Normothermic ex vivo lung perfusion(NEVLP)has emerged as a modernized organ preservation tech-nique that allows for detailed assessment of donor lung function prior to transplantation.The main goal of this study was to identify potential biomarkers of lung function and/or injury during a prolonged(19 h)NEVLP procedure using in vivo solid-phase microextraction(SPME)technology followed by liquid chromatography-high resolution mass spectrometry(LC-HRMS).The use of minimally invasive in vivo SPME fibers for repeated sampling of biological tissue permits the monitoring and evaluation of biochemical changes and alterations in the metabolomic profile of the lung.These in vivo SPME fibers were directly introduced into the lung and were also used to extract metabolites(on-site SPME)from fresh perfusate samples collected alongside lung samplings.A subsequent goal of the study was to assess the feasibility of SPME as an in vivo method in metabolomics studies,in comparison to the traditional in-lab metabolomics workflow.Several upregulated biochemical pathways involved in pro-and anti-inflammatory responses,as well as lipid metabolism,were observed during extended lung perfusion,especially between the 11th and 12th hours of the procedure,in both lung and perfusate samples.However,several unstable and/or short-lived metabolites,such as neuroprostanes,have been extracted from lung tissue in vivo using SPME fibers.On-site monitoring of the metabolomic profiles of both lung tissues through in vivo SPME and perfusate samples on site throughout the prolonged NEVLP procedure can be effectively performed using in vivo SPME technology.

Adjuvant chemotherapy improves the survival outlook for patients undergoing operations for lung metastases caused by colorectal cancer(CRC).However,a multidisciplinary approach that evaluates several factors related to patient and tumor characteristics is necessary for managing chemotherapy treatment in metastatic CRC patients with lung disease,as such factors dictate the timing and drug regimen,which may affect treatment response and prognosis.In this study,we explore the potential of spatial metabolomics for evaluating metabolic phenotypes and therapy outcomes during the local de-livery of the anticancer drug,oxaliplatin,to the lung.12 male Yorkshire pigs underwent a 3 h left lung in vivo lung perfusion(IVLP)with various doses of oxaliplatin(7.5,10,20,40,and 80 mg/L),which were administered to the perfusion circuit reservoir as a bolus.Biocompatible solid-phase microextraction(SPME)microprobes were combined with global metabolite profiling to obtain spatiotemporal infor-mation about the activity of the drug,determine toxic doses that exceed therapeutic efficacy,and conduct a mechanistic exploration of associated lung injury.Mild and subclinical lung injury was observed at 40 mg/L of oxaliplatin,and significant compromise of the hemodynamic lung function was found at 80 mg/L.This result was associated with massive alterations in metabolic patterns of lung tissue and perfusate,resulting in a total of 139 discriminant compounds.Uncontrolled inflammatory response,abnormalities in energy metabolism,and mitochondrial dysfunction next to accelerated kynurenine and aldosterone production were recognized as distinct features of dysregulated metabolipidome.Spatial pharmacometabolomics may be a promising tool for identifying pathological responses to chemotherapy.

In vivo lung perfusion(IVLP)is a novel isolated lung technique developed to enable the local,in situ administration of high-dose chemotherapy to treat metastatic lung cancer.Combination therapy using folinic acid(FOL),5-fluorouracil(F),and oxaliplatin(OX)(FOLFOX)is routinely employed to treat several types of solid tumours in various tissues.However,F is characterized by large interpatient variability with respect to plasma concentration,which necessitates close monitoring during treatments using of this compound.Since plasma drug concentrations often do not reflect tissue drug concentrations,it is essential to utilize sample-preparation methods specifically suited to monitoring drug levels in target organs.In this work,in vivo solid-phase microextraction(in vivo SPME)is proposed as an effective tool for quantitative therapeutic drug monitoring of FOLFOX in porcine lungs during pre-clinical IVLP and intravenous(Ⅳ)trials.The concomitant extraction of other endogenous and exogenous small molecules from the lung and their detection via liquid chromatography coupled to high resolution mass spec-trometry(LC-HRMS)enabled an assessment of FOLFOX's impact on the metabolomic profile of the lung and revealed the metabolic pathways associated with the route of administration(IVLP vs.Ⅳ)and the therapy itself.This study also shows that the immediate instrumental analysis of metabolomic samples is ideal,as long-term storage at-80 ℃ results in changes in the metabolite content in the sample extracts.

Development of a novel in vivo lung perfusion(IVLP)procedure allows localized delivery of high-dose doxorubicin(DOX)for targeting residual micrometastatic disease in the lungs.However,DOX delivery via IVLP requires careful monitoring of drug level to ensure tissue concentrations of this agent remain in the therapeutic window.A small dimension nitinol wire coated with a sorbent of biocompatible morphology(Bio-SPME)has been clinically evaluated for in vivo lung tissue extraction and determina-tion of DOX and its key metabolites.The in vivo Bio-SPME-IVLP experiments were performed on pig model over various(150 and 225 mg/m2)drug doses,and during human clinical trial.Two patients with metastatic osteosarcoma were treated with a single 5 and 7 μg/mL(respectively)dose of DOX during a 3-h IVLP.In both pig and human cases,DOX tissue levels presented similar trends during IVLP.Human lung tissue concentrations of drug ranged between 15 and 293 μg/g over the course of the IVLP procedure.In addition to DOX levels,Bio-SPME followed by liquid chromatography-mass spectrometry analysis generated 64 metabolic features during endogenous metabolite screening,providing information about lung status during drug administration.Real-time monitoring of DOX levels in the lungs can be per-formed effectively throughout the IVLP procedure by in vivo Bio-SPME chemical biopsy approach.Bio-SPME also extracted various endogenous molecules,thus providing a real-time snapshot of the physi-ology of the cells,which might assist in the tailoring of personalized treatment strategy.