A SORT1-associated transcriptional signature links adipogenic plasticity, stemness and drug-resistance programs in 3D glioblastoma spheroids: dissection of ligand-dependent and ligand-independent arms

Three-dimensional (3D) culture of glioblastoma (GBM) cells recapitulates tumour-niche features lost in 2D monolayers, including stemness, metabolic plasticity and therapy resistance. By creating hydrophobic compartments that sequester lipophilic chemotherapeutics, increased adipogenic reprogramming and lipid synthesis correlate with poor treatment response and increased drug tolerance in GBM. Sortilin (SORT1), a Vps10p-domain sorting receptor involved in lipoprotein handling and receptor trafficking, has been implicated in cancer stem cell biology and chemoresistance. Here, we used focused PCR arrays to investigate whether SORT1 links lipid remodeling to a chemoresistance-associated GBM molecular signature by comparing U87 GBM cells grown as 2D monolayers or 3D spheroids treated with non-targeting siRNA (siScr), SORT1-targeted siRNA (siSORT1), or the Vps10p-domain antagonist AF38469. Upregulation of angiogenic markers (VEGFA, HIF1A), adipogenic/lipogenic regulators (PPARG, DDIT3, ADIPOQ, CEBPD, FOXO1), and GBM stemness/drug-resistance markers (PROM1/CD133, SOX2, NANOG, CD44, ABCG2, ABCB5, TAZ, KITLG), indicated that 3D growth activates a lipid program with chemoresistance-linked cancer stem cells (CSC) features. Both siSORT1 and AF38469 reduced spheroid size and repressed the adipogenic module, supporting a role for SORT1 in regulating adipogenesis and lipid-synthesis programs in GBM spheroids. In contrast, the CSC module was more sensitive to SORT1 silencing (18/27 genes) than to AF38469 treatment (10/27 genes), with CXCL8, CD38, ZEB2, SOX2, NANOG and CD44 emerging as siSORT1-dependent but AF38469-insensitive transcripts. Principal component analysis confirmed this dual-module organization as siSORT1 and AF38469 jointly displaced the adipogenesis profile, whereas only siSORT1 clearly separated the CSC profile. These findings are consistent with a model in which SORT1 behaves as a state-persistence node associating adipogenic/lipogenic reprogramming with CSC-associated and drug-resistance-associated transcriptional programs, a model that remains to be tested with direct functional assays in 3D U87 spheroids. SORT1 functions appear to segregate into a ligand-dependent arm targetable by AF38469 and a ligand-independent scaffold-like arm requiring SORT1 expression. Strategies that can circumvent the acquired SORT1-regulated CSC/drug resistance phenotype are discussed. 3D spheroid culture of U87 glioblastoma cells induces a coordinated adipogenic and stem cell transcriptional program. SORT1 pharmacological and genetic perturbations reveal a bifurcation of the SORT1-associated transcriptional interactome relevant to glioblastoma drug-resistance-associated transcriptional programs. The SORT1-dependent transcriptional state is organised into six functionally coherent modules, providing a modular blueprint for combinatorial therapeutic targeting. Module-level scoring supports, at systems level, that the bifurcation operates on entire transcriptional modules - lipid/metabolism + drug-resistance machinery as a co-regulated ligand-dependent arm, and stemness + plasticity + developmental backbone as a scaffold-dependent, persistence arm. An AF38469 phenocopy (mimicry) index provides quantitative support for the bifurcation as the pharmacological inhibitor reproduces the SORT1 genetic-silencing effect nearly perfectly on the lipid/metabolic module but fails to reproduce it on the developmental backbone.