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Summary: Although aberrant activation of the KRAS and PI3K pathway alongside TP53 mutations account for frequent aberrations in human gastric cancers, neither the sequence nor the individual contributions of these mutations have been clarified. Here, we establish an allelic series of mice to afford conditional expression in the glandular epithelium of KrasG12D;Pik3caH1047R or Trp53R172H and/or ablation of Pten or Trp53. We find that KrasG12D;Pik3caH1047R is sufficient to induce adenomas and that lesions progress to carcinoma when also harboring Pten deletions. An additional challenge with either Trp53 loss- or gain-of-function alleles further accelerated tumor progression and triggered metastatic disease. While tumor-intrinsic STAT3 signaling in response to gp130 family cytokines remained as a gatekeeper for all stages of tumor development, metastatic progression required a mutant Trp53-induced interleukin (IL)-11 to IL-6 dependency switch. Consistent with the poorer survival of patients with high IL-6 expression, we identify IL-6/STAT3 signaling as a therapeutic vulnerability for TP53-mutant gastric cancer.

Abstract Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Abstract Although gastric cancer is a leading cause of cancer-related deaths, systemic treatment strategies remain scarce. Here, we report the pro-tumorigenic properties of the crosstalk between intestinal tuft cells and type 2 innate lymphoid cells (ILC2) that is evolutionarily optimized for epithelial remodeling in response to helminth infection. We demonstrate that tuft cell-derived interleukin 25 (IL25) drives ILC2 activation, inducing the release of IL13 and promoting epithelial tuft cell hyperplasia. While the resulting tuft cell - ILC2 feed-forward circuit promotes gastric metaplasia and tumor formation, genetic depletion of tuft cells or ILC2s, or therapeutic targeting of IL13 or IL25 alleviates these pathologies in mice. In gastric cancer patients, tuft cell and ILC2 gene signatures predict worsening survival in intestinal-type gastric cancer where ~40% of the corresponding cancers show enriched co-existence of tuft cells and ILC2s. Our findings suggest a role for ILC2 and tuft cells, along with their associated cytokine IL13 and IL25 as gatekeepers and enablers of metaplastic transformation and gastric tumorigenesis, thereby providing an opportunity to therapeutically inhibit early-stage gastric cancer through repurposing antibody-mediated therapies.

Abstract Over the past decade, immune checkpoint inhibitor (ICI) therapy has been established as the standard of care for many types of cancer, but the strategies employed have continued to evolve. Recently, much clinical focus has been on combining targeted therapies with ICI for the purpose of manipulating the immune setpoint. The latter concept describes the equilibrium between factors that promote and those that suppress anti-cancer immunity. Besides tumor mutational load and other cancer cell-intrinsic determinants, the immune setpoint is also governed by the cells of the tumor microenvironment and how they are coerced by cancer cells to support the survival and growth of the tumor. These regulatory mechanisms provide therapeutic opportunities to intervene and reduce immune suppression via application of small molecule inhibitors and antibody-based therapies against (receptor) tyrosine kinases and thereby improve the response to ICIs. This article reviews how tyrosine kinase signaling in the tumor microenvironment can promote immune suppression and highlights how therapeutic strategies directed against specific tyrosine kinases can be used to lower the immune setpoint and elicit more effective anti-tumor immunity.

Summary: Tumor-derived organoids are valuable for testing anti-cancer drugs in vitro, but existing lysis-based protocols for viability measurement are laborious and restricted at a single time point. Here, we provide a lysis-free protocol for longitudinal and rapid assessment of mouse gastric tumor organoid viability and growth. We describe organoid plating, viability assessment via luminescence measurement, quantification of organoid growth by microscopy imaging, and treatment of organoids with test compounds to evaluate the effects on viability and growth at various time points. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Gene-of-interest knockout organoids present a powerful and versatile research tool to study a gene’s effects on many biological and pathological processes. Here, we present a straightforward and broadly applicable protocol to generate gene knockouts in mouse organoids using CRISPR-Cas9 technology. We describe the processes of transient transfecting organoids with pre-assembled CRISPR-Cas9 ribonucleoprotein complexes, organoid cell sorting, and establishing clonal organoid culture pairs. We then detail how to confirm the knockout via Western blot analysis. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we provide a protocol for an intrasplenic injection model to establish pancreatic tumors in the mouse liver. We describe the steps to inject tumor cells into mouse spleen and to perform a splenectomy, followed by animal recovery and end point analysis of tumors in the liver. This model allows rapid and reproducible tumor growth in a clinically relevant metastatic site, providing a platform to evaluate the efficacy of anti-cancer drugs. This technique can be expanded to other cancer cell lines.For complete details on the use and execution of this protocol, please refer to Poh et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates that can develop within or adjacent to tumors, but protocols that can accurately identify and characterize TLSs are lacking. Here, we present a protocol for the in situ interrogation and characterization of TLSs in human and murine tissue sections using Opal™-tyramide signal amplification multiplex immunohistochemistry. This protocol enables simultaneous detection of up to 7 markers (6 antigens and a DAPI counterstain). We also describe a grading system to identify immature and mature TLSs. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Specific signalling thresholds of the WNT/β-catenin pathway affect embryogenesis and tissue homeostasis in the adult, with mutations in this pathway frequently occurring in cancer. Excessive WNT/β-catenin activity inhibits murine anterior development associated with embryonic lethality and accounts for the driver event in 80% of human colorectal cancers. Uncontrolled WNT/β-catenin signalling arises primarily from impairment mutation in the tumour suppressor gene APC that otherwise prevents prolonged stabilisation of β-catenin. Surprisingly, no inhibitor compounds for WNT/β-catenin signalling have reached clinical use in part owing to the lack of specific in vivo assays that discriminate between on-target activities and dose-limiting toxicities. Here, we present a simple in vivo assay with a binary outcome whereby the administration of candidate compounds to pregnant and phenotypically normal Apcflox/flox mice can rescue in utero death of Apcmin/flox mutant conceptus without subsequent post-mortem assessment of WNT/β-catenin signalling. Indeed, the phenotypic plasticity of born Apcmin/flox conceptus enables future refinement of our assay to potentially enable dosage finding and cross-compound comparisons. Thus, we show for the first time the suitability of endogenous WNT/β-catenin signalling during embryonic development to provide an unambiguous and sensitive mammalian in vivo model to assess the efficacy and bioavailability of potential WNT/β-catenin antagonists.

The gut epithelium not only provides a physical barrier to separate a noxious outside from a sterile inside but also allows for highly regulated interactions between bacteria and their products, and components of the immune system. Homeostatic maintenance of an intact epithelial barrier is paramount to health, requiring an intricately regulated and highly adaptive response of various cells of the immune system. Prolonged homeostatic imbalance can result in chronic inflammation, tumorigenesis and inefficient antitumor immune control. Here we provide an update on the role of innate lymphoid cells, macrophages and dendritic cells, which collectively play a critical role in epithelial barrier maintenance and provide an important linkage between the classical innate and adaptive arm of the immune system. These interactions modify the capacity of the gut epithelium to undergo continuous renewal, safeguard against tumor formation and provide feedback to the gut microbiome, which acts as a seminal contributor to cellular homeostasis of the gut.

Doublecortin-like kinase 1 (DCLK1) is a functional serine/threonine (S/T)-kinase and a member of the doublecortin family of proteins which are characterized by their ability to bind to microtubules (MTs). DCLK1 is a proposed cancer driver gene, and its upregulation is associated with poor overall survival in several solid cancer types. However, how DCLK1 associates with MTs and how its kinase function contributes to pro-tumorigenic processes is poorly understood. This review builds on structural models to propose not only the specific functions of the domains but also attempts to predict the impact of individual somatic missense mutations on DCLK1 functions. Somatic missense mutations in DCLK1 are most frequently located within the N-terminal MT binding region and likely impact on the ability of DCLK1 to bind to αβ-tubulin and to polymerize and stabilize MTs. Moreover, the MT binding affinity of DCLK1 is negatively regulated by its auto-phosphorylation, and therefore mutations that affect kinase activity are predicted to indirectly alter MT dynamics. The emerging picture portrays DCLK1 as an MT-associated protein whose interactions with tubulin heterodimers and MTs are tightly controlled processes which, when disrupted, may confer pro-tumorigenic properties.

The tumor microenvironment (TME) is composed of a heterogenous population of cells that exist alongside the extracellular matrix and soluble components. These components can shape an environment that is conducive to tumor growth and metastatic spread. It is well-established that stromal cancer-associated fibroblasts (CAFs) in the TME play a pivotal role in creating and maintaining a growth-permissive environment for tumor cells. A growing body of work has uncovered that tumor cells recruit and educate CAFs to remodel the TME, however, the mechanisms by which this occurs remain incompletely understood. Recent studies suggest that the signal transducer and activator of transcription 3 (STAT3) is a key transcription factor that regulates the function of CAFs, and their crosstalk with tumor and immune cells within the TME. CAF-intrinsic STAT3 activity within the TME correlates with tumor progression, immune suppression and eventually the establishment of metastases. In this review, we will focus on the roles of STAT3 in regulating CAF function and their crosstalk with other cells constituting the TME and discuss the utility of targeting STAT3 within the TME for therapeutic benefit.

Aberrant expression of the oncoprotein c-Myc (Myc) is frequently observed in solid tumors and is associated with reduced overall survival. In addition to well-recognized cancer cell-intrinsic roles of Myc, studies have also suggested tumor-promoting roles for Myc in cells of the tumor microenvironment, including macrophages and other myeloid cells. Here, we benchmark Myc inactivation in tumor cells against the contribution of its expression in myeloid cells of murine hosts that harbor endogenous or allograft tumors. Surprisingly, we observe that LysMCre-mediated Myc ablation in host macrophages does not attenuate tumor growth regardless of immunogenicity, the cellular origin of the tumor, the site it develops, or the stage along the tumor progression cascade. Likewise, we find no evidence for Myc ablation to revert or antagonize the polarization of alternatively activated immunosuppressive macrophages. Thus, we surmise that systemic targeting of Myc activity may confer therapeutic benefits primarily through limiting Myc activity in tumor cells rather than reinvigorating the anti-tumor activity of macrophages.

Mast cells within the tumor microenvironment have controversial roles. Here, the authors show, using genetic mouse models, that in gastric cancer, mast cells at the periphery of the tumors are activated via cancer cell produced-IL33 and promote tumorigenesis by recruiting macrophages within the tumors.

Abstract Excessive signaling through gp130, the shared receptor for the interleukin (IL)6 family of cytokines, is a common hallmark in solid malignancies and promotes their progression. Here, we established the in vivo utility of bazedoxifene, a steroid analog clinically approved for the treatment of osteoporosis, to suppress gp130‐dependent tumor growth of the gastrointestinal epithelium. Bazedoxifene administration reduced gastric tumor burden in gp130Y757F mice, where tumors arise exclusively through excessive gp130/STAT3 signaling in response to the IL6 family cytokine IL11. Likewise, in mouse models of sporadic colon and intestinal cancers, which arise from oncogenic mutations in the tumor suppressor gene Apc and the associated β‐catenin/canonical WNT pathway, bazedoxifene treatment reduces tumor burden. Consistent with the proposed orthogonal tumor‐promoting activity of IL11‐dependent gp130/STAT3 signaling, tumors of bazedoxifene‐treated Apc‐mutant mice retain excessive nuclear accumulation of β‐catenin and aberrant WNT pathway activation. Likewise, bazedoxifene treatment of human colon cancer cells harboring mutant APC did not reduce aberrant canonical WNT signaling, but suppressed IL11‐dependent STAT3 signaling. Our findings provide compelling proof of concept to support the repurposing of bazedoxifene for the treatment of gastrointestinal cancers in which IL11 plays a tumor‐promoting role.

Asparagine and glutamine side-chains can form hydrogen-bonded ladders which contribute significantly to the stability of amyloid fibrils. We show, using the example of HET-s(218–289) fibrils, that the primary amide side-chain proton resonances can be detected in cross-polarization based solid-state NMR spectra at fast magic-angle spinning (MAS). J-coupling based experiments offer the possibility to distinguish them from backbone amide groups if the spin-echo lifetimes are long enough, which turned out to be the case for the glutamine side-chains, but not for the asparagine side-chains forming asparagine ladders. We explore the sensitivity of NMR observables to asparagine ladder formation. One of the two possible asparagine ladders in HET-s(218–289), the one comprising N226 and N262, is assigned by proton-detected 3D experiments at fast MAS and significant de-shielding of one of the NH2 proton resonances indicative of hydrogen-bond formation is observed. Small rotating-frame 15N relaxation-rate constants point to rigidified asparagine side-chains in this ladder. The proton resonances are homogeneously broadened which could indicate chemical exchange, but is presently not fully understood. The second asparagine ladder (N243 and N279) in contrast remains more flexible.

The Interleukin (IL-)1 family IL33 is best known for eliciting type 2 immune responses by stimulating mast cells (MCs), regulatory T-cells (Tregs), innate lymphoid cells (ILCs) and other immune cells. MCs and IL33 provide critical control of immunological and epithelial homeostasis in the gastrointestinal (GI) tract. Meanwhile, the role of MCs in solid malignancies appears tissue-specific with both pro and anti-tumorigenic activities. Likewise, IL33 signaling significantly shapes immune responses in the tumor microenvironment, but these effects remain often dichotomous when assessed in experimental models of cancer. Thus, the balance between tumor suppressing and tumor promoting activities of IL33 are highly context dependent, and most likely dictated by the mixture of cell types responding to IL33. Adding to this complexity is the promiscuous nature by which MCs respond to cytokines other than IL33 and release chemotactic factors that recruit immune cells into the tumor microenvironment. In this review, we integrate the outcomes of recent studies on the role of MCs and IL33 in cancer with our own observations in the GI tract. We propose a working model where the most abundant IL33 responsive immune cell type is likely to dictate an overall tumor-supporting or tumor suppressing outcome in vivo. We discuss how these opposing responses affect the therapeutic potential of targeting MC and IL33, and highlight the caveats and challenges facing our ability to effectively harness MCs and IL33 biology for anti-cancer immunotherapy.

Abstract Hyperpolarized silicon particles have been shown to exhibit long spin-lattice relaxation times at room temperature, making them interesting as novel MRI probes. Demonstrations of hyperpolarized silicon particle imaging have focused on large micron size particles (average particle size (APS) = 2.2 μm) as they have, to date, demonstrated much larger polarizations than nanoparticles. We show that also much smaller silicon-29 particles (APS = 55 ± 12 nm) can be hyperpolarized with superior properties. A maximum polarization of 12.6% in the solid state is reported with a spin-lattice relaxation time of 42 min at room temperature thereby opening a new window for MRI applications.

Oncolytic viruses (OV) and second mitochondrial activator of caspase (Smac)-mimetic compounds (SMC) synergistically kill cancer cells directly. Here, the authors show that SMC and OV therapies combination also synergize in vivo by promoting anticancer immunity through an increase in CD8+ T-cell response.

The gastric epithelium consists of tubular glandular units, each containing several differentiated cell types, and populations of stem cells, which enable the stomach to secrete the acid, mucus and various digestive enzymes required for its function. Very little is known about which cell signalling pathways are required for homeostasis of the gastric epithelium. Many diseases, such as cancer, arise as a result of deregulation of signalling pathways that regulate homeostasis of the diseased organ. Therefore, it is important to understand the biology of how normal conditions are maintained in a tissue to help inform the mechanisms driving disease in that same tissue, and to identify potential points of therapeutic intervention. Wnt signalling regulates several cell functions, including proliferation, differentiation and migration, and plays a crucial role during homeostasis of several tissues, including the intestinal epithelium. Wnt3a is required in the culture medium of gastric organoids, suggesting it is also important for the homeostasis of the gastric epithelium, but this has not been investigated in vivo. Here, we show that the Wnt receptor frizzled 7 (Fzd7), which is required for the homeostasis of the intestine, is expressed in the gastric epithelium and is required for gastric organoid growth. Gastric-specific loss of Fzd7 in the adult gastric epithelium of mice is deleterious and triggers rapid epithelial repopulation, which we believe is the first observation of this novel function for this tissue. Taken together, these data provide functional evidence of a crucial role for Wnt signalling, via the Fzd7 receptor, during homeostasis of the gastric epithelium.

The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5′-aza-2′-deoxycytidine’ treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not regulated by SOCS1 gene methylation or microRNA155 in these cells. In conclusion, we confirmed that IPF fibroblasts had lower levels of SOCS1 mRNA compared with control fibroblasts, but we were unable to determine the mechanism. Furthermore, although SOCS1 may be important in the fibrotic process, we were unable to find a significant role for SOCS1 in regulating fibroblast function.

The intestinal epithelium provides a barrier against commensal and pathogenic microorganisms. Barrier dysfunction promotes chronic inflammation, which can drive the pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC). Although the Signal Transducer and Activator of Transcription-3 (STAT3) is overexpressed in both intestinal epithelial cells and immune cells in IBD patients, the role of the interleukin (IL)-6 family of cytokines through the shared IL-6ST/gp130 receptor and its associated STAT3 signalling in intestinal barrier integrity is unclear. We therefore investigated the role of STAT3 in retaining epithelial barrier integrity using dextran sulfate sodium (DSS)-induced colitis in two genetically modified mouse models, to either reduce STAT1/3 activation in response to IL-6 family cytokines with a truncated gp130∆STAT allele (GP130∆STAT/+), or by inducing short hairpin-mediated knockdown of Stat3 (shStat3). Here, we show that mice with reduced STAT3 activity are highly susceptible to DSS-induced colitis. Mechanistically, the IL-6/gp130/STAT3 signalling cascade orchestrates intestinal barrier function by modulating cytokine secretion and promoting epithelial integrity to maintain a defence against bacteria. Our study also identifies a crucial role of STAT3 in controlling intestinal permeability through tight junction proteins. Thus, therapeutically targeting the IL-6/gp130/STAT3 signalling axis to promote barrier function may serve as a treatment strategy for IBD patients.

Triple-negative breast cancer (TNBC) represents 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes. Hence, new molecular targets for therapeutic intervention are necessary. Analyses of panels of human or mouse cancer lines derived from the same individual that differ in their cellular phenotypes but not in genetic background have been instrumental in defining the molecular players that drive the various hallmarks of cancer. To determine the molecular regulators of metastasis in TNBC, we completed a rigorous in vitro and in vivo characterisation of four populations of the MDA-MB-231 human breast cancer line ranging in aggressiveness from non-metastatic to spontaneously metastatic to lung, liver, spleen and lymph node. Single nucleotide polymorphism (SNP) array analyses and genome-wide mRNA expression profiles of tumour cells isolated from orthotopic mammary xenografts were compared between the four lines to define both cell autonomous pathways and genes associated with metastatic proclivity. Gene set enrichment analysis (GSEA) demonstrated an unexpected association between both ribosome biogenesis and mRNA metabolism and metastatic capacity. Differentially expressed genes or families of related genes were allocated to one of four categories, associated with either metastatic initiation (e.g. CTSC, ENG, BMP2), metastatic virulence (e.g. ADAMTS1, TIE1), metastatic suppression (e.g. CST1, CST2, CST4, CST6, SCNNA1, BMP4) or metastatic avirulence (e.g. CD74). Collectively, this model system based on MDA-MB-231 cells should be useful for the assessment of gene function in the metastatic cascade and also for the testing of novel experimental therapeutics for the treatment of TNBC. This article has an associated First Person interview with the first author of the paper.

Macrophages are a major component of the tumor microenvironment and orchestrate various aspects of immunity. Within tumors, macrophages can reversibly alter their endotype in response to environmental cues, including hypoxia and stimuli derived from other immune cells, as well as the extracellular matrix. Depending on their activation status, macrophages can exert dual influences on tumorigenesis by either antagonizing the cytotoxic activity immune cells or by enhancing antitumor responses. In most solid cancers, increased infiltration with tumor-associated macrophages (TAMs) has long been associated with poor patient prognosis, highlighting their value as potential diagnostic and prognostic biomarkers in cancer. A number of macrophage-centered approaches to anticancer therapy have been investigated, and include strategies to block their tumor-promoting activities or exploit their antitumor effector functions. Integrating therapeutic strategies to target TAMs to complement conventional therapies has yielded promising results in preclinical trials and warrants further investigation to determine its translational benefit in human cancer patients. In this review, we discuss the molecular mechanisms underlying the pro-tumorigenic programming of macrophages and provide a comprehensive update of macrophage-targeted therapies for the treatment of solid cancers.

Activation of the Wnt/β-catenin pathway occurs in the vast majority of colorectal cancers. However, the outcome of the disease varies markedly from individual to individual, even within the same tumor stage. This heterogeneity is governed to a great extent by the genetic make-up of individual tumors and the combination of oncogenic mutations. In order to express throughout the intestinal epithelium a degradation-resistant β-catenin (Ctnnb1), which lacks the first 131 amino acids, we inserted an epitope-tagged ΔN(1-131)-β-catenin-encoding cDNA as a knock-in transgene into the endogenous gpA33 gene locus in mice. The resulting gpA33ΔN-Bcat mice showed an increase in the constitutive Wnt/β-catenin pathway activation that shifts the cell fate towards the Paneth cell lineage in pre-malignant intestinal epithelium. Furthermore, 19% of all heterozygous and 37% of all homozygous gpA33ΔN-Bcat mice spontaneously developed aberrant crypt foci and adenomatous polyps, at frequencies and latencies akin to those observed in sporadic colon cancer in humans. Consistent with this, the Wnt target genes, MMP7 and Tenascin-C, which are most highly expressed in benign human adenomas and early tumor stages, were upregulated in pre-malignant tissue of gpA33ΔN-Bcat mice, but those Wnt target genes associated with excessive proliferation (i.e. Cdnn1, myc) were not. We also detected diminished expression of membrane-associated α-catenin and increased intestinal permeability in gpA33ΔN-Bcat mice in challenge conditions, providing a potential explanation for the observed mild chronic intestinal inflammation and increased susceptibility to azoxymethane and mutant Apc-dependent tumorigenesis. Collectively, our data indicate that epithelial expression of ΔN(1-131)-β-catenin in the intestine creates an inflammatory microenvironment and co-operates with other mutations in the Wnt/β-catenin pathway to facilitate and promote tumorigenesis.

The cells of the intestinal epithelium provide a selectively permeable barrier between the external environment and internal tissues. The integrity of this barrier is maintained by tight junctions, specialised cell-cell contacts that permit the absorption of water and nutrients while excluding microbes, toxins and dietary antigens. Impairment of intestinal barrier function contributes to multiple gastrointestinal disorders, including food hypersensitivity, inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Glycoprotein A33 (GPA33) is an intestinal epithelium-specific cell surface marker and member of the CTX group of transmembrane proteins. Roles in cell-cell adhesion have been demonstrated for multiple CTX family members, suggesting a similar function for GPA33 within the gastrointestinal tract. To test a potential requirement for GPA33 in intestinal barrier function, we generated Gpa33−/− mice and subjected them to experimental regimens designed to produce food hypersensitivity, colitis and CAC. Gpa33−/− mice exhibited impaired intestinal barrier function. This was shown by elevated steady-state immunosurveillance in the colonic mucosa and leakiness to oral TRITC-labelled dextran after short-term exposure to dextran sodium sulphate (DSS) to injure the intestinal epithelium. Gpa33−/− mice also exhibited rapid onset and reduced resolution of DSS-induced colitis, and a striking increase in the number of colitis-associated tumours produced by treatment with the colon-specific mutagen azoxymethane (AOM) followed by two cycles of DSS. In contrast, Gpa33−/− mice treated with AOM alone showed no increase in sporadic tumour formation, indicating that their increased tumour susceptibility is dependent on inflammatory stimuli. Finally, Gpa33−/− mice displayed hypersensitivity to food allergens, a common co-morbidity in humans with IBD. We propose that Gpa33−/− mice provide a valuable model to study the mechanisms linking intestinal permeability and multiple inflammatory pathologies. Moreover, this model could facilitate preclinical studies aimed at identifying drugs that restore barrier function.

The mammalian adult small intestinal epithelium is a rapidly self-renewing tissue that is maintained by a pool of cycling stem cells intermingled with Paneth cells at the base of crypts. These crypt base stem cells exclusively express Lgr5 and require Wnt3 or, in its absence, Wnt2b. However, the Frizzled (Fzd) receptor that transmits these Wnt signals is unknown. We determined the expression profile of Fzd receptors in Lgr5+ stem cells, their immediate daughter cells, and Paneth cells. Here we show Fzd7 is enriched in Lgr5+ stem cells and binds Wnt3 and Wnt2b. Conditional deletion of the Fzd7 gene in adult intestinal epithelium leads to stem cell loss in vivo and organoid death in vitro. Crypts of conventional Fzd7 knockout mice show decreased basal Wnt signaling and impaired capacity to regenerate the epithelium following deleterious insult. These observations indicate that Fzd7 is required for robust Wnt-dependent processes in Lgr5+ intestinal stem cells.

The extreme chemical and mechanical forces endured by the gastrointestinal tract drive a constant renewal of the epithelial lining. Stem cells of the intestine and stomach, marked by the cell surface receptor Lgr5, preserve the cellular status-quo of their respective tissues through receipt and integration of multiple cues from the surrounding niche. Wnt signalling is a critical niche component for gastrointestinal stem cells and we have previously shown that the Wnt receptor, Frizzled-7 (Fzd7), is required for gastric homeostasis and the function of Lgr5+ intestinal stem cells. Additionally, we have previously shown a requirement for the Wnt target gene Myc in intestinal homeostasis, regeneration and tumourigenesis. However, it is unknown whether Fzd7 or Myc have conserved functions in gastric Lgr5+ stem cells. Here we show that gastric Lgr5+ stem cells do not require Fzd7 or Myc and are able to maintain epithelial homeostasis, highlighting key differences in the way Wnt regulates homeostasis and Lgr5+ stem cells in the stomach compared to the intestinal epithelium. Furthermore, deletion of Myc throughout the epithelium of the gastric antrum has no deleterious effects suggesting therapeutic targeting of Myc in gastric cancer patients will be well tolerated by the surrounding normal tissue.

Inflammatory breast cancer is a rare, yet highly aggressive form of breast cancer, which accounts for less than 5% of all locally advanced presentations. The clinical presentation of inflammatory breast cancer often differs significantly from that of noninflammatory breast cancer; however, immunohistochemistry reveals few, if any, distinguishing features. The more aggressive triple-negative and HER2-positive breast cancer subtypes are overrepresented in inflammatory breast cancer compared with noninflammatory breast cancer, with a poorer prognosis in response to conventional therapies. Despite its name, there remains some controversy regarding the role of inflammation in inflammatory breast cancer. This review summarises the current molecular evidence suggesting that inflammatory signaling pathways are upregulated in this disease, including NF-κB activation and excessive IL-6 production among others, which may provide an avenue for novel therapeutics. The role of the tumor microenvironment, through tumor-associated macrophages, infiltrating lymphocytes, and cancer stem cells is also discussed, suggesting that these tumor extrinsic factors may help account for the differences in behavior between inflammatory breast cancer and noninflammatory breast cancer. While there are various novel treatment strategies already underway in clinical trials, the need for further development of preclinical models of this rare but aggressive disease is paramount.

The originally published version of this article contained an error in the spelling of the author Pankaj Tailor, which was incorrectly given as Pankaj Taylor. This has now been corrected in both the PDF and HTML versions of the article.

TRAF2 is a component of TNF superfamily signalling complexes and plays an essential role in the regulation and homeostasis of immune cells. TRAF2 deficient mice die around birth, therefore its role in adult tissues is not well-explored. Furthermore, the role of the TRAF2 RING is controversial. It has been claimed that the atypical TRAF2 RING cannot function as a ubiquitin E3 ligase but counterclaimed that TRAF2 RING requires a co-factor, sphingosine-1-phosphate, that is generated by the enzyme sphingosine kinase 1, to function as an E3 ligase. Keratinocyte-specific deletion of Traf2, but not Sphk1 deficiency, disrupted TNF mediated NF-κB and MAP kinase signalling and caused epidermal hyperplasia and psoriatic skin inflammation. This inflammation was driven by TNF, cell death, non-canonical NF-κB and the adaptive immune system, and might therefore represent a clinically relevant model of psoriasis. TRAF2 therefore has essential tissue specific functions that do not overlap with those of Sphk1.

Suppressor of cytokine signalling 3 (SOCS3) negatively regulates STAT3 activation in response to several cytokines such as those in the gp130-containing IL-6 receptor family. Thus, SOCS3 may play a major role in immune responses to pathogens. In the present study, the role of SOCS3 in M. tuberculosis infection was examined. All Socs3(fl/fl) LysM cre, Socs3(fl/fl) lck cre (with SOCS3-deficient myeloid and lymphoid cells, respectively) and gp130(F/F) mice, with a mutation in gp130 that impedes binding to SOCS3, showed increased susceptibility to infection with M. tuberculosis. SOCS3 binding to gp130 in myeloid cells conveyed resistance to M. tuberculosis infection via the regulation of IL-6/STAT3 signalling. SOCS3 was redundant for mycobacterial control by macrophages in vitro. Instead, SOCS3 expression in infected macrophages and DCs prevented the IL-6-mediated inhibition of TNF and IL-12 secretion and contributed to a timely CD4+ cell-dependent IFN-γ expression in vivo. In T cells, SOCS3 expression was essential for a gp130-independent control of infection with M. tuberculosis, but was neither required for the control of infection with attenuated M. bovis BCG nor for M. tuberculosis in BCG-vaccinated mice. Socs3(fl/fl) lck cre mice showed an increased frequency of γδ+ T cells in different organs and an enhanced secretion of IL-17 by γδ+ T cells in response to infection. Socs3(fl/fl) lck cre γδ+ T cells impaired the control of infection with M. tuberculosis. Thus, SOCS3 expression in either lymphoid or myeloid cells is essential for resistance against M. tuberculosis via discrete mechanisms.

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/beta-catenin pathway, we challenged the allele combinations by genetically restricting intracellular beta-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/beta-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/beta-catenin signaling capacity similar to that in the germline of the Apc(min) mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc(min) mice arise independently of intestinal tumorigenesis. Together, the present genotype-phenotype analysis suggests tissue-specific response levels for the Wnt/beta-catenin pathway that regulate both physiological and pathophysiological conditions.

High-resolution solid-state NMR is rapidly evolving into a tool for determining the structure of biomolecules in solid phase, thereby opening up avenues for studying systems which are difficult to access by other methods. In this contribution, we focus on assigning the resonances of a uniformly isotope-enriched peptide or protein to its primary structure. This represents a first step towards the goal of determining the complete three-dimensional structure of proteins by solid-state NMR. The assignment strategy relies on polarization-transfer experiments involving 13C and 15N spins. The prospects for structural studies based on such assignments are discussed.