Blocking Autophagy Supercharges Lung Cancer Immunotherapy Response

Non-small cell lung cancer (NSCLC) remains one of the leading causes of cancer death globally, and while PD-1 checkpoint inhibitors have transformed oncology, only a minority of patients respond to monotherapy. Combining antiangiogenic tyrosine kinase inhibitors (TKIs) like anlotinib with anti-PD-1 therapy has improved outcomes in some cancers but has been less consistent in NSCLC — a gap this study set out to help explain. The researchers hypothesized that autophagy, a cellular self-recycling process triggered by TKIs in tumor-supporting cells, blunts the therapeutic effect, and that inhibiting it could restore efficacy.

The team used two immunocompetent mouse NSCLC models: LLC (Lewis lung carcinoma, known to be anti-PD-1 resistant) and LA795 co-implanted with mesenchymal stem cells (MSCs) to create a CAF-rich tumor environment. Mice received anlotinib, the autophagy inhibitor chloroquine (CQ), and/or anti-PD-1 antibody (specific dosing details are in the full methods). The triple combination — anlotinib + CQ + anti-PD-1 — produced the strongest tumor growth inhibition in both models, with tumor volumes significantly smaller than any doublet combination. The triple combination also extended survival compared with anlotinib + anti-PD-1 alone.

Immunohistochemical and immunofluorescence analyses of tumor sections revealed the mechanistic underpinning. In triple-treated tumors, CD8+ T cell infiltration was markedly increased while α-SMA+ CAFs and CD163+ M2 macrophages were substantially reduced. TUNEL staining confirmed elevated apoptosis of both CAFs and M2 macrophages. These findings suggest that CQ sensitizes CAFs and immunosuppressive macrophages to anlotinib-induced cell death, converting a cold immune microenvironment into a hot one permissive to T cell attack. Autophagy markers Beclin-1 and LC3-II were elevated in tumors treated with anlotinib alone, confirming that TKI treatment activates autophagy in vivo — and that CQ suppresses this survival mechanism.

In vitro, CAF models were established using bone marrow MSCs (the exact induction protocol is described in the full methods). Anlotinib dose-dependently inhibited AKT and mTOR phosphorylation in CAFs, inducing autophagy (measured by Beclin-1 and LC3-II levels). Adding CQ to anlotinib-treated CAFs significantly enhanced apoptosis (Bax upregulation, Bcl-2 downregulation) compared with anlotinib alone. Conditioned medium experiments showed that autophagy-intact CAFs strongly promoted M2 polarization of macrophages via secreted factors, while autophagy-inhibited CAFs lost much of this immunosuppressive activity — a key mechanistic link to the in vivo immune findings.

To isolate the specific contribution of CAF autophagy, the team constructed LLC tumor-bearing mice co-implanted with CAFs transfected with ATG5-targeting siRNA (silencing a critical autophagy gene) versus non-targeting control siRNA. Mice carrying ATG5-knockdown CAFs showed significantly better responses to anti-PD-1 therapy versus control siRNA mice — directly validating that CAF autophagy is a central regulator of the immunosuppressive TME. The study provides a mechanistic rationale for clinical trials combining autophagy inhibitors like hydroxychloroquine with antiangiogenic TKIs and PD-1 blockade in NSCLC.