Clone rmp1 14

FVB/N mice and 129-E mice were purchased from Charles River Laboratories. Tumor cells were implanted in mice aged 7–9 weeks subcutaneously at optimal doses as indicated in figure legends. Tumor length and width were measured blindly by caliper and the volume calculated by the equation: 0.4×length×width^∧2. For immunotherapy studies, mice bearing ~50 mm³ tumors were randomized and treated with control, IT CCL21-DC (1×10⁶ cells), IP 200 µg of anti-PD-1 antibody (BioXcell, Clone RMP1-14) or combination as illustrated in the figures. IP administration of rat IgG2a (BioXcell, Clone 2A3) was used as an isotype control for anti-PD-1. For T cell egress studies, FVB/N mice bearing KPL-3M tumors were treated with fingolimod (2 mg/kg) every other day starting at day 5, 1 day prior to treatment of tumors with IT CCL21-DC (1×10⁶ cells). For secondary tumor challenge studies, mice were euthanized when tumor volume reached 1500 mm³. In vivo bioluminescence images were obtained by IVIS Spectrum imager 10 min after IP injection of D-luciferin (150 mg/kg). For DC trafficking studies, mock-DCs and CCL21-DCs were labeled with CellTracker Red CMTPX (Invitrogen) fluorescent dye per manufacturer’s protocol prior to IT injection.

Antibodies against PD1 were given IP 200 μg/mouse bi-weekly (Clone RMP1-14; BioxCell, West Lebanon, NH) and CTLA4 were given IP 200 μg/mouse bi-weekly (Clone 9D9 IgG1 and IgG2 a; kind gift from Bristol Myers Squibb). Diphtheria toxin (Sigma, St. Louis MO) was given IP 1 μg/mouse bi-weekly.

To establish orthotopic models, either 66cl4-shMYC-NC/1/3 or 4T1-MYC-vec/oe cells in 50 µL of DMEM medium were injected into the fourth mammary gland of 6-week-old female BALB/c or NSG mice (66cl4: 1×10⁶ cells; 4T1: 5×10⁵ cells). For CD8⁺ T cell depletion experiments, 6-week-old female BALB/c mice were treated with rat IgG2b (BioXCell, clone LTF-2, 100 µg injected intraperitoneally on day 1, followed by 50 µg on day 3 and 50 µg weekly for the remainder of the experiment) or anti-mouse CD8a (BioXCell, clone 2.43, 100 µg injected intraperitoneally on day 1, followed by 50 µg on day 3 and 50 µg weekly for the remainder of the experiment). For in vivo combination therapy, mice were treated with diluent or decitabine alone (S1200, Selleck, 0.8 mg/kg, intraperitoneal injection, every day) or in combination with isotype IgG (BioXcell, clone 2A3, 100 µg injected intraperitoneally, on days 3, 7, 10, 14) or anti-PD-1 antibody (BioXcell, clone RMP1-14, 100 µg injected intraperitoneally, on days 3, 7, 10, 14).
Tumor growth was measured 2–3 times per week using calipers. The primary tumors were harvested for analysis once they reached a 3–5 week time point or a volume of 1 or 2 cm³ using the formula: volume = (width² ×length)/2. Mice were humanely sacrificed after measurement. For experimental accuracy, these data were also included.

C57BL/6 mice were subcutaneously (s.c.) injected with 2×10⁴ Ret cells OE IL-6 or not. Tumor size was measured by a caliper and recorded three times per week. On a tumor length or width of 1.5 cm or any other termination criterion, mice were sacrificed and recorded as died. Tumor volume was calculated according to the formula: $volume=\frac{{width}^2*length}{2}$ .25 (link) After 3 weeks, mouse tumors were isolated for FACS analysis.
On the first signs of tumors, RET transgenic mice were separated into four groups containing equal numbers of males and females. One group received isotype control antibodies (clone 2A3, 12.5 mg/kg; and clone HRPN,10 mg/kg; both BioXcell), the second group was injected with anti-PD-1 antibodies (clone RMP1-14, 12.5 mg/kg; BioXcell). Other mice received anti-IL-6 antibodies (clone MP5-20F3, 10 mg/kg; BioXcell) or the combination of anti-PD-1 and anti-IL-6. Antibodies were injected intraperitoneally for 4 weeks, twice per week. Mice with any of the termination criteria were sacrificed and recorded as died. In another set of experiments, mice of the same groups were sacrificed after 4 weeks of therapy, and tumors were isolated for FACS analysis.