Nano-pulse stimulation (NPS) as a developing technology has been studied for minimally invasive, nonthermal local cancer elimination for more than a decade. Here we show that a single NPS treatment results in complete regression of the poorly immunogenic, metastatic 4T1-Luc mouse mammary carcinoma. Impressively, spontaneous distant organ metastases were largely prevented, even in those animals with incomplete tumor regression. All tumor-free mice were protected from secondary tumor cell challenge, demonstrating a vaccine-like effect. NPS treatment induced antitumor immunity, long-term memory T cells, destruction of tumor microenvironment and reversal of the massive increase of immune suppressor cells in the tumor microenvironment and blood. NPS-treated 4T1 cells exhibited release of damage-associated molecular patterns (DAMPs), including calreticulin, HMGB1 and ATP, and activated dendritic cells. Those findings suggest that NPS is a potent immunogenic cell death inducer that elicits antitumor immunity to prevent distant metastases in addition to local tumor eradication.

Nanopulse Stimulation (NPS) eliminates mouse and rat tumor types in several different animal models. NPS induces protective, vaccine-like effects after ablation of orthotopic rat N1-S1 hepatocellular carcinoma. Here we review some general concepts of NPS in the context of studies with mouse metastatic 4T1 mammary cancer showing that the postablation, vaccine-like effect is initiated by dynamic, multilayered immune mechanisms. NPS eliminates primary 4T1 tumors by inducing immunogenic, caspase-independent programmed cell death (PCD). With lower electric fields, like those peripheral to the primary treatment zone, NPS can activate dendritic cells (DCs). The activation of DCs by dead/dying cells leads to increases in memory effector and central memory T-lymphocytes in the blood and spleen. NPS also eliminates immunosuppressive cells in the tumor microenvironment and blood. Finally, NPS treatment of 4T1 breast cancer exhibits an abscopal effect and largely prevents spontaneous metastases to distant organs. NPS with fast rise–fall times and pulse durations near the plasma membrane charging time constant, which exhibits transient, high-frequency components (1/time = Hz), induce responses from mitochondria, endoplasmic reticulum, and nucleus. Such effects may be responsible for release of danger-associated molecular patterns, including ATP, calreticulin, and high mobility group box 1 (HMBG1) from 4T1-Luc cells to induce immunogenic cell death (ICD). This likely leads to immunity and the vaccine-like response. In this way, NPS acts as a unique onco-immunotherapy providing distinct therapeutic advantages showing possible clinical utility for breast cancers as well as for other malignancies.

Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. controls. The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.

It is now well established that ruthenium complexes are attractive alternatives to platinum-based anticancer agents. Most of the ruthenium compounds currently under investigation contain a single metal center. The synthesis of multinuclear analogues may provide access to novel complexes with enhanced biological activity. In this work, we have synthesized a set of three trinuclear complexes containing organometallic ruthenium fragments-(arene)RuCl-coordinated to a 2,4,6-tris(di-2-pyridylamino)-1,3,5-triazine core [(Arene = benzene (2), p-cymene (1), or hexamethylbenzene (3)]. The interaction of the complexes with DNA was extensively studied using a variety of biophysical probes as well as by molecular docking. The complexes bind strongly to DNA with apparent binding constants ranging from 2.20 to 4.79 × 10 M. The binding constants from electronic absorption titrations were an order of magnitude greater. The mode of binding to the nucleic acid was not definitively determined, but the evidence pointed to some kind of non-specific electrostatic interaction. None of the complexes displayed any significant antimicrobial activity against the organisms that were studied and exhibited anticancer activity only at high (> 100 μM) concentration.

Nano-pulse stimulation (NPS), previously called nsPEFs, induced a vaccine-like effect after ablation of orthotopic N1-S1 hepatocellular carcinoma (HCC), protecting rats from subsequent challenges with N1-S1 cells. To determine immunity, immune cell phenotypes were analyzed in naïve, treated and protected rats. NPS provides a positive, post-ablation immuno-therapeutic outcome by alleviating immunosuppressive T regulatory cells (Treg) in the tumor microenvironment (TME), allowing dendritic cell influx and inducing dynamic changes in natural killer cells (NKs), NKT-cells and T-lymphocytes in blood, spleen and liver. NPS induced specific increases in NKs and NKT-cells expressing CD8 and activation receptors CD314-NKG2D and CD161 (NK1.1) in the TME after treatment, as well as some variable changes in CD4+ and CD8+ effector (Tem) and central memory (Tem) lymphocytes in blood and spleen. After orthotopic challenge, CD8+ T-cells were cytotoxic, inducing apoptosis in N1-S1 cells; additionally, in contrast to post-treatment immune responses, CD4+ and CD8+ memory precursor effector cells (MPECs) and short-lived effector cells (SLECs) were present, while still including CD8+ CD161 NK cells, but not involving CD8+ CD314-NKG2D+ NKs. This immunity was N1-S1-specific and was sustained for at least 8 months. NPS vaccinates rats in vivo against HCC by activating innate and adaptive immune memory mechanisms that prevent HCC recurrence.