Recombinant Lactaptin Induces Immunogenic Cell Death and Creates an Antitumor Vaccination Effect in Vivo with Enhancement by an IDO Inhibitor

Abstract

Natural compounds of various origins are intensively investigated for their antitumor activity. Potential benefits of antitumor therapy can be achieved when cytotoxic agents kill cancer cells and these dying cancer cells drive adoptive immunity to the tumor. This strategy was successfully demonstrated for chemotherapeutic drugs that induce immunogenic type of cell death (ICD) with release of DAMPs (danger associated molecular patterns) and exposure of “eat me” signals. In this study, we demonstrated that recombinant human milk peptide lactaptin (RL2) induces death of cancer cells with ICD hallmarks in vitro with the release of ATP and high-mobility group box 1 protein (HMGB1) and exposure of calreticulin and HSP70 on the external cell membrane. RL2-treated cancer cells were efficiently engulfed by phagocytic cells. Using the syngeneic mouse model, we demonstrated that RL2-treated MX-7 rhabdomyosarcoma cells confer long-term immune-mediated protection against challenge with live MX-7 cells. We also analyzed the combinatorial antitumor effect of vaccination with RL2-treated cells and the inhibition of indoleamine 2,3-dioxygenase (IDO) with ethyl pyruvate. Compared to solo anti-tumor immunization with RL2-treated cells, additional chemical inhibition of IDO demonstrated better long-term antitumor responses than vaccination alone.

Dual regulation of osteosarcoma hypoxia microenvironment by a bioinspired oxygen nanogenerator for precise single-laser synergistic photodynamic/photothermal/induced antitumor immunity therapy

“…Considering the enhanced PDT ability observed in vitro , it is reasonable to assume that NGs can induce the PDT effect in vivo under single NIR laser irradiation. Besides, robust PDT as well as PTT can not only directly kill tumor cells but also induce immunogenic cell death (ICD), providing antitumor immunity and long-term immunological memory [ 40 ]. In light of this scenario, the antitumor efficiency of PA imaging-guided triple therapy (enhanced-PDT, PTT, and induced antitumor immunity therapy) were investigated in LM8 tumor-bearing mice.…”

Non-Thermal Plasma Application in Tumor-Bearing Mice Induces Increase of Serum HMGB1

“…The samples were prepared in according to the manufacturer’s protocols and their optical density was detected with a multichannel spectrophotometer Apollo LB912 (Berthold Technologies, TN, USA) at 450 nm (reference wavelength 620 nm). The total cellular HMGB1 was detected by Western blot as was described previously [ 38 ].…”

“…Serum cytokines were measured using the Multi-Analyte ELISAArray Kit MEM−004A (Qiagen, USA) where the level of IL−1α, Il−1β, IL−4, IL−6, IL−10, IL−12, IL−17α, INF- γ, TNF- α, G-CSF and GM-CSF can be assessed. Murine blood was isolated from the mice 1 h after the second CAP irradiation, and the serums were prepared as described previously [ 38 ]. Serum samples were diluted 8 times for subsequent analysis.…”

Immunogenic Cell Death in Cancer Therapy

“…Recombinant analog of lactaptin (RL2). Recent studies have shown that a recombinant analog of the human milk pro-apoptotic protein lactaptin (RL2) [ 79 , 80 ] can induce ICD in vitro by activating the whole cascade of immunogenic cell death markers and elicit an antitumor immune response in the prophylactic vaccination model [ 81 ]. Thus, in experiments on immunocompetent C3H/He mice, 43% of mice vaccinated with RL2-treated MX-7 murine rhabdomyosarcoma cells did not develop a tumor nodule after they had been re-challenged.…”

“…It is also worth mentioning that the growth rate of tumors that had actually developed was significantly lower compared to the control group. Ethyl pyruvate, an indoleamine 2,3-dioxygenase inhibitor, used in combination with cells incubated in the presence of RL2 potentiated the vaccination effect of RL2-treated cells by up to 60% [ 81 ].…”