Chromosomal instability (CIN) is the persistent reshuffling of cancer karyotypes via chromosome mis-segregation during cell division. In cancer, CIN exists at varying levels that have differential effects on tumor progression. However, mis-segregation rates remain challenging to assess in human cancer despite an array of available measures. We evaluated measures of CIN by comparing quantitative methods using specific, inducible phenotypic CIN models of chromosome bridges, pseudobipolar spindles, multipolar spindles, and polar chromosomes. For each, we measured CIN fixed and timelapse fluorescence microscopy, chromosome spreads, 6-centromere FISH, bulk transcriptomics, and single cell DNA sequencing (scDNAseq). As expected, microscopy of tumor cells in live and fixed samples correlated well (R=0.77; p<0.01) and sensitively detect CIN. Cytogenetics approaches include chromosome spreads and 6-centromere FISH, which also correlate well (R=0.77; p<0.01) but had limited sensitivity for lower rates of CIN. Bulk genomic DNA signatures and bulk transcriptomic scores, CIN70 and HET70, did not detect CIN. By contrast, single-cell DNA sequencing (scDNAseq) detects CIN with high sensitivity, and correlates very well with imaging methods (R=0.83; p<0.01). In summary, single-cell methods such as imaging, cytogenetics, and scDNAseq can measure CIN, with the latter being the most comprehensive method accessible to clinical samples. To facilitate comparison of CIN rates between phenotypes and methods, we propose a standardized unit of CIN: Mis-segregations per Diploid Division (MDD). This systematic analysis of common CIN measures highlights the superiority of single-cell methods and provides guidance for measuring CIN in the clinical setting.

BackgroundCancer associated fibroblasts (CAFs) promote tumorigenesis by secreting immunosuppressive cytokines, stimulating angiogenesis, and supporting the growth of tumor cells. Through their interactions with immune cells, CAFs are known to directly impact the functionality of T cells and macrophages. However, CAF interaction with dendritic cells (DCs) and DC progenitor cells and its impact on DC function is relatively understudied and was the main focus of this study.MethodsTwo types of coculture systems were used in this study. For the human system, fibroblasts from lung squamous cell carcinoma (LUSC) were cocultured with MUTZ3 cells (hematopoietic progenitor cells) in the presence of DC differentiation stimuli, sometimes followed by DC maturation stimuli. For the mouse coculture system, activated (YPSC-c) and inactivated (PSC-b) pancreatic stellate cells (PSCs) were isolated from the pancreas of C57BL/6 mice by the density gradient method and co-cultured in the presence of bone marrow cells in the presence of DC differentiation and maturation stimuli. For human tumor antigen processing and cross presentation assay MART1 peptide (10mer and 20mer) was used.ResultsCo-culture of human and murine hematopoietic progenitor cells with fibroblasts (human LUSC CAFs and murine PSC results in decrease in differentiation and maturation of DCs. DCs differentiated and matured in the presence of fibroblasts have impaired ability to process and present tumor antigen to T cells. In the presence of PSC fibroblasts DC differentiation from murine bone marrow cells is skewed more towards MDSC and macrophages. In contrast to inactivated PSC-b, activated PSC-c influence DC differentiation in a contact dependent manner. Furthermore, PSC-b and PSC-c show transcriptionally distinct signatures which translate to unique secretory profiles as measured by Luminex. Analysis of the conditioned media from the coculture demonstrated that PSC-c secrete (among others) CXCL1, IL6, and CCL5 chemo/cytokines. These and other factors may play an important role in mediating fibroblast induced suppression of DC differentiation from monocytes.ConclusionsOur study demonstrates that cancer associated fibroblasts, or their precursors directly impact DC differentiation and antigen presentation via cytokines that could be targeted therapeutically to improve DC expansion and activity in the tumor microenvironment.