This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15- CA-117: Tumor dormancy. Prostate cancer (PCa) metastasizes to the bone in ~90% of patients with advanced disease. We have reported dormant disseminated tumor cells (DTC) are present in 45% of patients with no evidence of disease (NED) >5 years after radical prostatectomy (RP). These dormant DTC represent a nidus for disease recurrence and their activation can lead to the replacement of bone marrow (BM), spinal cord compression, severe bone pain, cachexia and death. We hypothesize that by comparing the molecular profile of dormant and active DTC we will gain insight into dormancy and dormant cell activation. To test this hypothesis, we will use dormant tumor cells from our LuCaP PCa xenografts and dormant DTC from patient BM.
Our aims are (1) activate dormant DTC by altering the microenvironment in microfluidic chambers and then restore dormancy, and (2) characterize the transcriptome of dormant cells at a single cell level in the BM. To activate dormant tumor cells, we will use (a) LuCaP xenograft cells that are cytostatic in culture and (b) dormant DTC from patients who are NED >5 years post RP. Cells will be placed in microfluidic chambers coated with extracellular matrix from reactive BM stromal cells, and using conditioned media, we will stimulate proliferation and then reverse the process. Since BM aspirates from NED patients only yield ~10-20 DTC, these studies are only possible using microfluidics. The transcriptome of both the xenograft cells and DTC will be analyzed using AgilentTM arrays, real-time PCR, and statistical analyses. Next, we will amplify and array RNA using a population of 1, 5, 10, and 25 C4-2B cells in replicates of 10, to determine the representation, fidelity, and reproducibility of using one cell for AgilentTM array analysis. We will then apply the technique to amplify and profile the transcriptome of ten single DTC from BM aspirates of each of 5 patients with advanced metastatic disease and from each of 5 NED patients >5 years post RP. Data from these 100 single cell arrays will determine DTC heterogeneity and identify dormancy related genes. The individual DTC will be profiled by AgilentTM arrays using single cells or in pools of 10 DTC or less if the single cell methodologies don't show high fidelity. These data will be analyzed in combination with the array data from the aforementioned microfluidic assays and patient DTC. We expect that these data will advance our understanding of tumor cell dormancy in the BM of patients with PCa.
Prostate cancer metastasizes to the bone in approximately 90% of patients with advanced disease and dissemination of tumor cells to the bone can lead to replacement of bone marrow, spinal cord compression, fracture, severe bone pain, cachexia and death. The long latency period that occurs in some patients between initial treatment and evidence of metastases is attributed to tumor cell dormancy. These dormant disseminated tumor cells represent a nidus for disease recurrence and studying tumor cell dormancy will significantly impact our ability to, understand, identify and target tumor cells in individuals with no evidence of disease that may recur at a later point in time.
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