Macrophage-secreted factors promote invasiveness in somatotroph PitNETs: Cathepsin Z as a macrophage-associated...

Invasive somatotroph pituitary neuroendocrine tumors (PitNETs) exhibit poor clinical outcomes due to frequent recurrence and treatment resistance. The molecular mechanisms underlying their invasiveness remain incompletely understood. Cathepsin Z (CTSZ), a lysosomal protease highly expressed in tumor-associated macrophages (TAMs), promotes malignancy in various cancers. Our prior proteomics identified CTSZ overexpression in invasive somatotroph PitNETs correlating with adverse prognosis. This study aims to elucidate the mechanistic role of CTSZ in promoting tumor invasiveness and its interaction with TAMs.

Multiplex immunofluorescence determined CTSZ distribution in human somatotroph PitNET tissues. Functional assessment employed macrophage-conditioned medium co-culture with GH3 cells to evaluate proliferation, hormone secretion, and morphological changes. Mechanistic investigations were conducted through integrated transcriptomic profiling (RNA-seq) followed by targeted validation of identified pathways using RT-qPCR and Western blot analyses. Recombinant CTSZ protein was used to assess the direct effects of CTSZ on GH3 cells.

Pan-cancer data analysis revealed that CTSZ expression correlates with macrophage infiltration across multiple human cancers, providing exploratory background for this study. In somatotroph PitNETs, CTSZ expression co-localized with CD68⁺ macrophages and showed a positive correlation with the M2-like macrophage marker CD163 (R² = 0.422, P = 0.036). In vitro, treatment with Raw264.7 macrophages-conditioned medium induced morphological alterations and enhanced the proliferative, migratory, and invasive capacities, as well as GH secretion, of GH3 cells. However, recombinant CTSZ protein alone did not significantly affect GH3 cell proliferation or GH secretion. Macrophage-derived factors reduced autolysosome abundance and downregulated LC3-II expression, indicating modulated autophagic activity. Mechanistically, RNA-sequencing analysis suggested the involvement of the transforming growth factor-β (TGF-β) signaling pathway. Specifically, downregulation of Decorin (Dcn) attenuated its inhibitory effect on Tgf-β1, while reduced expression of the cell cycle inhibitor p15 released its suppression of cyclin-dependent kinase 4 (Cdk4), collectively promoting tumor progression.

Macrophage-secreted effector molecules collectively drive somatotroph PitNET progression through coordinated regulation of autophagy and TGF-β signaling. Although recombinant CTSZ alone did not directly enhance GH3 cell proliferation or GH secretion, CTSZ serves as a macrophage-associated marker of invasiveness. These findings reveal the macrophage-tumor microenvironment as a potential therapeutic target.