Background & AimsInterleukin (IL)33 is a recently described alarmin that is highly expressed in the gastric mucosa and potently activates Th2 immunity. It may play a pivotal role during Helicobacter pylori infection. Here, we delineate the role of IL33 in the normal gastric mucosa and in response to gastropathy.MethodsIL33 expression was evaluated in mice and human biopsy specimens infected with H pylori and in mice after dosing with aspirin. IL33 expression was localized in the gastric mucosa using immunofluorescence. Mice were given 1 or 7 daily doses of recombinant IL33 (1 μg/dose), and the stomach and the spleen responses were quantified morphologically, by flow cytometry and using quantitative reverse-transcription polymerase chain reaction and immunoblotting.ResultsIn mice, the IL33 protein was localized to the nucleus of a subpopulation of surface mucus cells, and co-localized with the surface mucus cell markers Ulex Europaeus 1 (UEA1), and Mucin 5AC (Muc5AC). A small proportion of IL33-positive epithelial cells also were Ki-67 positive. IL33 and its receptor Interleukin 1 receptor-like 1 (ST2) were increased 4-fold after acute (1-day) H pylori infection, however, this increase was not apparent after 7 days and IL33 expression was reduced 2-fold after 2 months. Similarly, human biopsy specimens positive for H pylori had a reduced IL33 expression. Chronic IL33 treatment in mice caused systemic activation of innate lymphoid cell 2 and polarization of macrophages to the M2 phenotype. In the stomach, IL33-treated mice developed transmural inflammation and mucous metaplasia that was mediated by Th2/signal transducer and activator of transcription 3 signaling. Rag-1-/- mice, lacking mature lymphocytes, were protected from IL33-induced gastric pathology.ConclusionsIL33 is highly expressed in the gastric mucosa and promotes the activation of T helper 2–cytokine–expressing cells. The loss of IL33 expression after prolonged H pylori infection may be permissive for the T helper 1–biased immune response observed during H pylori infection and subsequent precancerous progression.

Here, we show, using FGF2 as an example, that identification of factors responsible for the protumoral effects of infiltrating myeloid cells can be used to target established liver metastases. Such therapies could be utilized to limit disease progression and potentiate the effects of standard ablative therapies. (Hepatology 2017;65:1920-1935).

Lung cancer remains the leading cause of cancer‐related death due to poor treatment responses and resistance arising from tumour heterogeneity. Here, we show that adverse prognosis associated with epigenetic silencing of the tumour suppressor RASSF1A is due to increased deposition of extracellular matrix (ECM), tumour stiffness and metastatic dissemination in vitro and in vivo. We find that lung cancer cells with RASSF1A promoter methylation display constitutive nuclear YAP1 accumulation and expression of prolyl 4‐hydroxylase alpha‐2 (P4HA2) which increases collagen deposition. Furthermore, we identify that elevated collagen creates a stiff ECM which in turn triggers cancer stem‐like programming and metastatic dissemination in vivo. Re‐expression of RASSF1A or inhibition of P4HA2 activity reverses these effects and increases markers of lung differentiation (TTF‐1 and Mucin 5B). Our study identifies RASSF1A as a clinical biomarker associated with mechanical properties of ECM which increases the levels of cancer stemness and risk of metastatic progression in lung adenocarcinoma. Moreover, we highlight P4HA2 as a potential target for uncoupling ECM signals that support cancer stemness.

Regulation of the programming of tumour-associated macrophages (TAMs) controls tumour growth and anti-tumour immunity. We examined the role of FGF2 in that regulation. Tumours in mice genetically deficient in low-molecular weight FGF2 (FGF2 LMW) regress dependent on T cells. Yet, TAMS not T cells express FGF receptors. Bone marrow derived-macrophages from Fgf2 LMW−/− mice co-injected with cancer cells reduce tumour growth and express more inflammatory cytokines. FGF2 is induced in the tumour microenvironment following fractionated radiation in murine tumours consistent with clinical reports. Combination treatment of in vivo tumours with fractionated radiation and a blocking antibody to FGF2 prolongs tumour growth delay, increases long-term survival and leads to a higher iNOS + /CD206 + TAM ratio compared to irradiation alone. These studies show for the first time that FGF2 affects macrophage programming and is a critical regulator of immunity in the tumour microenvironment.

Emerging evidence suggests a role for radiation in eliciting anti‐tumour immunity. We aimed to investigate the role of macrophages in modulating the immune response to radiation. Irradiation to murine tumours generated from colorectal (MC38) and pancreatic (KPC) cell lines induced colony‐stimulating factor 1 (CSF‐1). Coincident with the elevation in CSF‐1, macrophages increased in tumours, peaking 5 days following irradiation. These tumour‐associated macrophages (TAMs) were skewed towards an immunosuppressive phenotype. Macrophage depletion via anti‐CSF (aCSF) reduced macrophage numbers, yet only achieved tumour growth delay when combined with radiation. The tumour growth delay from aCSF after radiation was abrogated by depletion of CD8 T cells. There was enhanced recognition of tumour cell antigens by T cells isolated from irradiated tumours, consistent with increased antigen priming. The addition of anti‐PD‐L1 (aPD‐L1) resulted in improved tumour suppression and even regression in some tumours. In summary, we show that adaptive immunity induced by radiation is limited by the recruitment of highly immunosuppressive macrophages. Macrophage depletion partly reduced immunosuppression, but additional treatment with anti‐PD‐L1 was required to achieve tumour regression.

We tested whether the T helper (Th) type 2 (Th2) cell agonist and allergenic ligand IL-33 was associated with eosinophilic esophagitis (EoE) development in a pediatric cohort and whether IL-33 protein could induce disease symptoms in mice. Biopsies from EoE patients or controls were used to measure IL-33 mRNA and protein expression. Increased expression of IL-33 mRNA was found in the esophageal mucosa in EoE. IL-33 protein was detected in cells negative for CD45, mast cells, and epithelial cell markers near blood vessels. Circulating levels of IL-33 were not increased. The time course for IL-33 gene expression was quantified in an established Aspergillus fumigatus allergen mouse model of EoE. Because IL-33 induction was transient in this model and chronicity of IL-33 expression has been demonstrated in humans, naive mice were treated with recombinant IL-33 for 1 wk and esophageal pathology was evaluated. IL-33 application produced changes consistent with phenotypically early EoE, including transmural eosinophilia, mucosal hyperproliferation, and upregulation of eosinophilic genes and chemokines. Th2 cytokines, including IL-13, along with innate lymphoid cell group 2, Th1/17, and M2 macrophage marker genes, were increased after IL-33 application. IL-33-induced eosinophilia was ablated in IL-13 null mice. In addition, IL-33 induced a profound inhibition of the regulatory T cell gene signature. We conclude that IL-33 gene expression is associated with pediatric EoE development and that application of recombinant protein in mice phenocopies the early clinical phase of the human disease in an IL-13-dependent manner. IL-33 inhibition of esophageal regulatory T cell function may induce loss of antigenic tolerance, thereby providing a mechanistic rationale for EoE development.

BackgroundColorectal cancer (CRC) liver metastasis is highly unfavorable for patient outcome and is a leading cause of cancer-related death. Pre-clinical research of CRC liver metastasis predominately utilizes CRC cell lines grown in tissue culture. Here, we demonstrate that CRC liver metastases organoids derived from human specimens recapitulate some aspects of human disease.MethodsHuman CRC liver metastases pathological specimens were obtained following patient consent. Tumor disaggregates were plated and organoids were allowed to expand. CRC markers were identified by immunofluorescence. Stem cell genes were analysed by QPCR and flow cytometry. Response to drug therapy was quantified using time-lapse imaging and MATLAB analysis.ResultsOrganoids showed global expression of the epithelial marker, EpCAM and the adenocarcinoma marker, CEA CAM1. Flow cytometry analysis demonstrated that organoids express the stem cell surface markers CD24 and CD44. Finally, we demonstrated that CRC liver metastases organoids acquire chemotherapy resistance and can be utilized as surrogates for drug testing.ConclusionThese data demonstrate that CRC liver metastases organoids recapitulate some aspects of human disease and may provide an invaluable resource for investigating novel drug therapies, chemotherapy resistance and mechanism of metastasis.

Chemotherapy-resistant dormant colorectal cancer cells are a differentiated cell that can de-differentiate and proliferate, accounting for tumor recurrence. Itraconazole induces all tumor cells, including those dormant, to proliferate then enter senescence and is effective in multiple preclinical assays.