A key step for metastatic outgrowth involves the generation of a deeply altered microenvironment (niche) that supports the malignant behavior of cancer cells. The complexity of the metastatic niche has posed a significant challenge in elucidating the underlying programs driving its origin. Here, by focusing on early stages of breast cancer metastasis to the lung in mice, we describe a cancer-dependent chromatin remodeling and activation of developmental programs in alveolar type 2 (AT2) cells within the niche. We show that metastatic cells can prime AT2 cells into a reprogrammed multilineage state. In turn, this cancer-induced reprogramming of AT2 cells promoted stem-like features in cancer cells and enhanced their initiation capacity. In conclusion, we propose the concept of \"reflected stemness\" as an early phenomenon during metastatic niche initiation, wherein metastatic cells reprogram the local tissue into a stem-like state that enhances intrinsic cancer-initiating potential, creating a positive feedback loop where tumorigenic programs are amplified.

Lung diseases rank third in terms of mortality and represent a significant economic burden globally. Scientists have been conducting research to better understand respiratory diseases and find treatments for them. An ideal in vitro model must mimic the in vivo organ structure, physiology, and pathology. Organoids are self-organizing, three-dimensional (3D) structures originating from adult stem cells, embryonic lung bud progenitors, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). These 3D organoid cultures may provide a platform for exploring tissue development, the regulatory mechanisms related to the repair of lung epithelia, pathophysiological and immunomodulatory responses to different respiratory conditions, and screening compounds for new drugs. To create 3D lung organoids in vitro, both co-culture and feeder-free methods have been used. However, there exists substantial heterogeneity in the organoid culture methods, including the sources of AT2 cells, media composition, and feeder cell origins. This article highlights the currently available methods for growing AT2 organoids and prospective improvements to improve the available culture techniques/conditions. Further, we discuss various applications, particularly those aimed at modeling human distal lung diseases and cell therapy.

Sex differences exist for many lung pathologies, including COVID-19 and pulmonary fibrosis, but the mechanistic basis for this remains unclear. Alveolar type 2 cells (AT2s), which play a key role in alveolar lung regeneration, express the X-linked Ace2 gene that has roles in lung repair and SARS-CoV-2 pathogenesis, suggesting that X chromosome inactivation (XCI) in AT2s might impact sex-biased lung pathology. Here we investigate XCI maintenance and sex-specific gene expression profiles using male and female AT2s. Remarkably, the inactive X chromosome (Xi) lacks robust canonical Xist RNA \"clouds\" and less enrichment of heterochromatic modifications in human and mouse AT2s. We demonstrate that about 68% of expressed X-linked genes in mouse AT2s, including Ace2, escape XCI. There are genome-wide expression differences between male and female AT2s, likely influencing both lung physiology and pathophysiologic responses. These studies support a renewed focus on AT2s as a potential contributor to sex-biased differences in lung disease.

OBJECTIVE: Lung type 2 alveolar cells, by secreting surfactant to lower surface tension, contribute to enhance lung compliance. Stretching, as a result of lung expansion, triggers type 1 alveolar cell to release ATP, which in turn stimulates Ca2+-dependent surfactant secretion by neighboring type 2 cells. In this report, we studied ATP-triggered Ca2+ signaling in human alveolar type 2 A549 cells.
METHODS: Ca2+ signaling was examined using microfluorimetric measurement with fura-2 as fluorescent dye.
RESULTS: Ca2+ oscillations triggered by ATP relied on inositol 1,4,5-trisphosphate-induced Ca2+ release and store-operated Ca2+ entry. Pathological conditions such as influenza virus infection and diabetes reportedly inhibit sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that a very mild inhibition of SERCA by cyclopiazonic acid (CPA) sufficed to decrease Ca2+ oscillation frequency and the percentage of cells exhibiting Ca2+ oscillations. Ochratoxin A (OTA), an activator of SERCA, could prevent the suppressive effects by CPA. Inhibition of SERCA by hydrogen peroxide also suppressed Ca2+ oscillations. Interestingly, hydrogen peroxide-induced inhibition was prevented by OTA but aggravated by CDN1163, an allosteric activator of SERCA. CDN1163 also had an untoward effect of releasing intracellular Ca2+.
CONCLUSIONS: Different modes of activation of SERCA may determine the outcome of rescue of Ca2+ oscillations in case of SERCA inhibition in alveolar type 2 cells.

SARS-CoV-2 infection leads to severe lung damage due to pneumonia and, in more severe cases, leads to acute respiratory distress syndrome, or ARDS. This affects the viability of bronchoalveolar cells. An important role in the pathogenesis of these complications is the hyperactivation of the renin-angiotensin-aldosterone (RAA) pathway and induction of cytokine storm that occurs in an Nlrp3 inflammasome-dependent manner. To shed more light on the susceptibility of lung tissue to SARS-CoV-2 infection, we evaluated murine bronchioalveolar stem cells (BASC), alveolar type II cells (AT2), and 3D-derived organoids expression of mRNA encoding genes involved in virus entry into cells, components of RAA, and genes that comprise elements of the Nlrp3 inflammasome pathway. We noticed that all these genes are expressed by lung alveolar stem cells and organoids-derived from these cells. Interestingly, all these cells express a high level of ACE2 that, on the one hand, serves as an entry receptor for SARS-CoV-2 and, on the other, converts angiotensin II into its physiological antagonist, angiotensin 1-7 (Ang 1-7), which has been reported to have a protective role in lung damage. To shed more light on the role of Ang 1-7 on lung tissue, we exposed lung-derived BASC and AT2 cells to this mediator of RAA and noticed that it increases the proliferation of these cells. Based on this, Ang 1-7 could be employed to alleviate the damage to lung alveolar stem/progenitor cells during SARS-CoV-2 infection.

Development of effective treatment strategies for lung tissue destruction as seen in emphysema would greatly benefit from representative human in vitro models of the alveolar compartment. Studying how cellular cross talk and/or (altered) biomechanical cues affect alveolar epithelial function could provide new insight for tissue repair strategies. Preclinical models of the alveolus ideally combine human primary patient-derived lung cells with advanced cell culture applications such as breathing-related stretch, to reliably represent the alveolar microenvironment. To test the feasibility of such a model, we isolated primary alveolar type 2 cells (AEC2s) from patient-derived lung tissues including those from patients with severe emphysema, using magnetic bead-based selection of cells expressing the AEC2 marker HTII-280. We obtained pure alveolar feeder-free organoid cultures using a minimally modified commercial medium. This was confirmed by known AEC2 markers as well as by detection of lamellar bodies using electron microscopy. Following (organoid-based) expansion, cells were seeded on both cell culture inserts and the Chip-S1 Organ-Chip that has a flexible polydimethylsiloxane (PDMS) membrane enabling the application of dynamic stretch. AEC2s cultured for 7 days on inserts or the chip maintained expression of HTII-280, prosurfactant protein C (SP-C), SP-A and SP-B, and zonula occludens-1 (ZO-1) also in the presence of stretch. AEC2s cultured on the chip showed lower expression levels of epithelial-mesenchymal transition-related vimentin expression compared with static cultures on inserts. The combination of a straightforward culture method of patient-derived AEC2s and their application in microfluidic chip cultures supports successful development of more representative human preclinical models of the (diseased) alveolar compartment.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive deadly fibrotic lung disease with high prevalence and mortality worldwide. The therapeutic potential of mesenchymal stem cells (MSCs) in pulmonary fibrosis may be attributed to the strong paracrine, anti-inflammatory, anti-apoptosis and immunoregulatory effects. However, the mechanisms underlying the therapeutic effects of MSCs in IPF, especially in terms of alveolar type 2 (AT2) cells senescence, are not well understood. The purpose of this study was to evaluate the role of MSCs in NAD metabolism and senescence of AT2 cells in vitro and in vivo.
MSCs were isolated from human bone marrow. The protective effects of MSCs injection in pulmonary fibrosis were assessed via bleomycin mouse models. The senescence of AT2 cells co-cultured with MSCs was evaluated by SA-β-galactosidase assay, immunofluorescence staining and Western blotting. NAD+ level and NAMPT expression in AT2 cells affected by MSCs were determined in vitro and in vivo. FK866 and NAMPT shRNA vectors were used to determine the role of NAMPT in MSCs inhibiting AT2 cells senescence.
We proved that MSCs attenuate bleomycin-induced pulmonary fibrosis in mice. Senescence of AT2 cells was alleviated in MSCs-treated pulmonary fibrosis mice and when co-cultured with MSCs in vitro. Mechanistic studies showed that NAD+ and NAMPT levels were rescued in AT2 cells co-cultured with MSCs and MSCs could suppress AT2 cells senescence mainly via suppressing lysosome-mediated NAMPT degradation.
MSCs attenuate AT2 cells senescence by upregulating NAMPT expression and NAD+ levels, thus exerting protective effects in pulmonary fibrosis.

Palmitic acid (PA) is a saturated free fatty acid which, when being excessive, accounts for lipotoxicity. Using human lung A549 cells as a model for lung alveolar type 2 epithelial cells, we found that challenge of A549 cells with PA resulted in apoptotic cell death, as reflected by positive annexin V and PI staining, and also appearance of cleaved caspase-3. PA treatment also caused depletion of intracellular Ca2+ store, endoplasmic reticulum (ER) stress, and oxidative stress. Tannic acid (TA), a polyphenol present in wines and many beverages, alleviated PA-induced ER stress, oxidative stress and apoptotic death. Thus, our results suggest PA lipotoxicity in lung alveolar type 2 epithelial cells could be protected by TA.

Bronchopulmonary dysplasia (BPD) remains the most important respiratory morbidity of preterm infants with few effective preventive strategies. Administration of mesenchymal stem cells (MSC) was considered effective to prevent BPD via paracrine extracellular vesicles (EVs), while appropriate regimens of MSC-EVs and the mechanism remain unclear. Therefore, we established a hyperoxia-induced rat BPD model, and examined the effect of early intraperitoneal MSC-EVs with different doses on BPD. We found that MSC-EVs ameliorated hyperoxia-induced lung injury in a dose-dependent manner, and high-dose MSC-EVs ameliorated alveolar simplification and fibrosis. Also, MSC-EVs showed its beneficial effects on vascular growth and pulmonary hypertension. Primary AT2 cells were observed to transdifferentiate into AT1 cells when exposure to hyperoxia in vitro. Administration of MSC-EVs at the first-day culture significantly delayed the transdifferentiation of AT2 cells induced by hyperoxia. We further found that exposure to hyperoxia led to elevated expression of WNT5a mRNA and protein, a key agent in AT2 transdifferentiation, while MSC-EVs administration decreased it. Further study is warranted that MSC-EVs may delay the transdifferentiation of AT2 cells via WNT5a. These studies provide key preclinical evidence of MSC-EVs therapeutics on BPD and highlight the effect of MSC-EVs on suppressing the transdifferentiation of AT2 cells and its possible mechanism through downregulation of WNT5a.

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Although infection initiates in the proximal airways, severe and sometimes fatal symptoms of the disease are caused by infection of the alveolar type 2 (AT2) cells of the distal lung and associated inflammation. In this study, we develop primary human lung epithelial infection models to understand initial responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface (ALI) cultures of proximal airway epithelium and alveosphere cultures of distal lung AT2 cells are readily infected by SARS-CoV-2, leading to an epithelial cell-autonomous proinflammatory response with increased expression of interferon signaling genes. Studies to validate the efficacy of selected candidate COVID-19 drugs confirm that remdesivir strongly suppresses viral infection/replication. We provide a relevant platform for study of COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and emergent respiratory pathogens.