The impact of POSTN on tumor cell behavior and the tumor microenvironment in lung adenocarcinoma

Abstract

Background: The role of cancer-associated fibroblasts (CAFs) in modulating the anti-tumor immune response in lung adenocarcinoma (LUAD) remains elusive, primarily due to the heterogeneous nature of these cells. This heterogeneity muddles the understanding of their impact on immunotherapy effectiveness.

Methods: We utilized the LUAD single-cell dataset to precisely classify tumor cells and CAFs. By employing CSOmap, we predicted cell interactions and reconstructed the three-dimensional spatial organization, highlighting the close association of myofibroblasts with specific tumor cell subsets. A prognostic signature based on myofibroblast-specific genes was developed and validated to predict LUAD patient survival. In vivo, we conducted subcutaneous tumorigenesis assays in mice, treating with PD-L1 and the POSTN inhibitor RGD to assess the combined effects of POSTN pathway blockade and immunotherapy on tumor growth and immune cell dynamics. For analyzing the tumor microenvironment, we used flow cytometry and multiplex immunofluorescence staining. In vitro, with cell lines like A549, H1299, and RAW264.7, we investigated POSTN's role in macrophage recruitment and polarization. Through ELISA, Western blot, and immunofluorescence staining, we explored how POSTN acts via ITGB3, providing a more comprehensive understanding of its mechanism in LUAD.

Results: Our analysis discerned six distinct tumor cell subsets, with cluster 1 displaying pronounced cellular communication with myofibroblasts, evidenced by spatial accessibility in three dimensions. The myofibroblast-specific genomic signature was established and confirmed as a robust, independent prognostic indicator. Among the signature genes, CTHRC1, POSTN, and MMP11 emerged as high-variant genes in myofibroblasts, identified via the FindAllMarkers function in Seurat. Of these, only POSTN's differential expression correlated with LUAD prognosis, with high POSTN expression being indicative of poor patient outcomes. In vitro, recombinant POSTN was observed to enhance tumor invasiveness, motility, and proliferation, while attenuating apoptosis and fostering an EMT phenotype. Additionally, Transwell assays showed that rPOSTN could induce macrophage infiltration via ITGB3 and drive M2 polarization via the PI3K-Akt-JNK pathway. Importantly, blocking the POSTN pathway augmented the efficacy of PD-L1 inhibitors. In vivo, in a mouse subcutaneous tumorigenesis model, the combination of POSTN pathway blockade with PD-L1 inhibitor treatment notably inhibited tumor growth and changed the tumor microenvironment's immune cell composition, with an increase in CD8+ T cells and a favorable shift in the M1/M2 macrophage ratio.

Conclusion: This study sheds light on the intricate interplay between tumor cells and myofibroblasts in LUAD, pinpointing the pivotal role of the highly mutated gene POSTN. It underscores POSTN's instrumental role in manipulating the tumor microenvironment, primarily by promoting EMT and inhibiting apoptosis in lung cancer cells, alongside enhancing macrophage recruitment and fostering M2 polarization. These insights provide a foundation for enriching immunotherapy strategies, particularly through the inhibition of the POSTN pathway in LUAD.

Keywords: Immunotherapy; Lung adenocarcinoma; Macrophages polarization; Myofibroblasts; POSTN.