Researchers have developed a novel therapeutic strategy for pancreatic cancer using bacteria-assisted delivery of aptamer-drug conjugates (ApDCs). The study, published in Nature, demonstrates that this approach enhances drug penetration, modulates the tumor microenvironment, and boosts antitumor immunity in preclinical models. The innovative therapy combines the targeting ability of aptamers with the tumor-penetrating capabilities of bacteria to deliver cytotoxic drugs specifically to pancreatic cancer cells, addressing a critical unmet need in this challenging disease.
Engineering of Aptamer-Drug Conjugate-Loaded Bacteria
The researchers constructed a functionalized bacterium, VNP@Sgc8c-MMAE, by conjugating the aptamer Sgc8c, which targets protein tyrosine kinase 7 (PTK7), to the drug monomethyl auristatin E (MMAE) and loading it onto the attenuated Salmonella typhimurium strain VNP20009. PTK7 was validated as a relevant target in pancreatic cancer through immunohistochemical (IHC) staining, survival analysis, and comparative expression studies using The Cancer Genome Atlas (TCGA) data, confirming its high expression in pancreatic cancer tissues and correlation with poorer disease-free survival.
The conjugation process utilized click chemistry to ensure efficient and stable binding of the ApDC to the bacterial surface. Spectrophotometric measurements estimated approximately 1.5 x 10^7 Sgc8c-MMAE molecules per VNP20009 bacterium. Stability assays showed that the bacterial surface-grafted ApDC exhibited enhanced stability, retaining over 60% of the aptamers intact after 48 hours, compared to the rapid degradation of free ApDCs.
In Vitro Validation of Targeting and Cytotoxicity
In vitro studies confirmed that VNP@Sgc8c-MMAE specifically binds to pancreatic cancer cells. Laser scanning confocal microscopy (LSCM) revealed increased bacterial association with Miapaca-2 cells when using VNP@Sgc8c-MMAE compared to naive VNP20009. A Yopro-PI assay demonstrated that VNP@Sgc8c-MMAE triggered 20-60% cell death (necrosis and apoptosis) in pancreatic cancer cells, significantly more than standalone VNP20009 or Sgc8c-MMAE. CCK-8 assays further confirmed the heightened, dose-dependent cytotoxicity of VNP@Sgc8c-MMAE across multiple pancreatic cancer cell lines (Panc-1, Miapaca-2, and KPC1199).
Enhanced Tumor Colonization and Penetration In Vivo
In vivo imaging of Miapaca-2 tumor-bearing mice showed that VNP@Sgc8c-MMAE displayed a threefold higher signal in tumor locations compared to controls at 72 hours post-injection. Biodistribution analysis revealed that nearly 90% of VNP@Sgc8c-MMAE colonized the tumor at 72 hours, demonstrating enhanced tumor-specific localization. The VNP carrier also notably enhanced ApDC stability, maintaining its presence in the tumor area longer than unmodified nucleic acid drugs.
Confocal microscopy of tumor tissue sections from KPC1199 subcutaneous mouse models demonstrated that VNP@Sgc8c-MMAE facilitated deeper penetration of the drug into the tumor, with some bacteria and drugs colocalized at a significant distance (~1000 μm) from the tumor's edge. In contrast, the distribution of the drug alone, Sgc8c-MMAE, was predominantly confined to a region within 30 μm of the tumor's edge.
Modulation of the Tumor Microenvironment and Immune Response
Histological analysis revealed decreased staining for α-SMA and Masson in the VNP@Sgc8c-MMAE group, suggesting a loosening of the dense tumor microenvironment. Immunofluorescence assays showed an enrichment of T cells in the tumor center after injection of VNP@Sgc8c-MMAE. scRNA-seq analysis demonstrated a significant increase in cytotoxic CD8+ T cells and helper CD4+ T cells, along with a reduction in myofibroblastic CAFs, indicating an enhanced immune response and a more favorable tumor microenvironment.
Synergistic Therapeutic Efficacy In Vivo
In vivo efficacy studies in KPC1199 subcutaneous pancreatic tumor models showed that VNP-containing formulations consistently impeded tumor progression, with the VNP@Sgc8c-MMAE group displaying superior antitumor activity compared to controls. Flow cytometry analysis revealed a significant increase of CD3+ CD4+ T cells in the VNP@Sgc8c-MMAE group. ELISA results showed elevated levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in plasma and tumor tissues, suggesting a heightened antitumor immune response.
In a Patient-Derived Xenograft (PDX) model, VNP@Sgc8c-MMAE demonstrated a marked therapeutic advantage over the isolated Sgc8c-MMAE group, even in nude mice with a diminished immune response. In an in-situ carcinoma model, the VNP@Sgc8c-MMAE group exhibited significant antitumor effects, with tumors weighing only 30 to 35% of those in the Sgc8c-MMAE group. Survival curves suggested no adverse effects, and cytokine profiles indicated that VNP@Sgc8c-MMAE triggers a strong innate immune response and enhances adaptive immunity.
Flow cytometry analysis of tumor tissues from the in-situ carcinoma model demonstrated a significant increase in the infiltration of CD45+ immune cells, enhanced infiltration of CD4+ T cells, a marked decrease in CD4+ Tregs, a significant increase in mature dendritic cells, and an elevated presence of neutrophils in the VNP@Sgc8c-MMAE treated group. The ratio of M1-type to M2-type macrophages increased, and CD8+ T cells exhibited higher expression of GzmB, T-bet, IFN-γ, and TNF-α, indicating enhanced cytotoxic activity and a stronger Th1 immune response.
These comprehensive findings highlight the potential of bacteria-assisted ApDC delivery as a synergistic therapeutic strategy for pancreatic cancer, improving drug delivery, modulating the tumor microenvironment, and enhancing antitumor immunity.
