Quantifying Cell Death Induced by the NLRC4 Inflammasome.

The Nod-like Receptor (NLR) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that sense cytosolic bacterial proteins. NAIP ligation induces its association with NLRC4, leading to the assembly of the NAIP/NLRC4 inflammasome, which induces the activation of the caspase-1 protease. Caspase-1 then cleaves pro-interleukin (IL)-1ß, pro-IL-18, and gasdermin D and induces a form of pro-inflammatory cell death, pyroptosis. These processes culminate in host defense against bacterial infection. Here we describe methods for activating NAIP/NLRC4 inflammasome signalling in human and murine macrophages and quantifying inflammasome-induced cell death.

Screening for CD19-specific chimaeric antigen receptors with enhanced signalling via a barcoded library of intracellular domains.

The immunostimulatory intracellular domains (ICDs) of chimaeric antigen receptors (CARs) are essential for converting antigen recognition into antitumoural function. Although there are many possible combinations of ICDs, almost all current CARs rely on combinations of CD3𝛇, CD28 and 4-1BB. Here we show that a barcoded library of 700,000 unique CD19-specific CARs with diverse ICDs cloned into lentiviral vectors and transduced into Jurkat T cells can be screened at high throughput via cell sorting and next-generation sequencing to optimize CAR signalling for antitumoural functions. By using this screening approach, we identified CARs with new ICD combinations that, compared with clinically available CARs, endowed human primary T cells with comparable tumour control in mice and with improved proliferation, persistence, exhaustion and cytotoxicity after tumour rechallenge in vitro. The screening strategy can be adapted to other disease models, cell types and selection conditions, and could be used to improve adoptive cell therapies and to expand their utility to new disease indications.

Cross-species transcriptomic signatures predict response to MK2 inhibition in mouse models of chronic inflammation.

Inflammatory bowel diseases (IBDs) are genetically complex and exhibit significant inter-patient heterogeneity in disease presentation and therapeutic response. Here, we show that mouse models of IBD exhibit variable responses to inhibition of MK2, a pro-inflammatory serine/threonine kinase, and that MK2 inhibition suppresses inflammation by targeting inflammatory monocytes and neutrophils in murine models. Using a computational approach (TransComp-R) that allows for cross-species comparison of transcriptomic features, we identified an IBD patient subgroup that is predicted to respond to MK2 inhibition, and an independent preclinical model of chronic intestinal inflammation predicted to be non-responsive, which we validated experimentally. Thus, cross-species mouse-human translation approaches can help to identify patient subpopulations in which to deploy new therapies.

Cell surface integrin α5ß1 clustering negatively regulates receptor tyrosine kinase signaling in colorectal cancer cells via glycogen synthase kinase 3.

As a key process within the tissue microenvironment, integrin signaling can influence cell functional responses to growth factor stimuli. We show here that clustering of integrin α5ß1 at the plasma membrane of colorectal cancer-derived epithelial cells modulates their ability to respond to stimulation by receptor tyrosine kinase (RTK)-activating growth factors EGF, NRG and HGF, through GSK3-mediated suppression of Akt pathway. We observed that integrin α5ß1 is lost from the membrane of poorly organized human colorectal tumors and that treatment with the integrin-clustering antibody P4G11 is sufficient to induce polarity in a mouse tumor xenograft model. While adding RTK growth factors (EGF, NRG and HGF) to polarized colorectal cancer cells induced invasion and loss of monolayer formation in 2D and 3D, this pathological behavior could be blocked by P4G11. Phosphorylation of ErbB family members as well as MET following EGF, NRG and HGF treatment was diminished in cells pretreated with P4G11. Focusing on EGFR, we found that blockade of integrin α5ß1 increased EGFR phosphorylation. Since activity of multiple downstream kinase pathways were altered by these various treatments, we employed computational machine learning techniques to ascertain the most important effects. Partial least-squares discriminant analysis identified GSK3 as a major regulator of EGFR pathway activities influenced by integrin α5ß1. Moreover, we used partial correlation analysis to examine signaling pathway crosstalk downstream of EGF stimulation and found that integrin α5ß1 acts as a negative regulator of the AKT signaling cascade downstream of EGFR, with GSK3 acting as a key mediator. We experimentally validated these computational inferences by confirming that blockade of GSK3 activity is sufficient to induce loss of polarity and increase of oncogenic signaling in the colonic epithelial cells.

An interspecies translation model implicates integrin signaling in infliximab-resistant inflammatory bowel disease.

Anti-tumor necrosis factor (anti-TNF) therapy resistance is a major clinical challenge in inflammatory bowel disease (IBD), due, in part, to insufficient understanding of disease-site, protein-level mechanisms. Although proteomics data from IBD mouse models exist, data and phenotype discrepancies contribute to confounding translation from preclinical animal models of disease to clinical cohorts. We developed an approach called translatable components regression (TransComp-R) to overcome interspecies and trans-omic discrepancies between mouse models and human subjects. TransComp-R combines mouse proteomic data with patient pretreatment transcriptomic data to identify molecular features discernable in the mouse data that are predictive of patient response to therapy. Interrogating the TransComp-R models revealed activated integrin pathway signaling in patients with anti-TNF-resistant colonic Crohn's disease (cCD) and ulcerative colitis (UC). As a step toward validation, we performed single-cell RNA sequencing (scRNA-seq) on biopsies from a patient with cCD and analyzed publicly available immune cell proteomics data to characterize the immune and intestinal cell types contributing to anti-TNF resistance. We found that ITGA1 was expressed in T cells and that interactions between these cells and intestinal cell types were associated with resistance to anti-TNF therapy. We experimentally showed that the α1 integrin subunit mediated the effectiveness of anti-TNF therapy in human immune cells. Thus, TransComp-R identified an integrin signaling mechanism with potential therapeutic implications for overcoming anti-TNF therapy resistance. We suggest that TransComp-R is a generalizable framework for addressing species, molecular, and phenotypic discrepancies between model systems and patients to translationally deliver relevant biological insights.

Substrate-based kinase activity inference identifies MK2 as driver of colitis.

Inflammatory bowel disease (IBD) is a chronic and debilitating disorder that has few treatment options due to a lack of comprehensive understanding of its molecular pathogenesis. We used multiplexed mass spectrometry to collect high-content information on protein phosphorylation in two different mouse models of IBD. Because the biological function of the vast majority of phosphorylation sites remains unknown, we developed Substrate-based Kinase Activity Inference (SKAI), a methodology to infer kinase activity from phosphoproteomic data. This approach draws upon prior knowledge of kinase-substrate interactions to construct custom lists of kinases and their respective substrate sites, termed kinase-substrate sets that employ prior knowledge across organisms. This expansion as much as triples the amount of prior knowledge available. We then used these sets within the Gene Set Enrichment Analysis framework to infer kinase activity based on increased or decreased phosphorylation of its substrates in a dataset. When applied to the phosphoproteomic datasets from the two mouse models, SKAI predicted largely non-overlapping kinase activation profiles. These results suggest that chronic inflammation may arise through activation of largely divergent signaling networks. However, the one kinase inferred to be activated in both mouse models was mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2 or MK2), a serine/threonine kinase that functions downstream of p38 stress-activated mitogen-activated protein kinase. Treatment of mice with active colitis with ATI450, an orally bioavailable small molecule inhibitor of the MK2 pathway, reduced inflammatory signaling in the colon and alleviated the clinical and histological features of inflammation. These studies establish MK2 as a therapeutic target in IBD and identify ATI450 as a potential therapy for the disease.

Proteogenomic Network Analysis of Context-Specific KRAS Signaling in Mouse-to-Human Cross-Species Translation.

The highest frequencies of KRAS mutations occur in colorectal carcinoma (CRC) and pancreatic ductal adenocarcinoma (PDAC). The ability to target downstream pathways mediating KRAS oncogenicity is limited by an incomplete understanding of the contextual cues modulating the signaling output of activated K-RAS. We performed mass spectrometry on mouse tissues expressing wild-type or mutant Kras to determine how tissue context and genetic background modulate oncogenic signaling. Mutant Kras dramatically altered the proteomes and phosphoproteomes of preneoplastic and neoplastic colons and pancreases in a context-specific manner. We developed an approach to statistically humanize the mouse networks with data from human cancer and identified genes within the humanized CRC and PDAC networks synthetically lethal with mutant KRAS. Our studies demonstrate the context-dependent plasticity of oncogenic signaling, identify non-canonical mediators of KRAS oncogenicity within the KRAS-regulated signaling network, and demonstrate how statistical integration of mouse and human datasets can reveal cross-species therapeutic insights.

Multivariate Computational Analysis of Gamma Delta T Cell Inhibitory Receptor Signatures Reveals the Divergence of Healthy and ART-Suppressed HIV+ Aging.

Even with effective viral control, HIV-infected individuals are at a higher risk for morbidities associated with older age than the general population, and these serious non-AIDS events (SNAEs) track with plasma inflammatory and coagulation markers. The cell subsets driving inflammation in aviremic HIV infection are not yet elucidated. Also, whether ART-suppressed HIV infection causes premature induction of the inflammatory events found in uninfected elderly or if a novel inflammatory network ensues when HIV and older age co-exist is unclear. In this study we measured combinational expression of five inhibitory receptors (IRs) on seven immune cell subsets and 16 plasma markers from peripheral blood mononuclear cells (PBMC) and plasma samples, respectively, from a HIV and Aging cohort comprised of ART-suppressed HIV-infected and uninfected controls stratified by age (≤35 or ≥50 years old). For data analysis, multiple multivariate computational algorithms [cluster identification, characterization, and regression (CITRUS), partial least squares regression (PLSR), and partial least squares-discriminant analysis (PLS-DA)] were used to determine if immune parameter disparities can distinguish the subject groups and to investigate if there is a cross-impact of aviremic HIV and age on immune signatures. IR expression on gamma delta (γδ) T cells exclusively separated HIV+ subjects from controls in CITRUS analyses and secretion of inflammatory cytokines and cytotoxic mediators from γδ T cells tracked with TIGIT expression among HIV+ subjects. Also, plasma markers predicted the percentages of TIGIT+ γδ T cells in subjects with and without HIV in PSLR models, and a PLS-DA model of γδ T cell IR signatures and plasma markers significantly stratified all four of the subject groups (uninfected younger, uninfected older, HIV+ younger, and HIV+ older). These data implicate γδ T cells as an inflammatory driver in ART-suppressed HIV infection and provide evidence of distinct "inflamm-aging" processes with and without ART-suppressed HIV infection.

The colonic epithelium plays an active role in promoting colitis by shaping the tissue cytokine profile.

Inflammatory bowel disease (IBD) is a chronic condition driven by loss of homeostasis between the mucosal immune system, the commensal gut microbiota, and the intestinal epithelium. Our goal is to understand how these components of the intestinal ecosystem cooperate to control homeostasis. By combining quantitative measures of epithelial hyperplasia and immune infiltration with multivariate analysis of inter- and intracellular signaling, we identified epithelial mammalian target of rapamycin (mTOR) signaling as a potential driver of inflammation in a mouse model of colitis. A kinetic analysis of mTOR inhibition revealed that the pathway regulates epithelial differentiation, which in turn controls the cytokine milieu of the colon. Consistent with our in vivo analysis, we found that cytokine expression of organoids grown ex vivo, in the absence of bacteria and immune cells, was dependent on differentiation state. Our study suggests that proper differentiation of epithelial cells is an important feature of colonic homeostasis because of its effect on the secretion of inflammatory cytokines.

In vivo systems biology approaches to chronic immune/inflammatory pathophysiology.

Systems biology offers an emphasis on integrative computational analysis of complex multi-component processes to enhance capability for predictive insights concerning operation of those processes. The immune system represents a prominent arena in which such processes are manifested for vital roles in physiology and pathology, encompassing dozens of cell types and hundreds of reciprocal interactions. Chronic, debilitating pathologies involving immune system dysregulation have become recognized as increasing in incidence over recent decades. While clinical consequences of immune dysregulation in such pathologies are well characterized, treatment options remain limited and focus on ameliorating symptoms. Because it is difficult to recapitulate more than a severely limited facet of the immune system in vitro, application of systems biology approaches to autoimmune and inflammatory pathophysiology in vivo has opened a new door toward discerning disease sub-groups and developing associated stratification strategies for patient treatment. In particular, early instances of these approaches have demonstrated advances in uncovering previously under-appreciated dysregulation of signaling networks between immune system and tissue cells, raising promise for improving upon current therapeutic approaches.

A Chimeric Egfr Protein Reporter Mouse Reveals Egfr Localization and Trafficking In Vivo.

EGF receptor (EGFR) is a critical signaling node throughout life. However, it has not been possible to directly visualize endogenous Egfr in mice. Using CRISPR/Cas9 genome editing, we appended a fluorescent reporter to the C terminus of the Egfr. Homozygous reporter mice appear normal and EGFR signaling is intact in vitro and in vivo. We detect distinct patterns of Egfr expression in progenitor and differentiated compartments in embryonic and adult mice. Systemic delivery of EGF or amphiregulin results in markedly different patterns of Egfr internalization and trafficking in hepatocytes. In the normal intestine, Egfr localizes to the crypt rather than villus compartment, expression is higher in adjacent epithelium than in intestinal tumors, and following colonic injury expression appears in distinct cell populations in the stroma. This reporter, under control of its endogenous regulatory elements, enables in vivo monitoring of the dynamics of Egfr localization and trafficking in normal and disease states.

Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer.

We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.

Clustering of integrin α5 at the lateral membrane restores epithelial polarity in invasive colorectal cancer cells.

Apicobasolateral polarity is a fundamental property of epithelial cells, and its loss is a hallmark of cancer. Integrin-mediated contact with the extracellular matrix defines the basal surface, setting in motion E-cadherin-mediated cell-cell contact, which establishes apicobasolateral polarity. Role(s) for lateral integrins in this polarization process and the consequences of their disruption are incompletely understood. We show that addition of an integrin ß1-activating monoclonal antibody, P4G11, to invasive colorectal cancer cells in three-dimensional type 1 collagen reverts the invasive phenotype and restores apicobasolateral polarity. P4G11 induces clustering of integrin α5ß1 at lateral, intercellular surfaces. This leads to deposition and polymerization of fibronectin and recruitment of paxillin to sites of lateral integrin α5ß1 clustering and is followed by tight junction formation, as determined by ZO-1 localization. Inducible elimination of integrin α5 abrogates the epithelial-organizing effects of P4G11. In addition, polymerization of fibronectin is required for the effects of P4G11, and addition of polymerized superfibronectin is sufficient to induce tight junction formation and apicobasolateral polarization. In the normal human colon, we show that integrin α5 localizes to the lateral membrane of terminally differentiated colonocytes and that integrin α5 staining may be reduced in colorectal cancer. Thus we propose a novel role for integrin α5ß1 in regulating epithelial morphogenesis.

Shed urinary ALCAM is an independent prognostic biomarker of three-year overall survival after cystectomy in patients with bladder cancer.

Proteins involved in tumor cell migration can potentially serve as markers of invasive disease. Activated Leukocyte Cell Adhesion Molecule (ALCAM) promotes adhesion, while shedding of its extracellular domain is associated with migration. We hypothesized that shed ALCAM in biofluids could be predictive of progressive disease. ALCAM expression in tumor (n = 198) and shedding in biofluids (n = 120) were measured in two separate VUMC bladder cancer cystectomy cohorts by immunofluorescence and enzyme-linked immunosorbent assay, respectively. The primary outcome measure was accuracy of predicting 3-year overall survival (OS) with shed ALCAM compared to standard clinical indicators alone, assessed by multivariable Cox regression and concordance-indices. Validation was performed by internal bootstrap, a cohort from a second institution (n = 64), and treatment of missing data with multiple-imputation. While ALCAM mRNA expression was unchanged, histological detection of ALCAM decreased with increasing stage (P = 0.004). Importantly, urine ALCAM was elevated 17.0-fold (P < 0.0001) above non-cancer controls, correlated positively with tumor stage (P = 0.018), was an independent predictor of OS after adjusting for age, tumor stage, lymph-node status, and hematuria (HR, 1.46; 95% CI, 1.03-2.06; P = 0.002), and improved prediction of OS by 3.3% (concordance-index, 78.5% vs. 75.2%). Urine ALCAM remained an independent predictor of OS after accounting for treatment with Bacillus Calmette-Guerin, carcinoma in situ, lymph-node dissection, lymphovascular invasion, urine creatinine, and adjuvant chemotherapy (HR, 1.10; 95% CI, 1.02-1.19; P = 0.011). In conclusion, shed ALCAM may be a novel prognostic biomarker in bladder cancer, although prospective validation studies are warranted. These findings demonstrate that markers reporting on cell motility can act as prognostic indicators.

Induction of lateral lumens through disruption of a monoleucine-based basolateral-sorting motif in betacellulin.

Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically (156)EEMETL(161)). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancer-associated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization.

Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer.

The epithelial-to-mesenchymal transition (EMT) transcriptional program is characterized by repression of E-cadherin (CDH1) and induction of N-cadherin (CDH2), and mesenchymal genes like vimentin (VIM). Placenta-specific 8 (PLAC8) has been implicated in colon cancer; however, how PLAC8 contributes to disease is unknown, and endogenous PLAC8 protein has not been studied. We analyzed zebrafish and human tissues and found that endogenous PLAC8 localizes to the apical domain of differentiated intestinal epithelium. Colon cancer cells with elevated PLAC8 levels exhibited EMT features, including increased expression of VIM and zinc finger E-box binding homeobox 1 (ZEB1), aberrant cell motility, and increased invasiveness. In contrast to classical EMT, PLAC8 overexpression reduced cell surface CDH1 and upregulated P-cadherin (CDH3) without affecting CDH2 expression. PLAC8-induced EMT was linked to increased phosphorylated ERK2 (p-ERK2), and ERK2 knockdown restored cell surface CDH1 and suppressed CDH3, VIM, and ZEB1 upregulation. In vitro, PLAC8 directly bound and inactivated the ERK2 phosphatase DUSP6, thereby increasing p-ERK2. In a murine xenograft model, knockdown of endogenous PLAC8 in colon cancer cells resulted in smaller tumors, reduced local invasion, and decreased p-ERK2. Using MultiOmyx, a multiplex immunofluorescence-based methodology, we observed coexpression of cytosolic PLAC8, CDH3, and VIM at the leading edge of a human colorectal tumor, supporting a role for PLAC8 in cancer invasion in vivo.

Elevated ALCAM shedding in colorectal cancer correlates with poor patient outcome.

Molecular biomarkers of cancer are needed to assist histologic staging in the selection of treatment, outcome risk stratification, and patient prognosis. This is particularly important for patients with early-stage disease. We show that shedding of the extracellular domain of activated leukocyte cell adhesion molecule (ALCAM) is prognostic for outcome in patients with colorectal cancer (CRC). Previous reports on the prognostic value of ALCAM expression in CRC have been contradictory and inconclusive. This study clarifies the prognostic value of ALCAM by visualizing ectodomain shedding using a dual stain that detects both the extracellular and the intracellular domains in formalin-fixed tissue. Using this novel assay, 105 patients with primary CRCs and 12 normal mucosa samples were evaluated. ALCAM shedding, defined as detection of the intracellular domain in the absence of the corresponding extracellular domain, was significantly elevated in patients with CRC and correlated with reduced survival. Conversely, retention of intact ALCAM was associated with improved survival, thereby confirming that ALCAM shedding is associated with poor patient outcome. Importantly, analysis of patients with stage II CRC showed that disease-specific survival is significantly reduced for patients with elevated ALCAM shedding (P = 0.01; HR, 3.0), suggesting that ALCAM shedding can identify patients with early-stage disease at risk of rapid progression.

Leading malignant cells initiate collective epithelial cell invasion in a three-dimensional heterotypic tumor spheroid model.

Solid tumors consist of genetically and phenotypically diverse subpopulations of cancer cells with unique capacities for growth, differentiation, and invasion. While the molecular and microenvironmental bases for heterogeneity are increasingly appreciated, the outcomes of such intratumor heterogeneity, particularly in the context of tumor invasion and metastasis, remain poorly understood. To study heterotypic cell-cell interactions and elucidate the biological consequences of intratumor heterogeneity, we developed a tissue-engineered multicellular spheroid (MCS) co-culture model that recapitulates the cellular diversity and fully three-dimensional cell-cell and cell-matrix interactions that characterize human carcinomas. We found that "invasion-competent" malignant cells induced the collective invasion of otherwise "invasion-incompetent" epithelial cells, and that these two cell types consistently exhibited distinct leader and follower roles during invasion. Analysis of extracellular matrix (ECM) microarchitecture revealed that malignant cell invasion was accompanied by extensive ECM remodeling including matrix alignment and proteolytic track-making. Inhibition of cell contractility- and proteolysis-mediated matrix reorganization prevented leader-follower behavior and malignant cell-induced epithelial cell invasion. These results indicate that heterogeneous subpopulations within a tumor may possess specialized roles during tumor progression and suggest that complex interactions among the various subpopulations of cancer cells within a tumor may regulate critical aspects of tumor biology and affect clinical outcome.

Tuning three-dimensional collagen matrix stiffness independently of collagen concentration modulates endothelial cell behavior.

Numerous studies have described the effects of matrix stiffening on cell behavior using two-dimensional synthetic surfaces; however, less is known about the effects of matrix stiffening on cells embedded in three-dimensional in vivo-like matrices. A primary limitation in investigating the effects of matrix stiffness in three dimensions is the lack of materials that can be tuned to control stiffness independently of matrix density. Here, we use collagen-based scaffolds where the mechanical properties are tuned using non-enzymatic glycation of the collagen in solution, prior to polymerization. Collagen solutions glycated prior to polymerization result in collagen gels with a threefold increase in compressive modulus without significant changes to the collagen architecture. Using these scaffolds, we show that endothelial cell spreading increases with matrix stiffness, as does the number and length of angiogenic sprouts and the overall spheroid outgrowth. Differences in sprout length are maintained even when the receptor for advanced glycation end products is inhibited. Our results demonstrate the ability to de-couple matrix stiffness from matrix density and structure in collagen gels, and that increased matrix stiffness results in increased sprouting and outgrowth.