Abstract P2-02-07: Collection, high-resolution imaging, and single cell isolation of circulating tumor cells from patient derived xenograft models using the AccuCyte® – CyteFinder® system
Authors: Ramirez AB , Sahay D , Lewis MT , Schiff R , Stilwell JL , Trivedi M , Kaldjian EP 
Published: May 2016

Background: Patient-derived xenograft (PDX) models of human tumors offer many advantages over traditional cell line xenograft models and other mouse models of cancer. A PDX model may be used to randomize a given patient's tumor to multiple treatment regimens in order to predict treatment responses. When PDX models are grouped, they represent a clinical trial "cohort" for testing new therapies and identifying biomarkers of response. One such biomarker is circulating tumor cells (CTCs), which provide a window of the metastatic process. CTCs have been reported in several PDX models, further supporting their clinical relevance. Thus, PDX models may also be used to study the utility of CTC analysis to inform treatment decisions. However, most current CTC technologies intended for use with human samples cannot be used with the small blood volume from mice. The objective of our study was to adapt the AccuCyte® – CyteFinder® (AC-CF) system to detect CTCs from low volumes of mouse blood, and apply this method for the analysis of CTCs in a PDX model, including individual cell retrieval for molecular analysis. Methods: The AC-CF PDX process was modified to include a red blood cell lysis step instead of the density-based separation for the removal of red blood cells. The isolated cells were spread onto microscope slides using a stabilization solution, stained by multi-color immunofluorescence, and visualized by the CF high-resolution multi-channel fluorescence scanner. Automated image analysis identified CTCs, which was followed by single cell retrieval. For optimization of the assay, BT474 breast cancer cells were spiked into blood from a tumor-free control mouse ( approx. 500 cells in 250 µl). Slides with BT474 cells were used to test sensitivity by using antibodies against human cytokeratins (pan-CK), epithelial cell adhesion molecule (EpCAM), and erbB family growth factor receptors (EGFR and HER2) to detect the spiked-in cells. Assay specificity was tested by using antibodies specific for the mouse isoform of CD45. The antibody panel was tested on blood samples from 6 mice carrying small (300-400 mm3) tumors of the breast cancer PDX model (BCM-4888) previously published to have CTCs. Results: BT474 were identified by their large nuclei, positive staining with human specific antibodies against pan-CK, EpCAM, and EGFR/HER2 markers, and negative staining for mouse CD45. BT474 were detected in approximately the same amount as were spiked in. CTCs were identified in the blood of all 6 PDX mice tested. We found 1-6 CTCs per 330 µl of blood, and clusters of CTCs were also identified in 4 mice. Overall, these findings agree with published data on this PDX model. Single CTCs will be isolated using the CytePicker® retrieval module for single cell sequencing to confirm the human origin of these cells. These results along with ongoing work on additional PDX models will be presented at the meeting. Conclusion: The modified AC-CF process is a simple and sensitive method of analyzing small volumes of blood for CTC detection and isolation, features that are critical for the longitudinal analysis of CTCs in PDX models of cancer.

Citation Format: Ramirez AB, Sahay D, Lewis MT, Schiff R, Stilwell JL, Trivedi M, Kaldjian EP. Collection, high-resolution imaging, and single cell isolation of circulating tumor cells from patient derived xenograft models using the AccuCyte® – CyteFinder® system. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-07.