(P 365) MULTIMODAL ANALYSIS OF NUCLEAR AND GENOMIC ORGANIZATION IN RHABDOMYOSARCOMA AND NORMAL MYOGENIC CELLS

Objective: Rhabdomyosarcoma (RMS), the most common soft-tissue pediatric sarcoma, is a tumor of skeletal muscle. A specific PAX-FOXO gene fusion is present in a subset of RMS tumors. The gene fusion status of RMS has significant effects on cellular biology and affects clinical outcomes. Myogenesis is the process by which proliferative myoblasts terminally differentiate into myotubes. As a tumor of skeletal muscle, RMS has been suggested to represent an arrested state of development between myoblast and myotube, resulting in continued proliferation.
Though RMS and normal myogenic cells share many similarities, such as gene expression and genome-wide binding patterns of myogenic regulatory factors, similarities and differences in higher-order chromatin structure and nuclear organization are less understood. Partial wave spectroscopic (PWS) microscopy can provide a quantitative assessment of chromatin heterogeneity in both individual and populations of live cells. We have recently shown myogenesis-dependent changes that are absent in RMS cells of the organization of specific chromosomes using fluorescent in situ hybridization.

Methods: To compare higher-order chromatin organization and nuclear organization within normal and RMS cells, normal myocytes and cell culture models of RMS were used. Normal proliferating myoblasts and terminally differentiated myotubes were compared to both PAX-FOXO fusion positive and negative RMS cell lines: fusion negative RD and fusion positive RH30 cells. Cells were imaged on glass bottomed dishes imprinted with a cell location grid enabling the tracking of specific nuclei throughout the imaging process. After live-cell PWS quantification of specific nuclei, cells were fixed, and fluorescence in-situ hybridization (FISH) for chromosomes 2 and 18 was performed. In addition, ATAC-Seq, RNA-Seq and Hi-C was performed in these cell types to characterize DNA accessibility, gene expression and DNA-DNA interactions, respectively.

Results: Initial analysis of population-level live-cell PWS data demonstrated statistically significant differences between all four cell types, with myotubes exhibiting the highest degree of heterogeneity, followed by myoblasts, then RDs, and finally RH30s (Figure 1). Imaging after FISH was performed has demonstrated that we can visualize specific chromosomes in individual cells of all cell types and analyze chromosomal organization data in the context of cell-specific PWS measurements.

Conclusion: Our initial analysis demonstrates that the terminally differentiated normal myotubes exhibit greater measures of chromatin heterogeneity compared to the proliferative myoblasts on the population level, potentially representing the significant changes in gene expression that accompany the morphological and cell cycle changes seen with differentiation. Interestingly, despite the known chromosomal complexity of the RMS tumor cell lines, they actually exhibited a lower measured level of heterogeneity compared to normal cells, with the fusion-positive tumor cells exhibiting the lowest level of all analyzed cell types. Further work will relate next-generation sequencing results with the imaging analysis of these cells.