IL-33 increases ST2+ Tregs and promotes metastatic tumour growth in the lungs in an amphiregulin-dependent manner.

Regulatory T cells (Tregs) facilitate primary and metastatic tumour growth through the suppression of anti-tumour immunity. Emerging evidence suggests a distinct role for Tregs in mediating tissue repair and barrier integrity in the lungs by IL-33 mediated production of the growth factor amphiregulin (AREG). Dependent on the type of cancer and local microenvironment, AREG may induce tumour cell proliferation, invasion, migration or resistance to apoptosis by signaling through the epidermal growth factor receptor (EGFR). We have found that IL-33 is dramatically increased in and around metastatic tumour foci in the lungs of mice bearing orthotopic murine mammary tumours. We observed that Tregs express significantly more of the IL-33 receptor, ST2, relative to conventional T cells, that ST2+ Tregs accumulate in the lungs of metastatic tumour-bearing mice, and that ST2+ Tregs produce significantly more AREG than ST2- Tregs. The intranasal administration of recombinant IL-33 increased the proportion of AREG producing ST2+ Tregs and enhanced the level of phosphorylated EGFR in the metastatic lungs. While recombinant AREG did not impact mammary tumour cell proliferation in vitro despite inducing a dose-dependent increase in phosphorylated EGFR, intranasal administration of AREG resulted in a ten-fold increase in pulmonary metastatic tumour burden in vivo. Further, the intranasal administration of recombinant IL-33 significantly increased metastatic tumour burden in the lungs in an amphiregulin-dependent manner. These data identify ST2+ Tregs as a microenvironmental source of AREG in the lungs of mice with orthotopic metastatic mammary tumours and highlight an important role for AREG in promoting metastatic tumour growth in the lungs.

Transgenic IGF-IR overexpression induces mammary tumors with basal-like characteristics, whereas IGF-IR-independent mammary tumors express a claudin-low gene signature.

Molecular profiling has allowed a more precise classification of human cancers. With respect to breast cancer, this approach has been used to identify five subtypes; luminal A, luminal B, HER2-enriched, basal-like and claudin-low. In addition, this approach can be used to determine the type of tumor represented by particular cell lines or transgenic animal models. Therefore, this approach was utilized to classify the mammary tumors that develop in MTB-IGFIR transgenic mice. It was determined that the primary mammary tumors, which develop due to elevated expression of the type I insulin-like growth factor receptor (IGF-IR) in mammary epithelial cells, most closely resemble murine tumors with basal-like or mixed gene expression profiles and with human basal-like breast cancers. Downregulation of IGF-IR transgene in MTB-IGFIR tumor-bearing mice leads to the regression of most of the tumors, followed by tumor reappearance in some of the mice. These tumors that reappear following IGF-IR transgene downregulation do not express the IGF-IR transgene and cluster with murine mammary tumors that express a mesenchymal gene expression profile and with human claudin-low breast cancers. Therefore, IGF-IR overexpression in murine mammary epithelial cells induces mammary tumors with primarily basal-like characteristics, whereas tumors that develop following IGF-IR downregulation express a gene signature that most closely resembles human claudin-low breast tumors.

Differential cytokine responses in human and mouse lymphatic endothelial cells to cytokines in vitro.

BACKGROUND: Inflammatory cytokines dysregulate microvascular function, yet how cytokines affect lymphatic endothelial cells (LEC) are unclear. METHODS AND RESULTS: We examined effects of TNF-α, IL-1 beta, and IFN-gamma on LEC proliferation, endothelial cell adhesion molecule (ECAM) expression, capillary formation, and barrier changes in murine (SV-LEC) and human LECs (HMEC-1a). RESULTS: All cytokines induced ICAM-1, VCAM-1, MAdCAM-1, and E-selectin in SV-LECs; TNF-α, IL-1 beta; and IFN-gamma induced ECAMs (but not MAdCAM-1) in HMEC-1a. IL-1 beta increased, while IFN-gamma and TNF-α reduced SV-LEC proliferation. While TNF-α induced, IFN-gamma decreased, and IL-1 beta did not show any effect on HMEC-1a proliferation. TNF-α, IL-1 beta, and IFN-gamma each reduced capillary formation in SV-LEC and in HMEC-1a. TNF-α and IL-1 beta reduced barrier in SV-LEC and HMEC-1a; IFN-gamma did not affect SV-LEC barrier, but enhanced HMEC-1a barrier. Inflammatory cytokines alter LEC growth, activation and barrier function in vitro and may disturb lymphatic clearance increasing tissue edema in vivo. CONCLUSION: Therapies that maintain or restore lymphatic function (including cytokines blockade), may represent important strategies for limiting inflammation.

Investigation of multidrug resistance in cultured human renal cell carcinoma cells by 31P-NMR spectroscopy and treatment survival assays.

KTCTL-26 and KTCTL-2 are renal cell carcinoma (RCC) lines with high and low expression of P-170 glycoprotein, respectively. Inherent differences between the two cell lines in terms of phosphate metabolites and growth characteristics in culture were examined for possible association with multidrug resistance (MDR). Differences in response to drug treatment were investigated for 40 h incubations with various doses of vinblastine (VBL) alone or as cotreatments with various concentrations of the calcium antagonist diltiazem (DIL) and/or interferon-alpha (IFN-alpha). Treatment effects were quantitated using the MTT survival assay and 31P magnetic resonance spectroscopy (MRS) to determine phosphate metabolite profiles in intact cells. KTCTL-2 and KTCTL-26 cells exhibited significant inherent differences in phosphocholine, glycerophosphocholine, glycerophosphoethanolamine, and phosphocreatine levels. KTCTL-26 cells were more sensitive than KTCTL-2 to 0.011 mircroM VBL alone (87% vs. 102% survival) or to 0.011 microM BL + 10 microM DIL (55% vs. 80% survival). The latter treatment resulted in a significant decrease in the ratio of phosphocholine to glycerophosphocholine in KTCTL-26 cells but no significant changes in phosphate metabolites in KTCTL-2 cells. Metabolomic 31P MRS detects different metabolite profiles for RCC cell lines with different MDR phenotypes and may be useful for noninvasive characterization of tumors in a clinical setting.

31P MRS of human tumor cells: effects of culture media and conditions on phospholipid metabolite concentrations.

31P magnetic resonance spectroscopy has been used to determine phosphate metabolite profiles in five human tumor cell lines in culture and as solid tumor xenografts in nude mice. Significant differences between cell lines, in particular in their phospholipid metabolite levels, were observed. The largest differences between metabolite profiles in vivo and in culture were observed for cell lines which exhibit low phosphoethanolamine levels in culture. One of these lines, the colon carcinoma CX-1, was studied in more detail in both incubated and perfused DMEM cultures with variation of the concentrations of glucose, choline and ethanolamine. Highly significant alterations of phospholipid metabolite concentrations and UDP-hexoses (primarily UDP-GlcNAc and UDP-GalNAc) were observed as a function of the precursor concentrations, culture time or perfusion time. A strong interaction between phospholipid metabolic pathways and UDP-hexose pathways could be demonstrated.