Mcd45

For humanized OCCC PDX model, 5th passage of a previously described ARID1A-mutated PDX (I832fs*)^{65 (link)} were transplanted to ovarian bursa sac of humanized-BLT mice. Three weeks after transplantation, mice were randomized into the following four treatment groups: vehicle plus IgG control (Bio X Cell, Cat#: BE0090, 5 mg/kg, 3 times per week, i.p.), simvastatin (5 mg/kg, 3 times per week, i.p.) plus IgG control, vehicle control plus anti-PD-L1 antibody (R&D Systems, Cat#: MAB10348, 5 mg/kg, 3 times per week, i.p.), and combination of simvastatin and anti-PD-L1 antibody. After 2 weeks treatment, mice were then euthanized, and tumor burden were compared using tumor weight as a surrogate in each of the treatment groups. Exhaustion of infiltrated human T cells in tumors were measured by flow cytometry (BD Biosciences, Symphony) using antibodies against mCD45 (BD Biosciences, Cat#: 560510), hCD45 (BD Biosciences, Cat#: 564586), hCD3 (BD Biosciences, Cat#: 612895), hCD4 (BD Biosciences, Cat#: 563550), hCD8 (BD Biosciences, Cat#: 565310), PD1 (BD Biosciences, Cat#: 563789), LAG3 (BD Biosciences, Cat#: 565720) and Fixable Viability Stain (BD Biosciences, Cat#: 564406). Data were analyzed with FlowJo software (version 10.0).

For SKM-1 competition assays, live cells were washed with FACS buffer and stained with DAPI before running on a FACSCanto (BD Biosciences). For GM-CSF stimulation experiments, cells were starved overnight, incubated with zombie violet for 20mins, washed, and stimulated with varying concentrations of GM-CSF for 15 minutes. Immediately after stimulation, paraformaldehyde was added to a final concentration of 1.6% and cells were fixed for 10 mins. Cells were then washed with PBS and permeabilized using 2mL of ice-cold 95% methanol. After washing off methanol, cells were stored in FACS buffer until analysis. On the day of analysis, cells were stained with pSTAT5 antibody (BD Biosciences) for 15 minutes, washed and run on a FACSCanto. For PDX experiments, cells were resuspended in 50μL FACS buffer with 2μL of both human and mouse FCR blocking antibody and incubated for 10mins. An antibody cocktail comprised of hCD45, mCD45, hCD3, hCD33, and hCD34(BD Biosciences) was added to each tube, incubated for 15 minutes and washed with FACS buffer. Cells were run on an LSRII (BD Biosciences). Data was analyzed in FlowJo(RRID:SCR_008520).