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Page 8 of 28 Cheng et al. Cancer Drug Resist. 2025;8:46

Several signaling pathways contribute to hypoxia-induced checkpoint upregulation, including signal
transducer and activator of transcription 3 (STAT3), NF-κB, TGF-β, and elf5B. Under hypoxia, pyruvate
kinase M2 phosphorylates STAT3, and loss of von Hippel-Lindau protein further activates it. Activated
STAT3 inhibits HIF-1α degradation and promotes its biosynthesis [102] . In the hypoxic microenvironment,
phosphorylated STAT3 interacts with PD-L1, facilitating its nuclear translocation. Nuclear PD-L1 acts as a
transcriptional mediator, upregulating immunosuppressive molecules such as PD-L1 and PD-L2 [103,104] .
NF-κB is also activated under hypoxia, leading to enhanced expression of PD-L1 and other immune
checkpoint molecules through regulation of proinflammatory cytokines such as TNF-α and IL-6 [105] . Both
STAT3 and NF-κB can directly bind the PD-L1 promoter to induce transcription [106] . Hypoxia amplifies
TGF-β activity, which promotes PD-L1 expression via PI3K/AKT-mediated suppression of glycogen
synthase kinase 3β, reducing PD-L1 degradation . eIF5B, enriched under hypoxia, enhances PD-L1 mRNA
[107]
translation [108,109] . HIF-1 also amplifies HLA-G transcription and protein synthesis via hypoxia regulatory
elements in the HLA-G promoter and exon 2 . It directly binds genes encoding CD137 or indirectly
[99]
upregulates CD137 via CD3/CD28 signaling [110,111] . Hypoxia induces a soluble form of CD137 that
predominates over membrane-bound forms in tumor cells, blocking CD137L-mediated T cell
costimulation .
[101]

Regulatory mechanisms of hypoxia-induced angiogenesis in immune suppression
HIF-1α acts as a transcription factor promoting tumor angiogenesis. The resulting morphologically
abnormal blood vessels impair oxygen delivery and exacerbate tumor hypoxia, creating a vicious cycle.
Moreover, these disordered tumor vasculatures contribute to resistance to immunotherapy .
[36]

Malformed neovessels in tumors disrupt anticancer immune responses at multiple levels and reduce the
efficacy of immunotherapy. Abnormal tumor vasculature impairs the adhesion of immune cells to
endothelial cells and forms a barrier that restricts immune cell infiltration into the tumor. Endothelial cells
further inhibit immune cell adhesion through intracellular sequestration or by suppressing the transcription
of endothelial adhesion molecules (EAMs) . Vascular-associated factors - including pro-angiogenic factors,
[112]
inflammatory cytokines, and chemokines - downregulate EAM expression in tumor-associated endothelial
cells, impairing interactions between T cells and the endothelium. TNF-α and IL-1β activate endothelial cells
to initiate immune cell adhesion [113,114] , while bFGF and VEGF counteract proinflammatory cytokine-induced
adhesion by downregulating intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1
(VCAM-1), and E-selectin [115] . VEGF-A also upregulates the death mediator FAS ligand (FASL), which
selectively eliminates effector T cells but spares Tregs, resulting in reduced intratumoral CD8 T cells and an
+
abundance of Tregs . Additionally, tumor-associated endothelial cells can overexpress inhibitory molecules
[116]
such as galectin-1 and endothelin B receptor, further blocking immune cell infiltration. Galectin-1 induces T
cell apoptosis, and its high expression correlates with reduced T lymphocyte recruitment [117] . Endothelin B
receptor maintains vascular homeostasis, and its integration with endothelin-1 disrupts T cell-endothelial
adhesion, thereby limiting T cell infiltration .
[118]

Hypoxia also upregulates PD-L1 expression on tumor endothelium, causing T cells to become functionally
anergic within the tumor vascular lumen before entering the TME [119] . Hypoxia enhances IDO activity in
endothelial cells, leading to tryptophan catabolism into immunosuppressive metabolites and promoting the
generation of FoxP3 Tregs and tolerogenic dendritic cells through aryl hydrocarbon receptor signaling [120,121] .
+
Hypoxia-induced VEGF functions as a potent immunosuppressive cytokine. VEGF binding to VEGFR1
inhibits dendritic cell maturation by suppressing NF-κB signaling, thereby impairing T cell priming [122] .
VEGF also inhibits T cell function by promoting the accumulation and proliferation of MDSCs, which in
turn induce M2 macrophages and Tregs through IL-10 and IFN-γ secretion, reduce cell adhesion factor
expression, interfere with immune cell extravasation, and deplete L-arginine and cystine [123] . Tox, a high

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