In vivo tumorigenicity assays

Tumour studies were performed using 5‐ to 6‐week‐old male NOD.Cg‐Prkdc ^scid Il2rg ^tm1Wjl/SzJ (NSG) mice, sourced from the Francis Crick Institute common colony and housed in individually ventilated cages. Intradermal injection was performed following procedures described previously 38. Briefly, 350,000 DBTRG‐05MG cells, expressing either EMX2 cDNA or an RFP expressing control, were injected intradermally into both flanks of three 5‐week male NSG mice for each condition. After appearance, tumour size was measured weekly using digital callipers and volume was calculated as L*W ²/2, where L = longest edge of tumour and W = shortest edge of tumour. For orthotropic brain tumours, NSG mice were anesthetised, and upon surgical exposure, a syringe needle was inserted into the striatum (1.5 mm lateral of the bregma, 2.5 mm deep) 88. 300,000 DBTRG‐05MG cells expressing either EMX2 cDNA or an RFP expressing control were delivered to five mice for each condition. Three mice (one RFP control and two EMX2) were removed from the study early from health complications, with no tumours evident at endpoint. Tumour appearance was monitored by magnetic resonance imaging (MRI) from 3 weeks. Tumour volume was measured from MRI anatomical axial scans using ImageJ software and calculated as the average area of tumours in mm² from consecutive sections multiplied by the depth (0.32‐mm section interval multiplied by number of sections). Mice were randomly allocated for injection, ensuring that animals of similar age were present in both conditions. Assessment of results was not performed blinding the investigator. Animal studies were conducted in accordance with the Francis Crick Institute project licence PPL 70/8167 approved by the Home Office.