Rpm1 14

EMT6-hHER2 and EMT6-WT cells were injected into the mammary gland of female BALB/c mice (8–10 weeks old). Once tumors reached an average volume of 80 mm³ (day0), mice were treated with T-PNU (1 mg/kg) and/or α-mouse PD1 (12.5 mg/kg) (RPM1–14, rat IgG2a, Biox Cell), T-DM1 (15 mg/kg), trastuzumab (20 mg/kg) or mitoxantrone (2 μg/kg) or as indicated in the figures or legends. Tumor volume was measured three times per week as 1 alone or in combination with α-PD1 at day 0, 2, 4, 7. For FACS analysis and CD45-positive cell isolation, T-PNU, trastuzumab or T-DM1 were given as single treatment once the tumors reached 80 mm³ at the concentration indicated above. Animals were euthanized 10 days after treatments, when tumors in the T-PNU cohort reached a volume of 30 mm³ suitable for tumor harvest and cell processing. For T cell depletion experiments, α-CD8 depleting antibodies (53–6.72, rat IgG2a, Bio X Cell) were given at day − 2, 0, and once a week for the next 4 weeks at 10 mg/kg. Mice were euthanized once tumors reached a volume of 1200 mm³. For re-challenge experiments, T-PNU-cured animals received EMT6-hHER2 (10⁶), EMT6-WT (2.5 × 10⁵) or T/SA Thy1.1 (2.5 × 10⁵) tumor cells by mammary fat pad injection and orthotopic tumor growth was measured for a further 70 days.

C57BL/6 mice were injected subcutaneously into the right flank with 500,000 syngeneic murine MC38 colorectal adenocarcinoma cells (kindly provided by Thomas Wirth, Medizinischen Hochschule Hannover) suspended in phenol red-free DMEM (without additives). Once tumors reached an average volume of 60–80 mm³ (day 12–15), mice were injected intraperitoneally with 200 μl of PBS suspended solutions of CD39 ASO at indicated doses, non-targeting control oligonucleotide (control oligo 1) (100 mg/kg) or left untreated. On day 9 post first compound injection, mice were euthanized and tumors, and in selected cases tumor draining lymph nodes were excised and processed for FACS analyses as outlined below. For tumor growth experiments EMT6 (obtained from ATCC) murine breast cancer cells were injected into the mammary gland of female Balb/c mice. Once tumors reached an average volume of 80 mm³ (Day 8), mice were injected intraperitoneally with 200 μl of PBS suspended solutions of CD39 ASO (20 mg/kg), non-targeting control oligonucleotide (control oligo 1) (20 mg/kg) and/or mouse anti-PD-1 (12.5 mg/kg) (RPM1–14, rat IgG2a, BioXCell) at indicated timepoints. Tumor volume was calculated according to the formula: D /2*d*d, with D and d being the longest and shortest tumor diameter in mm, respectively.

The B16-F10 tumour model was established by using the method described above. To establish the 4 T1 tumour model, we injected the resuspended 4 × 10⁵ 4 T1 cells in 0.1 mL PBS into the fourth pair of the mammary fat pad of BALB/C mice. When the tumour volumes of B16-F10 and 4 T1 tumour-bearing mice reached 120–180 mm³, the mice were randomly distributed into the following groups (n = 6): control, POG, anti-PD-1 (Bio X Cell RPM1–14, rat IgG2a) and a combination of POG and anti-PD-1 groups. The control group was treated with the vehicle alone (5% DMSO in 20% hydroxypropyl beta-cyclodextrin buffer). The POG group was administered intraperitoneally daily at 100 and 200 mg/kg for 14 days. Anti-PD-1 antibody (clone RMP1–14, Bio X Cell) or isotype control antibody (clone 2A3, rat IgG2a, Bio X Cell) was intraperitoneally given on days 11, 14, 17, 20 and 23 (200 μg/injection). Tumour volume was measured every 3 days. Tumour volume was calculated as length × width² / 2.