Background: Chemotherapy is a mainstay of tumor therapy, however, it is predominantly applied according to empirically developed recommendations derived from statistical relapse rates occurring years after the treatment in the adjuvant situation and from progression-free interval data in the metastatic situation, without any possibility of individually determining the efficacy in the adjuvant situation and with loss of time and quality of life in the metastatic situation if the drugs chosen are not effective. Here, we present a method to determine the efficiency of chemotherapeutic drugs using tumor cells circulating in blood as the part of the tumor actually available in the patient's body for chemosensitivity testing. Methodology/Principal Findings: After only red blood cell lysis, omitting any enrichment (analogous to other blood cell enumeration methods, including rare CD34 cells), the white cells comprising the circulating epithelial tumor cells (CETC) are exposed to the drugs in question in different concentrations and for different periods of time. Staining with a fluorescence-labeled anti-epithelial antibody detects both vital and dying tumor cells, distinguishing vital from dying cells through membrane permeability and nuclear staining with propidium iodide. Increasing percentages of dying tumor cells are observed dependent on time and concentration. The sensitivity can vary during therapy and was correlated with decrease or increase in CETC and clinical outcome. Conclusions/Significance: Thus, we are able to show that chemosensitivity testing of circulating tumor cells provides real-time information about the sensitivity of the tumor present in the patient, even at different times during therapy, and correlates with treatment success.

In spite of ample prognostic markers available in breast cancer, still a considerable proportion of patients with good prognostic markers suffers relapse whereas patients with poor prognostic markers may remain disease free. It would, therefore, be desirable to control, at the individual patient level, whether the applied therapy is effective. Our previous work indicates, that in cancer patients most of the epithelial cells circulating in peripheral blood (CETC) are part of the tumor and that the response of these cells reflects the response of the tumor to the applied therapies.Therefore, these cells were used to assay chemosensitivity in short time cultures analyzing the percent of cell killing during short time incubation and monitoring the decrease or increase in numbers of these cells during treatment with the respective agents providing a unique tool for therapy surveillance. Patients were prospectively analysed for the number of CETC before each new combination of chemotherapy. 1ml of blood was drawn into EDTA vials, red blood cells lysed and the white blood cell pellet stained with FITC-labelled anti-Epcam. Green fluorescent cells were detected by image analysis and dead cells excluded due to red PI fluorescence. Activity of individual compounds was determined using three different concentrations of each compound at 3h, 6h and 12hs and displayed as % cell killing. The in vitro results were then compared to in vivo reduction of CETCs and to the reduction of a marker lesion.215 patients have been investigated so far. Cell killing was dose and time dependent. The highest killing rates were observed with Epirubicin and Taxanes, agents which are known for their high activity in breast cancer. Less than 20% killing activity was termed marginal activity. In vitro sensitivitity was highly significantly predictive of in vivo CETC reduction. In some cases an increasing resistance could be shown to develop during repeated cycles of the same combination therapy. CETC reduction was correlated with a prolonged progression free survival.Thus this approach can in the future be used to test in advance the sensitivity of the circulating tumor cells to chemotherapy and at the same time monitor the current response of the cells to therapy in vivo in order to optimize and individualize therapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2044.

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Solid tumors are notorious for their ability to form lethal metastases, sometimes several decades following initial cancer diagnosis. Development of distant metastases is a result of the primary tumor shedding cells that travel via lymphatics and the blood to distant sites where they can form metastases. Platelets are known to specifically enhance tumor cells’ survival in the bloodstream by as yet poorly understood mechanisms. To study the interplay of platelets with circulating tumor cells, we implemented our published approach to label both circulating epithelial tumor cells and platelets. Blood samples were collected avoiding fixation from patients with non-metastatic cancer diagnoses and processed at 4 time points following blood collection. Circulating epithelial tumor cells were undetectable directly after blood collection but became visible after overnight storage at room temperature presumably due to release of platelets from the tumor cells. Our results suggest that platelets play a key role in masking circulating tumor cells. Masking may explain the difficulties in detection of these cells and prevention of their elimination by the immune system. Our unmasking approach detects sufficient numbers of circulating tumor cells to monitor the effect on blood tumor cells of different therapeutic measures, thus contributing to improved systemic therapies for cancer.