Assessment of a novel nanoparticle hyperthermia therapy in a murine model of osteosarcoma

Abstract

Objective: To evaluate the effects of nanoparticle hyperthermia therapy on monocyte function and tumor-derived factors associated with macrophage polarization in a murine osteosarcoma model.

Study design: Experimental study.

Animals: Female C3H mice.

Methods: Peripheral blood monocyte cell surface phenotype, monocyte chemotaxis, tumor messenger RNA expression, and survival were compared among osteosarcoma (OS)-bearing mice treated with nanoparticle hyperthermia therapy, OS-bearing mice with osteomyelitis, OS-bearing mice, vehicle control mice, and normal control mice.

Results: OS-bearing mice with osteomyelitis had a higher proportion of "nonclassical" monocytes (Ly6C^lo ) compared with all other experimental groups. There were alterations in monocyte expression of multiple chemokine receptors among experimental groups including CXCR2, CCR2, and CXCR4. Monocytes from OS-bearing mice treated with hyperthermia therapy exhibited greater chemotaxis compared with monocytes from OS-bearing mice with osteomyelitis.

Conclusion: OS likely induced alterations in monocyte phenotype and function. Nanoparticle hyperthermia therapy increased in vitro monocyte chemotaxis.

Clinical impact: Enhancing monocyte/macrophage function in dogs with OS may enhance antitumor immunity.

FIGURE 1

Induction of bacterial osteomyelitis. Mice were inoculated with luciferase-positive XEN36 S aureus. A, Osteomyelitis was confirmed after femoral inoculation by using luminescence live imaging in anesthetized mice (arrow). B, After mice were humanely killed, the femur was aseptically harvested and cultured. Luciferase-positive bacterial cultures confirmed the presence of pure luminescent XEN36 S aureus

FIGURE 2

Distribution of nanoparticles in tumors arising at injection sites and tumor histopathology. Photomicrographs at × 20 of hematoxylin and eosin-stained sections of injection site tumors. A, Neoplastic cells only. The subcutis contains a large expansile neoplasm that extends into the subjacent musculature. The overlying skin is intact. B, Neoplastic cells and magnetic cationic liposomes (MCL). The neoplasm arising from the subcutis has large areas of pale pink necrosis (asterisks) and intralesional black nanoparticle material (arrow). C, Neoplastic cells, MCL, and hyperthermia therapy. There is extensive coagulation necrosis (homogenous red staining), which is consistent with thermal injury, that extends from the epidermis into the panniculus and affects approximately 75% of the neoplasm. Note the uneven distribution of black nanoparticle material and areas of necrosis. Scale bars = 500 μm

FIGURE 3

Monocyte identification and phenotypic analysis by flow cytometry. A, Flow cytometry gating illustrates typical forward light scatter (FSC) vs side light scatter (SSC; left panel) and lack of Ly6G expression (Ly6G⁻, circled, right panel). B, The Ly6G⁻ monocytes (from A, circled) were then gated on the basis of Ly6C and CD11b expression for identification of classical (CD11b⁺ Ly6C^hi, upper right) and nonclassical (CD11b⁺ Ly6C^lo, middle right) monocytes

FIGURE 4

Comparison of monocyte chemotaxis among treatment groups. A transwell chemotaxis assay was used to assess monocyte migration. Visualization of monocytes on the transwell membrane (A, × 100 objective). Box and whisker plots compare the numbers of migrated monocytes counted on the transwell membrane in response to the chemoattractants MCP (B) and SDF (C). The horizontal line represents the median, the box represents the first and third quartiles, and the whiskers represent the minimum and maximum values. *P < .05. MCP, monocyte chemoattractant protein; SDF, stromal cell-derived factor