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Influence of the Anesthetic Technique on Circulating Extracellular Vesicles in Bladder Cancer Patients Undergoing Radical Cystectomy: A Prospective, Randomized Trial.

Author(s): Gluth L, Ochsenfarth C, Pham PNV, Wischermann JM, Komanek T, Roghmann F, Frey UH

Publication: Cells, 2023, Vol. 12, Page

PubMed ID: 37887347 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared EV characteristics, total miRNA concentration, and levels of tumor-associated miRNAs in plasma specimens of bladder cancer patients that were collected at different time points during radical cystectomy (prior to the administration of anesthesia, at anesthesia administration, 30 min after incision, and immediately after suturing); comparisons were also drawn among patients that received Propofol or sevoflurane for anesthesia.  Blood was collected from 51 patients with bladder cancer who were undergoing a radical cystectomy. The first specimen was collected before induction of anesthesia (baseline), a second after anesthesia was administered through a central venous catheter (CVC), a third 30 min after surgical incision, and a final specimen immediately following surgical suture.  All patients were anesthetized using fentanyl (2–4 μg/kg), and Propofol (2–mg/kg) and muscle relaxation using rocuronium (0.6–0.9 mg/kg) or succinylcholine (mg/kg), followed by a single intravenous administration of 2 g of ceftriaxone and 500 mg of metronidazole (30 min preoperatively) before establishing a CVC. Anesthesia (Propofol or sevoflurane) was then administered using a perfuser at the rate necessary to obtain a depth index of 45 (based on an ECG). Sevoflurane was also administered to a minimum alveolar concentration (MAC) of 0.8 to 1.2. After collection, blood specimens were immediately stored at 4°C for < 2 h. Plasma was separated by centrifugation at 2000 g for 15 min at 4°C, aliquoted into LoBind tubes, and stored at -80°C until EV isolation. EVs were isolated using ExoQuick-TC Exosome Precipitation Solution and filtered using Cytiva PD SpinTrap G-25 columns.  EV isolation was verified by Western blot using antibodies against the EV proteins ALIX, CD63, and Flotillin-1. The concentration and size profile of isolated EVs were quantified using nanoparticle tracking analysis. miRNAs were isolated from EVs using the mirVANA miRNA Isolation Kit, spiked with cel-miR-39-3p, and stored at -80°C. In total, 192 miRNAs were quantified in the EVs isolated from 10 patients (5 patients per anesthetic) using TaqMan low-density arrays, and miR-15a-5p, miR-17-5p, miR-21-5p, miR-451a, and cel-miR-39-3p were quantified in EVs from all patients using TaqMan real-time PCR assays.

   Summary of Findings:

   Randomized assignment of patients to the two anesthesia groups resulted in more women in the sevoflurane than the Propofol group (32% versus 8%, P=0.038), and a higher incidence of norepinephrine use during surgery in the Sevoflurane than Propofol group (P=0.03); no other differences in patient or intra-/post- operative characteristics were found. EVs were efficiently extracted from the plasma of both anesthesia groups; EV-specific proteins (ALIX, Flotillin and CD63) were detectable in EV suspensions and absent in EV-depleted plasma. The mean size of the EVs isolated from plasma collected prior to the administration of anesthesia was 113±12.7 nm. The average size of EVs was larger in specimens collected at anesthesia administration than prior to starting anesthesia (132±11.5 nm for Propofol and 120±8.87 nm for sevoflurane versus 113±12.7 nm, P=0.002), but 30 min after surgical excision the average EV size was smaller than in specimens collected at anesthesia administration (126 ±10.6 nm for Propofol and 116 ±12.7 for sevoflurane). On average, EVs were larger from patients who received Propofol compared to those who received sevoflurane at the onset of anesthesia and 30 min after surgical excision (P=0.002 and P=0.005, respectively). Average EV size was larger in plasma specimens collected immediately after surgical suture than those collected prior to administration of anesthesia, although EV size was comparable between specimens collected from patients receiving Propofol or sevoflurane.  Further analysis revealed a steady increase in particle concentration from baseline until completion of surgical sutures among patients who received Propofol (2.3-fold, P=0.0001 at administration of anesthesia; 2.9-fold, P<0.0001 30 min after surgical incision; and 3.6-fold, P=0.002 at suture). In patients receiving sevoflurane, the particle concentration was 1.9-fold higher than baseline after the administration of anesthesia (P<0.0001), and 1.8-fold higher 30 min after surgical incision (P<0.005), but only 70% of baseline immediately following surgical suture (P=0.0.16). Consequently, particle concentration was significantly higher in the Propofol group than the sevoflurane group when plasma specimens were collected 30 min after surgical incision or immediately after surgical suture (P=0.02 and P=0.001, respectively).

   Overall, levels of the 192 miRNAs evaluated were 12% higher after administration of anesthesia and 5% higher 30 minutes after surgical incision than baseline. When broken down by anesthesia type, there was a 30% increase in miRNA levels from baseline to the administration of anesthesia in patients receiving Propofol but a 6% decrease in patients receiving sevoflurane. Thirty minutes after surgical incision, miRNA levels were 9% higher than baseline in patients receiving Propofol but 9% lower than baseline in patients receiving sevoflurane. A total of 176 miRNAs were detected in patients who received Propofol but only 159 miRNAs were detected in patients who received sevoflurane. Levels that were significantly higher than baseline were observed for miR-17-5p, at the administration of anesthesia (5.39-fold, P=0.014) and immediately after surgical suture (39.8-fold, P=0.042), for miR-15a-5p 30 min after surgical incision  (9.7-fold, P=0.027), for miR-21-5p immediately after surgical suture (24.38-fold, P=0.029), and for miR-451a at the administration of anesthesia (1.75-fold, P=0.03). Importantly, miR-451 was higher than baseline at the administration of anesthesia only in samples collected from patients who received Propofol (2.5-fold, P=0.022), which resulted in significantly higher levels of miR-451 in patients receiving Propofol compared to those who received sevoflurane at that time point (P=0.032).

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Low density array
   Morphology	Light scattering
   Protein	Western blot
   Cell count/volume	Light scattering

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Anesthesia	Propofol Sevoflurane
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-15a-5p miR-17-5p miR-21-5p miR-451a Cel-miR-39-3p 192 miRNA array
   Biospecimen Acquisition	Time of biospecimen collection	Before induction of anesthesia (baseline) After induction of anesthesia 30 min after surgical incision Immediately following surgical suture

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