Abstract 4109: Unbiased detection of disseminated tumor cells in murine bone marrow
Authors: Valkenburg Kenneth C. , Hernandez James R. , Amend Sarah R. , Verdone James E. , Gorin Michael A. , Pienta Kenneth J. 
Published: July 2016

Approximately 20-30% of prostate cancer patients develop disease recurrence and metastasis years after initial therapy. This is thought to be largely due to the presence of growth-arrested and chemoresistant disseminated tumor cells (DTCs) in secondary sites, such as bone. Bone metastasis is found in 90-100% of prostate cancer patients who succumb to the disease. There are still many gaps in knowledge about the biological mechanisms by which DTCs home to bone, resist chemotherapy, become dormant, and escape dormancy to grow into clinical metastases. As such, it is important to be able to detect, quantify, and study bone marrow DTCs. In particular, it must be possible to do this in metastatic cancer mouse models, which are critical to study the process of tumor dissemination. DTC detection techniques currently exist, usually as either a positive selection or negative selection methodology. Positive selection techniques use markers or cell size to isolate and purify tumor cells out of the bone marrow. Positive selection markers are generally epithelial-specific, such as EpCam, E-cadherin, or Cytokeratin, and therefore may miss cells that lose epithelial marker expression and may gain mesenchymal markers. DTCs can also be as small as or smaller than white blood cells, meaning that positive selection based on size may miss some DTCs. Negative selection enriches for DTCs by removing blood and bone marrow cells from the population, usually using cell-specific markers. A popular strategy is CD45-based depletion, which removes white blood cells, and theoretically leaves behind DTCs. In our hands, this strategy causes loss of DTCs in the depletion process. To capture these heterogeneous and rare DTCs, we have developed a strategy to detect DTCs in murine bone marrow in an un-biased manner. The procedure entails removal of the bone marrow via centrifugation from the long bones (femur and tibia) of mice that have been injected with cancer cells (the injection site may vary depending on the experimental setup). The bone marrow then undergoes red blood cell lysis, and further centrifugation. The white blood cells are then counted, and the bone marrow is spread onto glass slides. The cells on the slide are fixed, permeabilized, and stained (immunofluorescence and RNA fluorescent in situ hybridization can be used). The staining can include any type of marker, including epithelial, mesenchymal, disease-specific, species-specific, or other biologically interesting markers, such as cell cycle markers. The unbiased nature of this procedure is based on the lack of positive or negative selection based on cell size or protein expression. Some DTC loss is noted in this protocol, due to the centrifugation and staining steps, but the cell population on the slide should include all DTC types. Notably, this protocol can be used to detect human or mouse cells in the mouse bone marrow and can thus be used in immune-compromise and immune-competent mouse models of metastasis.

Citation Format: Kenneth C. Valkenburg, James R. Hernandez, Sarah R. Amend, James E. Verdone, Michael A. Gorin, Kenneth J. Pienta. Unbiased detection of disseminated tumor cells in murine bone marrow. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4109.