(C-111) Immuotherapy on Chip to Study T-Cell Transmigration toward Tumor

Introduction:: The tumor microenvironment (TME) contributes to the tumor niche in highly complex ways, including tumor growth, immune response, and drug delivery1. Tissue chips based on vascularization have successfully simulated tumor fate with vascular structures, drug delivery, and immune cell trafficking in vitro. Many studies have used angiogenesis to promote the delivery of anticancer drugs and immune cells in the TME^2,3. One primary obstacle is ensuring that tumor models accurately replicate the interactions between vessels and tumor spheroids4. The capillary network can transfer immune cells from the main blood vessels to the outflow and recruit immune cells through cytokines secreted by the tumors or the immune response5. The current approaches focused on angiogenesis cannot generate vascularized tumors effectively because of the "dead ends" in the vessel network near the tumor. Therefore, alternative strategies to vascular-supported tumor models are needed.

We presented a microfluidic 3D vascular-supported tumor model consisting of the endothelial lumen and vessel network to deliver immune cells. Generating interstitial flow can assist the vessel assembly and form a perfusable network. To mimic the tumor structure supported by blood vessels, we deposited the tumor spheroids to the outlet of the vessel network, which is embedded in the hydrogel. Our study illustrates the process of T cell transport through the vessel network and interacting with tumor spheroids. Furthermore, we evaluate the cytotoxicity of PD-1/PD-L1 immune checkpoint inhibition after T cell activation.


Materials and Methods:: The human colorectal carcinoma cell line, HCT116, was used to form tumor spheroids by the AggreWell. Human umbilical vein endothelial cord cells (HUVECs) were mixed with the 5mg/mL fibrinogen from bovine plasma and 5U/mL thrombin and loaded into the gel region of the microfluidic device. The endothelium lumen was generated by the HUVECs monolayer, and then the tumor spheroids were loaded, allowing attachment to the vessel network outlet. To stimulate the T cells, the supplement of anti-CD3 and anti-CD28 was inducted into the medium. The inertial flow driven by the hydraulic pressure helps the vessel network formation. Afterward, T cells were introduced into the chip, allowing the T cells to transport and aggregate around tumor spheroids for 48 hours. Meanwhile, the PD-1/PD-L1 immune checkpoint inhibitor, IB3, was added to the medium to activate the immune response further. The tumor viability was evaluated by Calcein AM and PI live-dead staining for immune cytotoxicity.


Results, Conclusions, and Discussions:: The microfluidic 3D vascular-supported tumor model described in the study uses interstitial flow to promote the assembly and perfusion of the vessel network. The model also allows the tumor to adhere to the vessel network outlets, facilitating the transportation of T cells toward the tumor within a high density of vessel network. This finding suggests that dense capillary vessels are crucial for immune cell delivery and immune repositioning.

In our preliminary experiments, we investigated the effects of immune checkpoint inhibition on both tumor cells and T cells and evaluated the delivery and efficacy of immunotherapy in T cells. After 48 hours of T cell activation, we found that the activated T cells exhibited higher cytotoxicity towards the tumor cells, resulting in a 3.6-fold increase in cytotoxicity compared to the groups without T cell activation. In addition, when the PD-1/PD-L1 inhibitor IB3 was used, a significant decrease in tumor viability was observed, 1.6 times lower than those without IB3 groups. Combining T cell activation and immune checkpoint blockade reduced tumor viability to less than 60%.

In conclusion, we demonstrate a vessel-tumor-on-chip model that simulates the interaction between endothelial, cancer, and immune cells. It represents the process of T-cell transportation from different vessel-tumor compositions. By incorporating immune checkpoint inhibition, the platform can assess the effectiveness of immunotherapy. This innovative approach of employing the vessel-supported tumor platform has promising possibilities in screening and evaluating immunotherapy's significance in the preclinical stage.


Acknowledgements (Optional): :

References (Optional): : 1. Liu, X. et al. Tumor-on-a-chip: from bioinspired design to biomedical application. Microsyst Nanoeng 7, 50 (2021).

2. Ayuso, J. M. et al. Evaluating natural killer cell cytotoxicity against solid tumors using a microfluidic model. Oncoimmunology 8, 1553477 (2019).

3. Kim, D. et al. Vascularized Lung Cancer Model for Evaluating the Promoted Transport of Anticancer Drugs and Immune Cells in an Engineered Tumor Microenvironment. Adv. Healthc. Mater. 11, e2102581 (2022).

4. Gurski, L. A., Jha, A. K., Zhang, C., Jia, X. & Farach-Carson, M. C. Hyaluronic acid-based hydrogels as 3D matrices for in vitro evaluation of chemotherapeutic drugs using poorly adherent prostate cancer cells. Biomaterials 30, 6076–6085 (2009).

5. Lee, W. S., Yang, H., Chon, H. J. & Kim, C. Combination of anti-angiogenic therapy and immune checkpoint blockade normalizes vascular-immune crosstalk to potentiate cancer immunity. Exp. Mol. Med. 52, 1475–1485 (2020).