Intrasplenic inoculation

1 × 10⁵ 4T1-Luc cells were inoculated into the spleen parenchyma of BALB/c mice under general anaesthesia.

Unless otherwise stated, primary tumours and lungs were weighed at necropsy. For IVIS imaging, mice were injected intraperitoneally with 150 mg kg⁻¹ d-luciferin (Caliper Life Sciences) in 100 μL and mice imaged in vivo using an IVIS imaging chamber (IVIS Illumina II). Luminescence measurements (photons/second/cm²) were acquired over 1 min and analysed using the Living Image software (PerkinElmer) using a constant size region of interest over the tissues. Alternatively, 5 min after d-luciferin injection, dissected lungs, or livers were imaged ex vivo and quantified as total flux values from organs.

Where indicated, primary tumours were dissociated using a mouse tumour dissociation kit (130-096-730, Miltenyi Biotec) with the 37C_m_TDK_2 programme on the gentleMACS Octo Dissociator (Miltenyi Biotec). The resulting cell pellet was resuspended in Red Blood Cell Lysis buffer (Sigma) for 5 min, spun and resuspended in FACS buffer (PBS with 5% FBS) containing a 1:100 dilution of anti-mouse CD16/CD32 block for 10 min at room temperature. Cells were stained with directly conjugated CD45, CD31, F4/80 and PDGFRα antibodies for 30 min at 4 °C. Cells were co-stained with DAPI (1:10,000) to exclude dead cells, washed with PBS and sorted on a FACS Aria III. Sorted populations were defined as tumour cells (RFP+), fibroblasts (PDGFRα+), endothelial cells (PDGFRα−), macrophages (CD45+/F4/80+), other immune cells (CD45+/F4/80−) and negative for all other markers.