DOI: 10.5061/dryad.83bk3j9xn
title: Data from: Desmoplastic stroma restricts T cell extravasation and mediates immune exclusion and immunosuppression in solid tumors

description: In this study, we used our modified precision-cut tumor slice assay of
 tumors treated with CAR T cells in vivo prior to harvest and harvested at
 different time-points post-treatment, which can better reflect the
 behavior of CAR T cells in the TME in vivo.
description: Precision-cut tumor slice-based
 real-time two-photon microscopy Tumor slices
 were prepared as described previously, with modifications. In brief,
 tumors were embedded in 5% low-gelling-temperature agarose (Sigma-Aldrich)
 prepared in PBS. Tumors were cut with a vibratome (Leica VT1200S
 vibratome) in a bath of ice-cold PBS. The thickness of the slices was 500
 μm. Live tumor slices were stained with AF647-anti-mouse CD90.2
 (BioLegend) at a concentration of 10 μg/mL for 15 minutes at 37°C and were
 then transferred to 0.4-mm organotypic culture inserts (Millipore) in
 35-mm Petri dishes containing 1 mL RPMI-1640 (without phenol red;
 ThermoFisher) before imaging. Imaging fresh slices of mouse
 tumors was performed using Leica SP8-MP upright multiphoton microscope
 with Coherent Chameleon Vision II MP laser equipped with a 37°C
 thermostatic chamber. Tumor slices were secured with a stainless-steel
 ring slice anchor (Warner Instruments) and perfused at a rate of 0.3
 mL/min with a solution of RPMI (without phenol red), bubbled with 95%
 O<sub>2</sub> and 5% CO<sub>2</sub>. Images were
 systematically acquired at 6 different regions within the tumor with a 20×
 (1.0 NA) water immersion lens and a Coherent Chameleon laser at 880 nm/25
 mW. The following filters were used for fluorescence detection: CFP
 (483/32), GFP (535/30), AF647 (685/40) and tdTomato (610/75). For
 four-dimensional analysis of cell migration, a 70-90 μm z-stack at 5 μm
 step size was acquired for 2 hours, alternating between six fields every
 30 seconds. Videos were made by compressing the z information into a
 single plane with the max intensity z projection of Imaris and LAS X
 software. Image analysis
 was performed at PennVet Imaging Core (University of Pennsylvania). A 3D
 image analysis was performed on x, y, and z planes using Imaris 7.4
 software. First, superficial planes from the top of the slice to 15 μm in
 depth were removed to exclude T cells located near the cut surface.
 Cellular motility parameters were then calculated using Imaris. Tracks
 &gt;10% of the total recording time were included in the analysis.
 When a drift in the x, y dimension was noticed, it was corrected using the
 “Correct 3D Drift” plug-in in FIJI-ImageJ. CAR T cell number and motility
 were quantified in different tumor regions, including stroma-rich and
 tumor-nest regions. These regions were identified by visual inspection of
 immunofluorescence images. Stroma-rich region was defined by high
 CD90.2<sup>+</sup> area with a clear border between stromal
 cells and mCerulean<sup>+</sup> tumor cells. Tumor-rich region
 was defined by high Cerulean<sup>+</sup> area and stromal
 cells were interlaced with Cerulean<sup>+</sup> tumor cells.
 Fluorescence intensities were determined in regions of interest using
 FIJI-ImageJ. The number of T cells in defined regions was quantified using
 the Analyze Particles function of FIJI-ImageJ from fluorescent images that
 were first thresholded and then converted to binary images. Collagen
 measurement was performed using CT-FIRE software (version 2.0 beta) (https://loci.wisc.edu/software/ctfire).
description: Imaris, ImageJ, FlowJo, LAX X, CT-FIRE