Low melting temperature agarose

Human glioblastoma tumor tissue was acquired with informed consent by all patients or their legally authorized representative, under an IRB approved protocol at the University of Colorado Hospital. All subsequent experiments were performed in accordance with IRB regulations. Human glioblastoma tissue from contrast enhancing regions was obtained intraoperatively utilizing surgical navigation technology^{43 (link)–45 (link)}. Slice culture generation is described elsewhere^{11 (link),46 (link)}. Briefly, tumor tissue was embedded in low melting temperature agarose (Invitrogen) and sliced 300–350 μm thick using a VT100S Vibratome (Leica). Tumor slices were cultured on PTFE inserts (Millipore) maintained in a humidified incubator (37 °C and 5% CO₂) using slice culture medium as described elsewhere^{11 (link)}. Pathology reports for patient samples (n = 7) provided population-level EGFR-amplification binary scoring, but did not assess levels in individual cells. EGFR amplification status was assessed via fluorescence in situ hybridization performed and scored by an accredited cytogenetics laboratory at the University of Colorado Hospital. All samples were IDH1 wild-type (assessed by IHC) and EGFRviii status was not assessed. Individual patient demographic and tumor genetic information is summarized in Supplemental Table 1.

Blood was collected by cardiac puncture and kept in a −80 °C refrigerator for further investigation. Bronchoalveolar lavage fluid (BALF) was collected by pushing 1 mL of normal saline solution (NSS) into the lung, and then, the lung fluid was gently drawn back and collected in microcentrifuge tubes. After heart perfusion, the right lung was clamped, and the left lung was inflated with 10% low-melting-temperature agarose (Invitrogen, Carlsbad, CA, USA) dissolved in a sterile normal saline solution (Terumo, Tokyo, Japan) into the trachea at a transpulmonary pressure of 25–30 cm H₂O, monitored by pressure manometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Then, the inflated lung was removed and fixed in 10% buffered formalin (Vidhyasom Co., Ltd., Bangkok, Thailand) for 24 h to examine histopathology of the lung. The right lung was removed and kept in −80 °C refrigerator for further biochemical analysis.