R32 - Engineering In Vitro Vasculature Model to Evaluate Tumor Endothelium-Mediated CAR-T Cell Infiltration

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Shijia Sun

Undergraduate Researcher at USC Laboratory for Integrative Biosystems Engineering
University of Southern California, United States

Co-Author(s)

CK

Cem Kilic, MS

PhD Student
University of Southern California
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: : T-cells engineered to express chimeric antigen receptors (CARs) have led to major advances in treating hematological malignancies, but hypoxic tumor microenvironment (TME) reduces T-cell efficacy in solids tumors, including glioblastoma (GBM). Characterized by hypervascularity, GBM tumors show low levels of immune cell infiltration as vasculature associated with hypoxic tumor regions is characterized by abnormal vessel organization [1]. To improve CAR-T cell efficacy in solid tumors, a deeper understanding of tumor endothelium and immune cells is crucial, but current in vitro models often fail to fully capture these complex interactions [2]. To address this, we created a 3D culture platform incorporating a uniform monolayer of human umbilical vein endothelial cells (HUVECs) atop a 3D hypoxic cancer cell embedded hydrogel to simulate the hypoxic tumor-endothelium interface. The device not only enables on-device real-time transepithelial electrical resistance (TEER) measurement to quantify membrane permeability but also supports live imaging of the transwell component.

Materials and

Methods: : Device

Design: A device was designed with Fusion 360 to create the endothelial-tumor interface and created using micromilling. The device features a tumor gel island and pillars to hold the transwell in place so that the endothelial layer can directly contact the tumor gel (Fig 1A). The mold was then vapor polished, washed, sterilized by UV and coated before adding the tumor cells.
Endothelial-Tumor Co-Culture: Human umbilical vein endothelial cells (HUVECs) were seeded in porous transwells for 7-days culture to form confluent monolayer. On day 0, U251 GBM cells were embedded in collagen matrix to form tumor gels within the assembled devices. Transwell was then inserted into the device and pushed down to contact the surface of tumor hydrogel. Monolayer integrity was validated with calcein AM imaging, and transwells were subsequently immunostained with VE-cadherin to evaluate junctional organization. TEER was measured over consecutive days to assess monolayer permeability.

Results, Conclusions, and Discussions:: To establish a uniform monolayer across the transwell membrane, low-passage HUVECs were seeded at a density of 30,000 cells/cm² on Day -7. By Day 0, monolayer formation was confirmed via live imaging using 1.0 μM calcein AM staining and assessing barrier permeability through TEER measurements (Figure 1B). TEER value of monolayers prior to device assembly were 38.5 Ohm×cm2, which agrees with previous reports of HUVEC barrier integrity on transwell inserts [3].
Under hypoxic conditions, cancer cells upregulate pro-angiogenic factors such as vascular endothelial growth factor (VEGF), which can increase vascular permeability and disrupt endothelial integrity [4]. In this model, the extent of hypoxia within the tumor gel is related to cancer cell density in the hydrogel bulk. To assess the impact of tumor cell density on the endothelial barrier, 3D tumor models were created using U251 GBM cells embedded in 3 mg/mL collagen I hydrogels at two seeding densities (1 million cells/mL and 5 million cells/mL) seeded into the culture vessel, and the HUVEC-seeded transwells were positioned on top prior to gel crosslinking.
Calcein AM staining indicated monolayer viability was maintained through Day 5 for both tumor densities (Figure 1C). TEER measurements showed a sharper decline in barrier function in co-cultures with higher tumor cell density by day 2 accompanied by a more dramatic drop by day 5 (Figure 1D). Overall, these results confirmed the influence of cancer cell density within the tumor bulk on endothelial barrier function.
Here, we present a proof-of-concept for a novel, reproducible 3D endothelial–tumor co-culture platform using HUVECs, which enables real-time, on-device assessment of endothelial barrier function through TEER and live-cell imaging. To enhance physiological relevance, we will next incorporate a tri-culture system consisting of astrocytes, pericytes, and human brain microvascular endothelial cells on a transwell membrane to model the GBM-associated blood–brain barrier (BBB), helping us better recapitulate the GBM tumor vasculature in vitro.
Ultimately, we aim to integrate CAR T cells into the system to evaluate their interaction with the endothelial–tumor interface and quantify their infiltration into the tumor. This advanced platform will support the preclinical evaluation of immunotherapeutic strategies targeting solid tumors.

Acknowledgements and/or References (Optional): : [1] Campisi, M., Shin, Y., Osaki, T., Hajal, C., Chiono, V., & Kamm, R. D. (2018). 3D self-organized microvascular model of the human blood-brain barrier with endothelial cells, pericytes and astrocytes. Biomaterials, 180, 117–129. https://doi.org/10.1016/j.biomaterials.2018.07.014
[2] Straehla, J. P., Hajal, C., Safford, H. C., Offeddu, G. S., Boehnke, N., Dacoba, T. G., Wyckoff, J., Kamm, R. D., & Hammond, P. T. (2022). A predictive microfluidic model of human glioblastoma to assess trafficking of blood–brain barrier-penetrant nanoparticles. Proceedings of the National Academy of Sciences, 119(23), e2118697119. https://doi.org/10.1073/pnas.2118697119
[3] Kim, D., Eom, S., Park, S. M., Hong, H., & Kim, D. S. (2019). A collagen gel-coated, aligned nanofiber membrane for enhanced endothelial barrier function. Scientific reports, 9(1), 14915. https://doi.org/10.1038/s41598-019-51560-8
[4] Tzeng, H. T., & Huang, Y. J. (2023). Tumor Vasculature as an Emerging Pharmacological Target to Promote Anti-Tumor Immunity. International journal of molecular sciences, 24(5), 4422. https://doi.org/10.3390/ijms24054422