(F-222) Spatially Resolved Cellular Senescence Mapping in Healthy and NASH Human Liver

Introduction:: Cellular Senescence (SNC) is a fail-safe mechanism of cell proliferation inhibition in response to various cell stressors, including DNA damage, telomere dysfunction, oncogene activation, and organelle stress. Senescent cells in general and in non-alcoholic steatohepatitis (NASH) exhibit 4 hallmarks, including prolonged and generally irreversible cell cycle arrest, macromolecular damage, secretory features, and deregulated metabolism. Nuclear DNA damage, as the primary senescence establishment, activates a signaling cascade defined as DNA damage response (DDR). DDR response is characterized by phosphorylated histone H2AX (γH2AX), 53BP1 and MDC1. Prolonged DDR activation produces senescence features including cell cycle arrest that results in upregulation of p21 and p16 cell cycle inhibitors. Moreover, the resistance of these cells to apoptosis, upregulates metabolic changes (including senescence-associated-β-galactosidase (SA-β-gal) accumulation), a senescence-associated secretory phenotype (SASP) and high mobility group protein 1(HMGB1). Here, we spatially profile the epigenomic and proteomic landscape of senescence cells in healthy and NASH human liver using transposase-accessible chromatin sequencing (spatial-ATAC-seq) and spatial CITE-seq.


Materials and Methods:: Tissue sections of two fresh frozen (FF) tissue block in OCT from NASH and healthy human liver were obtained. Tissues were fixed using polymer form of formaldehyde (PFA). In spatial ATAC-seq, Tn5 transposition, and in spatial CITE-seq, cocktail of antibodies with their primers were applied in bulk on the tissue. Next, deterministic barcoding in tissue (DBiT-seq) device was assembled on top of the tissue to cross flow barcodes A (1-50) and B (1-50) in ligation buffer through microchannels. Finally, tagmented DNAs and barcoded proteins were captured by incubating the tissue in reverse crosslinking buffer with proteinase K overnight. The next day, a library was generated using Nextera Illumina kit and sent for the sequencing. The data were analyzed using a combined ArchR and in-house pipeline.


Results, Conclusions, and Discussions:: Results and Discussion: Spatial ATAC-seq and spatial CITE-seq were applied on both healthy and NASH human liver. Four senescence associated markers including γH2AX, P21, P16 and HMGB1 were investigated in each experiment. The chromatin accessibility of transcription factors (TF) of four SNC associated markers (H2AFX, CDKN2A, CDKN1A, and HMGB1) were spatially mapped in zones 1 and 2. An immune-fluorescent (IF) image of CYP2E1 (in red) and nucleus (in blue) validated zone 2 in the ROI of FF healthy human liver (Fig1.b). A beta gal staining image of FF NASH human liver defined the ROI of spatial CITE-seq (Fig1.c) and four SNC associated proteins were successfully mapped on NASH liver. Spatial ATAC-seq also mapped chromatin accessibility of four SNC associated transcription factors in the region of abundant P21 protein on NASH human liver.



Conclusion: The chromatin accessibility of four senescence associated transcription factors (H2AFX, CDKN2A, CDKN1A, and HMGB1) were mapped in zones 1 and 2 which indicated presence of senescence cells close to the portal veins and their relationship with stellate cells and hepatocytes in this region. On the other hand, the spatial ATAC-seq data visualized HMGB1 and CDKN1A in one spatial cluster while markers of H2AFX and CDKN2 appeared in another distinct clusters indicating their correlation with specific cell types. More investigation such as spatial transcriptomics and higher resolution of protein visualization using CODEX technology will be utilized to find the relation of these four SNC associated markers and their pathways related to NASH associated liver disease.




Acknowledgements (Optional): :

References (Optional): :