Immune-cold NSCLC tumors harbor tumor-associated macrophages with elevated expression of immunoglobulin genes.

Abstract

In the non-small cell lung cancer (NSCLC) tumor microenvironment (TME), tumor cells mediate inhibitory signals to key immune cells, promoting an immunosuppressive environment that facilitates tumor immune evasion. CD8 + cytotoxic T lymphocytes are central mediators of antitumor immunity, and tumors can be classified by immunohistochemistry (IHC) as immune-hot or immune-cold based on the abundance of tumor-infiltrating lymphocytes (TILs). Although cancer immunotherapy can boost lymphocyte functions and improve progression-free survival in patients with advanced NSCLC, only a minority of patients experience a durable clinical benefit. Tumor-associated macrophages (TAMs) are key immunoregulatory cells in the NSCLC microenvironment and capable of generating potent immunosuppressive signals. However, their functional roles in immune-hot versus immune-cold tumors remain poorly understood. In this study, we compared the transcriptional programs of TAMs from NSCLC patients with immune-hot or immune-cold tumors. We classified 11 surgically resected NSCLC tumors as immune‑hot or immune‑cold based on quantitative immunohistochemistry of CD4⁺ and CD8⁺ tumor‑infiltrating lymphocytes. TAMs were isolated from tumor and adjacent healthy tissue by fluorescence-activated cell sorting (FACS), followed by bulk RNA sequencing and differential gene expression analysis. TAMs from immune‑cold tumors exhibited a striking upregulation of genes involved in immunoglobulin-mediated immune responses. Additionally, these TAMs demonstrated increased expression of genes involved in extracellular matrix organization, including matrix metalloproteinases and collagen‑associated genes, suggesting enhanced matrix remodeling activity. These findings highlight TAMs' potential contribution to immunosuppression, stromal remodeling, and impaired lymphocyte infiltration. The TAM-mediated pathways identified here may represent actionable targets for future immunotherapeutic strategies aimed at reshaping the tumor microenvironment.