Tissue tek optimum cutting temperature medium

At 6 weeks post human CD4⁺ T cell transfer, NSG-DR1 mice were sacrificed by cervical dislocation, then whole-body perfusion with PBS + 50 U/ml heparin (Ratiopharm, Ulm, Germany) was conducted and the organs were extracted for ex vivo analysis. FACS analysis of freshly isolated cells (heart and spleen) was performed. The samples intended for RNA analysis were stored in RNAlater (Qiagen, Hilden, Germany) for 24 h and then stored at -80°C. Samples intended for histological analysis were embedded in Tissue-TeK optimum cutting temperature medium (Sakura Finetek, Alphen ann den Rijn, The Netherlands) and then stored at -80°C. Heart DNA extraction was performed from heart slices by trimming the Tissue-TeK content and performing tissue digestion and DNA purification using a GeneJET genomic DNA purification kit (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer’s instructions.

EBC‐1 xenografts (10⁶ cells injected subcutaneously on the right flank) were grown in 12‐week‐old female Rag2 common gamma‐null mice (Rag2^−/−γc^−/−, Taconic), and the growth of the tumors was regularly monitored by caliper measurements. Once tumors had reached a size of 300 mm³ (day 0), mice were randomly divided into four treatment groups: vehicle control (Solutol HS 15; BASF ChemTrade GmbH, Burgbernheim, Germany), tepotinib only (15 mg·kg⁻¹·day⁻¹per os, days 1–6), IR only (one single 6 Gy dose on day 3), and combination of tepotinib with IR. Radiation was delivered locally using the XStrahl 150 (Xstrahl Limited, Surrey, UK). Tumors were harvested 24 h after the last tepotinib treatment, frozen in Tissue‐Tek optimum cutting temperature medium (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands), and stored at −20 °C. Animal experiments were conducted in strict compliance with Swiss Federal guidelines and have been approved by Federal Food Safety and Veterinary Office.