Maximilian J. Waldner

Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis

Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn’s disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohn’s disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8ΔIEC) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8ΔIEC mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8ΔIEC mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-α, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn’s disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IECs from TNF-α-induced necroptotic cell death.

In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn's disease

As antibodies to tumor necrosis factor (TNF) suppress immune responses in Crohns disease by binding to membrane-bound TNF (mTNF), we created a fluorescent antibody for molecular mTNF imaging in this disease. Topical antibody administration in 25 patients with Crohns disease led to detection of intestinal mTNF+ immune cells during confocal laser endomicroscopy. Patients with high numbers of mTNF+ cells showed significantly higher short-term response rates (92%) at week 12 upon subsequent anti-TNF therapy as compared to patients with low amounts of mTNF+ cells (15%). This clinical response in the former patients was sustained over a follow-up period of 1 year and was associated with mucosal healing observed in follow-up endoscopy. These data indicate that molecular imaging with fluorescent antibodies has the potential to predict therapeutic responses to biological treatment and can be used for personalized medicine in Crohns disease and autoimmune or inflammatory disorders.

In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor.

BACKGROUND & AIMS Epidermal growth factor receptor (EGFR) is a therapeutic target for colorectal cancer (CRC). However, technical challenges have limited in vivo imaging of EGFR in CRCs. Confocal laser endomicroscopy (CLE) enables accurate microscopic visualization of CRC in patients during endoscopy. We evaluated the ability to use CLE in vivo for instantaneous molecular imaging of EGFR in CRC models. METHODS Tumors were grown in mice (n = 68) from human CRC cell lines that expressed high (SW480 cells) or low (SW620 cells) levels of EGFR. Tumors were visualized in vivo with a handheld CLE probe after injection of fluorescently labeled EGFR antibodies. EGFR-specific fluorescence was graded from 0 to 3+. Neoplastic and non-neoplastic specimens from human colorectal mucosa were examined. In vivo findings were correlated with histopathology, immunohistochemistry, and fluorescence microscopy analyses. RESULTS CLE analysis of cell cultures confirmed the different expression levels of EGFR between cell lines. In living animals, CLE differentiated EGFR expression levels between tumor cell limes (mean fluorescence, 1.92 +/- 0.22 [SW480] and 0.59 +/- 0.21 [SW620], P = .0004). CLE analysis of EGFR expression in human specimens allowed distinction of neoplastic from non-neoplastic tissues (mean fluorescence, 2.0 +/- 0.37 vs 0.25 +/- 0.16, respectively, P = .0035). CONCLUSIONS CLE can be used for in vivo, molecular analysis of CRC and to differentiate EGFR expression patterns in xenograft tumors and human tissue samples. Because CLE can be performed during endoscopy, in vivo molecular imaging might be used in diagnosis of CRC and to predict response to targeted therapies.

Interleukin-6 - A Key Regulator of Colorectal Cancer Development

Growing evidence proposes an important role for pro-inflammatory cytokines during tumor development. Several experimental and clinical studies have linked the pleiotropic cytokine interleukin-6 (IL-6) to the pathogenesis of sporadic and inflammation-associated colorectal cancer (CRC). Increased IL-6 expression has been related to advanced stage of disease and decreased survival in CRC patients. According to experimental studies, these effects are mediated through IL-6 trans-signaling promoting tumor cell proliferation and inhibiting apoptosis through gp130 activation on tumor cells with subsequent signaling through Janus kinases (JAKs) and signal transducer and activator of transcription 3 (STAT3). During recent years, several therapeutics targeting the IL-6/STAT3 pathway have been developed and pose a promising strategy for the treatment of CRC. This review discusses the molecular mechanisms and possible therapeutic targets involved in IL-6 signaling in CRC.

Characterization of the immunophenotypes and antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy

BackgroundWhile large-scale cancer genomic projects are comprehensively characterizing the mutational spectrum of various cancers, so far little attention has been devoted to either define the antigenicity of these mutations or to characterize the immune responses they elicit. Here we present a strategy to characterize the immunophenotypes and the antigen-ome of human colorectal cancer.ResultsWe apply our strategy to a large colorectal cancer cohort (n = 598) and show that subpopulations of tumor-infiltrating lymphocytes are associated with distinct molecular phenotypes. The characterization of the antigenome shows that a large number of cancer-germline antigens are expressed in all patients. In contrast, neo-antigens are rarely shared between patients, indicating that cancer vaccination requires individualized strategy. Analysis of the genetic basis of the tumors reveals distinct tumor escape mechanisms for the patient subgroups. Hypermutated tumors are depleted of immunosuppressive cells and show upregulation of immunoinhibitory molecules. Non-hypermutated tumors are enriched with immunosuppressive cells, and the expression of immunoinhibitors and MHC molecules is downregulated. Reconstruction of the interaction network of tumor-infiltrating lymphocytes and immunomodulatory molecules followed by a validation with 11 independent cohorts (n = 1,945) identifies BCMA as a novel druggable target. Finally, linear regression modeling identifies major determinants of tumor immunogenicity, which include well-characterized modulators as well as a novel candidate, CCR8, which is then tested in an orthologous immunodeficient mouse model.ConclusionsThe immunophenotypes of the tumors and the cancer antigenome remain widely unexplored, and our findings represent a step toward the development of personalized cancer immunotherapies.

Antibodies Against Tumor Necrosis Factor (TNF) Induce T-Cell Apoptosis in Patients With Inflammatory Bowel Diseases via TNF Receptor 2 and Intestinal CD14+ Macrophages

BACKGROUND & AIMS The anti-tumor necrosis factor (TNF) antibodies infliximab, adalimumab, and certolizumab pegol have proven clinical efficacy in Crohns disease. Here, we assessed the effects of anti-TNF antibodies on apoptosis in inflammatory bowel disease (IBD). METHODS CD14(+) macrophages and CD4(+) T cells were isolated from peripheral blood and lamina propria mononuclear cells from patients with IBD and control patients. Cell surface markers and apoptosis were assessed by immunohistology and fluorescence-activated cell sorting techniques. RESULTS Lamina propria CD14(+) macrophages showed significantly more frequent and higher membrane-bound TNF (mTNF) expression than CD4(+) T cells in IBD, whereas mTNF-dependent signaling proteins such as TNF receptor (TNFR) 2, TNFR-associated factor (TRAF) 2, and nuclear factor κB were induced in IBD mucosal CD4(+) T cells. Most anti-TNF antibodies did not induce T-cell apoptosis in purified peripheral or mucosal CD4(+) T cells. However, in contrast to etanercept, administration of all clinically effective anti-TNF antibodies resulted in a significant induction of T-cell apoptosis in IBD when lamina propria CD4(+) T cells expressing TNFR2(+) were cocultured with mTNF(+) CD14(+) intestinal macrophages. In contrast, no effects in control patients were noted. T-cell apoptosis in IBD occurred in vivo after treatment with adalimumab and infliximab, was critically dependent on TNFR2 signaling, and could be prevented via interleukin-6 signal transduction. Blockade of interleukin-6R signaling augmented anti-TNF-induced T-cell apoptosis in IBD. CONCLUSIONS Clinically effective anti-TNF antibodies are able to induce T-cell apoptosis in IBD only when mucosal TNFR2(+) T cells are cocultured with mTNF-expressing CD14(+) macrophages. The finding that anti-TNF antibodies induce apoptosis indirectly by targeting the mTNF/TNFR2 pathway may have important implications for the development of new therapeutic strategies in IBD.

The tumor microenvironment and Immunoscore are critical determinants of dissemination to distant metastasis

Local lymphatic vessel density and immune cytotoxicity prevent metastatic invasion. Managing metastasis Because of the poor prognosis of metastatic cancer, it is critical to determine exactly how different factors contribute to cancer spread. Mlecnik et al. examined the impact of tumor-intrinsic, microenvironmental, and immunological factors on tumor metastasis in colorectal cancer patients. They found that decreased presence of lymphatic vessels and reduced immune cytotoxicity were more strongly associated with the metastatic process than tumor-intrinsic factors such as chromosomal instability or cancer-associated mutations. These data support testing the Immunoscore as a biomarker to predict metastasis and guide therapy. Although distant metastases account for most of the deaths in cancer patients, fundamental questions regarding mechanisms that promote or inhibit metastasis remain unanswered. We show the impact of mutations, genomic instability, lymphatic and blood vascularization, and the immune contexture of the tumor microenvironment on synchronous metastases in large cohorts of colorectal cancer patients. We observed large genetic heterogeneity among primary tumors, but no major differences in chromosomal instability or key cancer-associated mutations. Similar patterns of cancer-related gene expression levels were observed between patients. No cancer-associated genes or pathways were associated with M stage. Instead, mutations of FBXW7 were associated with the absence of metastasis and correlated with increased expression of T cell proliferation and antigen presentation functions. Analyzing the tumor microenvironment, we observed two hallmarks of the metastatic process: decreased presence of lymphatic vessels and reduced immune cytotoxicity. These events could be the initiating factors driving both synchronous and metachronous metastases. Our data demonstrate the protective impact of the Immunoscore, a cytotoxic immune signature, and increased marginal lymphatic vessels, against the generation of distant metastases, regardless of genomic instability.

VEGF receptor signaling links inflammation and tumorigenesis in colitis-associated cancer

Inflammation drives expression of VEGFR2, which is expressed on and drives growth of tumor cells in colitis-associated cancer.

Functional Network Pipeline Reveals Genetic Determinants Associated with in Situ Lymphocyte Proliferation and Survival of Cancer Patients

Genomic alterations result in changes of immune cell densities within the tumor. There Goes the Neighborhood Just as a new homeowner will remodel when moving into a house, a tumor also alters the local microenvironment. One of the least tumor-friendly things in the tumor microenvironment is the immune response. Mlecnik et al. now examine the network of changes to cytokines that contribute to shaping the intratumoral immune response in colorectal tumors. The authors found changes in local expression of 13 cytokines. Deletion of one cytokine, interleukin 15, was associated with tumor recurrence and reduced patient survival, potentially by affecting the local proliferation of B and T cells. Their results show that chromosome instability contributes to the regulation of cytokines and downstream tumor immune response. The tumor microenvironment is host to a complex network of cytokines that contribute to shaping the intratumoral immune reaction. Chromosomal gains and losses, coupled with expression analysis, of 59 cytokines and receptors and their functional networks were investigated in colorectal cancers. Changes in local expression for 13 cytokines were shown. Metastatic patients exhibited an increased frequency of deletions of cytokines from chromosome 4. Interleukin 15 (IL15) deletion corresponded with decreased IL15 expression, a higher risk of tumor recurrence, and reduced patient survival. Decreased IL15 expression affected the local proliferation of B and T lymphocytes. Patients with proliferating B and T cells at the invasive margin and within the tumor center had significantly prolonged disease-free survival. These results delineate chromosomal instability as a mechanism of modulating local cytokine expression in human tumors and underline the major role of IL15. Our data provide further mechanisms resulting in changes of specific immune cell densities within the tumor, and the importance of local active lymphocyte proliferation for patient survival.

Colitis-associated cancer: the role of T cells in tumor development

Chronic inflammation severely increases the risk for cancer development as seen in patients with inflammatory bowel disease (IBD). Although the exact mechanisms of inflammation-associated tumor development remain to be shown, a role for the adaptive immune system has been implicated in colitis-associated cancer (CAC). In fact, CD4+ effector T cells, which promote chronic inflammation in IBD, create a tumor convenient environment, which can lead to cancer initiation, promotion, and progression. Thereby, the cytokines interleukin-6 and tumor necrosis factor-α constitute an important link between inflammation and tumor growth. Furthermore, cytotoxic CD8+ T cells, which usually are protective as part of the host antitumor immune response in sporadic cancer, can contribute to the aggravation of chronic inflammation and thereby support tumor development. In contrast, regulatory T cells, which have been shown to attenuate tumor immunosurveillance, act as potent suppressors of chronic inflammation and thus can have protective effects in CAC. This review discusses the role of the adaptive immune response and especially T cells in the pathogenesis CAC and possible implications for the therapeutic applications.