A PTN+ glioblastoma subclone orchestrates immune reprogramming to drive distant recurrence

Distant recurrence (DR) represents a distinct and lethal trajectory in glioblastoma (GBM), characterized by the colonization of spatially isolated neural niches. The mechanisms by which disseminated tumor cells remodel the microenvironment to overcome anatomical and immunological barriers remain poorly understood. This study aimed to decode the immune ecosystem of DR and elucidate the intercellular crosstalk driving this lethal dissemination.

We performed single-cell RNA sequencing (scRNA-seq) on paired primary and DR tumor specimens from GBM patients, alongside an independent local recurrence (LR) control cohort, to map dynamic cellular shifts. Bioinformatic analyses, including differential abundance testing (Milo), cell-cell communication inference (CellChat), and copy number variation (CNV) analysis, were integrated with clinical co-immunoprecipitation (Co-IP) validation, bidirectional in vitro co-culture assays, and an orthotopic xenograft mouse model.

Our high-resolution atlas revealed a profound remodeling of the microenvironment in DR lesions, characterized by a specific expansion of anti-inflammatory monocyte-derived tumor-associated macrophages (Anti_infl-TAM) and a paradoxical co-enrichment of CD8 + T cells and Tregs. Mechanistically, an OPC-like subpopulation of PTN+ tumor cells acted as a "paracrine architect," promoting Anti_infl-TAM polarization through the interaction between PTN and NCL. Co-IP assays confirmed direct physical binding of these two proteins in human DR tissues. Functional and bidirectional knockdown assays demonstrated that decoupling this cellular communication, achieved by targeting either PTN in tumor cells or NCL in macrophages, significantly attenuated glioma cell proliferation, migration, invasion, and macrophage polarization. In vivo, silencing PTN markedly reduced Anti_infl-TAM infiltration, suppressed intracranial tumor burden, and prolonged overall survival in tumor-bearing mice.

This study establishes that DR GBM is defined by a reconfigured immune niche rather than mere genetic drift. The paracrine crosstalk between PTN+ tumor cells and Anti_infl-TAM serves as a critical engine of this permissive soil, offering a promising therapeutic target to disrupt the supportive microenvironment in distant recurrent GBM.