In vivo treatment of antibodies or inhibitors

To determine whether Spp1^hi-TAMs are resistant to CSF1R blockade, mice were randomly divided into two groups when they developed CRPC (tumour volume of 100–200 mm³) and were administered intraperitoneally 1 mg anti-mouse CSF1R (AFS98, BioXCell) or the respective isotype-matched control (2A3, BioXCell) antibodies in 200 μl PBS. A maintenance dose of 0.5 mg in 200 μl PBS was given after 5 days. The myeloid composition was analysed by flow cytometry 2 days after the final injection.

For immune checkpoint inhibition, mice with established CRPC (tumour volume of 100–200 mm³) were randomly divided into four groups and subjected to intraperitoneal injection with these antibodies in 200 μl PBS every 3 days for a total of three injections: 200 μg anti-mouse CTLA-4 (24H2)⁶⁶ alone; 400 μg anti-mouse PD-1 (17D2)⁶⁷ alone; a combination of anti-mouse CTLA-4 and PD-1; or the respective IgG2a, κ isotype-matched control. Tumour burden was measured every 2–3 days after the initial injection until it reached 750 mm³, unless otherwise indicated.

To determine whether Spp1^hi-TAMs contribute to resistance to ICIs in vivo, mice with developed CRPC (tumour volume of 100–200 mm³) were randomly divided into three groups. They were administered with: a combination of anti-mouse CTLA-4 and PD-1 in 200 μl PBS injected intraperitoneally along with intratumoral injection of 1 × 10⁵ Spp1^hi-TAMs purified from digested CRPC (more than 350 mm³) of a mouse from the same cohort in 50 μl PBS; a combination of anti-mouse CTLA-4 and PD-1 in 200 μl PBS injected intraperitoneally along with 50 μl of PBS intratumorally; or the respective isotype-matched control antibody in 200 μl PBS injected intraperitoneally along with 50 μl PBS intratumorally. Antibodies were administered every 3 days for a total of three injections, and Spp1^hi-TAMs were adoptively transferred every 5 days for a total of two injections. Tumour growth was measured every 2–3 days after the initial injection until it reached 750 mm³, unless otherwise indicated. The lymphoid composition was analysed by flow cytometry one day after the final injection.

For blockade of adenosine receptors (A2ARs), mice with established CRPC (tumour volume, 100–200 mm³) were randomly divided into two groups. Ciforadenant (10 mg per kg, Corvus Pharmaceuticals) or DMSO vehicle control (Sigma-Aldrich) in 200 μl of injection solution was administered once daily through oral gavage for 10 consecutive days. The injection solution consisted of 10% ciforadenant (or DMSO medium) and 90% corn oil (MedchemExpress). Tumour growth was measured every 2–3 days after the initial injection.

To determine whether A2AR blockade enhances immunotherapy efficacy, mice with established CRPC (tumour volume, 100–200 mm³) were randomly divided into two groups. Ciforadenant (10 mg per kg, Corvus Pharmaceuticals) or DMSO vehicle control (Sigma-Aldrich) in 200 μl of injection solution described above was administered once daily by oral gavage for 10 consecutive days. Simultaneously, mice were injected intraperitoneally with 400 µg anti-mouse PD-1 or the respective isotype-matched control antibodies in 200 µl PBS every 3 days for a total of three injections. Tumour growth was monitored every 2–3 days after the initial injection. The lymphoid and myeloid compositions were analysed by flow cytometry 1–2 h after the eighth injection of ciforadenant (1 day after the final anti-mouse PD-1 antibody injection).

All comparisons within experiments were carried out using age-matched mice (6–10 weeks old) engrafted with the same stock of MyC-CaP throughout the study.