Publications by authors named "Karsten Brand"

28 Publications

  • Page 1 of 1

Macrophage ablation significantly reduces uptake of imaging probe into organs of the reticuloendothelial system.

Acta Radiol 2020 Aug 9:284185120943048. Epub 2020 Aug 9.

Department of Radiology, German Cancer Research Center, Heidelberg, Germany.

Background: Macrophages engulf particulate contrast media, which is pivotal for biomedical imaging.

Purpose: To introduce a macrophage ablation animal model by showing its power to manipulate the kinetics of imaging probes.

Material And Methods: The kinetics of a particulate computed tomography (CT) contrast media was compared in macrophage ablative mice and normal mice. Liposomes (size 220 µg), loaded with clodronate, were injected into the peritoneum of three C57BL/6 mice. On the third day, 200 µL of the particulate agent ExiTron nano 6000 were injected into three macrophage-ablative mice and three control mice. CT scans were acquired before and 3 min, 1 h, 6 h, and 24 h after the ExiTron application. The animals were sacrificed, and their spleens and livers removed. Relative CT values (CTV) were measured and analyzed.

Results: Liver and spleen enhancement of treated mice and controls were increasing over time. The median peak values were different with 225 CTV for treated mice and 582 CTV for controls in the liver ( = 0.032) and 431 CTV for treated and 974 CTV in controls in the spleen ( = 0.016).

Conclusion: Macrophage ablation leads to a decrease of enhancement in organs containing high numbers of macrophages, but only marginal changes in macrophage-poor organs. Macrophage ablation can influence the phagocytic activity and thus opens new potentials to investigate and manipulate the uptake of imaging probes.
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http://dx.doi.org/10.1177/0284185120943048DOI Listing
August 2020

ShRNA-mediated knock-down of CXCL8 inhibits tumor growth in colorectal liver metastasis.

Biochem Biophys Res Commun 2018 06 23;500(3):731-737. Epub 2018 Apr 23.

Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, D-69120, Heidelberg, Germany. Electronic address:

CXCL8 belongs to proinflammatory chemokines that are predominantly involved in neutrophil chemotaxis and degranulation. Several studies have suggested that secretion of CXCL8 from cancer cells have a profound effect on tumor microenvironment. In this study, in continuation to our previous work of understanding the global picture of invasion related genes in colorectal liver metastases, we clearly show an up-regulation of CXCL8 expression in the tumor cells at the invasion front as compared to the tumor cells in the inner parts of the tumor. Furthermore, ShRNA mediated down-regulation of CXCL8 resulted in inhibition of cell proliferation, viability and invasion in vitro and a near complete growth reduction of tumor in vivo. We showed that CXCL8 secreted by tumor cells at the invasion front were able to promote migration through angiogenesis by upregulating VEGFA and invasion via the AKT/GSK3β/β-catenin/MMP7 pathway by upregulating BCL-2 confirming the key role of CXCL8 during tumor progression.
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http://dx.doi.org/10.1016/j.bbrc.2018.04.144DOI Listing
June 2018

Tumoral Immune Cell Exploitation in Colorectal Cancer Metastases Can Be Targeted Effectively by Anti-CCR5 Therapy in Cancer Patients.

Cancer Cell 2016 Apr;29(4):587-601

Department of Medical Oncology, National Center for Tumor Diseases, University Hospital Heidelberg, 69120 Heidelberg, Germany; Tissue Imaging and Analysis Center, National Center for Tumor Diseases, BIOQUANT, University of Heidelberg, 69120 Heidelberg, Germany.

The immune response influences the clinical course of colorectal cancer (CRC). Analyzing the invasive margin of human CRC liver metastases, we identified a mechanism of immune cell exploitation by tumor cells. While two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10, CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5. CCR5 blockade in patient-derived functional in vitro organotypic culture models showed a macrophage repolarization with anti-tumoral effects. These anti-tumoral effects were then confirmed in a phase I trial with a CCR5 antagonist in patients with liver metastases of advanced refractory CRC. Mitigation of tumor-promoting inflammation within the tumor tissue and objective tumor responses in CRC were observed.
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http://dx.doi.org/10.1016/j.ccell.2016.03.005DOI Listing
April 2016

The role of perfusion effects in monitoring of chemoradiotherapy of rectal carcinoma using diffusion-weighted imaging.

Cancer Imaging 2013 Dec 11;13(4):548-56. Epub 2013 Dec 11.

Quantitative Imaging Based Disease Characterization (E011), German Cancer Research Center, Heidelberg, Germany.

Purpose: The aim of this study was to characterize and understand the therapy-induced changes in diffusion parameters in rectal carcinoma under chemoradiotherapy (CRT). The current literature shows conflicting results in this regard. We applied the intravoxel incoherent motion model, which allows for the differentiation between diffusion (D) and perfusion (f) effects, to further elucidate potential underlying causes for these divergent reports.

Materials And Methods: Eighteen patients with primary rectal carcinoma undergoing preoperative CRT were examined before, during, and after neoadjuvant CRT using diffusion-weighted imaging. Using the intravoxel incoherent motion approach, f and D were extracted and compared with postoperative tumor downstaging and volume.

Results: Initial diffusion-derived parameters were within a narrow range (D1 = 0.94 ± 0.12 × 10(-3) mm(2)/s). At follow-up, D rose significantly (D2 = 1.18 ± 0.13 × 10(-3) mm(2)/s; P < 0.0001) and continued to increase significantly after CRT (D3 = 1.24 ± 0.14 × 10(-3) mm(2)/s; P < 0.0001). The perfusion fraction f did not change significantly (f1 = 9.4 ± 2.0%, f2 = 9.4 ± 1.7%, f3 = 9.5 ± 2.7%). Mean volume (V) decreased significantly (V1 = 16,992 ± 13,083 mm(3); V2 = 12,793 ± 8317 mm(3), V3 = 9718 ± 6154 mm(3)). T-downstaging (10:18 patients) showed no significant correlation with diffusion-derived parameters.

Conclusions: Conflicting results in the literature considering apparent diffusion coefficient (ADC) changes in rectal carcinoma under CRT for patients showing T-downstaging are unlikely to be due to perfusion effects. Our data support the view that under effective therapy, an increase in D/ADC can be observed.
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http://dx.doi.org/10.1102/1470-7330.2013.0045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3864228PMC
December 2013

Chemotherapy-associated liver injury and its influence on outcome after resection of colorectal liver metastases.

Surgery 2014 Feb 5;155(2):245-54. Epub 2013 Dec 5.

Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany; Department of Visceral, Thoracic and Vascular Surgery, University of Dresden, Dresden, Germany.

Background: Advances in neoadjuvant therapy enabled novel strategies for treating resectable and initially unresectable colorectal cancer liver metastases. Although it is well known that chemotherapeutic agents cause certain types of liver parenchymal injury, the actual contribution of chemotherapy-associated hepatotoxicity to postoperative morbidity remains poorly defined. The aim of this study was to define all kinds of chemotherapy-associated liver injury and to examine its impact on postoperative morbidity.

Patients And Methods: We included 119 patients who were treated between 2002 and 2010. Chemotherapy-associated changes of the liver were subclassified in 11 different categories and correlated with postoperative morbidity with the ultimate aim of generating a liver injury risk score.

Results: On univariate analysis severity (P = .004) and localization of parenchymal inflammation (P = .04) were associated with morbidity. Steatosis did not correlate with postoperative outcome (P = .69), whereas steatohepatitis (as assessed by the nonalcoholic fatty liver disease activity score score) was related with morbidity (P = .03). On multivariate analysis, the severity of inflammation (95% confidence interval, 1.008-6.526; odds ratio, 2.56; P = .04) was significantly correlated with postoperative morbidity. The newly developed liver injury risk score was highly associated with postoperative complications (P = .006).

Conclusion: In this study, the induction of inflammation by conventional chemotherapy and its relevance for the development of clinical complications could be demonstrated. The proposed risk score for liver injury-related morbidity might help to better select patients eligible for an operation.
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http://dx.doi.org/10.1016/j.surg.2013.07.022DOI Listing
February 2014

Hepatic metastases of colorectal cancer are rather homogeneous but differ from primary lesions in terms of immune cell infiltration.

Oncoimmunology 2013 Apr;2(4):e24116

National Center for Tumor Diseases; Medical Oncology and Internal Medicine VI; University Hospital Heidelberg; Heidelberg, Germany ; Tissue Imaging and Analysis Center (TIGA); BIOQUANT; Heidelberg, Germany.

The immune system plays an important role in shaping the clinical course of colorectal cancer (CRC). However, it is still unclear how the immune infiltrates of primary CRC lesions and distant metastases by immune effector cells are related to each other. To address this issue, we quantified CD3, CD8 and granzyme B lymphocytes in primary CRC samples and corresponding liver metastases. This analysis showed that the prognostic predictions that can be drawn from the infiltration of immune cells in primary CRCs and their metastases are heterogeneous. To investigate whether such heterogeneity would also be observed within CRC hepatic metastases, the density of the immune infiltrate and cytokine production were assessed in opposite sides of the same metastatic lesion. In addition, tumor-infiltrating lymphocytes were assessed in sequential sections of the same metastatic lesion, with a spacing of 30 μm. In summary, consistent cell counts and cytokine levels were detected within the same lesion. The study of a case of synchronous metastases, however, suggested that different metastatic lesions within the same patient may be heterogeneous, perhaps indicating a major impact for local causes on tumor infiltration by immune cells. In summary, our study demonstrates a consistent degree of heterogeneity between primary tumors and hepatic metastases but an excellent intra-lesional homogeneity. These findings may be of key importance for patient stratification and the development of personalized strategies against CRC.
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http://dx.doi.org/10.4161/onci.24116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654605PMC
April 2013

Sequential metastases of colorectal cancer: Immunophenotypes and spatial distributions of infiltrating immune cells in relation to time and treatments.

Oncoimmunology 2012 Aug;1(5):593-599

National Center for Tumor Diseases, Department of Medical Oncology/Internal Medicine VI; University Hospital; Heidelberg, Germany.

The role of the immune system in the course of colorectal cancer has been elucidated in the last decade. While quantification of immune cell infiltrates within the resected specimen at diagnosis has a clear power to estimate the prognosis of the patient, the role of infiltrating immune cells within the metastatic situation and especially within the metastatic lesion itself requires further detailed analyses. Recent analyses of infiltrates in colorectal cancer liver metastases revealed a role for the infiltrate density not only for prognosis but also in the prediction of treatment response. This not only broadens the view on these infiltrates and indicates a systematic role of the local immunological microenvironment, but also raises the question how these infiltrates change during repeated courses of treatment (i.e., resection, chemotherapy, etc.). To address this question, sequential lung or sequential liver metastases of colorectal cancer patients were analyzed using whole slide image quantification after immunohistochemical staining against CD3, CD8, FOXP3, CD68 and Granzyme B. The clinical data and interventions were associated with each individual patient and the metastatic lesions. The resulting cell densities reveal a heterogeneous profile: after successful treatment of a metastatic lesion, the recurrent lesion can still have the same immunophenotype with similar cell distributions. In a situation of a favorable immune cell profile, this profile can return and apparently convey a similar favorable course throughout the disease. But also the opposite was found: the recurrent metastatic lesion could have a different profile with alterations in specific immune cell subsets over time. Further analyses are required to elucidate the different patterns and their associations to the treatment, the tumor cell phenotype and other dynamic factors. However, it is clear from this data however, that there is an immune cell plasticity that needs to be analyzed for individual patients.
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http://dx.doi.org/10.4161/onci.20179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3429563PMC
August 2012

Paracrine signalling in colorectal liver metastases involving tumor cell-derived PDGF-C and hepatic stellate cell-derived PAK-2.

Clin Exp Metastasis 2012 Jun 24;29(5):409-17. Epub 2012 Feb 24.

Department of General Pathology, Institute of Pathology, University Hospital Heidelberg, University of Heidelberg, Im Neuenheimer Feld 220/221, 69120 Heidelberg, Germany.

In a nude mouse model of colorectal liver metastases, we have identified a paracrine tumor cell/host cell signalling pathway that is apparently required for successful tumor growth. Whereas recombinant platelet derived growth factor-C (PDGF-C) and supernatants from PDGF-C secreting wild type LS174T colon carcinoma cells could rescue tumor promoting hepatic stellate cells (HSC) from growth inhibition by serum starvation, supernatants from LS174T colon carcinoma cells with reduced secretion of PDGF-C had much less effect on serum starved HSC. Autocrine growth inhibition of LS174T cells by PDGF-C knock-down was only marginal. In vivo, a prominent inhibition of liver metastasis was observed if PDGF-C was knocked-down in LS174T cells. By whole genome array analysis of host cells of the invasion front and subsequent immunohistochemical staining we identified p21 activated kinase-2 (PAK-2) as being strongly and specifically expressed by HSC. The above described effect of PDGF-C on HSC was found to be dependent on PAK-2 because in contrast to wild type HSC, silencing of PAK-2 in HSC only allowed for a partial PDGF-C-mediated rescue from serum starvation leading to only a slight increase of proliferation. These data indicate that PDGF-C promotes tumor growth via a growth promoting effect on HSC that is at least in part dependent on the presence of functional PAK-2.
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http://dx.doi.org/10.1007/s10585-012-9459-3DOI Listing
June 2012

Opposite effects of tissue inhibitor of metalloproteinases-1 (TIMP-1) over-expression and knockdown on colorectal liver metastases.

BMC Res Notes 2012 Jan 9;5:14. Epub 2012 Jan 9.

Institute of Pathology, University Hospital Heidelberg, University of Heidelberg, Im Neuenheimer Feld 220/1, 69120 Heidelberg, Germany.

Background: Tissue inhibitors of metalloproteinases (TIMPs) and the corresponding metalloproteinases are integral parts of the protease network and have been shown to be involved in cancer development and metastasis. Paradoxically, for TIMP-1, tumor promoting as well as tumor inhibitory effects have been observed.

Methods: To address this paradox, we utilized the BALB/c/CT26 mouse model that reliably leads to liver metastasis after splenic tumor cell injection and variegated the type of target cells for therapeutic intervention and the modalities of gene transfer. Since we have observed before that over-expression of TIMP-1 in liver host cells leads to efficient tumor growth inhibition in this model, we now examined whether targeting the tumor cells themselves will have a similar effect.

Results: In concordance with the earlier results, TIMP-1 over-expression in tumor cells led to a dramatic reduction of tumor growth as well. To evaluate any influence of treatment modality, we further examined whether TIMP-1 knockdown in the same animal model would have the opposite effect on tumor growth than TIMP-1 over-expression. Indeed, TIMP-1 knockdown led to a marked increase in tumor burden.

Conclusion: These data indicate that in the BALB/c/CT26 model, the modification of TIMP-1 has concordant effects irrespective of the type of target cell or the technique of modulation of TIMP-1 activity, and that TIMP-1 is unequivocally tumor inhibitory in this model.
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http://dx.doi.org/10.1186/1756-0500-5-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3284431PMC
January 2012

Down-regulation of CXCL1 inhibits tumor growth in colorectal liver metastasis.

Cytokine 2012 Jan 29;57(1):46-53. Epub 2011 Nov 29.

Department of General Pathology, University of Heidelberg, Im Neuenheimer Feld 220/221, 69120 Heidelberg, Germany.

As part of ongoing studies to obtain a global picture of invasion related events in colorectal liver metastases, here, we report our findings on gene expression of the pro-angiogenic subgroup of chemokines, the CXCL-ELR+ chemokines. Apart from their pro-angiogenic and chemoattractant function, these chemokines appear to also contribute to tumor cell transformation, growth and invasion. In our nude mouse model of colorectal liver metastases, we found CXCL1,2,3,5 and 8 (IL-8) to be up-regulated in the tumor cells of the invasion front as compared to the tumor cells in the inner parts of the tumor. ShRNA mediated down-regulation of the most prominently up-regulated group member, CXCL1/gro-alpha resulted in inhibition of cell viability, invasion and proliferation. In vivo, down-regulation of CXCL1 resulted in a nearly complete prevention of tumor growth in nude mice. Mechanistically, auto-regulatory mechanisms involving NF-kappaB and Akt appear to be involved in pro-tumorigenic functions of CXCL1.
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http://dx.doi.org/10.1016/j.cyto.2011.10.019DOI Listing
January 2012

Overexpression of ZEB2 at the invasion front of colorectal cancer is an independent prognostic marker and regulates tumor invasion in vitro.

Clin Cancer Res 2011 Dec 31;17(24):7654-63. Epub 2011 Oct 31.

Department of General, Visceral and Transplantation Surgery, Institute of Pathology, University of Heidelberg, and German Cancer Research Center, Division of Biostatistics, Heidelberg, Germany.

Purpose: Epithelial-to-mesenchymal transition (EMT) plays a pivotal role in tumor invasion and dissemination. EMT occurs predominantly at the tumor edge where it is induced by cytokines, the extracellular matrix environment, or hypoxia. In the tumor cell, it is further mediated by several transcription factors and microRNAs. The aim of this study was to explore the expression of EMT-associated genes at the invasive front in colorectal cancer and to evaluate their prognostic significance.

Experimental Design: We evaluated the expression of 13 EMT-associated genes at the invasion front of 30 colorectal liver metastases by quantitative real-time PCR. Immunostaining against zinc finger E-box-binding homeobox 2 (ZEB2) was carried out on 175 primary colorectal cancer specimens and 30 colorectal liver metastases and correlated to clinical and histopathologic data. DLD-1 cells were transfected with siRNA and subjected to migration and invasion assays.

Results: Gene expression analysis and immunohistochemistry showed an upregulation of ZEB2 at the invasion front in primary colorectal cancer and liver metastases. Overexpression of ZEB2 at the invasion front correlated significantly with tumor stage in primary colorectal cancer. Moreover, univariate and multivariate analysis revealed overexpression of ZEB2 at the invasion front as an independent prognostic marker for cancer-specific survival. Downregulation of ZEB2 by siRNA decreased the migration and invasion capacity of DLD-1 cells in vitro.

Conclusions: Overexpression of ZEB2 at the invasion front correlates with tumor progression and predicts cancer-specific survival in primary colorectal cancer. Therefore, ZEB2 may be interesting as biomarker and potential target for treatment of colorectal cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-2816DOI Listing
December 2011

Distinct types of tumor-initiating cells form human colon cancer tumors and metastases.

Cell Stem Cell 2011 Oct;9(4):357-65

Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.

Human colon cancer harbors a small subfraction of tumor-initiating cells (TICs) that is assumed to be a functionally homogeneous stem-cell-like population driving tumor maintenance and metastasis formation. We found unexpected cellular heterogeneity within the TIC compartment, which contains three types of TICs. Extensively self-renewing long-term TICs (LT-TICs) maintained tumor formation in serial xenotransplants. Tumor transient amplifying cells (T-TACs) with limited or no self-renewal capacity contributed to tumor formation only in primary mice. Rare delayed contributing TICs (DC-TICs) were exclusively active in secondary or tertiary mice. Bone marrow was identified as an important reservoir of LT-TICs. Metastasis formation was almost exclusively driven by self-renewing LT-TICs. Our results demonstrate that tumor initiation, self-renewal, and metastasis formation are limited to particular subpopulations of TICs in primary human colon cancer. We identify LT-TICs as a quantifiable target for therapies aimed toward eradication of self-renewing tumorigenic and metastatic colon cancer cells.
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http://dx.doi.org/10.1016/j.stem.2011.08.010DOI Listing
October 2011

Invasion front-specific expression and prognostic significance of microRNA in colorectal liver metastases.

Cancer Sci 2011 Oct 4;102(10):1799-807. Epub 2011 Aug 4.

Department of Surgery, University of Heidelberg, Heidelberg, Germany.

The tumor edge of colorectal cancer and its adjacent peritumoral tissue is characterized by an invasion front-specific expression of genes that contribute to angiogenesis or epithelial-to-mesenchymal transition. Dysregulation of these genes has a strong impact on the invasion behavior of tumor cells. However, the invasion front-specific expression of microRNA (miRNA) still remains unclear. Therefore, the aim of the present study was to investigate miRNA expression patterns at the invasion front of colorectal liver metastases. Laser microdissection of colorectal liver metastases was performed to obtain separate tissue compartments from the tumor center, tumor invasion front, liver invasion front and pure liver parenchyma. Microarray expression analysis revealed 23 miRNA downregulated in samples from the tumor invasion front with respect to the same miRNA in the liver, the liver invasion front or the tumor center. By comparing samples from the liver invasion front with samples from pure liver parenchyma, the tumor invasion front and the tumor center, 13 miRNA were downregulated. By quantitative RT-PCR, we validated the liver invasion front-specific downregulation of miR-19b, miR-194, let-7b and miR-1275 and the tumor invasion front-specific downregulation of miR-143, miR- 145, let-7b and miR-638. Univariate analysis demonstrated that enhanced expression of miR-19b and miR-194 at the liver invasion front, and decreased expression of let-7 at the tumor invasion front, is an adverse prognostic marker of tumor recurrence and overall survival. In conclusion, the present study suggests that invasion front-specific downregulation of miRNA in colorectal liver metastases plays a pivotal role in tumor progression.
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http://dx.doi.org/10.1111/j.1349-7006.2011.02023.xDOI Listing
October 2011

Increased apoptosis of host cells and tumor cells in the invasion front of colorectal liver metastases.

Anticancer Res 2011 Apr;31(4):1215-24

Institute of Pathology, University of Heidelberg, Heidelberg, Germany.

Background: The invasion front of colorectal liver metastases is an area of intensive tumor cell-host cell contact.

Materials And Methods: In a xenograft nude mouse model, we analyzed whether apoptosis induction is a prominent feature in this active area, perhaps offering new modalities of therapeutic intervention.

Results: Using global gene expression technology, an over-representation of apoptosis-related biological themes in the invasion front was observed. A combination of apoptosis-specific TUNEL/DAPI staining and cell type-specific staining showed that all examined cell types, including tumor cells, hepatocytes, endothelial cells, macrophages and hepatic stellate cells, displayed increased apoptosis in the invasion front. Evaluation of gene expression of the death receptor/ligand pairs TRAILR2 /TRAIL and FAS/FASL indicated that tumor cells overexpressed TRAILR2 and FAS, whereas host cells expressed TRAIL and FASL.

Conclusion: This data indicates that the invasion front of colorectal liver metastases is an area of prominent pro-apoptotic activity, involving known death receptor/ligand interactions.
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April 2011

A gene signature distinguishing CD133hi from CD133- colorectal cancer cells: essential role for EGR1 and downstream factors.

Pathology 2011 Apr;43(3):220-7

Division of Molecular Genetics, German Cancer Research Center, University of Heidelberg, Heidelberg, Germany.

Aims: In colorectal cancer (CRC), CD133 expression is an independent prognostic marker associated with adverse clinical outcome. The CD133 epitope AC133 allowed isolating stem cells from normal and cancerous tissues, although its use in colon was questioned. We aimed to identify differences between AC133 and AC133 cells.

Methods: We analysed the gene expression profiles of EpCAM/CEA/AC133 and EpCAM/CEA/AC133 cells from primary CRC and liver metastasis tissues (n = 5). Immunohistochemistry confirmed these results in a validation set.

Results: We identified 68 genes differentially expressed between both populations, including genes of notorious importance in CRC pathogenesis, and several candidates not previously shown to play a major role in CRC. Notably, EGR1 belonged to the most highly expressed genes in AC133 cells. In the validation set, the presence of EGR1 and CD133 correlated (r = 0.625). Since EGR1 regulates Wnt through up-regulation of TCF4, which induces stem cell marker LGR5, the potential association between LGR5, EGR1 and CD133 was investigated. The presence of LGR5 correlated with the presence of EGR1 and CD133. Strong signals for LGR5 were detected throughout tumour invasion fronts.

Conclusions: The study suggests a connection between CD133 and EGR1 and emphasises the importance of the EGR1/TCF4/CD133/LGR5 network in CRC.
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http://dx.doi.org/10.1097/PAT.0b013e328344e391DOI Listing
April 2011

Natural killer cells are scarce in colorectal carcinoma tissue despite high levels of chemokines and cytokines.

Clin Cancer Res 2011 Feb;17(4):678-89

Medical Oncology, National Center for Tumor Diseases, University Heidelberg, Germany.

Purpose: Tumor infiltrating T lymphocytes in colorectal cancer (CRC) have prognostic impact, but the role of natural killer (NK) cells in CRC tissue is unclear. The contribution of intratumoral cytokines and chemokines in shaping the composition of the inflammatory lymphocytic infiltrate is also unclear.

Experimental Design: In this study, localization and densities of NK and T cells within primary CRC, CRC liver metastases, adenomas, and normal tissues were analyzed on whole tissue sections from 112 patients. In a subset of these patients, the most important 50 cytokines and chemokines were quantified in paired serum, primary CRC and adjacent mucosa samples and in CRC liver metastases and correlated with NK and T-cell infiltration, respectively.

Results: The various compartments displayed characteristic differences like significantly higher chemokine concentrations in CRC tissue. Most importantly, despite high local chemokine levels, NK cells were generally scarce within CRC tumor tissues, independent of human leukocyte antigen (HLA) class I expression. Adjacent normal mucosa contained normal levels of NK cells. In contrast, corresponding T-cell numbers varied substantially and were positively correlated with higher chemokine levels.

Conclusions: Our findings indicate a distinct regulation of NK cells versus T cells in the CRC tumor microenvironment. NK-cell migration into CRC tumor tissue is obviously impaired early during tumor development by mechanisms that do not affect T-cell infiltration.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-2173DOI Listing
February 2011

Comparison of different surgical techniques in distal pancreatectomy: an experimental study in a porcine model.

Surg Innov 2011 Dec 8;18(4):329-37. Epub 2011 Feb 8.

University of Heidelberg, Heidelberg, Germany.

Aims: Different surgical transection methods have been used for distal pancreatectomy (DP), but none of them has yet achieved perfect results. This study compares 2 standard transection techniques with the alternative LigaSure technique.

Methods: Forty-eight pigs underwent a DP. Sixteen animals were operated on with a scalpel followed by hand suturing. Sixteen pigs received a DP using an Endo GIA, and the pancreas of 16 pigs was transected with LigaSure. The transection surface of remnant pancreas was observed for liquid collection and abscess on postoperative day 7.

Results: Operating time on the day of DP was significantly different, with a shorter operating time in the stapler and LigaSure groups. The morbidity on postoperative day 7 was similar in all groups.

Conclusion: In the present experimental animal study, LigaSure seems to be fast and safe as well as comparable with the standard transection and closure techniques in DP.
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http://dx.doi.org/10.1177/1553350610395032DOI Listing
December 2011

Annexin A2 as a differential diagnostic marker of hepatocellular tumors.

Pathol Res Pract 2011 Jan 23;207(1):8-14. Epub 2010 Oct 23.

Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

While improved imaging techniques have made it possible to detect focal liver lesions smaller than 1cm in diameter, differentiating benign lesions from hepatocellular carcinoma (HCC) still remains a challenge. To address this problem and obtain a definite diagnosis, needle core biopsies are often performed, leading to an increased need for supportive ancillary techniques in the histopathological assessment of highly differentiated hepatocellular tumors. Here we evaluate the diagnostic value of immunohistologically detected Annexin A2 (ANXA2) expression in highly differentiated liver tumors. ANXA2 is a calcium-dependent phospholipid-binding protein that has been linked to malignant transformation and HCC development. Our data show that adding sinusoidal ANXA2 expression to the already established marker panel (GPC3, GS, and HSP70) increases the accuracy for the detection of well-differentiated HCC (74% sensitivity, 100% specificity). In addition, in our series, the combinations ANXA2-GPC3 and ANXA2-GS performed better compared to the other established marker combinations. In conclusion, we suggest that adding ANXA2 to the established diagnostic marker panel increases the reliability and objectivity of HCC diagnosis in liver biopsies.
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http://dx.doi.org/10.1016/j.prp.2010.09.007DOI Listing
January 2011

Transcriptional activation of the beta-catenin gene at the invasion front of colorectal liver metastases.

J Pathol 2009 Jul;218(3):370-9

Department of General Pathology, University of Heidelberg, Germany.

beta-Catenin is a pivotal molecule of the Wnt-signalling pathway, involved in regulation of developmental and oncogenic processes as well as in intercellular adhesion. So far, beta-catenin has been thought to be regulated mainly at the protein level. Here, we provide evidence for a transcriptional mechanism of beta-catenin regulation at the invasion front of colorectal liver metastases. In a nude mouse/LS174T cell xenograft model of colorectal liver metastases, we observed beta-catenin up-regulation at the mRNA and protein levels and a 13.7-fold increase of beta-catenin promoter activity in the cancer cells of the invasion front. In addition, the promoter activity was five-fold higher in the interior of the tumour than in cells growing in cell culture. In vitro studies revealed binding of TCF-4 to the beta-catenin promoter and reduced promoter activity by over-expression of dominant negative TCF-4, or beta-catenin knock-down and increased activity by beta-catenin over-expression, indicating that beta-catenin acts as co-transcription factor of its own promoter. In 55% (7/13) of clinical specimens, beta-catenin mRNA was markedly elevated in the cancer cells of the invasion front. Elevation of mRNA was paralleled by increased nuclear and cytoplasmic beta-catenin protein concentrations. These data indicate that transcriptional regulation contributes to the dynamic changes of beta-catenin levels upon the confrontation of tumour cells with the host microenvironment.
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http://dx.doi.org/10.1002/path.2539DOI Listing
July 2009

Cross-species comparison of biological themes and underlying genes on a global gene expression scale in a mouse model of colorectal liver metastasis and in clinical specimens.

BMC Genomics 2008 Sep 29;9:448. Epub 2008 Sep 29.

Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220/1, 69120 Heidelberg, Germany.

Background: Invasion-related genes over-expressed by tumor cells as well as by reacting host cells represent promising drug targets for anti-cancer therapy. Such candidate genes need to be validated in appropriate animal models.

Results: This study examined the suitability of a murine model (CT26/Balb/C) of colorectal liver metastasis to represent clinical liver metastasis specimens using a global gene expression approach. Cross-species similarity was examined between pure liver, liver invasion, tumor invasion and pure tumor compartments through overlap of up-regulated genes and gene ontology (GO)-based biological themes on the level of single GO-terms and of condensed GO-term families. Three out of four GO-term families were conserved in a compartment-specific way between the species: secondary metabolism (liver), invasion (invasion front), and immune response (invasion front and liver). Among the individual GO-terms over-represented in the invasion compartments in both species were "extracellular matrix", "cell motility", "cell adhesion" and "antigen presentation" indicating that typical invasion related processes are operating in both species. This was reflected on the single gene level as well, as cross-species overlap of potential target genes over-expressed in the combined invasion front compartments reached up to 36.5%. Generally, histopathology and gene expression correlated well as the highest single gene overlap was found to be 44% in syn-compartmental comparisons (liver versus liver) whereas cross-compartmental overlaps were much lower (e.g. liver versus tumor: 9.7%). However, single gene overlap was surprisingly high in some cross-compartmental comparisons (e.g. human liver invasion compartment and murine tumor invasion compartment: 9.0%) despite little histolopathologic similarity indicating that invasion relevant genes are not necessarily confined to histologically defined compartments.

Conclusion: In summary, cross-species comparison on a global gene expression scale suggests the validity of an animal model representing the human situation. The actual yield of potential target genes depends on several variables including the animal model, choice of inclusion criteria, inherent species differences and histologic assessment.
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http://dx.doi.org/10.1186/1471-2164-9-448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2573898PMC
September 2008

Distinct functionality of tumor cell-derived gelatinases during formation of liver metastases.

Mol Cancer Res 2008 Mar;6(3):341-51

Klinikum rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, Munich, Germany.

The specific spatiotemporal role of the matrix metalloproteinase 2 (MMP-2) and MMP-9 (gelatinase) during metastasis is still under debate. Host cells have been described as major contributors to these MMPs during metastasis. Here, we show strong overexpression of MMP-2 and MMP-9 by tumor cells of clinical liver specimen of recurrent metachronous metastases, leading us to address the importance of tumor cell-derived MMP-2 or MMP-9 during liver metastasis. Thus far, distinction of their roles was impossible due to lack of inhibitors which can act exclusively on tumor cells or distinguish MMP-2 from MMP-9. We therefore used short hairpin RNA interference technology in the well-established syngeneic L-CI.5s lymphoma model, in which we could analyze the time course of experimental liver colonization (arrest/invasion of single tumor cells, outgrowth, and invasion within the parenchyma) in immunocompetent mice and correlate these steps with MMP-2 or MMP-9 expression levels. In parental tumor cells, MMP-9 expression closely correlated with the invasive phases of liver colonization, whereas MMP-2 expression remained unaltered. Specific knockdown of MMP-9 revealed a close correlation between invasion-dependent events and tumor cell-derived MMP-9 expression. In contrast, knockdown of MMP-2 did not significantly alter the metastatic potential of the cells but led to a marked inhibition of metastatic foci growth. These findings explain the efficacy of gelatinase-specific synthetic inhibitors on invasion and growth of tumor cells and attribute distinct functions of MMP-2 and MMP-9 to aspects of liver metastasis.
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http://dx.doi.org/10.1158/1541-7786.MCR-07-2032DOI Listing
March 2008

Tissue inhibitor of metalloproteinases-1 promotes liver metastasis by induction of hepatocyte growth factor signaling.

Cancer Res 2007 Sep;67(18):8615-23

Institut für Experimentelle Onkologie und Therapieforschung, Universität München, Munich, Germany.

Balanced expression of proteases and their inhibitors is one prerequisite of tissue homeostasis. Metastatic spread of tumor cells through the organism depends on proteolytic activity and is the death determinant for cancer patients. Paradoxically, increased expression of tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of several endometalloproteinases, including matrix metalloproteinases and a disintegrin and metalloproteinase-10 (ADAM-10), in cancer patients is negatively correlated with their survival, although TIMP-1 itself inhibits invasion of some tumor cells. Here, we show that elevated stromal expression of TIMP-1 promotes liver metastasis in two independent tumor models by inducing the hepatocyte growth factor (HGF) signaling pathway and expression of several metastasis-associated genes, including HGF and HGF-activating proteases, in the liver. We also found in an in vitro assay that suppression of ADAM-10 is in principle able to prevent shedding of cMet, which may be one explanation for the increase of cell-associated HGF receptor cMet in livers with elevated TIMP-1. Similar TIMP-1-associated changes in gene expression were detected in livers of patients with metastatic colorectal cancer. The newly identified role of TIMP-1 to create a prometastatic niche may also explain the TIMP-1 paradoxon.
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http://dx.doi.org/10.1158/0008-5472.CAN-07-0232DOI Listing
September 2007

Biological profiling of gene groups utilizing Gene Ontology.

Genome Inform 2005 ;16(1):106-15

Institute for Theoretical Biology, Humboldt University Berlin, Germany.

Increasingly used high throughput experimental techniques, like DNA or protein microarrays give as a result groups of interesting, e.g. differentially regulated genes which require further biological interpretation. With the systematic functional annotation provided by the Gene Ontology the information required to automate the interpretation task is now accessible. However, the determination of statistical significance of a biological process within these groups is still an open question. In answering this question, multiple testing issues must be taken into account to avoid misleading results. Here we present a statistical framework that tests whether functions, processes or locations described in the Gene Ontology are significantly enriched within a group of interesting genes when compared to a reference group. First we define an exact analytical expression for the expected number of false positives that allows us to calculate adjusted p-values to control the false discovery rate. Next, we demonstrate and discuss the capabilities of our approach using publicly available microarray data on cell-cycle regulated genes. Further, we analyze the robustness of our framework with respect to the exact gene group composition and compare the performance with earlier approaches. The software package GOSSIP implements our method and is made freely available at http://gossip.gene-groups.net/.
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January 2006

Global analysis of host tissue gene expression in the invasive front of colorectal liver metastases.

Int J Cancer 2006 Jan;118(1):74-89

Institute of Biology, Humboldt University Berlin, Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Host cell reactions are a crucial determinant for tumor invasion. We analyzed on a genomewide scale gene expression differences between microdissected tissues taken from unaffected liver tissue of a human colorectal tumor (LS174) growing in the livers of nude mice and tissue from the host part of the invasive front. Due to the low degree of interspecies cross-hybridization of 15% as determined on Affymetrix microarrays, our xenograft model allowed for the distinction of genes of murine versus human origin even if the respective tissues could not be isolated separately. Using the gene ontology (GO) classification, we were able to determine patterns of up- and downregulated genes in the liver part of the invasive front. We observed a pronounced overrepresentation, e.g., of the GO terms "extracellular matrix," "cell communication," "response to biotic stimulus," "structural molecule activity" and "cell growth," indicating a very pronounced host cell response to tumor invasion. On the single gene level, hepatic stellate cell (HSC) activation markers were overrepresented in the liver part of the invasion front. Immunohistochemistry and qPCR confirmed an activation of HSC as well as an increased number of HSC in the invasive front as compared to the noninvaded liver tissue. In summary, our data demonstrate the feasibility of an interspecies differential gene expression approach on a genomewide scale.
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http://dx.doi.org/10.1002/ijc.21307DOI Listing
January 2006

Adenovirus-mediated overexpression of tissue inhibitor of metalloproteinases-1 in the liver: efficient protection against T-cell lymphoma and colon carcinoma metastasis.

J Gene Med 2004 Nov;6(11):1228-37

Institute for Biology, Humboldt University Berlin, at the Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Background: Matrix metalloproteinases (MMPs) are critical for metastasis of tumor cells. Tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural MMP inhibitor, was shown to reduce metastasis in different models. Here, we investigated whether increased TIMP-1 levels in the liver achieved by adenoviral gene transfer will effectively inhibit liver metastasis of two independent tumor cell lines.

Method: TIMP-1 was transferred with adenoviral vectors into the livers of DBA/2 and Balb/c mice, which were subsequently challenged by hematogenous experimental metastases of the T-cell lymphoma cell line L-CI.5s or the colorectal carcinoma cell line CT-26, respectively.

Results: MMP-9 expression in the liver was induced upon metastasis in both tumor types. Adenoviral gene transfer led to high transduction efficacy as indicated by lacZ expression in 60% of hepatocytes. TIMP-1, a key inhibitor of MMP-9, was expressed at 10(5)-fold higher levels by adenoviral gene transfer as compared with levels achieved in TIMP-1 transgenic mice, previously shown to be inefficient to reduce T-cell lymphoma metastasis. High local and systemic (serum) levels of TIMP-1 led to substantial (94%) reduction of T-cell lymphoma and colorectal carcinoma (73%) experimental liver metastasis.

Conclusions: Adenoviral gene transfer led to systemic and local TIMP-1 levels sufficient to inhibit metastasis of a highly aggressive T-cell lymphoma, pointing at the requirement of threshold levels for effective anti-metastatic efficacy. This approach was also efficient in a colon carcinoma solid tumor model. We propose that viral gene transfer of TIMP-1 can provide a suitable defense strategy to prevent metastatic spread to the liver.
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http://dx.doi.org/10.1002/jgm.637DOI Listing
November 2004

Eradication of glioblastoma, and breast and colon carcinoma xenografts by Hsp70 depletion.

Cancer Res 2002 Dec;62(24):7139-42

Apoptosis Laboratory, Institute for Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen, Denmark.

Heat shock protein 70 (Hsp70) is an antiapoptotic chaperone protein highly expressed in human tumors. Here we demonstrate that locoregional application of adenovirus expressing antisense Hsp70 cDNA (Ad.asHsp70) eradicates orthotopic xenografts of glioblastoma and breast carcinoma, as well as s.c. xenografts of colon carcinoma in immunodeficient mice. Ad.asHsp70-treated tumors showed massive apoptosis-like cell death and recruitment of macrophages. Human monocyte-derived macrophages effectively removed the corpses of Ad.asHsp70-treated tumor cells in vitro. Interestingly, both tumor cell death and phagocytosis were caspase-independent. Thus, Hsp70 appears as a promising target for the treatment of cancers resistant to classic caspase-mediated apoptosis.
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December 2002

Cancer gene therapy with tissue inhibitors of metalloproteinases (TIMPs).

Authors:
Karsten Brand

Curr Gene Ther 2002 May;2(2):255-71

Custos Biotech GmbH, Biomedical Research Campus Berlin-Buch, R. Rössle Str. 10, 13125 Berlin, Germany.

Matrix metalloproteinases (MMPs) are of crucial importance for the invasive behavior of primary tumors and their metastases. MMP activity is regulated by the four naturally occurring tissue inhibitors of metalloproteinases (TIMPs). It has been shown that overexpression of TIMPs in tumors of various origins leads to reduced tumor growth and formation of metastases. More recently, antitumor efficacy by in vivo gene transfer of TIMPs has been reported in several clinically relevant animal models. This review analyses the therapeutic potential of the TIMPs from a cancer gene therapeutic point of view with particular emphasis on cell culture and in vivo data.
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http://dx.doi.org/10.2174/1566523024605564DOI Listing
May 2002

YB-1 relocates to the nucleus in adenovirus-infected cells and facilitates viral replication by inducing E2 gene expression through the E2 late promoter.

J Biol Chem 2002 Mar 11;277(12):10427-34. Epub 2002 Jan 11.

Institut für Experimentelle Onkologie und Therapieforschung, Technische Universität München, Klinikum Rechts der Isar, München 81675, Germany.

The adenovirus early proteins E1A and E1B-55kDa are key regulators of viral DNA replication, and it was thought that targeting of p53 by E1B-55kDa is essential for this process. Here we have identified a previously unrecognized function of E1B for adenovirus replication. We found that E1B-55kDa is involved in targeting the transcription factor YB-1 to the nuclei of adenovirus type 5-infected cells where it is associated with viral inclusion bodies believed to be sites of viral transcription and replication. We show that YB-1 facilitates E2 gene expression through the E2 late promoter thus controlling E2 gene activity at later stages of infection. The role of YB-1 for adenovirus replication was demonstrated with an E1-minus adenovirus vector containing a YB-1 transgene. In infected cells, AdYB-1 efficiently replicated and produced infectious progeny particles. Thus, adenovirus E1B-55kDa protein and the host cell factor YB-1 act jointly to facilitate adenovirus replication in the late phase of infection.
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http://dx.doi.org/10.1074/jbc.M106955200DOI Listing
March 2002