Single cell RNA sequencing of dormant and proliferating prostate cancer cells from murine bone metastases

Using the immunocompetent bone-metastatic RM1 murine model of prostate cancer, the transriptomes of proliferating (PKH26-) eCFP+/luc2+ RM1 single cells (referred to as RM1536) were compared to dormant (PKH26+) RM1536 single cells dervied from bioluminescence imaging-verified bone metastases in mice at ~d16 post-intracardiac injection of PKH26 labelled RM1536 cells Overall design: Following intracardiac injection of PKH26-labeled RM1 cells expressing cerulean (eCFP) and luciferase (luc2; identified as RM1536) into C57BL/6 mice, dormant (occurring at a rate of ~2-6 in 3x10e7 bone marrow cells in ~1 in 8 tumor-bearing mice) and proliferating single RM1 cells were FACS-isolated from bone metastases ~16 days post-injection. Single cell RNA-seq of 28 dormant (PKH+) and 32 proliferating (PKH-) cells was performed and BASiCS normalisation employed. Pre- and post-normalization of all samples revealed no significant batch effects and potential technical noise was corrected after normalization. Confirmation of RM1 markers (eCFP and luc2) demonstrated the authenticity of more than 83% of single tumor cells from bone. Of 2410 differentially expressed genes in dormant cells, 1609 were identified by BASiCs as having no residual over-dispersion within dormant and proliferating groups, validating the absence of spurious signals in mean gene expression shifts. Stratification of DEGs enriched in dormant cells compared to proliferating cells isolated from the same hosts (counts appear under the file name 4_mice_of_interest, indicating dormant and proliferating cells derived from the same host, with 4 independent hosts included in analysis ) confirmed that differential gene expression did not result from unequal representation of dormant cells derived from a single mouse and proliferating cell from a separate host. Rather they encompass unbiased population-level changes evident across multiple tumor-bearing animals. Dormant (PKH+) and proliferating (PKH-) RM1 cells were isolated from 8 independent animals at different time-points of metastasis ~16 days post-intracardiac injection across independent experiments.