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Cancer Cells and their Microenvironment
The Cancer Session is dedicated to providing presentations and a discussion forum on solid tumors, their microenvironment and pathophysiology. Special emphasis is devoted to new strategies for a better understanding of tumor formation, progression, and metastasis. This session focuses particularly on cancer research with cytometric approaches. Through the eyes of a biochemist and a physician, the overall idea of this session is the “bench to bedside” principle - the direct transfer of laboratory bench results to clinical diagnostics and therapies for the benefit of patients.
Tumor biology in breast cancer - what do we need to know clinically?
University Hospital of Cologne, Germany
In order to understand how knowledge about tumor cell properties is utilized for clinical purposes, breast cancer constitutes a good example since in-depth understanding of breast cancer biology is already being used clinically in several ways. Breast cancer is the most common malignancy in women in industrialized countries with about 1 out of 8-10 women being affected during their life-time. The majority of all patients who present without manifest metastases, i.e. 70-80%, will be cured today by a multidisciplinary therapy concept. Yet, even at primary diagnosis, breast cancer is considered a systemic disease where single tumor cells may have already spread which cannot be detected by conventional technologies. Thus, systemic therapy is a key element in treating this disease.
The knowledge of tumor cell biology impacts clinical decision making in three different ways: prognostic factors allow forecasting the course of the disease, predictive factors enable assessment of response to therapy, and last but not least, individual biological properties of the tumor cells are utilized as therapy targets in breast cancer.
Next to conventional staging criteria such as tumor size, nodal involvement, and grade, steroid hormone receptor status (ER, PR) and HER2 status are the most important biological properties which are determined in every breast cancer. They also serve as therapy targets with anti-hormonal (endocrine) therapy being standard in hormone-receptor positive disease and anti-HER2 agents, trastuzumab and lapatinib being approved for HER2-positive disease. Usually, these markers are determined immunohistochemically in primary tumor tissue. However, single cell analysis is gaining clinical relevance, in early (eBC) as well as metastatic breast cancer (mBC).
Bone marrow (BM) micrometastases are a known prognostic factor in eBC with BM+ patients having a poor outcome. Yet, because of the additional clinical burden to patients, BM sampling has not been widely accepted as part of clinical routine. Moreover, proof of benefit from therapeutic interventions in patients with BM+ eBC is still lacking. More recently, circulating tumor cells (CTC) have been proposed as a prognostic marker in mBC and eBC. In particular, German investigators such as the SUCCESS study group or the DETECT consortium have been instrumental in integrating single cell analysis into clinical practice. Questions that are currently being addressed are not just the mere number of circulating cells as a prognostic marker but more importantly their biological properties as potential targets for therapy. Since immunohistochemically determined ER, PR, and HER2 status may differ in about 10-20% between primary tumor and metastasis, alternative techniques such as single cell analysis are currently been investigated in order to indicate targeted therapy in metastatic disease. As a proof of principle, the DETECT III trial will evaluate the benefit from anti-HER2 therapy in mBC with HER2-positive CTC but a HER2 negative primary tumor.
In conclusion, tumor heterogeneity makes in-depth understanding of tumor biology mandatory for clinical decision making. Properties of single tumor cells will thus become increasingly important, not just as prognostic or predictive markers, but more importantly for choosing targeted therapies and maybe also for elucidating therapy resistance.
Is ErbB4/HER4 a cancer drug target?
University of Turku, Finland
Our aim is to determine the role of the receptor tyrosine kinase ErbB4 as a factor regulating carcinogenesis and as a clinically relevant cancer drug target. ErbB4 is a member the ErbB subfamily of RTKs that serve as receptors for epidermal growth factor (EGF)-like ligands. There are four ErbBs of which the two first-discovered, EGFR (= ErbB1) and ErbB2, are well-documented human oncogenes and cancer drug targets. Indeed, several therapeutic antibodies and small molecular weight tyrosine kinase inhibitors that block EGFR and/or ErbB2 are currently in use in clinical practise. However, the role of ErbB4 in carcinogenesis and its potential as a therapeutic target have remained controversial and poorly understood.
We have identified four alternatively spliced isoforms ErbB4. Our recent in vitro and in vivo findings indicate that a specific ErbB4 isoform represents a promising cancer drug target. This isoform is capable of signaling via a novel mechanism involving proteolytic cleavage of the receptor and direct regulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) by an intracellular receptor fragment. Importantly, our data also indicate that inhibition of another, non-cleavable, ErbB4 isoform enhances growth. Thus, pharmaceutical inhibition of ErbB4 could be either beneficial or disadvantageous depending on the specific ErbB4 isoform present. Our hypothesis is that ErbB4 inhibitors should be developed to selectively block the function of the cleavable isoform. Determining the role of ErbB4 isoforms in cancer biology is relevant and necessary also as i) some of the kinase inhibitor drugs currently used in the clinic block ErbB4 activity as an off-target effect, and ii) pan-ErbB inhibitors targeted against all ErbBs (including ErbB4) are being developed by the pharmaceutical industry, while no solid information exists to predict the consequences of ErbB4 inhibition for the clinical outcome.
Multicolor flow cytometry in humanized tumor mice – A powerful method to study advanced cancer therapy in a novel in vivo model
1University of Regensburg, Germany; 2German Research Center for Environmental Health, Munich, Germany
The immunological impact on antibody based anti-cancer therapies remains incompletely understood due to the lack of appropriate animal models for in-vivo analysis. Therefore we generated a novel humanized tumor mouse (HTM) model by concurrent transplantation of human hematopoietic stem cells and human breast cancer cells in neonatal NOD- scid IL2Rγnull (NSG) mice. This co-transplantation is an extension of the already existing humanized mouse model in which a fully functional human immune system develops upon CD34+ cell transplantation. These humanized mice served already as a powerful in-vivo tool for the study of a variety of human infectious diseases (e.g. in HIV, EBV, and Dengue fever), of human haematopoiesis or graft versus host disease (GvHD).
In HTM, five weeks after intrahepatic co-transplantation of human tumor cells and human hematopoietic cells, a functional human immune system in all organs and early tumor cell dissemination in bone marrow and lung had been analyzed using multi-color flow cytometry. Three months post transplant tumor cell effusions and macroscopic tumors associated to liver or spleen were found and in addition disseminated cells in different lymphoid and non-lymphoid organs were measurable.
Ongoing immune responses were determined by flow cytometry using a LSR-II flow cytometer (BD Biosciences), detecting specific T cell maturation and tumor cell specific T cell activation. In addition, Natural Killer cell accumulation and activation was observed in HTM which was further enhanced upon IL-15 treatment facilitating the possibility of immune cell modulation in, e.g. ADCC based immunotherapeutic approaches.
This novel mouse model makes it possible to combine transfer of MHC mismatched tumor cells together with human hematopoietic stem cells resulting in a solid coexistence and interaction without evidence for rejection. Besides immunohistochemistry, quantitative PCR, and other molecular biological methods, flow cytometry is the major technique for characterizing of the ongoing immune response and the assessment of efficiency during cytokine manipulation.
Overall, humanized tumor mice represent a novel, powerful in-vivo model that unprecedently permits the investigation of the human immune defense against cancer, in particular with respect to antibody based targeted therapies and might reveal new strategies to overcome therapy failure in non responder patients.
in-vivo Tissue Classification by Hyperspectral Imaging
1University of Bonn, Germany; 2University of Marburg, Germany
Aim: To establish hyperspectral imaging for in vivo classification of human mucosal surfaces.
Material and methods: The larynx as a well-defined anatomical region was chosen as a prototypical surgical test area. The standard intra-operative setting for microlaryngoscopies was modified by using a polychromatic light source and a synchronous triggered monochromatic CCD-camera. Image stacks were analyzed by established software tools for principal component analysis and unsupervised hyperspectral classification.
Results: Sequential illumination of the same field of view by stepwise increasing wavelengths (390nm – 680nm, with 10nm steps) yielded a hyperspectral image datacube of the mucosa. These lambda-image stacks could be analyzed and classified by commercially available software. In principal component analysis, images at 590nm – 680nm loaded most onto the first principal component. Typically, the first principal component contained 95% of the total information. Unsupervised hyperspectral classification clustered the data thus highlighting areas of altered mucosa.
Conclusion: The technology of hyperspectral imaging can be applied to mucosal surfaces. This approach opens the way to analyze spectral characteristics of histologically different lesions in order to build up a spectral library and to allow non-touch optical biopsy.