High-Throughput 3D In Vitro Tumor Vasculature Model for Real-Time Monitoring of Immune Cell Infiltration and Cytotoxicity

Abstract

Recent advances in anticancer therapy have shown dramatic improvements in clinical outcomes, and adoptive cell therapy has emerged as a type of immunotherapy that can modulate immune responses by transferring engineered immune cells. However, a small percentage of responders and their toxicity remain as challenges. Three-dimensional (3D) in vitro models of the tumor microenvironment (TME) have the potential to provide a platform for assessing and predicting responses to therapy. This paper describes an in vitro 3D tumor model that incorporates clusters of colorectal cancer (CRC) cells around perfusable vascular networks to validate immune-cell-mediated cytotoxicity against cancer cells. The platform is based on an injection-molded 3D co-culture model and composed of 28 microwells where separate identical vascularized cancer models can be formed. It allows robust hydrogel patterning for 3D culture that enables high-throughput experimentation. The uniformity of the devices resulted in reproducible experiments that allowed 10× more experiments to be performed when compared to conventional polydimethylsiloxane (PDMS)-based microfluidic devices. To demonstrate its capability, primary natural killer (NK) cells were introduced into the vascularized tumor network, and their activities were monitored using live-cell imaging. Extravasation, migration, and cytotoxic activity against six types of CRC cell lines were tested and compared. The consensus molecular subtypes (CMS) of CRC with distinct immune responses resulted in the highest NK cell cytotoxicity against CMS1 cancer cells. These results show the potential of our vascularized tumor model for understanding various steps involved in the immune response for the assessment of adoptive cell therapy.

Vascularized Lung Cancer Model for Evaluating the Promoted Transport of Anticancer Drugs and Immune Cells in an Engineered Tumor Microenvironment

“…[ 53 ] Furthermore, tumor cells are known to retard or degrade vascularization in a tumor‐vasculature coculture model. [ 54 ] Although various factors may contribute to angiogenesis, we presume that the tumor‐secreted factors delivered by flow can suppress the growth and maturation of the main blood vessel. In the tumor‐only condition, the main blood vessel grew, suggesting the importance of flow‐mediated delivery of soluble factors in blood vessel growth and maturation.…”

A multi-organ-on-chip to recapitulate the infiltration and the cytotoxic activity of circulating NK cells in 3D matrix-based tumor model

“…For instance, the cultivation of cancer cells and immune cells in two separated compartments, connected through microchannels within the same planar chip, allows the establishment of a biochemical gradient for immune cell recruitment from the first chamber toward the side chamber, hosting tumor cells. However, most of these tumor-on-chip are used either in static conditions ( Hsu et al, 2012 ; Businaro et al, 2013 ; Parlato et al, 2017 ; Pavesi et al, 2017 ; Lee et al, 2018 ; Guo et al, 2019 ; Um et al, 2019 ; Yu et al, 2019 ; Ren et al, 2020 ; Ayuso et al, 2021 ), or through simple gravity-driven flow ( Song et al, 2021 ), or perfusion with very low fluid flow rates, being far from physiological conditions. For instance, in a tumor-on-chip model, Aung et al perfused T cells applying a fluid flow rate of 50 ul/hr (corresponding to 0,8 ul/min) ( Aung et al, 2020 ); similarly, in an immune system-on-chip recently developed by Goyal et al, immune cells were cultured through a flow rate of 60 ul/hr (corresponding to 1 ul/min) ( Goyal et al, 2022 ).…”

“…Importantly, the over-miniaturization of the “classical” microfluidic devices, beside failing in recapitulating the biological and clinical features of TME, possibly leads to the underrepresentation of the tumor heterogeneity occurring into the clinic. Moreover, this also carries some technical limitations related to downstream biochemical assays and to small volumes/bubbles handling ( Ayuso et al, 2021 ; Song et al, 2021 ): indeed, the use of very small cell numbers (e.g., 1,000 cells/spheroid ( Ayuso et al, 2019 ), 2,500 cells/well ( Gopal et al, 2021 )) and/or very small volumes (e.g., 10–20 ul containing 10 5 -2 × 10 5 cells ( Ren et al, 2020 )) are not always suitable for standard analytical methods such as immunofluorescence and flow cytometry analysis. Consequently, the user adaptation to a different cell culture technology, with a less comfortable handling with respect to the standard cultures and a narrowed range of analytical methods (i.e., often confined to cell imaging) make these microfluidic devices not easy to adopt in conventional laboratory practices.…”

Microfluidic devices: The application in TME modeling and the potential in immunotherapy optimization

“…Microfluidic technology promoted the development of a complicated vascularized in vitro model to imitate the TME. In a microfluidic platform with three parallel microchannels, modeling the tumor vasculature through the generation of blood vessel networks formed with endothelial cells, fibroblasts, and colorectal cancer (CRC) cells within the central channel ( Figure 2A ) ( Song et al, 2021 ). And then introduced NK cells into the vessel via the side channel.…”

“…ii) The blood vessel networks were formed with endothelial cells, fibroblasts, and CRC cells within the central channel. Reproduced from Song et al ( Song et al, 2021 ) Copyright 2021 Song, Choi, Koh, Park, Yu, Kang, Kim, Cho and Jeon. (B) A network platform with interconnected microfluidic channels for modeling a highly vascularized system.…”