Abstract 2985: DNA-directed patterning as a tool to study prostate cancer-bone marrow niche interactions at single cell and system level

Transforming cancer from a terminal diagnosis to a chronic or curable disease rests on predicting and preempting disease trajectory. This is particularly true in prostate cancer, in which a disseminated tumor cell (DTC) may reside in the bone marrow and remain dormant for years, only to awaken into metastatic cancer. Current in vitro and in vivo systems are incapable of uncovering the full network of interactions between DTCs and the microenvironment, as they lack either the complexity or the ability to recapitulate accurately human disease progression. By harnessing the power of an innovative DNA-directed cell patterning method, high-throughput, combinatorial single-cell studies that range from probing the individual contributions of different microenvironmental cell types to creating in vitro microenvironments of increasing complexity can be performed to study the conditions that lead PCa cells to remain dormant or to proliferate. To investigate individual contributions of the different bone cell types found to influence the proliferative or dormant tendencies of prostate cancer cells, DNA-directed patterning was used to create large-scale arrays of different combinations of single microenvironment cells (e.g. osteoblasts, osteoclasts, osteocytes, endothelial cells, immune cells) and prostate cancer cells. Briefly, different amine-terminated 20-nucleotide sequences were patterned on slides using photolithography. Cells were then tagged with the complimentary oligo and introduced to DNA-patterned regions, whereupon hybridization leads to the patterning of the cells. This technique was used to localize single bone cells (MC3T3 and OCT454 representing osteoblasts and osteocytes, respectively), cells of hematopoietic lineage (RAW264.7 to represent macrophages and osteoclasts), or endothelial cells (HUVEC) in proximity to prostate cancer cells (PC-3). Immunofluorescence staining determined cellular changes. An in vitro niche was then fabricated and validated for the ultimate use of examining the broader system of interactions between the cells of the bone marrow and prostate cancer cells. Cellular phenotypes and behavior were verified using RNAscope (in situ hybridization) and immunofluorescence staining. Finally, a fluorescently-labeled prostate cancer cell line (PC-3) was patterned along with bone cell types and imaged over the course of several days to assess proliferation, thereby determining the microenvironment influence on these cells. A typically high