the study was approved by the central cambridge research ethics committee and was compliant with the health research authority. the study adhered to the principles of the declaration of helsinki and the guidelines for good clinical practice. participants did not receive financial compensation and gave informed consent. frozen tumour samples were embedded in a hydroxypropyl methylcellulose / polyvinylpyrrolidone hydrogel, and 10 µm - thick cryo - sections were obtained. sections were thaw - mounted onto superfrost microscope slides for desorption electrospray ionization ( desi ) and imc experiments ( thermo scientific ), while sections prepared for maldi - msi were thaw - mounted onto conductive ito - coated slides ( bruker daltonik ). the sections were dried immediately and sealed in vacuum pouches for storage at −80 °c. human tumour samples were treated with uv - c light before msi analysis to minimize aerosolization of potential pathogens. decontamination was performed in a sensor - controlled uv chamber ( opsytec dr. grobel ) at 250 mj cm. desi - msi analysis was performed on a q - exactive mass spectrometer ( thermo scientific ) equipped with an automate d 2d - desi ion source ( prosolia ). the spectrometer was used with a home - made swagelok desi sprayer and a mixture of 95 % methanol, 5 % water delivered at a flow rate of 1. 5 µl min and nebulized with nitrogen at a backpressure of 6 bar. human tumour samples were analysed in the mass range from 70 to 280 m / z with a mass resolution of 140, 000 at 200 m / z. the injection time was set at 500 ms, and data were acquired with a pixel size of 65 µm. following analysis, sections were stained with h & e and co - registered with the desi - ms images. the resulting. raw files were converted into. mzml files using proteowizard msconvert ( v. 3. 0. 4043 ) with the built - in peak picking algorithm and subsequently compiled to an. imzml file ( imzml converter, v. 1. 3 ). all subsequent data processing was performed in scils lab ( v. 2022b, bruker daltonik ). maldi - msi analysis was performed using a rapiflex tissuetyper instrument ( bruker daltonik ) operated in negative ion detection mode. 9-aminoacridine, prepared in an 80: 20 methanol - to - water ratio, was used as a matrix and spray - deposited using an automate d spray system ( m3 - sprayer, htx technologies ). mass spectra were acquired from 180 to 1, 000 m / z with a pixel size of 40 µm. a total of 350 laser shots were summed up per pixel. for all experiments, the laser was operated with a repetition rate of 10 khz. raw data were directly uploaded and processed with scils lab ( v. 2022b ) software. desi and maldi data and images were normalized to the total ion current to compensate for signal variation during the course of the experiments, and acquisition parameters and data processing were identical for human tissue, neurospheres embedded in matrigel and xenograft sections. snap - frozen tumour samples were homogenized with 5 µl mg of 2 m perchloric acid. the extract was centrifuged at 13, 000 g for 15 min, and the ph of the supernatant was adjusted to 7. 0 using 2 m koh. extracts were lyophilized and dissolved in 550 µl deuterium oxide containing methylene diphosph onic acid at 100 nmol g tissue, which was added as a chemical shift and intensity standard. proton - decoupled p nmr spectra were acquired with more than 8, 000 repetitions into 32, 768 data points with an 11 µs 90° pulse, a repetition time of 7. 2 s and a spectral width of 57 ppm ( 14, 006 hz ). arterial blood collected during intra - operative infusion was centrifuged at 2, 000 g and 4 °c for 20 min to collect the plasma, which was snap - frozen and stored at −80 °c. samples were thawed on wet ice and aliquots diluted 50 - fold with cold methanol: acetonitrile: water ( 50: 30: 20 ) in chilled tubes, vortexed for 10 min and centrifuged at 21, 100 g for 10 min at 4 °c, and then the supernatants stored at −80 °c. on the day of analysis, supernatants were centrifuged and aliquots transferred to a 96 - well plate for analysis by hydrophilic interaction liquid chromatography ( hilic ) high - resolution mass spectrometry ( hrms ). the hilic – hrms system consisted of a shimadzu nexera x2 uhplc and sciex 6600 tripl e tof mass spectrometer, using a sequant zic - philic 5 µm 150 × 2. 1 mm column ( with a zic - philic 20 × 2. 1 mm guard column ) at 45 °c. the liquid chromatography gra dien t started at 80 % acetonitrile and 20 % 20 mm ammonium carbonate ( ph 9. 4 ), changing to 20 % acetonitrile over 17 min at 200 µl min, with a further 11. 5 min of column re - equilibration. each sample was injected twice for analysis in both positive and negative electrospray ionization with a full - scan m / z range of 75 – 1, 000. data were acquired using sciex analyst tf and processed using sciex multiquant software. extracted ion chromatograms were generated from the theoretical m / z ± 20 ppm. peak integration was reviewed manually, and the peak area of each metabolite and isotope was exported. primary human cell lines were derived at addenbrooke ’ s hospital, cambridge, uk, as described previously. tissue collection was approved by a regional ethics committee ( rec18 / ee / 0283 ) and was compliant with the uk human tissue act 2004. resected tissue samples were washed with hanks ’ balanced salt solution and minced using sterile razor blades, followed by digestion with accutase ( sigma - aldrich ) for 60 min at 37 °c. single cells were isolated by filtration through a 40 µm filter ( falcon ). cells were centrifuged at 350 g, 21 °c, and the pellet was incubated with 2 – 3 ml of red blood cell lysis buffer ( sigma - aldrich ) for 5 min at room temperature ( 21 °c ). following centrifugation, cells were seeded at a density of 15, 000 cells per cm in serum - free neurobasal a medium ( gibco ) supplemented with b27, n2, egf and fgf growth factors ( sigma - aldrich ). cells were allowed to form aggregates, and the medium was changed 3 days post derivation. for neurosphere formation, cells were seeded in ultra low attachment 96 - well plates ( corning ) at a density of 10, 000 cells per well. sphere diameter was monitored using an incucyte microscope ( sartorius ). spheres were embedded in 150 μl matrigel ( corning ) domes in 24 - well plates ( corning ). the domes were covered with 2 ml of fresh neurobasal medium and grown for 24 h. the domes were then washed three times with pbs before 2 ml of fresh glucose - free neurobasal medium supplemented with 25 mm [ u - c ] glucose was added. following incubation for 24 h, the medium was removed and the domes were washed three times with ice - cold pbs and then snap - frozen in liquid nitrogen. the domes were sectioned to a thickness of 10 µm and analysed using desi - msi and maldi - msi, as described above. black, clear - bottomed 96 - well plates ( corning ) pre - coated with ecm ( merck ) were seeded at 5, 000 cells per well and incubated at 37 °c in 200 μl of neurobasal medium supplemented with growth factors, as described above. the medium was then replaced with either fresh medium or medium containing the following drug concentrations: azd2014, 1 μm; gefitinib, 1 μm; imatinib, 10 μm. the cells were incubated for 72 h before re - imaging using the incucyte microscope, and cell numbers were determined using the sartorius cell confluency module. at the end of the incubation, the medium was aspirate d, and 100 μl of pbs containing 3 μm propidium io dide was added to each well to measure necrotic cell death. the plates were imaged using the incucyte 10× cell - by - cell module using the red channel. the experiment was repeated three times with six technical replicates for each drug treatment. neurospheres in matrigel domes were grown at 37 °c, with one plate incubated in atmospheric o 2 and the other grown in 0. 5 % o 2, 0. 5 pa ( avatar, xcellbio ).