Microengineered transplantation of human solid tumors for in vitro studies of CAR T immunotherapy
Data files
Aug 08, 2025 version files 8.41 GB
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Extended_Data_Figs_3_and_4.zip
116.26 MB
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Main_Fig_1.zip
73.52 MB
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Main_Fig_2.zip
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Main_Fig_4.zip
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Main_Fig_5.zip
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Main_Fig_6.zip
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README.md
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SI_Fig_25.zip
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SI_Fig_26.zip
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SI_Fig_27.zip
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SI_Fig_3.zip
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SI_Fig_6.zip
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SI_Fig_7.zip
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SI_Fig_8.zip
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SI_Fig_9.zip
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Abstract
Treatment of solid malignancies using chimeric antigen receptor (CAR) T cells remains a significant challenge, but current efforts to advance this therapy are challenged by our limited capacity to probe and understand cancer-immune interactions in human solid tumors. Here, we present a microengineered platform for in vitro modeling of malignant solid tumors during CAR T therapy. This system makes it possible to vascularize human tumor explants and perfuse them with blood-borne immune cells in a controlled manner. We first present a microphysiological model of human lung adenocarcinomas infused with CAR-T cells and show how this system can be used to simulate, visualize, and interrogate tumor-directed trafficking and effector function of CAR T cells. We then demonstrate the proof-of-principle of testing a chemokine-directed CAR T cell engineering strategy in a model of malignant pleural mesothelioma and validating our in vitro assessment using a matching in vivo mouse model. Finally, we describe a potential therapeutic target discovered by single-cell RNA sequencing that can be pharmacologically modulated to increase the efficacy of CAR T cells for lung adenocarcinoma, for which we also present specific biomarkers identified by global metabolomics analysis. We believe that the bioengineering principle demonstrated here will make important contributions to developing new capabilities for preclinical studies of adoptive cell therapies for cancer and other complex diseases.
Dataset DOI: 10.5061/dryad.mw6m9068f
Description of the data and file structure
Microscopic fluorescent images of human tumor constructs, microengineered blood vessels, and infused human T cells were collected in a microengineered human solid tumor model. Each folder corresponds to an individual figure in the related manuscript and contains subfolders of figure panels and/or subfolders of treatment conditions that are also described in detail below. Images were collected at various time points and treatment conditions as indicated in the folder path and file names. The filenaming convention for the image files is defined as follows: Figure number+panel label+(Tumor group, T cell group, or treatment group)+Construct ID+Culture time+(Tumor detail)+(Days post tumor transplantation)+(T cell detail)+(Days post T cell infusion)+(additional information such as imaging channel number).jpg/tif
Files and variables
File: Main_Fig_1.zip
Description: Raw images for Main Figs 1e and 1f. Fig. 1. Microengineered platform for in vitro transplantation and prolonged maintenance of human solid tumors. e, Representative fluorescence micrographs showing blood vessel development in the device over time. The dashed white circles show the location of closed culture wells. Scale bars, 250 µm (multi-well view), 100 μm (single-well view and image of human lung fibroblasts stained magenta), and 5 µm (inset showing the cross-section of a blood vessel). f, Quantification of total branch length (per engineered construct) (top) and the average diameters of microvessels (bottom) during the vascularization process in our microengineered platform. Data are presented as mean ± SEM (n = 8-17).
File: Extended_Data_Figs_3_and_4.zip
Description: Raw images for Extended Data Figs 3 and 4. Extended Data Fig. 3. Pharmacological inhibition of CAR T-endothelial interactions mediated by CD38-PECAM1 signaling. Fluorescence micrographs of single meso-tumors infused with meso-CAR T cells without drug treatment (Untreated), treated with Daratumumab (0.5 or 10 μg/ml), or IgG isotype control antibody (10 μg/ml IgG). Blood vessels are not shown in these images. Extended Data Fig. 4. Pharmacological inhibition of CAR T-endothelial interactions mediated by LTB-LTBR signaling. Fluorescence micrographs of single meso-tumors infused with meso-CAR T cells without drug treatment (Untreated), treated with Baminercept (0.5 or 10 μg/ml), or IgG isotope control antibody (10 μg/ml IgG). Blood vessels are not shown in these images.
File: Main_Fig_2.zip
Description: Raw images for Main Figs 2i, 2k, 2p, and 2r. Fig. 2. In vitro modeling of CAR T cell-tumor interactions. Quantification of tissue area occupied by CAR T cells (i) and tumor area normalized to that prior to CAR T cell infusion (k). p, Immunostaining of ICAM-1 expression and quantification of tissue area stained positive for ICAM-1. r, Visualization of apoptotic tumor cells at Day 5 post CAR T cell infusion and quantification of caspase 3 expression.
File: Main_Fig_4.zip
Description: Raw images for Main Figs 4d-f. Fig. 4. In vitro and in vivo analysis of human meso-CAR T cells armored with CCR2. d, Representative tiled images of EMMeso-tumors prior to and after T cell infusion. The three types of T cells were derived from one healthy donor. e,f, Quantification and comparison of T cell-occupied hydrogel area (e) and tumor area (f) at different time points.
File: Main_Fig_5.zip
Description: Raw images for Main Figs 5c-e. Fig. 5. Transplantation and CAR T cell treatment of human mesothelioma explants. c, Quantification of the percentage of vessel-covered area, total branch length (per engineered construct), total number of vessel junctions (per engineered construct), and the average diameter of microvessels in the CDX tumor (open circle) and patient-derived explant (closed diamond) models during tumor vascularization in our device. d, Representative tiled micrographs showing vascularized tumor constructs infused with non-transduced T cells (NTD) or meso-CAR T cells (meso-CAR). e, Quantification and comparison of T cell infiltration into the tumor compartment and surrounding stroma within the regions of interest.
File: SI_Fig_6.zip
Description: Raw images for Supplementary Fig 6a, 6b. SI Figure 6. Overexpression of mesothelin in A549 and meso-CAR design. a, Fluorescence images show mesothelin expression (red) by GFP-expressing A549 cells cultured in 2D monolayers and in A549-CDX tumors. b, Quantification of mesothelin expression.
File: Main_Fig_6.zip
Description: Raw images for Main Figs 6g-i. Fig. 6. Identification of new therapeutic targets through analysis of ligand-receptor interactions in CAR T cell-treated tumor models. g, Confocal micrographs of single tumors treated with CAR T cells and different concentrations of LAF237. Blood vessels are not shown in these images. h, Images of CAR T cell-infused tumors at Day 26. i, Quantification and comparison of tumor area and CAR T cell-occupied hydrogel area.
File: SI_Fig_3.zip
Description: Raw images for Supplementary Fig 3. SI Figure 3. Effect of initial tumor size on tumor vascularization. Confocal micrographs showing vascularization of single lung tumors with different initial sizes in our device.
File: SI_Fig_7.zip
Description: Raw images for Supplementary Fig 7. SI Figure 7. Growth of lung tumors and their responses to CAR-T cells. Fluorescence time-lapse images of meso- (left) and control (right) tumors in the device prior to (days 1 to 11) and after (days 14 to 19) meso-CAR T cell infusion. Compared to the control group, the post-infusion meso-tumor model is seen with noticeably higher levels of CAR T cell adhesion and retention in tumor masses.
File: SI_Fig_8.zip
Description: Raw images for Supplementary Fig 8. SI Figure 8. Analysis of lung tumors infused with non-transduced T cells. Confocal micrographs of single meso-tumor constructs treated with non-transduced (NTD) T cells or meso-CAR-T cells.
File: SI_Fig_9.zip
Description: Raw images for Supplementary Fig 9. SI Figure 9. Analysis of chemokine production. Array screening of 38 chemokines using device effluent samples collected from microengineered constructs containing blood vessels only (vessel only control), vascularized control tumors (Control tumor model), and vascularized meso-tumors (Meso-tumor model).
File: SI_Fig_25.zip
Description: Raw images for Supplementary Fig 25. SI Figure 25. Effects of LAF237 on tumor growth during CAR-T infusion. Confocal micrographs of meso-CAR T cell-infused lung tumors treated with different concentrations of LAF237 during long-term culture.
File: SI_Fig_26.zip
Description: Raw images for Supplementary Fig 26. SI Figure 26. Effects of LAF237 alone on tumor growth. Confocal micrographs of lung tumors during daily treatment with LAF237 in the absence of CAR T cells.
File: SI_Fig_27.zip
Description: Raw images for Supplementary Fig 27. SI Figure 27. Effect of blocking CXCR3 on the activity of CAR-T cells. Fluorescence micrographs of single meso-tumors infused with meso-CAR T cells without drug treatment (no LAF237), treated with LAF237 at 1000 nM, 1000 nM of LAF237 and anti-CXCR3 antibody, or 1000 nM of LAF237 with isotype-matched control antibody. Blood vessels are not shown in these images.
Human subjects data
Primary mesothelioma explants (Meso explant) were derived and obtained from tumors of malignant pleural mesothelioma patients undergoing potentially curative surgery. The tumor explants were de-identified before processing and transplantation into our microengineered constructs, and informed consent was obtained from all patients.