br Cytotoxicity Assay br Human colorectal cancer cell
Human colorectal cancer cell lines HCT-116 and HT29 were plated at 3 103 Oxaliplatin per well in cell-specific media supplemented with 5% FBS and 1% antibiotic-antimycotic solution in 96-well flat-bottom plates. The following day, cells were infected with virus at MOIs 1 and 0.01.
For the next 5 days, cell survival was determined by comparing absorp-tion of infected cells with uninfected cells after 3-h incubation with CellTiter 96 AQueous One Solution Cell Proliferation Assay per man-ufacturer’s protocol (Promega, Madison, WI, USA).
Viral Growth Assays
A total of 5 105 of HCT-116 and HT-29 cells were seeded in six-well plates. The following day, cells were counted and infected at an MOI of 0.01 PFU per cell with CF33-hNIS and CF33-GFP in a total volume of 0.5 mL medium containing 2.5% FBS. After 1 h, the inoculum was aspirated and fresh medium was added to each well, and plates were returned to the incubator. At the indicated times, cells were scraped into the medium and subjected to three rounds of freeze-thaw to ensure complete cell lysis and virus release. Viral titers in the lysates were determined by standard plaque assay. Lysates were serially diluted and used to infect 24-well plates of CV-1 cells. One hour after infection, 1 mL of medium containing 1% methyl-cellulose was added to each well. Forty-eight hours post-infection, 250 mL 0.5% (w/v) crystal violet was added to each well, and the plates were left at room temperature overnight. The next day, the plates were washed and plaques were counted.
Cell Death Analysis
Flow Cytometry. For Annexin V, caspase-3, or PI staining, cells were infected with CF33-NIS at an MOI of 5 in 3 mL of medium supple-mented with 2.5% FBS. After 1-h incubation, cells were washed twice with PBS before addition of complete growth medium. Cells were then harvested at 18 and 48 h and stained with Annexin V, propi-dium iodide, or caspase-3 antibody per manufacturer’s instructions (FITC Annexin V/Dead cell apoptosis kit, category no. V13242 [Invitrogen]; Live/Dead Fixable far red dead cell stain kit, category no. L34973; PE Active Caspase-3 Apoptosis kit, category no. 550914 [BD PharMingen]). Stained cells were then analyzed on a BD Accuri C6 Flow Cytometer (BD Biosciences).
TUNEL Assay. Cells were plated at 2 105 cells/well in an eight-well chamber slide (category no. 154534; Thermo Fisher Scientific). The following day, cells were infected with CF33-hNIS at MOI 0.01 pfu/ cell. After 18 or 48 h of infection, TUNEL reaction was performed us-ing In Situ Cell Death Detection kit (Roche; category no. 11684795910) following the manufacturer’s instructions; then cells were blocked in Tris-NaCl-blocking buffer, and stained with anti-vaccinia virus antibody (ab35219; Abcam) and a secondary Alexa Fluor 594-conjugated goat anti-Rabbit IgG H&L (ab150080; Abcam).
Three independent assays were used to monitor ICD: ATP level assay, flow cytometric assessment of calreticulin levels, and detection of HMGB1 release.
Secretion of ATP. Cells were infected at MOI 5 and were incubated for 16 h. After incubation, supernatants were collected, ATP levels were determined according to the manufacturer’s instructions (ATP
Molecular Therapy: Oncolytics
Determination Kit, category no. A22066; Invitrogen), and lumines-cence was measured with a TECAN microplate reader (Life Sciences).
Calreticulin. Cells were infected with CF33-hNIS (MOI 5) for 16 h. 1 106 cells were resuspended in PBS with 2% FBS and stained with isotype control (EPR25A; Abcam) or anti-calreticulin antibody (EPR3924; Abcam). After staining, FACS analysis was performed.