Single-cell transcriptomics reveals the landscape of immune phenotypes in pituitary neuroendocrine tumors

<h4>Background</h4>Immune cells critically influence pituitary neuroendocrine tumor (PitNET) progression and therapeutic responses. However, their composition and functional dynamics remain unclear. This study aimed to delineate the immune heterogeneity and intercellular communication networks within PitNETs.<h4>Methods</h4>This study conducted single-cell RNA sequencing (scRNA-seq) on 22 fresh PitNET samples obtained from surgical patients at Beijing Tiantan Hospital between September 2023 and January 2024, with classification based on the expression of key transcription factors: pituitary-specific transcription factor 1 (PIT1), steroidogenic factor 1 (SF1), and T-box transcription factor 19 (TPIT). Following cell type identification, cell-cell communication analysis revealed specific intercellular interactions, which were validated by multiplex immunohistochemistry (mIHC), flow cytometry, and in vitro co-culture assays.<h4>Results</h4>The scRNA-seq analysis revealed significant cellular heterogeneity and a lineage-specific immune landscape, including cancer-associated fibroblasts (CAFs), neutrophils, T cells, natural killer cells, and myeloid cells. Specifically, CAF subset distribution was highly lineage-dependent. Collagen-expressing CAF3 was significantly enriched in the PIT1 lineage compared to both TPIT and SF1 lineages. In contrast, inflammatory CAF5 was predominantly found in the TPIT lineage relative to the PIT1 and SF1 lineages. Furthermore, CD4 + regulatory T (Treg) and T follicular helper (Tfh) cells were significantly enriched in the PIT1 lineage relative to the TPIT and SF1 lineages, as validated by both flow cytometry and mIHC data. Cellular communication analysis revealed notable interactions between Treg/Tfh cells and macrophages/microglia that were mediated by the cytotoxic T-lymphocyte-associated protein 4 (CTLA4)-CD86 pathway. Subsequent mIHC assays confirmed spatial colocalization of Treg cells with macrophages/microglia. Complementing these findings, in vitro co-culture assays demonstrated functional CTLA4-CD86 signaling specifically between Treg cells and macrophages, providing deeper insights into the complex cellular crosstalk within the PitNET tumor microenvironment (TME).<h4>Conclusions</h4>The three major PitNET lineages exhibited distinct immune cell frequencies, with the PIT1 lineage notably enriched in Treg and Tfh cells, as well as collagen-producing CAF3. Furthermore, a novel immunosuppressive interaction between Treg cells and macrophages, mediated by the CTLA4-CD86 pathway, was identified, suggesting its potential regulatory role within the TME.