Lyovec

Sorted monocyte subsets were transferred to 5ml polypropylene tubes, spun down and suspended in cultured RPMI medium [RPMI with L-glutamine (Corning Cellgro, Manassas, VA USA) supplemented with 10% FBS and 1X (50U) Penicillin-Streptomycin (Invitrogen, Carlsbad, CA USA)]. Cells were plated at 30,000 cells/well in 96-well V-bottom plates. TLR and RIG-I agonists were added at the following concentrations: LPS-TLR4 (0.5 μg/ml) or CLO97-TLR7/8 (1 μg/ml) or 5′pppRNA-RIG-I (500 ng/ml). Optimal concentrations of different TLR agonists were selected based on median production of IL-6 and IFNα and a ≥ 85% survival rate of monocytes. All PRR ligands were purchased commercially (InvivoGen, San Diego, CA USA) except for 5′-pppRNA, which was custom synthesized, as previously described (13 (link)). Monocytes cultured in medium alone were used as a control for LPS and CLO97. Stimulation by 5′-pppRNA required the use of a cationic transfection agent LyoVec (InvivoGen) so medium plus LyoVec alone was used as a control. Monocytes were cultured for up to 24 hrs at 37°C in a 5% CO₂-humidified environment.

For in vitro LDAs, colorectal cancer cells treated or not with 5-AZA-CdR for one day, or transfected with 0.5ug/ml of poly(I:C) low and high molecular weight in LyoVec, (InvivoGen) or treated with flagellin 1ug/ml or transfected 0.5 ug/ml of cGAMP in LyoVec (Invivogen) for 3 days were dissociated into single cells. Cells were then seeded in 96-well plates at the indicated cell doses (1 cell, 10 cells/, 100 cells, and 1000 cells/well). SytoxBlue was used to exclude dead cells. For each cell dose, at least 18 wells were seeded with cells, and for the lower cell doses, at least 72 wells were plated. 4 weeks later, wells containing spheres were scored, and the number of positive wells was used to calculate the frequency of sphere-forming units using the Extreme Limiting Dilution Analysis (ELDA) software (http://bioinf.wehi.edu.au/software/elda/index.html), provided by the Walter and Eliza Hall Institute (Hu and Smyth, 2009 (link)).
For in vivo LDAs, LIM1215 cells were treated with and without 5-AZA-CdR for 24 hours. Following, single cells suspension was obtained and diluted serially to the desired cell doses. Cells were injected subcutaneously into the flanks of NSG mice. The number of tumors formed out of the number of sites injected was scored to determine the frequency of colorectal CICs calculated using the ELDA software.