bims-meluca 2019-11-03 papers

on Metabolism of non-small cell lung carcinoma

Issue of 2019–11–03
twelve papers selected by
the Muñoz-Pinedo/Nadal (PReTT) lab, L’Institut d’Investigació Biomèdica de Bellvitge and Cristina Muñoz Pinedo, L’Institut d’Investigació Biomèdica de Bellvitge

Glucose and glutamine metabolism in relation to mutational status in NSCLC histological subtypes.
In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer.
Dietary 2-deoxy-D-glucose impairs tumour growth and metastasis by inhibiting angiogenesis.
PINK1 depletion sensitizes non-small cell lung cancer to glycolytic inhibitor 3-bromopyruvate: Involvement of ROS and mitophagy.
Prolyl hydroxylase 3 involvement in lung cancer progression under hypoxic conditions: association with hypoxia-inducible factor-1α and pyruvate kinase M2.
Mutations in the Antioxidant KEAP1/NRF2 Pathway Define an Aggressive Subset of NSCLC Resistant to Conventional Treatments.
A lactate-induced Snail/STAT3 pathway drives GPR81 expression in lung cancer cells.
Associations between Folate Metabolism Enzyme Polymorphisms and Lung Cancer: A Meta-Analysis.
Prognostic impact of the Controlling Nutritional Status score in patients with non-small cell lung cancer treated with pembrolizumab.
Prognostic Impact of Postoperative Skeletal Muscle Decrease in Non-Small Cell Lung Cancer.
Prognostic importance of 18F-fluorodeoxyglucose uptake by positron emission tomography for stage III non-small cell lung cancer treated with definitive chemoradiotherapy.
Heme Oxygenase 1 dampens the macrophage sterile inflammasome response and regulates its components in the hypoxic lung.

Thorac Cancer. 2019 Oct 30.

Glucose and glutamine metabolism in relation to mutational status in NSCLC histological subtypes.

Tineke W H Meijer, Monika G Looijen-Salamon, Jasper Lok, Michel van den Heuvel, Bastiaan Tops, Johannes H A M Kaanders, Paul N Span, Johan Bussink.

   BACKGROUND: Both hypoxia and oncogenic mutations rewire tumor metabolism. In this study, glucose and glutamine metabolism-related markers were examined in stage I - resectable stage IIIA non-small cell lung cancer (NSCLC). Furthermore, expression of metabolism-related markers was correlated with mutational status to examine mutations associated with rewired tumor metabolism.
METHODS: Mutation analysis was performed for 97 tumors. Glucose and glutamine metabolism-related marker expression was measured by immunofluorescent staining (protein) and qPCR (mRNA) (n = 81).
RESULTS: Glutamine metabolism-related markers were significantly higher in adeno- than squamous cell NSCLCs. Glucose transporter 1 (GLUT1) protein expression was higher in solid compared to lepidic adenocarcinomas (P < 0.01). In adenocarcinomas, mRNA expression of glutamine transporter SLC1A5 correlated with tumor size (r(p) = 0.41, P = 0.005). Furthermore, SLC1A5 protein expression was significantly higher in adenocarcinomas with worse pTNM stage (r(s) = 0.39, P = 0.009). EGFR-mutated tumors showed lower GLUT1 protein (P = 0.017), higher glutaminase 2 (GLS2) protein (P = 0.025) and higher GLS2 mRNA expression (P = 0.004), compared to EGFR wild-type tumors. GLS mRNA expression was higher in KRAS-mutated tumors (P = 0.019). TP53-mutated tumors showed higher GLUT1 expression (P = 0.009).
CONCLUSIONS: NSCLC is a heterogeneous disease, with differences in mutational status and metabolism-related marker expression between adeno- and squamous cell NSCLCs, and also within adenocarcinoma subtypes. GLUT1 and SLC1A5 expression correlate with aggressive tumor behavior in adenocarcinomas but not in squamous cell NSCLCs. Therefore, these markers could steer treatment modification for subgroups of adenocarcinoma patients. TP53, EGFR and KRAS mutations are associated with expression of glucose and glutamine metabolism-related markers in NSCLC.

Keywords:  Glutamine metabolism; glycolysis; mutational status; non-small cell lung cancer

DOI:  https://doi.org/10.1111/1759-7714.13226
Nature. 2019 Oct 30.

In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer.

Milica Momcilovic, Anthony Jones, Sean T Bailey, Christopher M Waldmann, Rui Li, Jason T Lee, Gihad Abdelhady, Adrian Gomez, Travis Holloway, Ernst Schmid, David Stout, Michael C Fishbein, Linsey Stiles, Deepa V Dabir, Steven M Dubinett, Heather Christofk, Orian Shirihai, Carla M Koehler, Saman Sadeghi, David B Shackelford.

Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours.

DOI: https://doi.org/10.1038/s41586-019-1715-0
Eur J Cancer. 2019 Oct 24. pii: S0959-8049(19)30722-1. [Epub ahead of print]123 11-24

Dietary 2-deoxy-D-glucose impairs tumour growth and metastasis by inhibiting angiogenesis.

Saurabh Singh, Sanjay Pandey, Amanpreet Singh Chawla, Anant Narayan Bhatt, Bal Gangadhar Roy, Daman Saluja, Bilikere S Dwarakanath.

  Accumulating evidence suggests the antiangiogenic potential of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) among the anticancerous properties of this drug. In the present studies, we investigated the antiangiogenic effects of dietary 2-DG on tumour (Lewis lung carcinoma [LLC]) as well as ionising radiation-induced angiogenesis in mouse models. Dietary 2-DG reduced the serum vascular endothelial growth factor levels (∼40%) in LLC-bearing mice along with a significant inhibition of tumour growth and metastases. In vivo Matrigel plug assays showed significant decrease in vascularisation, Fluorescein isothiocyanate (FITC)-dextran fluorescence and factor VIII-positive cells in the plugs from 2-DG-fed mice, supporting the notion that dietary 2-DG significantly suppresses the tumour-associated and radiation-induced angiogenesis. 2-DG inhibited the glucose usage and lactate production as well as ATP levels of human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner, accompanied by growth inhibition and loss of viability in vitro. Furthermore, 2-DG inhibited the capillary-like tube formation in Matrigel as well as migration and transwell invasion by HUVECs, which are functional indicators of the process of angiogenesis. These results suggest that dietary 2-DG inhibits processes related to angiogenesis, which can impair the growth and metastasis of tumours.

Keywords:  2-Deoxy-D-glucose; Energy restriction; Lewis lung carcinoma; Radiation-induced angiogenesis; Tumour-induced angiogenesis

DOI:  https://doi.org/10.1016/j.ejca.2019.09.005
Pharmacol Rep. 2019 Aug 14. pii: S1734-1140(18)30740-0. [Epub ahead of print]71(6): 1184-1189

PINK1 depletion sensitizes non-small cell lung cancer to glycolytic inhibitor 3-bromopyruvate: Involvement of ROS and mitophagy.

Katherine Dai, Daniel P Radin, Donna Leonardi.

   BACKGROUND: Despite significant strides in understanding the pathophysiology of non-small cell lung cancer (NSCLC), these neoplasms typically present with intrinsic chemo- and radiotherapeutic resistance. Transcriptomic analyses of patient NSCLC tumors stratified by survival times have identified the PTEN-induced putative kinase 1 (PINK1) as a molecular governor of tumor aggressiveness and patient survival time. PINK1 has been shown to confer neuroprotection in models of Parkinson Disease by ensuring proper mitochondrial turnover (mitophagy), the upkeep of ATP production and sequestering of reactive oxygen species (ROS).
METHODS: We utilized an shRNA against PINK1 and the glycolytic inhibitor 3-BP to assess effects on NSCLC viability via MTS cell viability assay. ATP levels, caspase-9 activation, mitophagy and ROS production were determined with standardly available kits. Cytochrome c cellular localization and phosphorylated parkin levels were determined using an ELISA.
RESULTS: Our results demonstrate that PINK1 depletion in the NSCLC cell line A549 via shRNA, reduced cancer cell proliferation, increased cell death, reduced ATP production, inhibited mitophagy and increased ROS and caspase-9-dependent apoptosis. PINK1 depleted cells were more susceptible to the glycolytic inhibitor 3-bromopyruvate (3-BP), which further perturbed ATP production. PINK1 depletion and 3-BP synergistically increased ROS production, caspase-9-dependent apoptosis and additively repressed mitophagy.
CONCLUSIONS: These results suggest that PINK1 depletion alters energetic metabolism and confers sensitivity to agents that inhibit glycolysis. Targeting accelerated tumor cell metabolism may prove useful in the clinical setting while sparing non-malignant tissue.

Keywords:  3-bromopyruvate; Mitophagy; Parkin; Pink1; ROS

DOI:  https://doi.org/10.1016/j.pharep.2019.08.002
J Thorac Dis. 2019 Sep;11(9): 3941-3950

Prolyl hydroxylase 3 involvement in lung cancer progression under hypoxic conditions: association with hypoxia-inducible factor-1α and pyruvate kinase M2.

Xiao Chu, Ming Xiang, Liang Feng, Hui Liu, Chao Zhou.

   Background: Previous studies have suggested that the functions of prolyl hydroxylase 3 (PHD3) in tumor growth, apoptosis and angiogenesis are essentially dependent on hypoxia-inducible factor (HIF)-1α signaling. Nevertheless, whether PHD3 represents a promising tumor suppressor target remains to be clarified. To provide insight into the therapeutic potential of PHD3 in lung cancer, this study examined the effects of PHD3 expression on HIF-1α and pyruvate kinase M2 (PKM2), as well as on lung cancer cell proliferation, migration, and invasion.
Methods: The model of hypoxia was established in A549 and SK-MES-1 cells with 200 µM CoCl2 treatment, and verified by western blot and immunocytochemical staining. The expression levels of PKM2 and HIF-1α were determined by western blot after overexpression or depletion of PHD3 in A549 and SK-MES-1 cells. In addition, cell viability, migration and invasion were measured, respectively.
Results: Establishment of hypoxia in A549 and SK-MES-1 cells resulted in significant decreases in PHD3 expression and remarkable increase in PKM2 expression in 24 hrs. Overexpression of PHD3 in A549 and SK-MES-1 cells decreased HIF-1α and PKM2 expression. In contrast, PHD3 knockdown increased HIF-1α and PKM2 (P<0.05). In addition, the viability, migration and invasion of A549 and SK-MES-1 cells were significantly decreased with PHD3 overexpression, but dramatically increased with PHD3 depletion (P<0.05).
Conclusions: PHD3 is involved in lung cancer progression, and might be a promising therapeutic target for cancers.

Keywords:  Prolyl hydroxylase 3 (PHD3); hypoxia-inducible factor-1α (HIF-1α); lung cancer; pyruvate kinase M2 (PKM2)

DOI:  https://doi.org/10.21037/jtd.2019.08.124
J Thorac Oncol. 2019 Nov;pii: S1556-0864(19)30672-0. [Epub ahead of print]14(11): 1881-1883

Mutations in the Antioxidant KEAP1/NRF2 Pathway Define an Aggressive Subset of NSCLC Resistant to Conventional Treatments.

Ernest Nadal, Ramon Palmero, Cristina Muñoz-Pinedo.

DOI: https://doi.org/10.1016/j.jtho.2019.08.005
Biochim Biophys Acta Mol Basis Dis. 2019 Oct 28. pii: S0925-4439(19)30299-6. [Epub ahead of print] 165576

A lactate-induced Snail/STAT3 pathway drives GPR81 expression in lung cancer cells.

Qiang Xie, Zhanzhan Zhu, Yuanpeng He, Zhijian Zhang, Yao Zhang, Yixuan Wang, Jincheng Luo, Tianchou Peng, Feng Cheng, Jiguang Gao, Yuxiang Cao, Huijun Wei, Zhihao Wu.

  Highly expressed G protein-coupled receptor 81 (GPR81), a receptor for lactate, is emerging as a critical regulator of tumor growth and metastasis. However, the mechanistic basis for its highly expression in cancer cells remains elusive. Here we report that tumor-derived lactate transcriptionally regulates GPR81 expression. We demonstrated that the transcriptional response of GPR81 to lactate is mediated by Signal transducer and activator of transcription 3 (STAT3). Mechanistically, lactate upregulates transcriptional factor Snail and induces the assembly of Snail/EZH2/STAT3 complex. Within this ternary complex, STAT3 activity is strongly enhanced. Consequently, the activated STAT3 by lactate directly binds GPR81promoter and activates its expression. These findings shed light on the transcriptional mechanism by which GPR81 expression is regulated in cancer cells, and provides mechanistic insight into how aberrant signaling and continually high lactate levels due to metabolic switch may yield a feed-forward/self-enabling loop to promote tumor progression.

Keywords:  GPR81; Lactate; Lung cancer; STAT3; Snail

DOI:  https://doi.org/10.1016/j.bbadis.2019.165576
Nutr Cancer. 2019 Oct 29. 1-8

Associations between Folate Metabolism Enzyme Polymorphisms and Lung Cancer: A Meta-Analysis.

Fangshan Chen, Ting Wen, Qian Lv, Furong Liu.

Some previous studies already investigated potential associations between folate metabolism enzyme polymorphisms and lung cancer (LC). However, the results of these studies were inconsistent. Thus, we performed this meta-analysis to explore associations between folate metabolism enzyme polymorphisms and LC in a larger pooled population. Systematic literature research of PubMed, WOS, Embase and CNKI was performed to identify eligible studies. Review Manager Version 5.3.3 was used to conduct statistical analyses. Totally 37 genetic association studies were included for analyses. The pooled analyses showed that MTRR rs1801394 (dominant model: p = 0.01; recessive model: p = 0.04; allele model: p = 0.005) and MTHFR rs1801133 (dominant model: p = 0.008; recessive model: p = 0.0003; allele model: p = 0.0002) polymorphisms were both significantly associated with susceptibility to LC in overall population. Subgroup analyses revealed similar significant findings for MTHFR rs1801133 polymorphism in East Asians. Significant associations with LC were also observed for MTRR rs1801394 and MTHFR rs1801133 polymorphisms in smokers. In conclusion, this meta-analysis indicated that MTRR rs1801394 was significantly associated with LC in smokers, and MTHFR rs1801133 polymorphisms was also significantly associated with LC in smokers and East Asians. These results suggested that these two polymorphisms could be used to identify individuals at high risk of developing LC in certain populations.

DOI: https://doi.org/10.1080/01635581.2019.1677924
J Thorac Dis. 2019 Sep;11(9): 3757-3768

Prognostic impact of the Controlling Nutritional Status score in patients with non-small cell lung cancer treated with pembrolizumab.

Taro Ohba, Shinkichi Takamori, Ryo Toyozawa, Kaname Nosaki, Yasuhiro Umeyama, Naoki Haratake, Naoko Miura, Masafumi Yamaguchi, Kenichi Taguchi, Takashi Seto, Mototsugu Shimokawa, Mitsuhiro Takenoyama.

   Background: Pembrolizumab, an anti-programmed cell death-1 (PD-1) monoclonal antibody, has been shown to yield a durable response and significant survival benefit in some non-small cell lung cancer (NSCLC) patients. Recent studies have shown that the Controlling Nutritional Status (CONUT) score, a novel nutritional index, can be useful for predicting the prognosis in some malignancies. However, its usefulness in predicting the clinical outcome of immune-checkpoint inhibitor (ICI) treatment in patients with NSCLC has not been clarified. The aim of this study was to investigate the clinical significance of the CONUT score in NSCLC patients treated with pembrolizumab.
Methods: We conducted a retrospective analysis of the clinical data of 32 patients with advanced NSCLC who received pembrolizumab monotherapy. A cut-off CONUT score of 2 was used to categorize patients into low and high CONUT groups. We evaluated the relation between the clinicopathological factors including CONUT score and neutrophil-to-lymphocyte ratio (NLR) and the prognosis.
Results: Twenty-two patients were classified into the low CONUT score group, while 10 were classified into the high CONUT score group. In the univariate and multivariate analyses, the number of prior treatments and the CONUT score were found to independently predict progression-free survival (PFS) (P<0.05), while the CONUT score as well as NLR was an independent prognostic factor for overall survival (P<0.05). In addition, in patients who received pembrolizumab as a first-line treatment, a high CONUT score was associated with a significantly worse PFS and overall survival in comparison to a low CONUT score.
Conclusions: The CONUT score has potential application as a predictor of the therapeutic effect and the prognosis of NSCLC patients treated with pembrolizumab. Our findings suggest that in addition to the programmed cell death ligand 1 expression level, the CONUT may also be a useful indicator for selecting NSCLC patients who may benefit from ICI treatment.

Keywords:  Controlling Nutritional Status (CONUT); Pembrolizumab; neutrophil-to-lymphocyte ratio (NLR); non-small cell lung cancer (NSCLC)

DOI:  https://doi.org/10.21037/jtd.2019.09.29
Ann Thorac Surg. 2019 Oct 23. pii: S0003-4975(19)31595-4. [Epub ahead of print]

Prognostic Impact of Postoperative Skeletal Muscle Decrease in Non-Small Cell Lung Cancer.

Shinkichi Takamori, Tetsuzo Tagawa, Gouji Toyokawa, Mototsugu Shimokawa, Fumihiko Kinoshita, Yuka Kozuma, Taichi Matsubara, Naoki Haratake, Takaki Akamine, Fumihiko Hirai, Hiroshi Honda, Yoshihiko Maehara.

BACKGROUND: Preoperative skeletal muscle loss was reported to be associated with a postoperative poor prognosis in non-small cell lung cancer (NSCLC) patients. The aim of this study was to elucidate the relationship between the change in skeletal muscle mass following surgery and the postoperative outcomes in NSCLC patients.
METHODS: The data analyzed 204 NSCLC patients who had undergone curative lung resection and whose preoperative and postoperative (1 year) computed tomography images were available. The skeletal muscle area (SMA) at the 12th thoracic vertebra level was used. Post/pre ratio was defined as postoperative normalized SMA (cm2/m2) divided by preoperative normalized SMA. The cut-off value was set to post/pre ratio = 0.9. Neutrophil-lymphocyte ratio, platelet-lymphocyte ratio, modified Glasgow prognostic score, and prognostic nutritional index were used to estimate change in the nutritional status.
RESULTS: Seventy patients (34.3%) were classified into the SMA-decreased group. Low body mass index was significantly associated with the SMA-decreased patients (p = 0.019). The SMA-decreased status was found to be an independent prognostic factor for overall survival and disease-free survival (p < 0.001 and p = 0.001, respectively). The SMA-decreased status was significantly associated with the postoperative exacerbation of neutrophil-lymphocyte ratio, platelet-lymphocyte ratio, modified Glasgow prognostic score, and prognostic nutritional index (p = 0.009, p = 0.026, p = 0.003, and p = 0.013, respectively).
CONCLUSIONS: Skeletal muscle loss after surgery is significantly associated with poor postoperative outcomes in NSCLC patients. Further studies are needed for investigating the clinical impact of postoperative nutritional intervention.

DOI: https://doi.org/10.1016/j.athoracsur.2019.09.035
Rev Esp Med Nucl Imagen Mol. 2019 Oct 23. pii: S2253-654X(19)30059-9. [Epub ahead of print]

Prognostic importance of 18F-fluorodeoxyglucose uptake by positron emission tomography for stage III non-small cell lung cancer treated with definitive chemoradiotherapy.

G Kanyilmaz, B Benli Yavuz, M Aktan, O Sahin.

   OBJECTIVES: Survival heterogeneity exists among patients with non-small cell lung cancer (NSCLC), even within the same stage. We aimed to evaluate the prognostic role of pre-treatment maximum standardized uptake value (SUVmax) in patients treated with definitive concurrent chemoradiotherapy for stage III NSCLC.
MATERIALS AND METHODS: Between 2010 and 2017, 103 patients with stage III NSCLC who underwent 18F-FDG PET/CT at the time of diagnosis were included in the study.
RESULTS: Higher tumor stages were correlated with higher pre-treatment SUVmax of lymph nodes (LNs) (p=0.005) but were not correlated with higher SUVmax of primary tumor (p=0.2). The median SUVmax of LNs was 2.84, 8.06, and 11.11 in stage IIIa, IIIb and IIIc, respectively. Higher nodal stage was also correlated with higher SUVmax of LNs (p=0.01). According to ROC analysis, there was no significant cut-off value of SUVmax observed for primary tumor, therefore continuous variables were used for survival analyses. The best SUVmax cut-off was 3.5 for the LNs, therefore the SUVmax of LNs was evaluated as both a dichotomous and a continuous variable. Pre-treatment SUVmax of primary tumor did not predict survival outcomes but both the continuous and dichotomous variables of SUVmax of LNs predicted recurrence free survival and overall survival. Nodal stage (N0-2 vs. N3) and AJCC stage (IIIa vs IIIb vs. IIIc) were the other prognostic factors.
CONCLUSIONS: Pre-treatment SUVmax of LNs had prognostic value in patients treated with definitive concurrent chemoradiotherapy for stage III NSCLC. In future trials, pre-treatment SUVmax of the LNs would serve as a guide for patients who might benefit from more aggressive treatments.

Keywords:  Chemoradiotherapy; Cáncer de pulmón de células no pequeñas; Non-small cell lung cancer; PET/CT; PET/TC; Prognosis; Pronóstico; Quimiorradioterapia

DOI:  https://doi.org/10.1016/j.remn.2019.04.006
Am J Physiol Lung Cell Mol Physiol. 2019 Oct 30.

Heme Oxygenase 1 dampens the macrophage sterile inflammasome response and regulates its components in the hypoxic lung.

Sally H Vitali, Angeles Fernandez-Gonzalez, Janhavi Nadkarni, April Kwong, Chase Rose, S Alex Mitsialis, Stella Kourembanas.

  Exposure to hypoxia causes an inflammatory reaction in the mouse lung and this response can be modulated by overexpressing the hypoxia-inducible stress-response enzyme, heme oxygenase-1 (HO-1). We hypothesized that the inflammasome activity may be a central pathway by which HO-1 controls pulmonary inflammation following alveolar hypoxia. Therefore, we investigated whether HO-1 controls inflammasome activation by altering its expression in macrophages primed with classical NLRP3 inducers, and in murine lungs lacking HO-1 and exposed to acute hypoxia. We found that lack of HO-1 activated LPS and ATP-treated bone marrow derived-macrophages (BMDMs) causing an increase in secreted levels of cleaved IL-1B, IL-18, and caspase-1, markers of increased inflammasome activity, whereas HO-1 overexpression suppressed IL-1B, NLRP3, and IL-18. The production of cleaved IL-1B and the activation of caspase-1 in LPS and ATP-primed macrophages were inhibited by hemin, and two of the HO-1 enzymatic products (bilirubin and carbon monoxide (CO)). Exposure of mice to hypoxia induced the expression of several inflammasome mRNA components (IL-1B, Nlrp3 and Caspase-1) and this was further augmented by HO-1 deficiency. This pronounced inflammasome activation was detected as increased protein levels of ASC, IL-18, pro-caspase-1, and cleaved caspase-1 in the lungs of hypoxic mice. Systemically, Hmox1 deficient mice showed increased basal levels of IL-18 that were further increased after 48 hours of hypoxic exposure. Taken together, these finding points to a pivotal role for HO-1 in the control of baseline and hypoxic inflammasome signaling, perhaps through the antioxidant properties of bilirubin and CO's pleiotropic effects.

Keywords:  Heme oxygenase-1; Hypoxia; Inflammasome; Macrophage; lung

DOI:  https://doi.org/10.1152/ajplung.00074.2019