High-resolution computed tomography (HRCT) of the chest is commonly used in the diagnosis of a variety of lung diseases. Structural changes associated with clinical characteristics of disease may also define specific disease-associated physiologic states that may provide insights into disease pathophysiology. Gene expression profiling is potentially a useful adjunct to HRCT to identify molecular correlates of the observed structural changes. However, it is difficult to directly access diseased distal airway or lung parenchyma routinely for profiling studies. Previously, we have profiled bronchial airway in normal-appearing epithelial cells at the mainstem bronchus, detecting distinct gene expression alterations related to the clinical diagnosis of chronic obstructive pulmonary disease (COPD) and lung cancer. These gene expression alterations offer insights into the molecular events related to diseased tissue at more distal airways and in the parenchyma, which we hypothesize are due to a field-of-injury effect. Here, we expand this prior work by correlating airway gene expression to COPD and bronchiectasis phenotypes defined by HRCT to better understand the pathophysiology of these diseases. Additionally, we classified pulmonary nodules as malignant or benign by combining HRCT nodule imaging characteristics with gene expression profiling of the nasal airway. First, we collected brushing samples from the main-stem bronchus and assessed gene expression alterations associated with COPD phenotypes defined by K-means clustering of HRCT-based imaging features. We found three imaging clusters, which correlated with incremental severity of COPD: preserved, interstitial predominant, and emphysema predominant. 357 genes were differentially expressed between the normal and the emphysema predominant clusters. Functional analysis of the differentially expressed genes suggests a possible induction of inflammatory processes and repression of T-cell related biologic pathways, in the emphysema predominant cluster. We then discovered gene expression alterations associated with radiographic evidence of bronchiectasis (BE), an underdiagnosed obstructive pulmonary disease with unclear pathophysiology. We found 655 genes were differentially expressed in bronchial epithelium from individuals with radiographic evidence of BE despite none of the study participants having a clinical BE diagnosis. In addition to biological pathways that had been previously associated with BE, novel pathways that may play important roles in BE initiation were also discovered. Furthermore, we leveraged an independent single-cell RNA-sequencing dataset of the bronchial epithelium to explore whether the observed gene expression alterations might be cell-type dependent. We computationally detected an increased presence of ciliated and deuterosomal cells, as well as a decreased presence of basal cells in subjects with widespread radiographic BE, which may reflect a shift in the cellular landscape of the airway during BE initiation. Finally, we identified gene expression alterations within the nasal epithelium associated with the presence of malignant pulmonary nodules. A computational model was constructed for determining whether a nodule is malignant or benign that combines gene expression and imaging features extracted from HRCT. Leveraging data from single-cell RNA sequencing, we found genes increased in patients with lung cancer are expressed at higher levels within a novel cluster of nasal epithelial cells, termed keratinizing epithelial cells. In summary, we leveraged gene expression profiling of the proximal airway and discovered novel biological pathways that potentially drive the structural changes representative of physiologic states defined by chest HRCT in COPD and BE. This approach may also be combined with chest HRCT to detect weak signals related to malignant pulmonary nodules. / 2024-02-03T00:00:00Z

Serotonin has been widely explored in the brain. Recently, there have been new findings on how serotonin works in the periphery. Serotonin is introduced to the periphery by the enterochromaffin cells and metabolized by the liver and lung. Studies have shown that serotonin plays a role in controlling lung cancer. However, the mechanism by which it initiates tumor formation has not been fully explored. Cell viability was measured in several lung adenocarcinoma cell lines treated with serotonin to study this effect. In GFP-labelled cells, fluorescence intensity was measured for quantification of cell viability. Our data showed an overall increase in viability when serotonin concentration was increased, which is significant because it shows that serotonin affects lung cancer progression. We will look at how serotonin works on tumor cells compared to endothelial cells and its effect on immune system activation. This study hopes to inspire future anti-angiogenesis and immunotherapy studies of lung cancer by understanding this interaction.

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Megakaryocytes (MK) are responsible for platelet biogenesis, which is thought to occur canonically in the adult bone marrow (BM) and in the fetal liver during development. However, emerging evidence highlights the lung as a previously underappreciated residence for MKs that may significantly contribute to circulating platelet mass. While a diversity of cells specific to the BM are known to promote the maturation and trafficking of MKs, little investigation into the impact of the lung niche on the development and function of MKs has been done. Here, we describe the application of single cell RNA sequencing (scRNA-Seq) coupled with histological, ploidy and flow cytometric analyses to profile primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Transcriptional profiling demonstrated that lung MKs have a unique signature distinct from their hematopoietic counterparts with lung MKs displaying enrichment for maturation markers, potentially indicating a propensity for more efficient platelet production. Reciprocally, fetal lung MKs also showed the robust expression of cytokines and growth factors known to promote lung development. Lastly, lung MKs possess an enrichment profile skewed towards roles in immunity and inflammation. These findings highlight the existence of a lung-specific MK phenotype and support the notion that the lung plays an independent role in the development and functional maturation of MKs. In addition to MKs, the lung houses many resident hematopoietic cells, including hematopoietic stem and progenitor cells (HSPCs). The existence of lung HSPCs suggests that the differentiation and development of lung resident hematopoietic cells may occur in-situ. To investigate the potential role the lung has in instructing site specific hematopoiesis, we employed explant cultures of murine and human fetal lungs. This displayed adherent endothelial cells transitioning into floating hematopoietic cells, suggesting that the fetal lung is a source of hemogenic endothelial cells that have the functional capacity to undergo endothelial to hematopoietic transition (EHT) to produce HSPCs. Flow cytometric and functional assessment of fetal lung explants showed the production of HSPCs that expressed key EHT and pre-HSPC markers. Expression profiles revealed by scRNA-Seq and small molecule modulation demonstrated that fetal lung EHT is reliant on canonical EHT signaling pathways. These findings suggest that functional HECs are present in the fetal lung, thus establishing this location as a potential extramedullary site of de-novo hematopoiesis. Overall, these findings suggest that the lung may have a greater role in instructing tissue specific hematopoiesis and/or overall hematopoietic development.

Disease identification is one of the most important aspects of a physician's duties. Radiologists play a very important role in disease recognition based on the increasing use of diagnostic images. Nowadays, medical scanning devices such as MRI and CT produce thousands of images per patient which makes the radiologist's job even more onerous; indeed radiologists look at approximately 50,000 images per day leading to fatigue and a higher probability for missing smaller lesions. Therefore it is critical to assist radiologists in their duties.In this PhD work, research is focused on developing segmentation-based mathematical algorithms and computer programs for automatically characterizing lung CT images. There are two kinds of segmentation methods; the first group contains the methods that find edges of all objects in the image and the second group contains the methods that focus on one object in the image. By assessing many segmentation methods and based on the concept of this project, the level set method, from the second group, has the capability to accurately find the boundary of an object in medical images. Although this method does not need any threshold for segmenting an object in an image, it does require the setting of seven parameters. Genetic algorithms were employed to optimize seven parameters of the level set method for use as a boundary detection method. A streamlined automatic mechanism, essential for successful and fast segmenting processes, provided the level set method with a good initial contour.This segmentation step was fundamental for further measurements such as bronchial lumen diameter and wall thickness measurement. The developed program automatically measures airway lumen diameter with exceptional repeatability. Also, by simulating manual methods used by radiologists for measuring luminal wall thickness, the automated fitting method consistently finds the wall thickness at the thinnest part, minimizing partial volume problems. The current standard for measuring luminal wall thickness is the full-width at half-maximum method. The technique formulated here is more accurate and reproducible and can be performed automatically. To find the lumen airway tapering, in order to recognize some prominent lung diseases, a method for tracing of an airway through various CT slices was developed. These measurements are of critical importance in the understanding of a number of lung diseases including asthma and COPD; this tracing step was fundamental for bronchial bifurcation angle measurement. This tracing method was extended to detect and follow bifurcation branches. Then, an estimation method for finding a fitted line through airway center points was developed. For the last portion of this PhD work an approach for measuring lung airway bifurcation angle from CT datasets, which is important in lung diseases such as asthma, was created. The current goal, characterizing lung CT images, was achieved as a working form of this software development. This mathematical approach has been shown to be accurate with phantom studies. The ultimate goal of this work was to develop software for recognizing key lung diseases and to compare serially (weeks or months apart) acquired images to assess any progress, regression, or stability in a disease. / Thesis / Doctor of Philosophy (PhD)

Chemotherapy-associated thrombosis is a common complication in cancer patients. Cancer patients have a 5- to 7-fold increased risk for a thrombotic event compared to healthy individuals. While the overall risk for a thrombotic event in lung cancer patients is approximately 1.4%, the rates of thrombosis vary depending on the stage of the disease and the chemotherapeutic agents used. Activation of coagulation after initiation of chemotherapy has been reported in clinical studies. However, the mechanisms by which lung cancer chemotherapy agents modulate coagulation in lung cancer patients are not completely understood. The focus of this thesis is to investigate the mechanisms by which chemotherapy agents cisplatin, carboplatin, gemcitabine, and paclitaxel (in platinum-based combinations) induce procoagulant effects utilizing in vitro and in vivo approaches. First, we investigated the mechanisms by which lung cancer chemotherapy modulates cell-surface tissue factor (TF) activity on endothelial cells (HUVEC), monocytes, and non-small cell lung carcinoma (NSCLC) A549 cells. We observed that treatment of all three cell lines with platinum-based lung cancer chemotherapy increased cell surface TF activity. We found that the increased TF activity on chemotherapy-treated monocytes was due to increased phosphatidylserine (PS) exposure, whereas the increased TF activity on HUVEC and A549 cells was due to protein disulfide isomerase (PDI)-mediated decryption of TF. These studies demonstrate that lung cancer chemotherapy agents can exert procoagulant effects by increasing PS exposure and by inducing TF decryption on healthy and tumour cells. Next, we determined the effects of lung cancer chemotherapy on the generation of microparticles (MP) and the impact of MPs on thrombin generation. Our in vitro and in vivo studies demonstrate that lung cancer chemotherapy agents increase the generation of TF- and PS-positive MPs from tumour cells and that the MPs contribute to thrombin generation in a FVII-dependent manner. We also investigated the role of cell-free DNA (CFDNA) in mediating procoagulant effects induced by lung cancer chemotherapy agents. We found that lung cancer chemotherapy agents induce CFDNA release from healthy host neutrophils and that this leads to additional generation of thrombin by the intrinsic pathway of coagulation. Lastly, CFDNA levels have been shown to increase in cancer models through formation of neutrophil extracellular traps (NETs). Formation of NETs by NETosis, a process by which neutrophils release extracellular web-like structures composed of DNA, histones, and granular proteins, is dependent on histone citrullination by protein arginine deaminase-4 (PAD-4). In addition, PAD4 inhibition prevents NET formation. Therefore, we wanted to demonstrate that the neutrophil-derived CFDNA release induced by lung cancer chemotherapy is PAD4-dependent. Chemotherapy treatment of PAD4 knockout mice failed to increase CFDNA levels. Furthermore, chemotherapy-treatment did not increase thrombin generation in PAD4 knockout mice. This suggests that chemotherapy-induced CFDNA release occurs through NETosis. In conclusion, lung cancer chemotherapy leads to increased thrombin generation which occurs through increased TF decryption, MP generation, and CFDNA release. Therefore, lung cancer chemotherapy results in simultaneous activation of the extrinsic and intrinsic pathways of coagulation. These studies provide novel insight into the mechanisms of lung cancer chemotherapy-associated thrombosis. / Thesis / Doctor of Philosophy (PhD)

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