The tumor microenvironment (TME), where cancer cells reside, plays a crucial role in cancer progression and metastasis. It maintains an immunosuppressive state in many tumors and regulates the differentiation of precursor monocytes into M1 (anti-tumor)- and M2 (pro-tumor)-polarized macrophages, and greatly reduces anticancer drug and nanoparticle delivery. As a result, the effectiveness of recently developed chemo- and/or nanotechnology-mediated immune and magnetic nanoparticle hyperthermia (mNPH) therapies is inhibited significantly. One of the ways to overcome this limitation is to use E. coli phagelysate as a primer to modify the tumor microenvironment by switching tumor-associated M2 macrophages to anti-tumor M1 macrophages, and initiate the infiltration of tumor-associated macrophages (TAMs). Recently, bacteriophages and phage-induced lysed bacteria (bacterial phagelysates—BPLs) have been shown to be capable of modifying the tumor-associated environment. Phage/BPL-coated proteins tend to elicit strong anti-tumor responses from the innate immune system, prompting phagocytosis and cytokine release. It has also been reported that the microenvironments of bacteriophage- and BPL-treated tumors facilitate the conversion of M2-polarized TAMS to a more M1-polarized (tumoricidal) environment post-phage treatment. This paper demonstrates the feasibility and enhanced efficacy of combining E. coli phagelysate (EcPHL) and mNPH, a promising technology for treating cancers, in a rodent model. Specifically, we illustrate the EcPHL vaccination effect on the TME and mNP distribution in Ehrlich adenocarcinoma tumors by providing the tumor growth dynamics and histology (H&E and Prussian blue) distribution of mNP in tumor and normal tissue.

BACKGROUND.The tumor microenvironment (TME) has an essential role in cancer development. TME reduces the drug and nanoparticle delivery in tumor tissue and minimizes the treatment outcomes. The new therapeutic strategies of TME reprogramming have shown great promise. One such strategy is TME modulation by the E. coli phage-lysate (EcPHL) vaccination with TME modification and enhancement of magnetic nanoparticles (mNP) uptake by tumor tissue. OBJECTIVES In the current study, we aimed to evaluate the effects of EcPHL vaccination on TME and mNP distribution in the tumor tissue. METHODS Experiments were carried out on 20-25 g BALB/c male mice. EcPHL intraperitoneal vaccinations (0,25 ml, 3x, with 3-day intervals) were initiated after 6 days of the Ehrlich ascites carcinoma (EAC) cells subcutaneous inoculation and intratumoral administration of mNP (5 mg/cm3) -on the 4 th day after the last administration of EcPHL. Tumor volume was measured using a Vernier caliper. Tumor tissue samples, and the spread of mNPs in tumor tissue were estimated using microscope Leica DM6B, Motic Easy Scan Pro scanner (Ultrahigh NA APO 20X [NA 0,75], resolution 40X:0,25 mm/pixel). RESULTSThe mean tumor volume (MTV) was significantly reduced in EcPHL-vaccinated mice. The morphological investigation has shown an increased number of immunocompetent cells, basically macrophages, around the atypical cells in the tumor stroma, infiltrating the inflammatory regions and necrotic areas in the center of the tumor tissue. The inflammatory infiltrations with macrophages were prevalent in the necrotic areas. After mNP injection, the pigment was spread at a distance from the injection site as concentrated masses in extracellular regions of tumor tissue. Areas of inflammatory infiltration and concentration of iron-containing cells were significantly higher in the EcPHL-vaccinated mice compared to unvaccinated, and iron-containing macrophages were detected at a much greater distance from the injection site. CONCLUSIONS EcPHL stimulates anticancer responses, inhibits tumor growth, and enhance the distribution of mNP in tumor tissue. KEYWORDS Cancer; E. coli phage-lysate (EcPHL); magnetic/iron nanoparticles (mNP); Tumor microenvironment (TME).