A Computational Strategy for the Rapid Identification and Ranking of Patient-Specific T Cell Receptors Bound to Neoantigens.

T cell receptor (TCR) recognition of a peptide-major histocompatibility complex (pMHC) is crucial for adaptive immune response. The identification of therapeutically relevant TCR-pMHC protein pairs is a bottleneck in the implementation of TCR-based immunotherapies. The ability to computationally design TCRs to target a specific pMHC requires automated integration of next-generation sequencing, protein-protein structure prediction, molecular dynamics, and TCR ranking. A pipeline to evaluate patient-specific, sequence-based TCRs to a target pMHC is presented. Using the three most frequently expressed TCRs from 16 colorectal cancer patients, the protein-protein structure of the TCRs to the target CEA peptide-MHC is predicted using Modeller and ColabFold. TCR-pMHC structures are compared using automated equilibration and successive analysis. ColabFold generated configurations require an ≈2.5× reduction in equilibration time of TCR-pMHC structures compared to Modeller. The structural differences between Modeller and ColabFold are demonstrated by root mean square deviation (≈0.20 nm) between clusters of equilibrated configurations, which impact the number of hydrogen bonds and Lennard-Jones contacts between the TCR and pMHC. TCR ranking criteria that may prioritize TCRs for evaluation of in vitro immunogenicity are identified, and this ranking is validated by comparing to state-of-the-art machine learning-based methods trained to predict the probability of TCR-pMHC binding.

Organotypic stromal cells impact endothelial cell transcriptome in 3D microvessel networks.

Endothelial cells line all major blood vessels and serve as integral regulators of many functions including vessel diameter, cellular trafficking, and transport of soluble mediators. Despite similar functions, the phenotype of endothelial cells is highly organ-specific, yet our understanding of the mechanisms leading to organ-level differentiation is incomplete. We generated 3D microvessel networks by combining a common naïve endothelial cell with six different stromal cells derived from the lung, skin, heart, bone marrow, pancreas, and pancreatic cancer. Single cell RNA-Seq analysis of the microvessel networks reveals five distinct endothelial cell populations, for which the relative proportion depends on the stromal cell population. Morphologic features of the organotypic vessel networks inversely correlate with a cluster of endothelial cells associated with protein synthesis. The organotypic stromal cells were each characterized by a unique subpopulation of cells dedicated to extracellular matrix organization and assembly. Finally, compared to cells in 2D monolayer, the endothelial cell transcriptome from the 3D in vitro heart, skin, lung, and pancreas microvessel networks are more similar to the in vivo endothelial cells from the respective organs. We conclude that stromal cells contribute to endothelial cell and microvessel network organ tropism, and create an endothelial cell phenotype that more closely resembles that present in vivo.

Organ-on-a-chip model of vascularized human bone marrow niches.

Bone marrow niches (endosteal and perivascular) play important roles in both normal bone marrow function and pathological processes such as cancer cell dormancy. Unraveling the mechanisms underlying these events in humans has been severely limited by models that cannot dissect dynamic events at the niche level. Utilizing microfluidic and stem cell technologies, we present a 3D in vitro model of human bone marrow that contains both the perivascular and endosteal niches, complete with dynamic, perfusable vascular networks. We demonstrate that our model can replicate in vivo bone marrow function, including maintenance and differentiation of CD34+ hematopoietic stem/progenitor cells, egress of neutrophils (CD66b+), and niche-specific responses to doxorubicin and granulocyte-colony stimulating factor. Our platform provides opportunities to accelerate current understanding of human bone marrow function and drug response with high spatial and temporal resolution.

Isolating and characterizing lymphatic endothelial progenitor cells for potential therapeutic lymphangiogenic applications.

Lymphatic dysfunction is associated with the progression of several vascular disorders, though currently, there are limited strategies to promote new lymphatic vasculature (i.e., lymphangiogenesis) to restore lost lymphatic function. One promising approach to stimulate lymphangiogenesis involves delivering endothelial progenitor cells (EPCs), which are naturally involved in de novo blood vessel formation and have recently been identified to include a lymphatic subpopulation. However, the contribution of lymphatic EPCs in lymphangiogenesis is not clear and challenges with maintaining the activity of transplanted EPCs remain. Thus, the objective of this study was to isolate lymphatic EPCs from human umbilical cord blood and characterize their role in the initial stages of blood or lymphatic vasculature formation. Furthermore, this study also tested the applicability of alginate hydrogels to deliver lymphatic EPCs for a possible therapeutic application. We postulated and confirmed that blood and lymphatic EPC colonies could be isolated from human umbilical cord blood. Additionally, EPC populations responded to either angiogenic or lymphangiogenic growth factors and could stimulate their respective mature endothelial cells in vasculature models in vitro. Finally, lymphatic EPCs maintained their ability to promote lymphatic sprouts after prolonged interactions with the alginate hydrogel microenvironment. These results suggest EPCs have both a blood and a lymphatic population that have specific roles in promoting revascularization and highlight the potential of alginate hydrogels for the delivery of lymphatic EPCs. STATEMENT OF SIGNIFICANCE: Despite the potential therapeutic benefit of promoting lymphatic vasculature, lymphangiogenesis remains understudied. One appealing strategy for promoting lymphangiogenesis involves delivering lymphatic endothelial progenitor cells (EPCs), which are a subpopulation of EPCs involved in de novo vessel formation. Here, we investigate the role of isolated blood and lymphatic EPC subpopulations in promoting the early stages of vascularization and the utility of alginate hydrogels to deliver lymphatic EPCs. We determined that EPCs had two populations that expressed either blood or lymphatic markers, could stimulate their respective mature vasculature in tissue constructs and that alginate hydrogels maintained the therapeutic potential of lymphatic EPCs. We anticipate this work could support promising biomaterial applications of EPCs to promote revascularization, which could have many therapeutic applications.

Tumor-on-a-chip platform to investigate progression and drug sensitivity in cell lines and patient-derived organoids.

Most cancer treatment strategies target cell proliferation, angiogenesis, migration, and intravasation of tumor cells in an attempt to limit tumor growth and metastasis. An in vitro platform to assess tumor progression and drug sensitivity could provide avenues to enhance our understanding of tumor metastasis as well as precision medicine. We present a microfluidic platform that mimics biological mass transport near the arterial end of a capillary in the tumor microenvironment. A central feature is a quiescent perfused 3D microvascular network created prior to loading tumor cells or patient-derived tumor organoids in an adjacent compartment. The physiological delivery of nutrients and/or drugs to the tumor then occurs through the vascular network. We demonstrate the culture, growth, and treatment of tumor cell lines and patient-derived breast cancer organoids. The platform provides the opportunity to simultaneously and dynamically observe hallmark features of tumor progression including cell proliferation, angiogenesis, cell migration, and tumor cell intravasation. Additionally, primary breast tumor organoids are viable in the device for several weeks and induce robust sprouting angiogenesis. Finally, we demonstrate the feasibility of our platform for drug discovery and personalized medicine by analyzing the response to chemo- and anti-angiogenic therapy. Precision medicine-based cancer treatments can only be realized if individual tumors can be rapidly assessed for therapeutic sensitivity in a clinically relevant timeframe (⪅14 days). Our platform indicates that this goal can be achieved and provides compelling opportunities to advance precision medicine for cancer.