Distinct Tumor Immune Responses to Nanopulse Stimulation Determine Immunity

Nanopulse stimulation (NPS) induces immune-mediated vaccine effects (in situ vaccination) after ablation of orthotopic rat liver (75-78%) and mouse breast tumors (80-95%). More recently, 4T1-luc immunome mechanisms showed that NPS selectively targeted activated T-regulatory cells (Tregs, TGFβ+, 4-1BB+) by apoptosis with a decrease in activated/naïve T-reg ratio from 2.0 (day 0, control) to 0.5 (day 3). NPS also rapidly eliminated myeloid-derived suppressor cells (MDSC, CD11b+ Gr1+), and tumor-associated macrophages (TAMs, CD11b+ F4/80+) by apoptosis. These changes relieve immunosuppression in the tumor microenvironment (TME). There was also a stronger NPS bias for apoptotic reduction of suppressive Tregs vs. T-effector cells leading to a 2.7-fold increase in the ratio of resident memory CD8+ CD103+ T-cells to CD4 Tregs. This increased ratio coincided with significant increases in CD11c+ dendritic cell (DC) numbers expressing costimulatory receptors (CD80+ CD86+ and MHC II+). These findings define immunome changes that empower immunity and identify fragile Tregs and other immunosuppressor cells as therapeutic NPS targets in the 4T1-luc model.
Ongoing B16f10 melanoma studies show that, like the 4T1-luc tumors, NPS decreased T-regs and MDSCs. However, there were significant increases in activated M2 tumor-associated macrophages (M2-TAMs, CD11b+, F4/80+ with costimulatory receptors), thus reducing but not eliminating the immunosuppressive TME. There were increases of TME DC expressing costimulatory receptors; however, limited DC TME infiltration reduced the potential for antigen presentation. CD8+ cells increased in the TME, but central and effector memory cell numbers are low, and many were PD1+, suggesting a more anergic rather than an activated TME. Unlike immunity in the liver rat model, TME NK cell responses were meager in the B16f10 model. Overall, these responses allude to a narrower potential for immunity. Accordingly, for B16f10 tumors treated with conditions that induce immune responses in orthotopic rat liver and mouse breast cancer models, NPS ablates about 50% of B16f10 tumors and causes vaccine effects in about 50% of challenged mice. To enhance ablation and immune induction in the B16f10 model, ongoing studies injects carbon nanotubes (CNTs) functionalized with anti-PD-L1. Compared to control, CNT alone deceased NPS conditions by 3-fold in vitro and appear superior in vivo.