BACKGROUND: Airway basal cells have been shown to play important roles in lung disease, prompting their use in research involving 3-dimensional (3D) organoid culture. Mouse tracheal basal cells (MTBC) represent a key model in understanding the differentiation of airway basal cells in normal development and diseased states. The isolation, expansion, and culture media of basal cells can affect their capacity to maintain stemness and properly differentiate, thereby introducing unknown experimental variables. As MTBC use in 3D organoid culture proceeds, there is an increasing need for comparative studies to identify unknown experimental variables. In this study, we examined the effects of MTBC expansion media on the differentiation and tracheosphere-forming ability in 3D culture. METHODS: MTBC were isolated from mouse tracheas, and three commonly used airway basal cell media were compared during MTBC expansion in cell culture (hereafter referred to as Media 1, 2, and 3). Following expansion, MTBC were cultured identically in 3D conditions to analyze tracheosphere-forming capability. Image analyses were performed to characterize tracheosphere count, size, and morphology, while metabolic and transcriptomic signatures were analyzed to assess MTBC differentiation in 3D culture. RESULTS: Expansion of MTBC in the three distinct media resulted in subsequent 3D tracheospheres that displayed unique metabolite and gene expression profiles, accompanied by overall changes in tracheosphere count and size. MTBC expanded in Medium 1 displayed lower levels of TCA cycle intermediates, along with lowered expression of differentiation markers Foxj1 and Scgb1a1, compared to MTBC expanded in Media 2 or 3. Pathway analysis showed an upregulation of idiopathic pulmonary fibrosis signaling pathways in tracheospheres cultured from MTBC expanded in Media 2 or 3. CONCLUSION: This study identified several key differences in tracheosphere-forming ability of MTBC resulting from MTBC expansion media and highlights the importance of transparency in cell culture protocols, particularly those involving stem cells or 3D culture.

Changes in the oxidative (redox) environment accompany idiopathic pulmonary fibrosis (IPF). S-glutathionylation of reactive protein cysteines is a post-translational event that transduces oxidant signals into biological responses. We recently demonstrated that increases in S-glutathionylation promote pulmonary fibrosis, which was mitigated by the deglutathionylating enzyme glutaredoxin (GLRX). However, the protein targets of S-glutathionylation that promote fibrogenesis remain unknown. In the present study we addressed whether the extracellular matrix is a target for S-glutathionylation. We discovered increases in COL1A1 (collagen 1A1) S-glutathionylation (COL1A1-SSG) in lung tissues from subjects with IPF compared with control subjects in association with increases in ERO1A (endoplasmic reticulum [ER] oxidoreductin 1) and enhanced oxidation of ER-localized PRDX4 (peroxiredoxin 4), reflecting an increased oxidative environment of the ER. Human lung fibroblasts exposed to TGFB1 (transforming growth factor-β1) show increased secretion of COL1A1-SSG. Pharmacologic inhibition of ERO1A diminished the oxidation of PRDX4, attenuated COL1A1-SSG and total COL1A1 concentrations, and dampened fibroblast activation. Absence of Glrx enhanced COL1A1-SSG and overall COL1A1 secretion and promoted the activation of mechanosensing pathways. Remarkably, COL1A1-SSG resulted in marked resistance to collagenase degradation. Compared with COL1, lung fibroblasts plated on COL1-SSG proliferated more rapidly and increased the expression of genes encoding extracellular matrix crosslinking enzymes and genes linked to mechanosensing pathways. Overall, these findings suggest that glutathione-dependent oxidation of COL1A1 occurs in settings of IPF in association with enhanced ER oxidative stress and may promote fibrotic remodeling because of increased resistance to collagenase-mediated degradation and fibroblast activation.

In patients with breast cancer, lower bone mineral density increases the risk of bone metastasis. Although the relationship between bone-matrix mineralization and tumour-cell phenotype in breast cancer is not well understood, mineralization-induced rigidity is thought to drive metastatic progression via increased cell-adhesion forces. Here, by using collagen-based matrices with adjustable intrafibrillar mineralization, we show that, unexpectedly, matrix mineralization dampens integrin-mediated mechanosignalling and induces a less proliferative stem-cell-like phenotype in breast cancer cells. In mice with xenografted decellularized physiological bone matrices seeded with human breast tumour cells, the presence of bone mineral reduced tumour growth and upregulated a gene-expression signature that is associated with longer metastasis-free survival in patients with breast cancer. Our findings suggest that bone-matrix changes in osteogenic niches regulate metastatic progression in breast cancer and that in vitro models of bone metastasis should integrate organic and inorganic matrix components to mimic physiological and pathologic mineralization.

Bone metastasis is a leading cause of breast cancer-related deaths and often initiated by tumor cell dissemination to osteogenic niches. During new bone formation, osteoblasts first deposit osteoid, the collagen I-rich, unmineralized component of bone ECM, within which carbonated hydroxyapatite nanoparticles subsequently form. However, it remains elusive how bone matrix mineralization dictates tumor cell phenotype due in part to the lack of relevant model systems. Using biofunctional, collagen I-based bone matrix models with physiological, intrafibrillar mineralization, we show that mineralization inhibits proliferation, while inducing a stem-like phenotype in tumor cells. These changes were due to reduced mechanosignaling contradicting the conventional assumption that increased rigidity caused by mineralization stimulates metastatic progression. Our findings are translationally relevant as the presence of mineral reduced tumor growth in vivo and upregulated a gene signature that correlated with decreased patient mortality. Our results could help explain why decreased bone mineral density increases the risk for bone metastasis in patients and highlight that bone metastasis models should integrate organic and inorganic matrix components in a manner that mimics physiological mineralization.

AbstractTumour‐derived microvesicles (MVs) serve as critical mediators of cell‐to‐cell communication in the tumour microenvironment. So far, the underlying mechanisms of MV biogenesis, especially how key tumorigenesis signals such as abnormal EGF signalling regulates MV release, remain unclear. Here, we set out to establish reliable readouts for MV biogenesis and then explore the molecular mechanisms that regulate MV generation. We found that Rho family small G protein Cdc42 is a convergent node of multiple regulatory signals that occur in MV biogenesis. The binding of activated GTP‐bound Cdc42 and its downstream effector, Ras GTPase‐activating‐like protein 1 (IQGAP1), is required for MV shedding. Activated Cdc42 maintains sustained EGF signalling by inhibiting the internalization of cell surface receptors, including EGFR and the VEGF oligomer, VEGF90K, and then facilitates MV release. Subsequently, we further demonstrated that blocking these signalling pathways using the corresponding mutants effectively reduced MV shedding and significantly inhibited MV‐promoted in vivo tumour angiogenesis. These findings reveal a complex regulation of MV shedding by tumour cells, shedding light on the regulatory mechanism of MV biogenesis, and potentially contributing to strategies that target MVs in cancer therapy.

The lung is comprised of more than 40 distinct cell types that support a complex 3-dimensional (3D) architecture that is required for efficient lung function. Loss of this proper architecture can accommodate and promote lung disease, highlighting researchers' growing need to analyze lung structures in detail. Additionally, in vivo cellular and molecular response to chemical and physical signals, along with the recapitulation of gene-expression patterns, can be lost during the transition from complex 3D tissues to 2D cell culture systems. Therefore, technologies that allow for the investigation of lung function under normal and disease states utilizing the entirety of the lung architecture are required to generate a complete understanding of these processes. Airway cell-derived organoids that can recapitulate lung structure and function ex vivo while being amenable to experimental manipulation, have provided a new and exciting model system to investigate lung biology. In this perspective, we discuss emerging technologies for culturing lung-derived organoids, techniques to visualize organoids using high-resolution microscopy and the resulting information extracted from organoids supporting research focused on lung function and diseases.

Glutathione is a major redox buffer, reaching millimolar concentrations within cells and high micromolar concentrations in airways. While glutathione has been traditionally known as an antioxidant defense mechanism that protects the lung tissue from oxidative stress, glutathione more recently has become recognized for its ability to become covalently conjugated to reactive cysteines within proteins, a modification known as S-glutathionylation (or S-glutathiolation or protein mixed disulfide). S-glutathionylation has the potential to change the structure and function of the target protein, owing to its size (the addition of three amino acids) and charge (glutamic acid). S-glutathionylation also protects proteins from irreversible oxidation, allowing them to be enzymatically regenerated. Numerous enzymes have been identified to catalyze the glutathionylation/deglutathionylation reactions, including glutathione S-transferases and glutaredoxins. Although protein S-glutathionylation has been implicated in numerous biological processes, S-glutathionylated proteomes have largely remained unknown. In this paper, we focus on the pathways that regulate GSH homeostasis. S-glutathionylated proteins, and glutaredoxins, and we review methods required toward identification of glutathionylated proteomes. Finally, we present the latest findings on the role of glutathionylation/glutaredoxins in various lung diseases: idiopathic pulmonary fibrosis, asthma, and chronic obstructive pulmonary disease.

The small GTPase cell division cycle 42 (CDC42) plays essential roles in neurogenesis and brain development. Previously, using murine embryonic P19 cells as a model system, we showed that CDC42 stimulates mTOR complex 1 (mTORC1) activity and thereby up-regulates transcription factors required for the formation of neural progenitor cells. However, paradoxically, although endogenous CDC42 is required for both the initial transition of undifferentiated P19 cells to neural progenitors and their ultimate terminal differentiation into neurons, ectopic CDC42 overexpression promotes only the first stage of neurogenesis (i.e. the formation of neuroprogenitors) and not the second phase (differentiation into neurons). Here, using both P19 cells and mouse embryonic stem cells, we resolve this paradox, demonstrating that two splice variants of CDC42, differing only in nine amino acid residues in their very C-terminal regions, play distinct roles in neurogenesis. We found that a CDC42 splice variant that has a ubiquitous tissue distribution, termed here as CDC42u, specifically drives the formation of neuroprogenitor cells, whereas a brain-specific CDC42 variant, CDC42b, is essential for promoting the transition of neuroprogenitor cells to neurons. We further show that the specific roles of CDC42u and CDC42b in neurogenesis are due to their opposing effects on mTORC1 activity. Specifically, CDC42u stimulated mTORC1 activity and thereby induced neuroprogenitor formation, whereas CDC42b worked together with activated CDC42-associated kinase (ACK) in down-regulating mTOR expression and promoting neuronal differentiation. These findings highlight the remarkable functional specificities of two highly similar CDC42 splice variants in regulating distinct stages of neurogenesis.

The mitochondrial enzyme glutaminase (GLS) is frequently up-regulated during tumorigenesis and is being evaluated as a target for cancer therapy. GLS catalyzes the hydrolysis of glutamine to glutamate, which then supplies diverse metabolic pathways with carbon and/or nitrogen. Here, we report that SIRT5, a mitochondrial NAD+-dependent lysine deacylase, plays a key role in stabilizing GLS. In transformed cells, SIRT5 regulates glutamine metabolism by desuccinylating GLS and thereby protecting it from ubiquitin-mediated degradation. Moreover, we show that SIRT5 is up-regulated during cellular transformation and supports proliferation and tumorigenesis. Elevated SIRT5 expression in human breast tumors correlates with poor patient prognosis. These findings reveal a mechanism for increasing GLS expression in cancer cells and establish a role for SIRT5 in metabolic reprogramming and mammary tumorigenesis.

Efforts to target glutamine metabolism for cancer therapy have focused on the glutaminase isozyme GLS. The importance of the other isozyme, GLS2, in cancer has remained unclear, and it has been described as a tumor suppressor in some contexts. Here, we report that GLS2 is upregulated and essential in luminal-subtype breast tumors, which account for >70% of breast cancer incidence. We show that GLS2 expression is elevated by GATA3 in luminal-subtype cells but suppressed by promoter methylation in basal-subtype cells. Although luminal breast cancers resist GLS-selective inhibitors, we find that they can be targeted with a dual-GLS/GLS2 inhibitor. These results establish a critical role for GLS2 in mammary tumorigenesis and advance our understanding of how to target glutamine metabolism in cancer.

Obesity has been associated with increased severity of diagnoses for several types of cancer, and recent evidence suggests that the mechanism by which obese tissues contribute to cancer progression involves the extracellular matrix (ECM). Understanding the physicochemical differences between lean and obese ECM, and how cancer cells respond to these differences, promises therapeutic insight.

A key strategy targeting the metabolic changes underlying tumorigenesis involves inhibiting the glutaminase isozyme GLS to disrupt glutamine catabolism. However, identifying patient groups for this approach remains a challenge. Here, we define the roles of GLS and its less studied counterpart liver-type glutaminase (GLS2) in breast cancer. We find that GLS2 is upregulated and essential for TCA cycle anaplerosis, cell proliferation, and tumorigenesis in luminal-subtype breast cancers, which account for >70% of disease incidence, and that it mediates resistance to GLS-selective inhibitors. Although GLS is overexpressed in basal-subtype breast cancers, sensitivity to GLS inhibitors depends on glutamate efflux through the xCT antiporter. These findings establish an important role for GLS2 in tumorigenesis and suggest patient stratification approaches for glutaminase-targeted therapy.

Non-classical secretory vesicles, collectively referred to as extracellular vesicles (EVs), have been implicated in different aspects of cancer cell survival and metastasis. Here, we describe how a specific class of EVs, called microvesicles (MVs), activates VEGF receptors and tumour angiogenesis through a unique 90 kDa form of VEGF (VEGF90K). We show that VEGF90K is generated by the crosslinking of VEGF165, catalysed by the enzyme tissue transglutaminase, and associates with MVs through its interaction with the chaperone Hsp90. We further demonstrate that MV-associated VEGF90K has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor activation. However, treatment with an Hsp90 inhibitor releases VEGF90K from MVs, restoring the sensitivity of VEGF90K to Bevacizumab. These findings reveal a novel mechanism by which cancer cell-derived MVs influence the tumour microenvironment and highlight the importance of recognizing their unique properties when considering drug treatment strategies.

The Rho family small GTPase Cdc42 has been implicated in a wide range of cellular functions including the establishment of cell polarity and the remodeling of the actin cytoskeletal architecture, resulting in the tight regulation of cell growth and survival during developmental processes. The complete knock-out of Cdc42 in the mouse is embryonic-lethal, and its targeted deletion in various tissues has been shown to disrupt tissue homeostasis. Thus far, in most studies, the targeted deletion of Cdc42 occurred during embryogenesis. Here, we have used a conditional gene deletion strategy in mice to probe the specific role of Cdc42 during adult mammary gland function. Cdc42 conditional-knock-out females were unable to adequately nourish their pups, due to a disorganized epithelial compartment within their mammary glands. A closer examination showed that their mammary epithelial cells were not able to maintain functional alveolar lumens, due to an inability to establish normal apical/basal epithelial polarity, as well as proper cell-cell contacts. Loss of these essential epithelial characteristics led to a premature sloughing off of the Cdc42-null epithelial cells. Overall our findings demonstrate that Cdc42 plays essential roles in mammary gland function post pregnancy, where it helps to establish proper epithelial cell polarity and tissue homeostasis during lactation.