Vs 6063

Mice were inoculated intravenously (i.v.) with high doses (hd, 1–10 × 10⁶) or i.p. with low doses (ld, 2,5–5 × 10⁴) of freshly egressed T. gondii tachyzoites. To evaluate the effect of inflammation on parasite loads, T. gondii-infected CD1 mice were inoculated i.p. with LPS (serotype 011:B4, Sigma) at 0.08 or 0.2 mg/kg/day for seven or 5 days, respectively, starting 1 day before infection or treated subcutaneously with hydrocortisone (Cortisol, Sigma-Aldrich) at 20 or 40 mg/kg/day for 6 days, starting 3 days before T. gondii infection. For assessment of BBB permeability, control mice were injected i.p. with LPS (2 mg/kg) and sacrificed 6 h post-inoculation. T. gondii-infected mice were inoculated i.p. with the FAK inhibitor VS-6063 (defactinib; Selleckchem) at 50 mg/kg twice a day, starting 1 day before T. gondii infection.

PC9, HCC827 and H1975 cell lines were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Caicun Zhou at Tongji University School of Medicine provided PC9GR cells. HCC827GR cells were derived from HCC827 cells by exposure to Gefitinib, as previously described [24 (link)]. PC9 and PC9GR cells were routinely cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA). HCC827, HCC827GR, and H1975 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (Gibco). Cells were grown in a humidified incubator with 5% CO₂ at 37 °C.
Gefitinib was supplied by AstraZeneca (London, UK). Osimertinib and the FAK inhibitor VS-6063 were purchased from Selleck Chemicals (Houston, TX, USA).

Mice bearing bilateral MC38, B16-F10, or 4T1 tumors were subjected to RT in combination with other antitumor therapies. For MC38 or B16-F10 bilateral tumor-bearing ICAM-1 KO or WT C57BL/6 mice or 4T1 bilateral tumor-bearing BALB/c mice, the right hind flank tumors (primary tumors) were irradiated with a total dose of 24 Gy (three doses of 8 Gy) or non-irradiated.
For the anti-ICAM-1 blocking experiment, 50 μg of anti-ICAM-1 antibody (clone YN1/1.7.4, BioXcell, West Lebanon, NH) or an equal amount of IgG isotype-matched control was intratumorally injected into the left hind flank tumors on days 8, 10, 12, and 14. For the fingolimod (FTY720) treatment, MC38 tumor-bearing mice were injected (i.p.) with 40 μg of FTY720 on day 9 and 20 μg of FTY720 daily until day 28. For ACT, MC38 tumor-bearing ICAM-1 KO mice were administered (i.v.) activated CD8⁺ T cells from MC38 tumor-bearing ICAM-1 KO C57BL/6 donor mice (5 × 10⁶) or activated CD8⁺ T cells from MC38 or B16-F10 tumor-bearing C57BL/6 WT donor mice (5 × 10⁶) on days 10, 13, and 16. MC38 tumor-bearing WT mice were administered (i.v.) activated WT or ICAM-1 OE CD8⁺ T cells (5 × 10⁶) on days 12, 15, and 18. For small-molecule drug treatment, 4T1 tumor-bearing mice were injected (i.p.) with 200 µg of VS-6063 (Selleckchem, Houston, TX) every other day from days 10 to 22.

C57BL/6 mice bearing either single or bilateral MC38 tumors received three fractions of 8 Gy radiation to the right hind flank tumors or were non-irradiated. From days 8 to 14, mice were intratumorally administered PBS, anti-ICAM-1 antibody (50 µg), or IgG isotype-matched control (50 µg) every other day. On day 17, the mice were euthanized, and the tumors and spleens were collected.
In a separate study, BALB/c mice with either single or bilateral 4T1 tumors received three fractions of 8 Gy radiation to the right hind flank tumors or were non-irradiated. From days 10 to 14, they received i.p. injections of PBS control or 200 μg of VS-6063 (Selleckchem) every other day. On day 16, the mice were euthanized, and the tumors were harvested.
Tumor samples were mechanically minced and digested in RPMI-1640 medium (Invitrogen) supplemented with 10 U/mL collagenase I, 400 U/mL collagenase IV, and 30 U/mL DNase (Yuanye Bio-Technology, Shanghai, China) at 37°C for 1 h. The spleens were dissected and filtered through a strainer. Red blood cells in the digested tumor samples and spleens were lysed using erythrocyte lysis buffer (BioLegend). Tumor-infiltrating cells and spleen cells were resuspended in PBS containing 0.5% w/v bovine serum albumin (BSA). The collected cells were then stained and analyzed using flow cytometry.

SJP1602 (99% purity) was synthesized by Samjin Pharmaceutical Co. (Seoul, Republic of Korea). For in vitro studies, VS-6063 was sourced from Selleckchem (Houston, TX, USA) and for in vivo experiments, it was obtained from DAEJUNG Chemicals & Metals (Seoul, Republic of Korea). GSK2256098 was procured from MedKoo Biosciences (Morrisville, NC, USA). The cell culture media, HEPES buffer solution, 1X DPBS, and antibiotics were purchased from Gibco (Waltham, MA, USA). Fetal bovine serum (FBS) was acquired from Hyclone (Logan, MA, USA). Trypsin-EDTA solution and Dimethyl sulfoxide (DMSO) were both purchased from Sigma, with the latter specifically from Sigma-Aldrich (St. Louis, MO, USA). The culture dishes (100 mm), 96-well clear round-bottom ultra-low attachment microplates, 96-well white flat-bottom microplates, and Matrigel growth factor-reduced, phenol red-free were sourced from Corning (Corning, NY, USA). Lastly, the 3D CellTiter-Glo assay kit was bought from Promega (Madison, WI, USA).