Cytometry for improving and tailoring cancer therapy

Abstract

IN cancer therapy, the detailed understanding of the effects of drugs on signal transduction pathways in the target cells is of pivotal relevance in tailoring individualized therapeutic approaches. Furthermore, also new and more detailed diagnostics help to specifically design a therapy for the individual patient. Nowadays, flow cytometry becomes a key player in these endeavors. A characteristic of ‘‘stemness’’ of normal and tumor (1) stem cells is their ability to express transporter molecules that function as transporters to exclude certain dye molecules such as the vital DNA dye HOECHST3342 in all stem-like cells. This phenomenon allows the identification of stem cells based on reduced HOECHST33342 staining intensity (HOECHST side-population) providing also a convenient method for their further enrichment. Responsible for this exclusion is the ATPbinding cassette subfamily G member 2 (ABCG2) (2). This is a membrane-associated protein that is in humans encoded by the ABCG2 gene, and is assigned the cluster of differentiation w338ABCG2 (CDw338). ABCG2 is in tumor therapy also responsible for the resistance to chemotherapeutic agents such as mitoxantrone and may thereby indirectly render tumor stem cell survival (1,2). Survival of normal stem cells and neoplastic cells has been associated with the sonic hedgehog (Hh) signal transduction pathway. Hh is essential in embryonic development throughout the animal kingdom and in vertebrates its dysfunction leads to the loss of the left–right symmetry with congenital disorders such as cyclopia (single eye). Such developmental effects can be induced during embryogenesis by cyclopamine (11-deoxojervine) a plant-derived alkaloid from the corn lily (Varatrum californicum) that acts as Hh pathway inhibitor. In addition, cyclopamibe leads to the reduction of the HOECHST side-population in various cancer cells. Consequently, cyclopamine is now tested on many human tumors that overexpress Hh as a potential new cancer therapeutic. The interrelation of the Hh signaling pathway with ABCG2 activity has so far, not been elucidated in further detail. Now, Balbuena and colleagues (this issue, page 672) from research institutes in Pamplona and Barcelona, Spain, addressed this question. They analyzed the effect of cyclopamine on cell lines expressing a low level of HOECHST sidepopulation and ABCG2 transporter overexpressing cell lines that consisted mostly of side-population cells. The observation indicates that Hh pathway activity is also regulated by ABCG2 activity leading to stem cell and tumor cell protection against natural and cancer agents. These studies have clear implications for new preclinical and clinical therapy concepts. For treatment of other tumor entities, other therapeutic approaches have to be applied. In breast cancer, the human epidermal growth factor receptor 2 (EGFR2) (Her2/neu, ErbB2) on the tumor cells membrane is therapeutically targeted by monoclonal antibodies to inhibit cell growth or induce apoptotic cell death. Different types of these receptors form homodimers or heterodimers such as EGFR/Her2 as can be revealed by flow cytomeric assays such as proximity in situ ligation (3). Unfortunately, in the clinical praxis only a subgroup of the breast cancer patients respond to treatment with the commonly used antibody, trastuzumab. Some respond inefficiently and others may not respond at all. The reason for this heterogeneity is still an open question and has been addressed by Diermeier-Daucher and colleagues (this issue, page 684) from the University of Regensburg in Germany. The authors approached the question whether other factors that induce growth stimulating effects may affect antibody therapy