Tumor Treating Fields (TTFields) sensitize anti-PD1-resistant lung tumors to PD1 blockade and remodel myeloid and lymphoid states

Abstract

Background Primary resistance to programmed cell death protein 1 (PD1) blockade is sustained by defective antigen presentation, immunosuppressive myeloid programs, regulatory T-cell (Treg) dominance, and dysfunctional lymphocyte states. Tumor Treating Fields (TTFields) are non-ionizing alternating electric fields with established antiproliferative activity; however, whether they can overcome resistance to immune checkpoint blockade remains unclear. Methods The antitumor and immunomodulatory effects of TTFields were investigated in anti-PD1-resistant 344SQR murine lung adenocarcinoma cells and syngeneic tumors. Cells were exposed to TTFields for 24, 48, or 72 h and evaluated for proliferation, clonogenic survival, and transcriptional pathway changes. Tumor-bearing mice received control treatment, anti-PD1, matched heating control, TTFields, or their combinations. Tumor growth, survival, and pulmonary metastatic burden were assessed, and tumor-infiltrating immune populations were characterized by single-cell RNA sequencing and T-cell receptor clonotype analysis. Results TTFields suppressed tumor-cell proliferation and clonogenic survival in a time-dependent manner, with maximal inhibition at 48 h and partial recovery at 72 h. This biphasic response coincided with early suppression followed by reactivation of cell-cycle, metabolic, and interferon-related transcriptional programs. Anti-PD1 monotherapy showed minimal activity in vivo, whereas TTFields combined with anti-PD1 delayed tumor growth, reduced pulmonary metastases, and prolonged survival. Single-cell analysis showed that TTFields decreased Treg abundance, increased the CD8+ T-cell/Treg ratio, and shifted tumor-associated macrophages toward antigen-presenting and inflammatory states characterized by increased Cd86, H2-Ab1, Nos2, and Tnf and decreased Mrc1 and Tgfb1 expression. Combination treatment enriched Gzmk-dominant CD8+ T-cell and natural killer T-cell states, reduced Pdcd1 and Ctla4 expression, and promoted focused expansion of dominant T-cell clonotypes. Conclusions TTFields sensitized anti-PD1-refractory lung tumors to PD1 blockade and induced coordinated remodeling of tumor-cell fitness and the tumor immune microenvironment. These findings identify TTFields as a potential tumor-conditioning strategy for overcoming resistance to immune checkpoint blockade.