In silico model development and optimization of in vitro lung cell population growth.

Tissue engineering predominantly relies on trial and error in vitro and ex vivo experiments to develop protocols and bioreactors to generate functional tissues. As an alternative, in silico methods have the potential to significantly reduce the timelines and costs of experimental programs for tissue engineering. In this paper, we propose a methodology to formulate, select, calibrate, and test mathematical models to predict cell population growth as a function of the biochemical environment and to design optimal experimental protocols for model inference of in silico model parameters. We systematically combine methods from the experimental design, mathematical statistics, and optimization literature to develop unique and explainable mathematical models for cell population dynamics. The proposed methodology is applied to the development of this first published model for a population of the airway-relevant bronchio-alveolar epithelial (BEAS-2B) cell line as a function of the concentration of metabolic-related biochemical substrates. The resulting model is a system of ordinary differential equations that predict the temporal dynamics of BEAS-2B cell populations as a function of the initial seeded cell population and the glucose, oxygen, and lactate concentrations in the growth media, using seven parameters rigorously inferred from optimally designed in vitro experiments.

Bioengineering of vascularized porcine flaps using perfusion-recellularization.

Large volume soft tissue defects greatly impact patient quality of life and function while suitable repair options remain a challenge in reconstructive surgery. Engineered flaps could represent a clinically translatable option that may circumvent issues related to donor site morbidity and tissue availability. Herein, we describe the regeneration of vascularized porcine flaps, specifically of the omentum and tensor fascia lata (TFL) flaps, using a tissue engineering perfusion-decellularization and recellularization approach. Flaps were decellularized using a low concentration sodium dodecyl sulfate (SDS) detergent perfusion to generate an acellular scaffold with retained extracellular matrix (ECM) components while removing underlying cellular and nuclear contents. A perfusion-recellularization strategy allowed for seeding of acellular flaps with a co-culture of human umbilical vein endothelial cell (HUVEC) and mesenchymal stromal cells (MSC) onto the decellularized omentum and TFL flaps. Our recellularization technique demonstrated evidence of intravascular cell attachment, as well as markers of endothelial and mesenchymal phenotype. Altogether, our findings support the potential of using bioengineered porcine flaps as a novel, clinically-translatable strategy for future application in reconstructive surgery.

Extending the age criteria of lung transplant donors to 70+ years old does not significantly affect recipient survival.

OBJECTIVES: To determine the impact of older donor age (70+ years) on long-term survival and freedom from chronic lung allograft dysfunction in lung transplant (LTx) recipients. METHODS: A retrospective single-center study was performed on all LTx recipients from 2002 to 2017 and a modern subgroup from 2013 to 2017. Recipients were stratified into 4 groups based on donor lung age (<18, 18-55, 56-69, ≥70 years). Donor and recipient characteristics were compared using χ2 tests for differences in proportions and analysis of variance for differences in means. Univariable and multivariable Cox regression was used to describe differences in long-term survival and freedom from chronic lung allograft dysfunction. RESULTS: Between 2002 and 2017, 1600 LTx were performed, 98 of which were performed from donors aged 70 years or older. Recipients of 70+ years donor lungs were significantly older with a mean age of 55.5 ± 12.9 years old (P = .001) and had more Status 3 (urgent) recipients (37.4%, P = .002). After multivariable regression, there were no significant differences in survival or freedom from chronic lung allograft dysfunction between the 4 strata of recipients. CONCLUSIONS: Lung transplantation using donors 70 years old or older can be considered when all other parameters suggest excellent donor lung function without compromising short- or long-term outcomes.

Robotic Lobectomy Is Cost-effective and Provides Comparable Health Utility Scores to Video-assisted Lobectomy: Early Results of the RAVAL Trial.

OBJECTIVE: The aim of this study was to determine if robotic-assisted lobectomy (RPL-4) is cost-effective and offers improved patient-reported health utility for patients with early-stage non-small cell lung cancer when compared with video-assisted thoracic surgery lobectomy (VATS-lobectomy). BACKGROUND: Barriers against the adoption of RPL-4 in publicly funded health care include the paucity of high-quality prospective trials and the perceived high cost of robotic surgery. METHODS: Patients were enrolled in a blinded, multicentered, randomized controlled trial in Canada, the United States, and France, and were randomized 1:1 to either RPL-4 or VATS-lobectomy. EuroQol 5 Dimension 5 Level (EQ-5D-5L) was administered at baseline and postoperative day 1; weeks 3, 7, 12; and months 6 and 12. Direct and indirect costs were tracked using standard methods. Seemingly Unrelated Regression was applied to estimate the cost effect, adjusting for baseline health utility. The incremental cost-effectiveness ratio was generated by 10,000 bootstrap samples with multivariate imputation by chained equations. RESULTS: Of 406 patients screened, 186 were randomized, and 164 analyzed after the final eligibility review (RPL-4: n=81; VATS-lobectomy: n=83). Twelve-month follow-up was completed by 94.51% (155/164) of participants. The median age was 68 (60-74). There were no significant differences in body mass index, comorbidity, pulmonary function, smoking status, baseline health utility, or tumor characteristics between arms. The mean 12-week health utility score was 0.85 (0.10) for RPL-4 and 0.80 (0.19) for VATS-lobectomy ( P =0.02). Significantly more lymph nodes were sampled [10 (8-13) vs 8 (5-10); P =0.003] in the RPL-4 arm. The incremental cost/quality-adjusted life year of RPL-4 was $14,925.62 (95% CI: $6843.69, $23,007.56) at 12 months. CONCLUSION: Early results of the RAVAL trial suggest that RPL-4 is cost-effective and associated with comparable short-term patient-reported health utility scores when compared with VATS-lobectomy.

Association of Circulating Tumor DNA Testing Before Tissue Diagnosis With Time to Treatment Among Patients With Suspected Advanced Lung Cancer: The ACCELERATE Nonrandomized Clinical Trial.

Importance: Liquid biopsy has emerged as a complement to tumor tissue profiling for advanced non-small cell lung cancer (NSCLC). The optimal way to integrate liquid biopsy into the diagnostic algorithm for patients with newly diagnosed advanced NSCLC remains unclear. Objective: To evaluate the use of circulating tumor DNA (ctDNA) genotyping before tissue diagnosis among patients with suspected advanced NSCLC and its association with time to treatment. Design, Setting, and Participants: This single-group nonrandomized clinical trial was conducted among 150 patients at the Princess Margaret Cancer Centre-University Health Network (Toronto, Ontario, Canada) between July 1, 2021, and November 30, 2022. Patients referred for investigation and diagnosis of lung cancer were eligible if they had radiologic evidence of advanced lung cancer prior to a tissue diagnosis. Interventions: Patients underwent plasma ctDNA testing with a next-generation sequencing (NGS) assay before lung cancer diagnosis. Diagnostic biopsy and tissue NGS were performed per standard of care. Main Outcome and Measures: The primary end point was time from referral to treatment initiation among patients with advanced nonsquamous NSCLC using ctDNA testing before diagnosis (ACCELERATE [Accelerating Lung Cancer Diagnosis Through Liquid Biopsy] cohort). This cohort was compared with a reference cohort using standard tissue genotyping after tissue diagnosis. Results: Of the 150 patients (median age at diagnosis, 68 years [range, 33-91 years]; 80 men [53%]) enrolled, 90 (60%) had advanced nonsquamous NSCLC. The median time to treatment was 39 days (IQR, 27-52 days) for the ACCELERATE cohort vs 62 days (IQR, 44-82 days) for the reference cohort (P < .001). Among the ACCELERATE cohort, the median turnaround time from sample collection to genotyping results was 7 days (IQR, 6-9 days) for plasma and 23 days (IQR, 18-28 days) for tissue NGS (P < .001). Of the 90 patients with advanced nonsquamous NSCLC, 21 (23%) started targeted therapy before tissue NGS results were available, and 11 (12%) had actionable alterations identified only through plasma testing. Conclusions and Relevance: This nonrandomized clinical trial found that the use of plasma ctDNA genotyping before tissue diagnosis among patients with suspected advanced NSCLC was associated with accelerated time to treatment compared with a reference cohort undergoing standard tissue testing. Trial Registration: ClinicalTrials.gov Identifier: NCT04863924.

Multicentre positive deviance to reduce adverse events and length of stay after pulmonary resection.

BACKGROUND: Postoperative adverse events (AEs) following pulmonary resection enormously impact patient well-being, length of stay (LOS) and healthcare costs. Standardised AE data collection can be used to identify positive outliers demonstrating positive deviance (PD) who may be helpful to inform the best practice. Here, we describe our initial experience of a novel quality improvement process using PD to reduce LOS and AEs. METHODS: AE rates and LOS were collected from four centres (2014-2020) using a common dictionary. Surgeons repeatedly participated in 60 to 90 min seminars consisting of the following process: identify outcome and procedure targeted, review relevant best evidence literature, view all data anonymised by surgeon or centre (if multicentre), choose and reveal identity of best performance PD outliers, who discuss their management principles while all receive self-evaluation reports, followed by collegial discussion to generate consensus recommendations, voted by all. We assessed overall impact on AEs and LOS using aggregate data in a before/after analysis. RESULTS: A total of 131 surgeons (average 12/seminar) participated in 11 PD seminars (8 local and 3 multicentre), yielding 85 consensus recommendation (average 8/seminar). Median LOS following lobectomy decreased from 4.0 to 3.0 days (p=0.04) following local PD seminars and from 4.0 to 3.5 days (p=0.11) following multicentre seminars. Trends for reductions in multiple AE rates were also observed. CONCLUSION: While limited by the longitudinal design, these findings provide preliminary support for this data-driven, collegial and actionable quality improvement process to help standardise and improve patient care, and merits further more rigorous investigation.

Outcomes of lung transplantation from donors with a history of substance abuse.

OBJECTIVE: The study objective was to determine whether donor substance abuse (opioid overdose death, opioid use, cigarette or marijuana smoking) impacts lung acceptance and recipient outcomes. METHODS: Donor offers to a single center from 2013 to 2019 were reviewed to determine if lung acceptance rates and recipient outcomes were affected by donor substance abuse. RESULTS: There were 3515 donor offers over the study period. A total of 154 offers (4.4%) were opioid use and 117 (3.3%) were opioid overdose deaths. A total of 1744 donors (65.0%) smoked cigarettes and 69 donors (2.6%) smoked marijuana. Of smokers, 601 (35.0%) had less than 20 pack-year history and 1117 (65.0%) had more than 20 pack-year history. Substance abuse donors were younger (51.5 vs 55.2 P < .001), more often male (65.6 vs 54.8%, P < .001), more often White (86.2 vs 68.7%, P < .001), and had hepatitis C (8.3 vs 0.8%, P < .001). Donor acceptance was significantly associated with brain dead donors (odds ratio, 1.56, P < .001), donor smoking history (odds ratio, 0.56, P < .001), hepatitis C (odds ratio, 0.35, P < .001), younger age (odds ratio, 0.98, P < .001), male gender (odds ratio, 0.74, P = .004), and any substance abuse history (odds ratio, 0.50, P < .001), but not opioid use, opioid overdose death, or marijuana use. Recipient survival was equivalent when using lungs from donors who had opioid overdose death, who smoked marijuana, or who smoked cigarettes for less than 20 patient-years or more than 20 patient-years, and significantly longer in recipients of opioid use lungs. There was no significant difference in time to chronic lung allograft dysfunction for recipients who received lungs from opioid overdose death or with a history of opioid use, marijuana smoking, or cigarette smoking. CONCLUSIONS: Donor acceptance was impacted by cigarette smoking but not opioid use, opioid overdose death, or marijuana use. Graft outcomes and recipient survival were similar for recipients of lungs from donors who abused substances.

Influence of anterior tumor location on survival after resection of lung cancer invading the thoracic inlet (Pancoast tumors).

OBJECTIVE: Superior sulcus tumors are a challenging subset of non-small cell lung carcinomas invading the thoracic inlet. In this study, we determined whether the location of the tumor along the first rib had an influence on survival. METHODS: We performed a review of 92 consecutive patients undergoing surgery for non-small cell lung carcinomas invading the thoracic inlet between January 1996 and June 2021. Tumor location was categorized into anterior and posterior based on predefined zones. RESULTS: In total, 21 tumors were located anteriorly (23%) and 71 posteriorly (77%). The rate of R0 resection (81% vs 87%; P = .4) and pathological complete response to induction therapy (33% vs 37%; P = .8) were similar between locations. After a median follow-up of 5.8 years (range, 0.8-24 years), 49 patients died for an overall survival of 48% (95% CI, 38%-59%) at 5 years. The 5-year survival was favorably influenced by R0 (vs R1) resection (51% vs 29%; P = .02), pathological complete response (vs no pathological complete response) (69% vs 31%; P = .03), posterior (vs anterior) location (56% vs 22%; P = .01), and ≤60 (vs >60) years of age (61% vs 37%; P = .007). Compared with posterior tumors, anterior tumors were associated with higher risk of systemic recurrence and significantly greater survival benefit from pathological complete response. Anterior tumors remained an independent predictor of worse survival in multivariate analysis (hazard ratio, 2.3; 95% CI, 1.2-4.5; P = .01). CONCLUSIONS: The anatomical location of the tumor affects survival after resection of non-small cell lung carcinomas invading the thoracic inlet. Anterior tumors have greater propensity to metastasize and may derive greater benefit from optimal systemic therapy than posterior tumors.

Ex vivo lung evaluation of single donor lungs when the contralateral lung is rejected increases safe use.

OBJECTIVE: The decision to perform a single-lung transplant (SLT) when the contralateral donor lung is rejected is a challenging scenario. The introduction of ex vivo lung perfusion (EVLP) has improved donor lung assessment, and we hypothesize that it has improved SLT outcomes in this setting. METHODS: A retrospective single-center review of all SLTs performed between 2000 and 2017 was performed in which the years 2000 to 2008 were considered the "pre-EVLP era" and 2009 to 2017 the "EVLP era." Recipients of SLT lungs when the contralateral lung was declined were classified into 3 groups: (1) Pre-EVLP era, (2a) EVLP era but EVLP not used, and (2b) EVLP era and EVLP used. The outcomes of interest were survival, time-to-extubation, and intensive care unit and hospital stay. RESULTS: Among 1692 transplants between 2000 and 2017, 244 (14%) were SLT. SLT rate was similar between eras (pre-EVLP 16% vs EVLP 15%), but more SLTs were performed where the contralateral lung was declined in the EVLP era (pre-EVLP 32% vs EVLP 45%, P = .04). Lungs evaluated on EVLP had lower procurement partial pressure of oxygen and were more often from donation after cardiac death donors. Recipients were generally also sicker, with a greater proportion of rapidly deteriorating recipients. Despite this, outcomes were similar between eras with a trend towards lower 30-day mortality in the EVLP era. CONCLUSIONS: The availability of EVLP allowed for better evaluation of marginal single lungs when the contralateral was declined. This has led to increased use rates with preserved outcomes despite use of more extended criteria organs.

Plasma-first: accelerating lung cancer diagnosis and molecular profiling through liquid biopsy.

Introduction: Molecular profiling of tumor tissue is the gold standard for treatment decision-making in advanced non-small cell lung cancer (NSCLC). Results may be delayed or unavailable due to insufficient tissue, prolonged wait times for biopsy, pathology assessment and testing. We piloted the use of plasma testing in the initial diagnostic workup for patients with suspected advanced lung cancer. Methods: Patients with ⩽15 pack-year smoking history and suspected advanced lung cancer referred to the lung cancer rapid diagnostic program underwent plasma circulating-tumor DNA testing using a DNA-based mutation panel. Tissue testing was performed per standard of care, including comprehensive next-generation sequencing (NGS). The primary endpoint was time from diagnostic program referral to cancer treatment in stage IV NSCLC patients (Cohort A) compared to a contemporary cohort not enrolled in the study (Cohort B) and an historical pre-COVID cohort referred to the program between 2018 and 2019 (Cohort C). Results: From January to June 2021, 20 patients were enrolled in Cohort A; median age was 70.5 years (range 33-87), 70% were female, 55% Caucasian, 85% never smokers, and 75% were diagnosed with NSCLC. Seven had actionable alterations detected in plasma or tissue (4/7 concordant). Fusions, not tested in plasma, were identified by immunohistochemistry for three patients. Mean result turnaround time was 17.8 days for plasma NGS and 23.6 days for tissue (p = 0.10). Mean time from referral to treatment initiation was significantly shorter in cohort A at 32.6 days (SD 13.1) versus 62.2 days (SD 31.2) in cohort B and 61.5 days (SD 29.1) in cohort C, both p < 0.0001. Conclusion: Liquid biopsy in the initial diagnostic workup of patients with suspected advanced NSCLC can lead to faster molecular results and shorten time to treatment even with smaller DNA panels. An expansion study using comprehensive NGS plasma testing with faster turnaround time is ongoing (NCT04862924).

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor.

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient's quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells. This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor. Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

Extra-Pleural Pneumonectomy (EPP) in Children and Adults with Locally Advanced Sarcoma: A CanSaRCC Study.

Sarcoma can present as locally advanced disease involving pleura for which extra-pleural pneumonectomy (EPP) may be the only surgical option to ensure adequate local control. Data were collected on patients who underwent EPP between January 2009 and August 2021 at Princess Margret Hospital and SickKids (Toronto) using the CanSaRCC (Canadian Sarcoma Research and Clinical Collaboration). Ten patients with locally advanced sarcoma involving the pleura, aged 4 to 59 years (median 19.5 years) underwent EPP. Nine (90%) received pre-operative chemotherapy and eight (80%) achieved an R0 resection. Hemithoracic radiation was administered preoperatively (n = 6, 60%) or postoperatively (n = 4, 40%). Five (50%) patients were alive without disease at last follow-up (median 34.2 months) and time from EPP to last FU was median 29.2 months (range 2.2-87.5). Two patients (20%) had local recurrence, 4.3 and 5.8 months from EPP, and both died from progressive disease, 13.1 and 8.2 months from EPP, respectively. One patient died from brain metastasis (17 months), one died from radiation associated osteosarcoma (66 months), and one died from surgical complications (heart failure from constrictive pericarditis). EPP offers a feasible and life-prolonging surgical consideration for patients with locally advanced sarcoma involving the pleura in combination with chemotherapy and radiation. Consequently, EPP should be considered during multi-disciplinary tumor board discussions at high-volume centers.

Cell Inertia: Predicting Cell Distributions in Lung Vasculature to Optimize Re-endothelialization.

We created a transient computational fluid dynamics model featuring a particle deposition probability function that incorporates inertia to quantify the transport and deposition of cells in mouse lung vasculature for the re-endothelialization of the acellular organ. Our novel inertial algorithm demonstrated a 73% reduction in cell seeding efficiency error compared to two established particle deposition algorithms when validated with experiments based on common clinical practices. We enhanced the uniformity of cell distributions in the lung vasculature by increasing the injection flow rate from 3.81 ml/min to 9.40 ml/min. As a result, the cell seeding efficiency increased in both the numerical and experimental results by 42 and 66%, respectively.

Outcomes of lung transplantation at a Canadian center using donors declined in the United States.

OBJECTIVES: Donor lungs from the United States can be offered by US organ procurement organizations to Canada if no American centers accept them. The purpose of this study is to evaluate outcomes of patients undergoing transplant at a single center in Canada using declined lungs from the United States and to compare these outcomes to patients receiving lungs from Canadian donors. METHODS: A single-center retrospective review of recipients receiving lung transplantation between January 2009 and October 2019 was performed. An Organ Procurement and Transplantation Network standard transplant analysis and research-limited dataset as of August 17, 2021, was provided by the United Network for Organ Sharing. De-identified patient-level data were extracted from the standard transplant analysis and research file to identify lung offers made by US organ procurement organizations, declined by US lung centers, and transplanted by the University Health Network within the study time frame. We divided the analysis into 2 groups: recipients receiving donor lungs from Canada and recipients receiving donor lungs from the United States. Donor and recipient characteristics between the 2 groups were compared. Primary end point was proportional survival over a 10-year period. Secondary end points included 30-day mortality, intensive care unit and hospital length of stay, severe primary graft dysfunction, and incidence of chronic lung allograft dysfunction. RESULTS: During the study period, 1424 lung transplants were performed at our center. Of these, 124 (8.7%) were performed using donors from the United States. The incidence of transplants using US donors increased from 5% (5 out of 102) in 2009 to 15% (30 out of 200) in 2018. US donors were younger (aged 41 vs 47 years; P = .004), less likely to be from donors after cardiac death (9.6% vs 20%; P = .008), had higher use of ex vivo lung perfusion (EVLP, 46% vs 27%; P = .0002), and higher incidence of positive nucleic acid test for hepatitis C (16% vs 0.7%; P = .0001). Although the incidence of EVLP utilization was higher in the US lungs versus Canada lungs, more than half of US lungs (54%) proceeded directly to transplantation. Similar short- and long-term outcomes were observed between the 2 groups, including overall survival (hazard ratio, 1.12; 95% CI, 0.85-1.47; P = .40) CONCLUSIONS: Lung transplantation using donor lungs declined by multiple centers in the United States resulted in similar short- and long-term outcomes compared with donor lungs offered in Canada.

Integrative analysis of non-small cell lung cancer patient-derived xenografts identifies distinct proteotypes associated with patient outcomes.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. Only a fraction of NSCLC harbor actionable driver mutations and there is an urgent need for patient-derived model systems that will enable the development of new targeted therapies. NSCLC and other cancers display profound proteome remodeling compared to normal tissue that is not predicted by DNA or RNA analyses. Here, we generate 137 NSCLC patient-derived xenografts (PDXs) that recapitulate the histology and molecular features of primary NSCLC. Proteome analysis of the PDX models reveals 3 adenocarcinoma and 2 squamous cell carcinoma proteotypes that are associated with different patient outcomes, protein-phosphotyrosine profiles, signatures of activated pathways and candidate targets, and in adenocarcinoma, stromal immune features. These findings portend proteome-based NSCLC classification and treatment and support the PDX resource as a viable model for the development of new targeted therapies.

RAVAL trial: Protocol of an international, multi-centered, blinded, randomized controlled trial comparing robotic-assisted versus video-assisted lobectomy for early-stage lung cancer.

BACKGROUND: Retrospective data demonstrates that robotic-assisted thoracoscopic surgery provides many benefits, such as decreased postoperative pain, lower mortality, shorter length of stay, shorter chest tube duration, and reductions in the incidence of common postoperative pulmonary complications, when compared to video-assisted thoracoscopic surgery. Despite the potential benefits of robotic surgery, there are two major barriers against its widespread adoption in thoracic surgery: lack of high-quality prospective data, and the perceived higher cost of it. Therefore, in the face of these barriers, a prospective randomized controlled trial comparing robotic- to video-assisted thoracoscopic surgery is needed. The RAVAL trial is a two-phase, international, multi-centered, blinded, parallel, randomized controlled trial that is comparing robotic- to video-assisted lobectomy for early-stage non-small cell lung cancer that has been enrolling patients since 2016. METHODS: The RAVAL trial will be conducted in two phases: Phase A will enroll 186 early-stage non-small cell lung cancer patients who are candidates for minimally invasive pulmonary lobectomy; while Phase B will continue to recruit until 592 patients are enrolled. After consent, participants will be randomized in a 1:1 ratio to either robotic- or video-assisted lobectomy, and blinded to the type of surgery they are allocated to. Health-related quality of life questionnaires will be administered at baseline, postoperative day 1, weeks 3, 7, 12, months 6, 12, 18, 24, and years 3, 4, 5. The primary objective of the RAVAL trial is to determine the difference in patient-reported health-related quality of life outcomes between the robotic- and video-assisted lobectomy groups at 12 weeks. Secondary objectives include determining the differences in cost-effectiveness, and in the 5-year survival data between the two arms. The results of the primary objective will be reported once Phase A has completed accrual and the 12-month follow-ups are completed. The results of the secondary objectives will be reported once Phase B has completed accrual and the 5-year follow-ups are completed. DISCUSSION: If successfully completed, the RAVAL Trial will have studied patient-reported outcomes, cost-effectiveness, and survival of robotic- versus video-assisted lobectomy in a prospective, randomized, blinded fashion in an international setting. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02617186. Registered 22-September-2015. https://clinicaltrials.gov/ct2/show/NCT02617186.

Planar organization of airway epithelial cell morphology using hydrogel grooves during ciliogenesis fails to induce ciliary alignment.

Topographical cues are known to influence cell organization both in native tissues and in vitro. In the trachea, the matrix beneath the epithelial lining is composed of collagen fibres that run along the long axis of the airway. Previous studies have shown that grooved topography can induce morphological and cytoskeletal alignment in epithelial cell lines. In the present work we assessed the impact of substrate topography on the organization of primary human tracheal epithelial cells (HTECs) and human induced pluripotent stem cell (hiPSC)-derived airway progenitors and the resulting alignment of cilia after maturation of the airway cells under Air-Liquid-Interface (ALI) culture. Grooves with optimized dimensions were imprinted into collagen vitrigel membranes (CVM) to produce gel inserts for ALI culture. Grooved CVM substrates induced cell alignment in HTECs and hiPSC airway progenitors in submerged culture. Further, both cell types were able to terminally differentiate into a multi-ciliated epithelium on both flat and groove CVM substrates. When exposed to ALI conditions, HTECs lost alignment after 14 days. Meanwhile, hiPSC-derived airway progenitors maintained their alignment throughout 31 days of ALI culture. Interestingly, neither initial alignment on the grooves, nor maintained alignment on the grooves induced alignment of cilia basal bodies, an indication of the direction of ciliary beating direction in the airway cells. Planar organization of airway cells during or prior to ciliogenesis therefore does not appear to be a feasible strategy to control cilia organization and subsequent airway epithelial function and additional cues are likely necessary to produce cilia alignment.

Use of High-Rate Ventilation Results in Enhanced Recellularization of Bioengineered Lung Scaffolds.

While transplantation is a viable treatment option for end-stage lung diseases, this option is highly constrained by the availability of organs and postoperative complications. A potential solution is the use of bioengineered lungs generated from repopulated acellular scaffolds. Effective recellularization, however, remains a challenge. In this proof-of-concept study, mice lung scaffolds were decellurized and recellurized using human bronchial epithelial cells (BEAS2B). We present a novel liquid ventilation protocol enabling control over tidal volume and high rates of ventilation. The use of a physiological tidal volume (300 µL) for mice and a higher ventilation rate (40 breaths per minute vs. 1 breath per minute) resulted in higher cell numbers and enhanced cell surface coverage in mouse lung scaffolds as determined via histological evaluation, genomic polymerase chain reaction (PCR) analysis, and immunohistochemistry. A biomimetic lung bioreactor system was designed to include the new ventilation protocol and allow for simultaneous vascular perfusion. We compared the lungs cultured in our dual system to lungs cultured with a bioreactor allowing vascular perfusion only and showed that our system significantly enhances cell numbers and surface coverage. In summary, our results demonstrate the importance of the physical environment and forces for lung recellularization. Impact statement New bioreactor systems are required to further enhance the regeneration process of bioengineered lungs. This proof-of-concept study describes a novel ventilation protocol that allows for control over ventilation parameters such as rate and tidal volume. Our data show that a higher rate of ventilation is correlated with higher cell numbers and increased surface coverage. We designed a new biomimetic bioreactor system that allows for ventilation and simultaneous perfusion. Compared to a traditional perfusion only system, recellularization was enhanced in lungs recellularized with our new biomimetic bioreactor.

Short-Term Preclinical Application of Functional Human Induced Pluripotent Stem Cell-Derived Airway Epithelial Patches.

Airway pathologies including cancer, trauma, and stenosis lack effective treatments, meanwhile airway transplantation and available tissue engineering approaches fail due to epithelial dysfunction. Autologous progenitors do not meet the clinical need for regeneration due to their insufficient expansion and differentiation, for which human induced pluripotent stem cells (hiPSCs) are promising alternatives. Airway epithelial patches are engineered by differentiating hiPSC-derived airway progenitors into physiological proportions of ciliated (73.9 ± 5.5%) and goblet (2.1 ± 1.4%) cells on a silk fibroin-collagen vitrigel membrane (SF-CVM) composite biomaterial for transplantation in porcine tracheal defects ex vivo and in vivo. Evaluation of ex vivo tracheal repair using hiPSC-derived SF-CVM patches demonstrate native-like tracheal epithelial metabolism and maintenance of mucociliary epithelium to day 3. In vivo studies demonstrate SF-CVM integration and maintenance of airway patency, showing 80.8 ± 3.6% graft coverage with an hiPSC-derived pseudostratified epithelium and 70.7 ± 2.3% coverage with viable cells, 3 days postoperatively. The utility of bioengineered, hiPSC-derived epithelial patches for airway repair is demonstrated in a short-term preclinical survival model, providing a significant leap for airway reconstruction approaches.

Solid phase microextraction chemical biopsy tool for monitoring of doxorubicin residue during in vivo lung chemo-perfusion.

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 determination 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 performed 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 physiology of the cells, which might assist in the tailoring of personalized treatment strategy.