Doctor of Philosophy, Universitat Ulm (2010)
Immunobiology of colon cancer.
Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.
View details for DOI 10.1126/science.1259425
View details for PubMedID 26160952
Whereas cancers grow within host tissues and evade host immunity through immune-editing and immunosuppression, tumours are rarely transmissible between individuals. Much like transplanted allogeneic organs, allogeneic tumours are reliably rejected by host T cells, even when the tumour and host share the same major histocompatibility complex alleles, the most potent determinants of transplant rejection. How such tumour-eradicating immunity is initiated remains unknown, although elucidating this process could provide the basis for inducing similar responses against naturally arising tumours. Here we find that allogeneic tumour rejection is initiated in mice by naturally occurring tumour-binding IgG antibodies, which enable dendritic cells (DCs) to internalize tumour antigens and subsequently activate tumour-reactive T cells. We exploited this mechanism to treat autologous and autochthonous tumours successfully. Either systemic administration of DCs loaded with allogeneic-IgG-coated tumour cells or intratumoral injection of allogeneic IgG in combination with DC stimuli induced potent T-cell-mediated antitumour immune responses, resulting in tumour eradication in mouse models of melanoma, pancreas, lung and breast cancer. Moreover, this strategy led to eradication of distant tumours and metastases, as well as the injected primary tumours. To assess the clinical relevance of these findings, we studied antibodies and cells from patients with lung cancer. T cells from these patients responded vigorously to autologous tumour antigens after culture with allogeneic-IgG-loaded DCs, recapitulating our findings in mice. These results reveal that tumour-binding allogeneic IgG can induce powerful antitumour immunity that can be exploited for cancer immunotherapy.
View details for DOI 10.1038/nature14424
View details for PubMedID 25924063
Early detection of colonic polyps can prevent up to 90% of colorectal cancer deaths. Conventional colonoscopy readily detects the majority of premalignant lesions, which exhibit raised morphology. However, lesions that are flat and depressed are often undetected using this method. Therefore, there is a need for molecular-based contrast agents to improve detection rates over conventional colonoscopy. We evaluated a quenched fluorescent activity-based probe (qABP; BMV109) that targets multiple cysteine cathepsins that are overexpressed in intestinal dysplasia in a genetic model of spontaneous intestinal polyp formation and in a chemically induced model of colorectal carcinoma. We found that the qABP selectively targets cysteine cathepsins, resulting in high sensitivity and specificity for intestinal tumors in mice and humans. Additionally, the qABP can be administered by either intravenous injection or by local delivery to the colon, making it a highly valuable tool for improved detection of colorectal lesions using fluorescence-guided colonoscopy.
View details for DOI 10.1016/j.chembiol.2014.11.008
View details for Web of Science ID 000348508400017
View details for PubMedID 25579207
Chronic lymphocytic leukemia (CLL) cells fail to enter apoptosis in vivo as opposed to their non-malignant B-lymphocyte counterparts. The ability of CLL cells to escape apoptosis is highly dependent on their microenvironment. Compared to non-malignant B cells, CLL cells are more responsive to complex stimuli that can be reproduced in vitro by the addition of cytokines. To understand the molecular mechanism of the environment-dependent anti-apoptotic signaling circuitry of CLL cells, we quantified the effect of the SDF-1, BAFF, APRIL, anti-IgM, interleukin-4 (IL4) and secreted CD40L (sCD40L) on the survival of in vitro cultured CLL cells and found IL4 and sCD40L to be most efficient in rescuing CLL cells from apoptosis. In quantitative dose-response experiments using cell survival as readout, the binding affinity of IL4 to its receptor was similar between malignant and non-malignant cells. However, the downstream signaling in terms of the amount of STAT6 and its degree of phosphorylation was highly stimulated in CLL cells. In contrast, the response to sCD40L showed a loss of cooperative binding in CLL cells but displayed a largely increased ligand binding affinity. Although a high-throughput microscopy analysis did not reveal a significant difference in the spatial CD40 receptor organization, the downstream signaling showed an enhanced activation of the NF-kB pathway in the malignant cells. Thus, we propose that the anti-apoptotic phenotype of CLL involves a sensitized response for IL4 dependent STAT6 phosphorylation, and an activation of NF-kB signaling due to an increased affinity of sCD40L to its receptor.
View details for DOI 10.1002/ijc.28974
View details for PubMedID 24828787
Talactoferrin alfa (TLF) is a unique recombinant form of human lactoferrin. The hypothesized mechanism of action involves TLF binding to the intestinal endothelium inducing dendritic cell maturation and cytokine release leading to infiltration of tumor with monocytes and T-lymphocytes and inhibition of tumor growth.Based on promising phase II trial results, this correlative study was undertaken to examine immune mechanism of action of TLF in metastatic non-small cell lung cancer (NSCLC) patients.Talactoferrin was administered orally at 1.5 g bid weeks 1-12 with 2 weeks off on a 14-week cycle. Enrolled patients had a pathologic diagnosis of NSCLC previously treated with at least two lines of systemic treatment. Patients had core biopsy of tumor before initiation of talactoferrin and at week 7 on TLF. Flow cytometry and quantitative immunohistochemistry for immune correlates were performed on the biopsied specimens.Four patients with metastatic NSCLC were enrolled. The trial was halted pre-maturely in light of negative phase III trial results. For the two patients who had repeat on-treatment tumor biopsies, a consistent increase in monocytes as a percentage of total immune cells was observed. Otherwise, no clear trend of increase or decrease was observed in any other immune cell parameters compared to matched patient pre-treatment biopsies.Repeat biopsies for immune correlates by flow cytometry and quantitative immunohistochemistry in NSCLC patients are feasible. In the few patients sampled before trial closure, increased monocytes as a total percentage of the immune cell population within tumor was observed in response to TLF.
View details for DOI 10.3109/08923973.2013.864671
View details for Web of Science ID 000336735300013
Cancer-mediated immune dysfunction contributes to tumor progression and correlates with patient outcome. Metastasis to tumor draining lymph nodes (TDLNs) is an important step in breast cancer progression and is used to predict patient outcome and survival. Although lymph nodes are important immune organs, the role of immune cells in TDLNs has not been thoroughly investigated. We hypothesized that the host immune response in node negative (NN) patients is more intact and thereby can resist tumor invasion compared to node positive (NP) patients. As such, lymph node metastasis requires breakdown of the host immune response in addition to escape of cancer cells from the tumor. To investigate the immunological differences between NN and NP breast cancer patients, we purified and profiled immune cells from the three major compartments where cancer and immune cells interact: tumor, TDLNs and peripheral blood. Significant down-regulation of genes associated with immune-related pathways and up-regulation of genes associated with tumor-promoting pathways was consistently observed in NP patients' TDLNs compared to NN patients. Importantly, these signatures were seen even in NP patients' tumor-free TDLNs, suggesting that such immune changes are not driven solely by local tumor invasion. Furthermore, similar patterns were also observed in NP patients' tumor and blood immune cells, suggesting that immunological differences between NN and NP patients are systemic. Together, these findings suggest that alterations in overall immune function may underlie risk for LN metastasis in breast cancer patients.
View details for DOI 10.1002/ijc.27933
View details for Web of Science ID 000316824000009
Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA-mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.
View details for DOI 10.1371/journal.pgen.1003373
View details for PubMedID 23593011
Chronic lymphocytic leukemia is characterized by the accumulation of B cells that are resistant to apoptosis. This resistance is induced by pro-survival stimuli from the microenvironment. TCL1 and ATM are central to the pathogenesis of the disease and associated with more aggressive disease. Their protein products have recently been shown to physically interact in leukemic cells and to impact on NF-?B signaling, which is a key regulator of apoptosis. In the present study we show that TCL1 and ATM are significantly co-expressed and up-regulated in malignant cells compared to non-malignant B cells, and that expression of TCL1 is partially deregulated by aberrant DNA-methylation. In addition, complex external stimuli induce essentially similar TCL1 and ATM time-course kinetics. In line with a coordinative regulation of NF-?B signaling by TCL1, its knockdown induced apoptosis in primary leukemia cells. These findings suggest that both genes functionally cooperate to modulate similar apoptosis-related cellular pathways.
View details for DOI 10.3324/haematol.2012.070623
View details for Web of Science ID 000315415800021
View details for PubMedID 22875623
Dendritic cells (DCs) are important mediators of anti-tumor immune responses. We hypothesized that an in-depth analysis of dendritic cells and their spatial relationships to each other as well as to other immune cells within tumor draining lymph nodes (TDLNs) could provide a better understanding of immune function and dysregulation in cancer.We analyzed immune cells within TDLNs from 59 breast cancer patients with at least 5 years of clinical follow-up using immunohistochemical staining with a novel quantitative image analysis system. We developed algorithms to analyze spatial distribution patterns of immune cells in cancer versus healthy intra-mammary lymph nodes (HLNs) to derive information about possible mechanisms underlying immune-dysregulation in breast cancer. We used the non-parametric Mann-Whitney test for inter-group comparisons, Wilcoxon Matched-Pairs Signed Ranks test for intra-group comparisons and log-rank (Mantel-Cox) test for Kaplan Maier analyses.Degree of clustering of DCs (in terms of spatial proximity of the cells to each other) was reduced in TDLNs compared to HLNs. While there were more numerous DC clusters in TDLNs compared to HLNs,DC clusters within TDLNs tended to have fewer member DCs and also consisted of fewer cells displaying the DC maturity marker CD83. The average number of T cells within a standardized radius of a clustered DC was increased compared to that of an unclustered DC, suggesting that DC clustering was associated with T cell interaction. Furthermore, the number of T cells within the radius of a clustered DC was reduced in tumor-positive TDLNs compared to HLNs. Importantly, clinical outcome analysis revealed that DC clustering in tumor-positive TDLNs correlated with the duration of disease-free survival in breast cancer patients.These findings are the first to describe the spatial organization of DCs within TDLNs and their association with survival outcome. In addition, we characterized specific changes in number, size, maturity, and T cell co-localization of such clusters. Strategies to enhance DC function in-vivo, including maturation and clustering, may provide additional tools for developing more efficacious DC cancer vaccines.
View details for DOI 10.1186/1479-5876-11-242
View details for PubMedID 24088396
The discovery of immune cells with regulatory effects has created considerable excitement for their potential use in inducing tolerance to transplanted tissues. Despite the fact that these cells possess essential functions in vivo, attempts to translate them into effective clinical therapies has proved challenging due to a number of unanticipated complexities in their behavior. This article provides a broad summary of research done to understand the largest of the regulatory cell subtypes, namely CD4+Foxp3+ Regulatory T cells (T(Regs)). Special attention will be paid to current and future difficulties in using T(Regs) clinically, as well as room for improvement and innovation in this field.
View details for PubMedID 22180672
Chronic lymphocytic leukaemia (CLL) cells convert CD14(+) cells from patients into 'nurse-like' cells (NLCs). CLL cells can also convert CD14(+) peripheral blood mononuclear cells (PBMCs) from healthy donors into cells with morphological similarities to NLCs (CD14(CLL) -cells). However it is unclear whether only CLL cells induce this conversion process. This study showed that CD14(+) PBMCs from healthy donors could also be converted into differentiated cells (CD14(B) -cells) by non-malignant B-cells. In order to identify changes specifically induced by CLL cells, we compared gene expression profiles of NLCs, CD14(CLL) -cells and CD14(B) -cells. CD14(+) cells cultured with CLL cells were more similar to NLCs than those cultured with non-malignant B-cells. The most significant changes induced by CLL cells were deregulation of the antigen presentation pathway and of genes related to immunity. NLCs had reduced levels of lysozyme activity, CD74 and HLA-DR in-vitro while expression of inhibitory FCGR2B was increased. These findings suggest an impaired immunocompetence of NLCs which, if found in-vivo, could contribute to the immunodeficiency in CLL patients.
View details for DOI 10.1111/j.1365-2141.2011.08747.x
View details for Web of Science ID 000292648000008
View details for PubMedID 21615384
Immune modulatory drugs have been successfully used to treat patients with multiple myeloma (MM), myelodysplastic syndromes displaying loss of 5q (del5q MDS) and chronic lymphocytic leukemia (CLL). Immune modulatory drugs are used in first-line therapy in combination with functionally complementary compounds, but have also shown efficacy in refractory disease. However, their exact mode of action remains unclear. Here we describe the clinical impact of these compounds on MM, del5q MDS and CLL, discuss their mode of action with respect to intracellular targets, focus on the phenotypic changes that immune modulatory compounds induce in the tumor microenvironment and how they modulate the immune response.
View details for DOI 10.1016/j.ctrv.2011.05.004
View details for Web of Science ID 000295065200002
View details for PubMedID 21645972
Contemporary research on cellular signaling has undergone a shift of focus from qualitative measurements of single signaling pathways to high-throughput quantitation of comprehensive signaling networks. Notably, nuclear factor-kappaB (NFkappaB) is a family of transcription factors involved in immune and inflammatory responses, developmental processes, cellular growth and apoptosis and is deregulated in a number of disease states. We have established a chemiluminescent oligonucleotide-based enzyme-linked immunosorbent assay (co-ELISA) that is simple and quantitative. In contrast to currently used assays, it allows quantitation of all NFkappaB components (i.e., RelA, p50, p52, RelB and c-Rel). In addition, it can make use of whole extract and does not require cumbersome nuclear/cytosolic fractionation, saving time and resources. Co-ELISA has a 3.5- to 43-fold higher signal-over-noise ratio than currently available assays, whereas the percent relative standard deviation is 3- to 6-fold lower. Furthermore, the novel method is faster than electrophoretic mobility shift assay, not restricted to transfectable cells as is the case for luciferase reporter assays and 10 times more cost efficient than commercially available ELISA assays. Co-ELISA is a sensitive, fast and cost-efficient quantitation method for all DNA-binding NFkappaB proteins that can be used in high-throughput experimentation.
View details for DOI 10.1002/ijc.25054
View details for Web of Science ID 000278919000016
View details for PubMedID 19924814
Loss of a critical region in 13q14.3 [del(13q)] is the most common genomic aberration in chronic lymphocytic leukemia (CLL), occurring in more than 50% of patients (Stilgenbauer et al., Oncogene 1998;16:1891 - 1897, Dohner et al., N Engl J Med 2000;343:1910 - 1916). Despite extensive investigations, no point mutations have been found in the remaining allele that would inactivate one of the candidate tumor suppressor genes and explain the pathomechanism postulated for this region. However, the genes in the region are significantly down-regulated in CLL cells, more than would be expected by gene dosage, and recently a complex epigenetic regulatory mechanism was identified for 13q14.3 in non-malignant cells that involves asynchronous replication timing and monoallelic expression of candidate tumor suppressor genes. Here, we propose a model of a multigenic pathomechanism in 13q14.3, where several tumor suppressor genes, including the miRNA genes miR-16-1 and miR-15a, are co-regulated by the two long non-coding RNA genes DLEU1 and DLEU2 that span the critical region. Furthermore, we propose these co-regulated genes to be involved in the same molecular pathways, thereby also forming a functional gene cluster. Elucidating the molecular and cellular function of the 13q14.3 candidate genes will shed light on the underlying pathomechanism of CLL.
View details for DOI 10.1080/10428190902763509
View details for Web of Science ID 000265361100039
View details for PubMedID 19347735
The objective of the present study is to observe the effect of exogenous steroids, methyl testosterone (MT) and ethynyl estradiol (EEL) on gonadal differentiation and analyze its effect on the expression of several genes during testicular and ovarian differentiation in juvenile catfish. Exogenous hormone treatments (MT and EEL) were given by immersion at different days of hatching. The histological analysis revealed that the EEL- and MT-treatments resulted in the initiation of ovarian and testicular differentiation, respectively. This is further supported by specific expression of two forms of DMRT1 in the MT-treated group but not in the EEL-treated group at 47 days after hatching (dah). The reverse is true for the expression of ovarian aromatase. Results of the semi-quantitative RT-PCR show that brain aromatase transcript levels are high in 47 dah control (histologically female) and 47 dah EEL-treated fish, as compared to 47 dah MT-treated fish. At 60 dah, brain aromatase showed elevation in its expression. Interestingly, the expression pattern of 3 beta-HSD did not show any change in EEL- and MT-treated fish. The present study also provides a strategy to study sex differentiation, for those species where genetic sex population is unavailable.
View details for DOI 10.1007/s10695-006-0016-3
View details for Web of Science ID 000240362600008
View details for PubMedID 20035448