Digital spatial profiling reveals additive effects of triple therapy on tumor microenvironment: anti-PD-L1, anti-VEGF, and PARP inhibition in mouse models

Cancer Immunol Immunother. 2026 Feb 7;75(3):70. doi: 10.1007/s00262-026-04312-3.

Abstract

Background: Immune checkpoint inhibitors show limited efficacy against immune-desert tumors, including ovarian cancer. We investigated triple therapy combining anti-programmed cell death-ligand 1 (PD-L1) antibody, anti-vascular endothelial growth factor (VEGF) antibody, and Poly ADP-ribose polymerase inhibitor (PARPi) on tumor microenvironment using spatial profiling.

Methods: Two mouse models were employed: MC38 (immune-inflamed phenotype) and HM-1 (immune-desert phenotype). MC38 mice received anti-PD-L1 and anti-VEGF as monotherapy or dual combination. HM-1 mice received anti-PD-L1, anti-VEGF, and PARPi as monotherapy, dual combinations (anti-PD-L1 + anti-VEGF, anti-PD-L1 + PARPi, anti-VEGF + PARPi), or triple combination (anti-PD-L1 + anti-VEGF + PARPi). Spatial distribution of immune cells and the tumor microenvironment was analyzed using immunohistochemistry (CD8) and dual immunofluorescence (CD8/Granzyme B) with distance-based density quantification from tumor margins (0 to - 150, - 150 to - 300, - 300 to - 450 μm). High endothelial venule (HEV) formation was evaluated via CD31/MECA79 dual immunofluorescence.

Results: MC38 tumors responded to all treatments by day 10. Conversely, HM-1 tumors showed no response at day 10 but responded to two combination therapies by day 20: anti-PD-L1 + anti-VEGF (1.5-fold reduction, p = 0.04) and triple combination therapy (1.7-fold reduction, p = 0.03). In MC38, at - 150 to - 300 μm, anti-PD-L1 + anti-VEGF enhanced CD8 + Granzyme B + cells 1.9-fold versus Control (p = 0.01). In HM-1, at 0 to - 150 μm, triple therapy enhanced CD8 + Granzyme B + cells 2.8-fold (p = 0.02), while anti-PD-L1 + anti-VEGF increased CD8 + Granzyme B + cells 2.5-fold (p = 0.03). Both triple and anti-PD-L1 + anti-VEGF therapies induced CD31 + MECA79 + HEV formation (p < 0.01).

Conclusions: Triple therapy may overcome immune-desert ovarian cancer through additive HEV formation, enhancing cytotoxic CD8 + T cell infiltration into the tumor.

Keywords: Immune checkpoint inhibitor; Ovarian cancer; PARP inhibitor; Spatial profiling; Tumor microenvironment; VEGF inhibition.

MeSH terms

  • Animals
  • Antineoplastic Combined Chemotherapy Protocols* / pharmacology
  • Antineoplastic Combined Chemotherapy Protocols* / therapeutic use
  • B7-H1 Antigen* / antagonists & inhibitors
  • B7-H1 Antigen* / immunology
  • Cell Line, Tumor
  • Disease Models, Animal
  • Female
  • Humans
  • Immune Checkpoint Inhibitors* / pharmacology
  • Mice
  • Ovarian Neoplasms / drug therapy
  • Poly(ADP-ribose) Polymerase Inhibitors* / pharmacology
  • Poly(ADP-ribose) Polymerase Inhibitors* / therapeutic use
  • Tumor Microenvironment* / drug effects
  • Tumor Microenvironment* / immunology
  • Vascular Endothelial Growth Factor A* / antagonists & inhibitors
  • Vascular Endothelial Growth Factor A* / immunology

Substances

  • Vascular Endothelial Growth Factor A
  • B7-H1 Antigen
  • Poly(ADP-ribose) Polymerase Inhibitors
  • Immune Checkpoint Inhibitors