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.

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% O₂ and 5% CO₂. 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 >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⁺ area with a clear border between stromal cells and mCerulean⁺ tumor cells. Tumor-rich region was defined by high Cerulean⁺ area and stromal cells were interlaced with Cerulean⁺ 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).

Imaris, ImageJ, FlowJo, LAX X, CT-FIRE