Patients
The study was approved by the institutional review board of the University Duisburg-Essen (date of approval: 10.01.2018, code of approval: 17-7802-BO). Patients gave written informed consent ahead of surgery. All patients had either a verified or suspected lung cancer in stage I/II according to the 8th edition of the Union for the International Cancer Control TNM classification of malignant tumours [29], underwent complete preoperative staging, and had surgical resection recommended after interdisciplinary tumour board discussion. The protocol was designed to ensure that routine histopathological examination that was not hampered by the experiments.
Specimen preparation and isolated lung perfusion
Immediately after retrieval during surgery, the lobes were topically cooled with cold saline and flushed out both ante- and retrogradely with 1 L of cold (4 °C) buffered preservation solution (Perfadex Plus®, XVIVO, Göteborg, Sweden) with 5,000 IU of unfractionated heparin added. Silicone cannulas were sutured on the vascular and bronchial stumps with single running sutures (4/0 Prolene®, Johnson & Johnson Medical GmbH, Norderstedt, Germany). All procedures were performed by the same board certified thoracic surgeon with 5 years of experience. After adequate inflation with an Ambu-bag, the lungs were stored at 4 °C until ILP.
ILP was performed in the computed tomography (CT) room according to our institutional protocol [28]. Perfusion was achieved with a Cardiohelp pump (Maquet, Gettinge, Rastatt, Germany) and a modified extracorporeal membrane oxygenation circuit. The circuit was primed with 1220ml of hyper-oncotic acellular colloidal perfusate (32.8 g/L succinated gelatine; 32.8 g/L human albumin; 6.6 g/L glucose). After initial warming-up, lungs were ventilated (Dräger, Evita XL, Lübeck, Germany) in a protective manner according to the calculated tidal volume of the patient and the number of perfused lung segments (6ml/kg). Ventilation parameters were as follows: 8 bpm, ½ inspiratory/expiratory ratio, 0.4 fraction of inspired oxygen, and 5–10 cm H20 of positive end-expiratory pressure. Perfusion flow was maintained at 40% of the estimated cardiac output normalised on the number of perfused lung segments. Dynamic lung compliance was calculated according to following formula: Tidal volume/(peak airway pressure/positive end-expiratory pressure).
Transarterial lung embolisation
Before TPE, the explanted lung lobe was positioned in the CT gantry of a Somatom Definition AS (Siemens Healthineers, Forchheim, Germany) for repeated CT scans and CT perfusion imaging (CTPI) during the experiment (Figs. 1 and 2).
CT scans of the whole lobe were acquired under continuous ventilation with a slice thickness of 1mm and a pitch of 0.6. To optimise image quality, CareKVTM (preset 120kV) and CareDoseTM (preset 77mAs) were used. For CTPI, CT data were acquired continuously for 60 s with a time increment of 1 s and fixed CT acquisition parameters (100 kVp, 150 mAs, 10 mm slice thickness). Contrast agent (Ultravist® 300, Bayer Vital GmbH, Leverkusen, Germany) was administered using an automated contrast agent injector. Ten seconds after the start of continuous CT acquisition, a total of 15 mL contrast agent was injected followed a 15 mL saline flush with an injection rate of 2 mL/min using an automated syringe infusion pump (Injectomat® MC Agila, Fresenius Kabi, Bad Homburg vor der Höhe, Germany)
Lung perfusion was started at t0, and both flow and temperature were continuously increased until normothermia (37 °C) was reached. Ventilation and deoxygenation of the perfusate was started simultaneously. After and an initial CT scan (t30) of the lobe, baseline CTPI was performed. Then, (at t35) TPE was initiated with 10 mL of a mixture of 12.5 mL of contrast agent and 450 mg of DSM with a sphere size of 50 ± 7 μm in a solution of 7.5 mL (EmboceptTM S, PharmaCept GmbH, Berlin, Germany) at an injection rate of 2 mL/min via the arterial limb. This procedure was repeated at prespecified timepoints (t45, t60, and t75). After the assessment at t90, 500 units of alpha-amylase (Merck KGaA, Darmstadt, Germany) were injected at 2 mL/min into the arterial limb (diluted in 50 mL of perfusion solution) to hydrolyse the DSM. After complete administration of the amylase, two additional CT scans and CTPI were performed. Surgical biopsies of 1.0 to 2.5 cm in size were taken from the periphery of the lobe (Fig. 3), and functional assessments, i.e., pulmonary artery pressure (PAP), airway pressure, pulmonary vascular resistance (PVR), and a blood gas analysis were performed throughout the experiments according to the study timeline (Fig. 4). The perfusion was stopped after the last assessment.
Histopathological analysis
Histopathological specimens were stored directly after sampling in 4% neutral buffered formalin. For microscopy analysis, complete biopsy specimens were embedded and serial sections of 3 to 4 μm thickness were stained with haematoxylin and eosin. A pathologist, blinded to the timepoints of histopathological sampling, analysed all slides. At microscopy analysis, starch particles appeared as pale bluish spherical structures within blood vessels without any inflammatory reaction. Single DSM particles had a size of 20 to 30 μm and tended to aggregate with a cluster size > 150 μm. Affected small lung vessels had a size of 50 to 300 μm. Due to differences in lung parenchyma including emphysematous or atelectatic changes, an area-based quantitative analysis was considered as unfavourable. Therefore, a semiquantitative ordinal score was used to describe the extent of embolisation (0 = no particles; 1 = single particles, no aggregates; 2 = single particles, few aggregates (< 50% of all starch particles consisting of aggregates); 3 = ≥ 50% of starch particles appeared as large aggregates).
At the end of the procedure, the lobe resection specimens were processed for routine histopathological work-up.
CTPI analysis
Images of CTPI were analysed using the Syngo.CT Dynamic Angio workflow of the syngo.via software (VB40, Siemens Healthineers, Forchheim, Germany). In the upper, mid, and lower periphery of the lung lobe, three non-overlapping regions of interests (ROIs) were drawn with a size of 3 to 5 cm2 depending on the size of the lobe and a central ROI in the pulmonary artery for every CTPI acquired at t30, t45, t60, t75, t90, t105, and t120 for each patient to determine time to peak (TTP). To correct for differences caused by the length of the vascular anastomoses as well as artifacts caused by lung consolidation, TTP of ROI in the pulmonary artery was subtracted from the mean TTP value of the three ROI obtained in the upper, mid, and lower periphery of the lung lobe for each patient and each timepoint.
Statistical analysis
Data are presented as mean ± standard deviation or median and range (minimum and maximum) according to the normal/near-normal or non-normal distribution. Data plotting and analysis were performed with Graphpad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Linear regression analysis and one-way ANOVA were used to evaluate parameter dynamics throughout experiments. The 95% confidence intervals where computed and plotted on graphs. p values < 0.05 were considered as statistically significant. Due to the explorative nature of this study, no correction for multiple testing was performed.