Figure 4: Primary nuclear rupture and lamin expression are correlated with GBM tumor cell states.

A, scRNA-seq from 28 adult and pediatric GBM (n=24,131 cells) showing lamin RNA expression by GBM cell state. *p<0.05; t-test, two-sided, unpaired. B, Plots of scRNA-seq data showing putative markers of each cell state in GBM (*p<1x10⁻¹⁶) (t-test, two-sided, unpaired). C, Schematic of t-CyCIF experiment and markers to explore rupture and lineage correlations (generated with BioRender). D, Representative GBM t-CyCIF images showing cell state marker expression in distinct tumor cell populations, with reduced lamin A/C expression and increased PN rupture in NPC-like cells (DCX, ELAVL4) (white arrowheads) compared with NDRG1-expressing MES-like cells (white arrows) and HOPX-expressing AC-like cells (red arrowheads). TMA core image with ROI indicated (upper left). E, Two-dimensional representation of GBM cell states from t-CyCIF. Each quadrant represents a cell state (based on Neftel et al.), with position reflecting the relative score and indicated cell state scores for each cell and intensity values for the indicated markers. F, Composite representation of cell state groups in scRNA-seq data for validated markers. G, Plot of scores for each cell state mapped to individual tumor cells, showing an excellent correspondence of cell state designations at a single-cell level in t-CyCIF data. H, Correlation of manual gating of marker intensity and designation of cells according to core cell state markers and mapping of continuous cell state data. I, mapping of lamin A/C levels by cell state score shows that NPC-like cells exhibit the lowest lamin A/C expression. J, Quantification of PN rupture rate in t-CyCIF TMA data normalized to cell count. K, Quantification of mean nuclear Ki-67 expression in different cell states shows significantly higher proliferation rates in NPC-like cells compared to other populations (p<1x10⁻⁵⁰ for all comparisons). L,M, Quantification of cell state markers in 82 primary IDH-WT GBM comparing cells with ruptured and unruptured PN (L) and MN (M), showing enrichment of NPC cell state markers and EGFR in ruptured cells compared to the mean of all markers (**p<0.01) (t-test, two-sided, unpaired). N, two-dimensional mapping of EGFR expression by cell state shows that while AC-like cells exhibit the strongest overall expression, a secondary cluster of NPC-like cells also exhibits EGFR expression.