Lao, Y.-H. | bioRxiv Science

bioRxiv2022

An Immune Cell Recirculation-Enabled Microfluidic Array to Study Dynamic Immunotherapeutic Activity in Recapitulated Tumor Microenvironment

The efficacy of immunotherapeutic treatment protocols to enable immune cell mediated treatment of cancer is significantly modulated in the presence of tumor microenvironment (TME) which is a key factor in providing both a physical barrier and immunosuppressive stimuli. Herein, we developed a recirculating, high-throughput microfluidic cell array to capture these crucial players - cytotoxic T cells in circulation, endothelium, and tumor stroma. The system consisted of a three-layered cell array spatially emulating TME, with T cell circulation sustained via fluidic recirculating circuits. This allowed us to study the dynamic TME/circulation system and cancer cell response thereof. The system further revealed that tumor endothelium exhibited a hindrance to T cell infiltration into the breast cancer tumor compartment, which was alleviated when treated with anti-human PD-L1 antibody. The other key stromal component, cancer associated fibroblasts, further attenuated T cell infiltration, and led to reduced apoptosis activity in cancer cells. These results confirm the capability of our tumor-on-a-chip system to recapitulate some key immune cell interactions with the reconstructed TME, along with demon-strating as the feasibility of using this system for high-throughput cancer immunotherapeutic screening.

bioengineering↗

bioRxiv2021

Mapping morphological malformation to genetic dysfunction in blood vessel organoids with 22q11.2 Deletion Syndrome

DiGeorge Syndrome, or 22q11.2 deletion syndrome (22q11.2 DS), is a genetic disorder caused by microdeletions in chromosome 22, impairing the function of endothelial cells (EC) and/or mural cells and leading to deficits in blood vessel development such as abnormal aortic arch morphology, tortuous retinal vessels, and tetralogy of Fallot. The mechanism by which dysfunctional endothelial cells and pericytes contribute to the vasculopathy, however, remains unknown. In this study, we used human blood vessel organoids (VOs) generated from iPSC of 22q11.2 DS patients to model the vascular malformations and genetic dysfunctions. We combined high-resolution lightsheet imaging and single-cell transcriptome analysis to link the genetic profile and vascular phenotype at the single-cell level. We developed a comprehensive analytical methodology by integrating deep learning-mediated blood vessel segmentation, network graph construction, and tessellation analysis for automated morphology characterization. We report that 22q11.2DS VOs demonstrate a smaller size with increased angiogenesis/sprouting, suggesting a less stable vascular network. Overall, clinical presentations of smaller vascular diameter, less connected vasculature, and increased branch points were recapitulated in 22q11.2DS VOs. Single-cell transcriptome profiling showed heterogeneity in both 22q11.2DS and control VOs, but the former demonstrated alterations in endothelial characteristics that are organ-specific and suggest a perturbation in the vascular developmental process. Intercellular communication analysis indicated that the vascular dysfunctions in 22q11.2 deletion were due to a lower cell-cell contact and upregulated extracellular matrix organization involving collagen and fibronectin. Voronoi diagram-based tessellation analysis also indicated that the colocalization of endothelial tubes and mural cells was different between control and 22q11.2 VOs, indicating that alterations in EC and mural interactions might contribute to the deficits in vascular network formation. This study illustrates the utility of VO in revealing the pathogenesis of 22q11.2DS vasculopathy.

bioengineering↗