C7025

The expression of CARs and TCRs in primary T cells were determined by flow cytometry using biotinylated protein L (ThermoFisher #29997) followed by fluorescently labeled streptavidin, or fluorescently labeled anti-murine TRBV antibody (Biolegend Cl:H57-597) (S1 Fig). All staining was carried out at 4°C, and median fluorescence intensity (MFI) was determined using a FACS Canto II flow cytometer (BD Biosciences). For T cell proliferation, transduced T cells were stained with CMFDA (Invitrogen C7025; fresh CMFDA working solution at 1 μM was made in LymphoONE^™ from the solid) and mixed with 25% of untransduced T cells that were not loaded with CMFDA on day 0. Mixed T cells were cocultured with target cells while being imaged on IncuCyte^® or ImageXpress^® Micro (IXM) for 3 days. On day 3, T cells were recovered from the imaging plate. T cells before and after co-culture were stained with anti-CD3 antibody (Biolegend 300439) without washing. Relative T cell proliferation was calculated using the ratio of CD3+/CMFDA+ vs. CD3+/CMFDA- populations within each sample, and was compared among different peptide concentrations and E:T ratios (S5 Fig).

For live cell invasion assay, the cells were stained with cell tracker red (C7025, Invitrogen, Carlsbad, CA, USA) and cell tracker green (C34552, Invitrogen, Carlsbad, CA, USA) in 5 µg/mL and incubated for 20 min at 37 °C. For immunostaining, Zonula occludens-1 (ZO-1) (40-2200), cluster of differentiation 31 (CD31) (MA3100), Ras homolog family member A (RhoA) (MA1-011), Ras-related C3 botulinum toxin substrate 1 (Rac1) (701942), hypoxia-inducible factor 1-alpha (HIF-1α) (MA1-516), and matrix metallopeptidase 9 (MMP-9) (MA5-15886) were purchased from Invitrogen and used following the manufacturer protocols. For nuclei staining, 4′,6-diamidino-2-phenylindole (DAPI) (D9542, Sigma-Aldrich, St. Louis, MO, USA) was used. The samples were rinsed with DPBS and fixed using 4% paraformaldehyde in DPBS for 10 min at room temperature (RT). After permeabilization using 0.1% Triton X-100 in DPBS for 15 min at RT, samples were blocked with 1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) in DPBS for 1 h at RT. Then, the samples were incubated with primary antibodies overnight at 4 °C. After washing with 0.1% BSA, the samples were incubated with secondary antibodies for 2 h at RT and nuclei were stained with 300 nM DAPI. A confocal laser scanning microscope (CLSM) (LSM 510-META, Zeiss, Oberkochen, Germany) was used for imaging and analyses.