME). Glioblastoma (GBM), one of the aggressive solid tumors, is characterized by a hypoxic, immunosuppressive TME that limits immune cell function. Hypoxia is a hallmark of solid tumors and contributes to the immunosuppressive TME by promoting cancer cell metabolic and immunologic reprogramming, ultimately leading to poor treatment outcomes. Thus, new strategies are needed to improve CAR T-cell functionality within the hostile TME.
To address this, we incorporated a workflow into an established hypoxic tumor model developed in our lab, enabling high-throughput, real-time tracking of CAR T-cell cytotoxicity and infiltration within both hypoxic and normoxic tumor environments. This assay platform provides a physiologically relevant preclinical tool for evaluating the efficacy of CAR T-cell therapies.

Materials and

Methods: : Engineering of UnaG-expressing U251 cancer cell lines
We engineered the U251 GBM cell line to conditionally express the oxygen-independent fluorescent protein UnaG under the control of a hypoxia-inducible factor 1 alpha (HIF1a) promoter (HRE-UnaG U251) (Figure 1a). This enables green UnaG fluorescence specifically under hypoxic conditions that activate HIF1a transcriptional activity, serving as a real-time hypoxia reporter within the TME.
Additionally, to monitor tumor cell dynamics and assess CAR T-cell cytotoxicity in real time, we generated a second U251 cell line constitutively expresses UnaG under the control of the PGK promoter (PGK-UnaG U251) (Figure 1a). This construct provides a fluorescent signal for tracking tumor cells regardless of oxygen levels.

Live imaging of T cells
T cells were stained using CellTracker Deep Red dye to enable live imaging. A total of 100k/80ul stained cells were added into each device through the side channels.

Hypoxic and normoxic tumor model
Hypoxic and normoxic tumor microfluidic devices were fabricated in our lab (Figure 1b).1 Hypoxia was generated by placing a polycarbonate (PC) film to the device center as an oxygen barrier. PGK-UnaG U251 cells in collagen gel was injected into the central island to form 3D tumors. After 48hrs, stained T cells were added through the side channels to simulate immune cell infiltration. Devices were incubated under the same conditions and were imaged every 24hrs to track either HIF1a reporter activity or T cell infiltration and cytotoxicity.

Results, Conclusions, and Discussions:: To validate the functionality of the hypoxic and normoxic microfluidic tumor models, we introduced U251 cells engineered to express UnaG under the control of a hypoxia-responsive HRE promoter. Tumors were established using these HRE-UnaG U251 cells, and UnaG fluorescence was monitored over time. In the hypoxic devices, we observed a progressive increase in green UnaG fluorescence originating from the tumor core, in contrast to the normoxic models, where fluorescence remained minimal (Figure 1c). These results confirm the development of hypoxia and HIF-1α–mediated transcriptional activation within the tumor microenvironment, demonstrating the physiological relevance of the model.
Next, to assess T cell-mediated cytotoxicity, PGK-UnaG U251 were utilized. The constitutive expression of UnaG was used as proxy for tumor viability, and a loss in fluorescence was interpreted as cancer cell death. The results confirmed that there was less tumor cell killing in the hypoxic device, as shown by retained green fluorescence signal over the 72hrs. The loss of the green signal in the normoxia devices validated CAR T cell cytotoxicity, which shows the negative impact of hypoxia on immune cell function (Figure 1d).

In this study, we utilized a previously developed microfluidic platform to investigate CAR T-cell function in real-time within oxygen-deprived TMEs. To validate device functionality, we employed U251 cells engineered with a hypoxia-responsive HRE-UnaG reporter to monitor HIF1a activity. A progressive increase in UnaG fluorescence from the tumor core confirmed the establishment of hypoxic conditions and the functionality of the hypoxia-responsive biosensor.
Following this validation, we assessed CAR T-cell activity within the platform and observed reduced cytotoxicity under hypoxic conditions, consistent with hypoxia-induced resistance. Together, these findings demonstrate that the platform effectively models key features of the hypoxic TME and its impact on CAR T-cell function.
Looking forward, this system offers a physiologically relevant framework for evaluating combination therapies aimed at overcoming hypoxia-associated immunosuppression. Ongoing efforts will focus on optimizing culture conditions to further enhance the fidelity of the immunosuppressive environment. Ultimately, this platform serves as a powerful tool for characterizing resistant tumor phenotypes and accelerating the development of more effective, targeted CAR T-cell therapies.

Acknowledgements and/or References (Optional): : This work has been supported by an NIH NCI grant R01CA220012, a STOP CANCER Marni Levine Memorial Research Career Development Award, a Ming Hsieh Institute Research Award, a USC Provost's undergraduate research fellowship, and USC Viterbi School of Engineering.

[1] Jeong Min Oh, Hydari Masuma Begum, Yao Lucia Liu, Yuwei Ren, and Keyue Shen
ACS Biomaterials Science & Engineering 2022 8 (7), 3107-3121
DOI: 10.1021/acsbiomaterials.2c00207