DOI: 10.5061/dryad.mw6m9068f
title: Microengineered transplantation of human solid tumors for in vitro studies of CAR T immunotherapy

description: 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.
description: # Microengineered transplantation of human solid tumors for in vitro
 studies of CAR T immunotherapy Dataset DOI:
 [10.5061/dryad.mw6m9068f](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.