Opposing roles for TGFß- and BMP-signaling during nascent alveolar differentiation in the developing human lung.

Alveolar type 2 (AT2) cells function as stem cells in the adult lung and aid in repair after injury. The current study aimed to understand the signaling events that control differentiation of this therapeutically relevant cell type during human development. Using lung explant and organoid models, we identified opposing effects of TGFß- and BMP-signaling, where inhibition of TGFß- and activation of BMP-signaling in the context of high WNT- and FGF-signaling efficiently differentiated early lung progenitors into AT2-like cells in vitro. AT2-like cells differentiated in this manner exhibit surfactant processing and secretion capabilities, and long-term commitment to a mature AT2 phenotype when expanded in media optimized for primary AT2 culture. Comparing AT2-like cells differentiated with TGFß-inhibition and BMP-activation to alternative differentiation approaches revealed improved specificity to the AT2 lineage and reduced off-target cell types. These findings reveal opposing roles for TGFß- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro.

HER2-Selective and Reversible Tyrosine Kinase Inhibitor Tucatinib Potentiates the Activity of T-DM1 in Preclinical Models of HER2-positive Breast Cancer.

The oncogenic receptor HER2 is overexpressed in many cancers, including up to 20% of breast cancers. Despite the availability of HER2-targeted treatments, patients' disease often progresses during therapy, underscoring the need for novel treatment strategies. The addition of tucatinib, a reversible, highly selective HER2 tyrosine kinase inhibitor (TKI), to treatment with trastuzumab and capecitabine significantly improved survival outcomes of patients with HER2-positive metastatic breast cancer, including those with active brain metastases. We rationalized that combining tucatinib with other HER2-targeting agents with complementary mechanisms of action would further increase efficacy against tumors. We characterized the activity of tucatinib with the antibody­drug conjugate T-DM1 in preclinical models of breast cancer, including HER2-positive breast cancer cells and patient-derived xenograft (PDX) models. Mechanistic details on tucatinib activity were obtained in internalization and catabolism studies. In combination, tucatinib and T-DM1 showed an enhanced, often synergistic, cytotoxic response and demonstrated improved antitumor activity in vivo, including in PDX models refractory to T-DM1 single-agent activity. Mechanistically, tucatinib mediated an increase in inactive HER2 molecules at the cell surface through inhibition of HER2 ubiquitination, resulting in increased internalization and catabolism of T-DM1. The combination was correlated with enhanced HER2 pathway inhibition, decreased proliferation, and increased apoptosis. In a xenograft model of brain metastasis, tucatinib penetrated intracranial tumor tissues, inhibiting tumor growth and improving survival. These results suggest that tucatinib may be the optimal TKI partner for HER2-targeted therapies and support clinical studies of its combination with T-DM1, including in patients with brain metastases. SIGNIFICANCE: The preclinical findings in breast cancer models presented here demonstrate that combining tucatinib with T-DM1 enhances the antitumor activity of either agent alone, supporting clinical studies of the combination in HER2-positive breast cancer, including in patients with brain metastases, which remains an important unmet medical need.

Regionally distinct progenitor cells in the lower airway give rise to neuroendocrine and multiciliated cells in the developing human lung.

Using scRNA-seq and microscopy, we describe a cell that is enriched in the lower airways of the developing human lung and identified by the unique coexpression of SCGB3A2/SFTPB/CFTR. To functionally interrogate these cells, we apply a single-cell barcode-based lineage tracing method, called CellTagging, to track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro. Lineage tracing reveals that these cells have a distinct differentiation potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. Lineage tracing results are supported by studies using organoids and isolated cells from the lower noncartilaginous airway. We conclude that SCGB3A2/SFTPB/CFTR cells are enriched in the lower airways of the developing human lung and contribute to the epithelial diversity and heterogeneity in this region.

Opposing roles for TGFß- and BMP-signaling during nascent alveolar differentiation in the developing human lung.

Alveolar type 2 (AT2) cells function as stem cells in the adult lung and aid in repair after injury. The current study aimed to understand the signaling events that control differentiation of this therapeutically relevant cell type during human development. Using lung explant and organoid models, we identified opposing effects of TGFß- and BMP-signaling, where inhibition of TGFß- and activation of BMP-signaling in the context of high WNT- and FGF-signaling efficiently differentiated early lung progenitors into AT2-like cells in vitro . AT2-like cells differentiated in this manner exhibit surfactant processing and secretion capabilities, and long-term commitment to a mature AT2 phenotype when expanded in media optimized for primary AT2 culture. Comparing AT2-like cells differentiated with TGFß-inhibition and BMP-activation to alternative differentiation approaches revealed improved specificity to the AT2 lineage and reduced off-target cell types. These findings reveal opposing roles for TGFß- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro .

FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.

Fibroblast growth factor (FGF) signaling is known to play an important role in lung organogenesis. However, we recently demonstrated that FGF10 fails to induce branching in human fetal lungs as is observed in mouse. Our previous human fetal lung RNA sequencing data exhibited increased FGF18 during the pseudoglandular stage of development, suggestive of its importance in human lung branching morphogenesis. Whereas it has been previously reported that FGF18 is critical during alveologenesis, few studies have described its implication in lung branching, specifically in human. Therefore, we aimed to determine the role of FGF18 in human lung branching morphogenesis. Human fetal lung explants within the pseudoglandular stage of development were treated with recombinant human FGF18 in air-liquid interface culture. Explants were analyzed grossly to assess differences in branching pattern, as well as at the cellular and molecular levels. FGF18 treatment promoted branching in explant cultures and demonstrated increased epithelial proliferation as well as maintenance of the double positive SOX2/SOX9 distal bud progenitor cells, confirming its role in human lung branching morphogenesis. In addition, FGF18 treated explants displayed increased expression of SOX9, FN1, and COL2A1 within the mesenchyme, all factors that are important to chondrocyte differentiation. In humans, cartilaginous airways extend deep into the lung up to the 12th generation of branching whereas in mouse these are restricted to the trachea and main bronchi. Therefore, our data suggest that FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.

Human Lung Organoid Culture in Alginate With and Without Matrigel to Model Development and Disease.

Human lung organoids (HLOs) are enabling the study of human lung development and disease by modeling native organ tissue structure, cellular composition, and cellular organization. In this report, we demonstrate that HLOs derived from human pluripotent stem cells cultured in alginate, a fully defined nonanimal product substrate, exhibit enhanced cellular differentiation compared with HLOs cultured in the commercially available Matrigel. More specifically, we observed an earlier onset and increase in the number of multiciliated cells, along with mucus producing MUC5AC+ goblet-like cells that were not observed in HLOs cultured in Matrigel. The epithelium in alginate-grown HLOs was organized in a pseudostratified epithelium with airway basal cells lining the basal lamina, but with the apical surface of cells on the exterior of the organoid. We further observed that HLOs cultured in Matrigel exhibited mesenchymal overgrowth that was not present in alginate cultures. The containment of the mesenchyme within HLOs in alginate enabled modeling of key features of idiopathic pulmonary fibrosis (IPF) by treatment with transforming growth factor ß (TGFß). TGFß treatment resulted in morphological changes including an increase in mesenchymal growth, increased expression of IPF markers, and decreased numbers of alveolar-like cells. This culture system provides a model to study the interaction of the mesenchyme with the epithelium during lung development and diseased states such as IPF.

Stable iPSC-derived NKX2-1+ lung bud tip progenitor organoids give rise to airway and alveolar cell types.

Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.

R-SPONDIN2+ mesenchymal cells form the bud tip progenitor niche during human lung development.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

Ionic liquids in analytical chemistry.

The role of ionic liquids (ILs) in analytical chemistry is increasing substantially every year. A decade ago there were but a handful of papers in this area of research that were considered curiosities at best. Today, those publications are recognized as seminal articles that gave rise to one of the most rapidly expanding areas of research in chemical analysis. In this review, we briefly highlight early work involving ILs and discuss the most recent advances in separations, mass spectrometry, spectroscopy, and electroanalytical chemistry. Many of the most important advances in these fields depend on the development of new, often unique ILs and multifunctional ILs. A better understanding of the chemical and physical properties of ILs is also essential.

Evaluating the use of tricationic reagents for the detection of doubly charged anions in the positive mode by ESI-MS.

The analysis of anions remains an important task for many areas of science, and new sensitive analytical methods continue to be of great interest. In this study, we present the use of 17 tricationic reagents for use as gas-phase ion pairing agents for divalent anions. When the anion pairs with the tricationic reagent, an overall positive charge is retained and enables detection by ESI-MS in the positive mode. The 17 tricationic reagents were made from 1 of 4 core structures and 7 terminal charged groups. The effect of these structural elements on the detection sensitivity of the complex is examined empirically. A comparison of signal-to-noise ratios achieved in positive and negative modes also is presented.

Evaluation of dicationic reagents for their use in detection of anions using positive ion mode ESI-MS via gas phase ion association.

Twenty-three different dications were investigated for their effectiveness in pairing with singly charged anions, thereby allowing the electrospray ionization mass spectrometry (ESI-MS) detection of anions as positively charged complexes. Nitrate, iodide, cyanate, monochloroacetate, benzenesulfonate, and perfluoro-octanoate were chosen as representative test anions as they differ in mass, size-to-charge ratio, chaotropic nature, and overall complexity. Detection limits were found using direct injection of the anion into a carrier liquid containing the dication. Detection limits are given for all six anions with each of the 23 dications. Each anion was easily detected at the ppb (microg/L) and often the ppt (ng/L) levels using certain dicationic reagents. The ability of dicationic reagents to pair with anions and produce ESI-MS signals varied tremendously. Indeed, only a few dications can be considered broadly useful and able to produce sensitive results. Liquid chromatography (LC)-ESI-MS also was investigated and used to show how varying the dicationic reagent produced significantly different peak intensities. Also, the use of tandem mass spectrometry can lead to even greater sensitivity when using imidazolium based dications.

A general, positive ion mode ESI-MS approach for the analysis of singly charged inorganic and organic anions using a dicationic reagent.

Anion analysis continues to be of great importance to many scientific and technical fields. We propose here a general and sensitive method of detecting singly charged anions by ESI-MS and LC-ESI-MS as positive ions. This method utilizes a dicationic reagent to form a complex with the anion that retains an overall positive charge for analysis by MS. Nitrate, thiocyanate, perchlorate, perfluorooctanoic acid (PFOA), halogenated acetic acids, and various other inorganic and organic anions and are investigated. The use of tandem mass spectrometry to enhance the detection limits of some of the anions is demonstrated. Chaotropic anions provided the lowest detection limits, with PFOA detected at the hundreds of femtograms level. Indeed, this single approach provides the lowest reported detection limits for a variety of anions, especially PFOA, nitrate, monochloroacetic acid, dichloroacetic acid, and bromochloroacetic acid, among others. The integrated areas and signal-to-noise ratios for five ions during a chromatographic run in both the positive and negative ion modes are compared. The ability of this method to detect differences in related ions is shown for four arsenic species. Finally, a tap water sample is analyzed for the anions in this study using the dicationic reagent method.

TIM-1 regulates macrophage cytokine production and B7 family member expression.

T-cell immunoglobulin mucin-1 (TIM-1) is associated with the regulation of T helper type 2 (Th2) immune responses and has been associated with asthma susceptibility. Previous studies have shown that administration of TIM-1 results in T cell hyperproliferation and increased Th2 cytokine secretion. TIM-1 has also been shown to bind to macrophages, but the effects of TIM-1 administration on macrophage activity have not been assessed. In this study we demonstrate that TIM-1 binds to the mouse macrophage cell line RAW 264.7. Stimulation of the RAW264.7 cells with TIM-1 increases nitric oxide production. A dramatic increase in the pro-inflammatory cytokines TNF-alpha and IL-6 is seen upon TIM-1 stimulation of RAW 264.7 cells. Additionally, there is a moderate increase in the immuno-modulatory cytokine IL-10 when RAW 264.7 cells are stimulated with TIM-1. TIM-1 stimulation also alters the expression of some members of the B7 family of co-stimulatory/co-inhibitory proteins. TIM-1 stimulation leads to increased B7-1, B7-H1, and PD-L2 expression, while inhibiting B7-H2 expression. These studies suggest that TIM-1 can regulate macrophage activation and alter the co-stimulatory properties of macrophages and thus may contribute to allergic inflammatory diseases such as asthma.