Tumor Presence Induces Global Immune Changes and Enhances Nanoparticle Clearance

Abstract

Long-circulating nanoparticles are essential for increasing tumor accumulation to provide therapeutic efficacy. While it is known that tumor presence can alter the immune system, very few studies have explored this impact on nanoparticle circulation. In this report, we demonstrate how the presence of a tumor can change the local and global immune system, which dramatically increases particle clearance. We found that tumor presence significantly increased clearance of PRINT hydrogel nanoparticles from the circulation, resulting in increased accumulation in the liver and spleen, due to an increase in M2-like macrophages. Our findings highlight the need to better understand interactions between immune status and nanoparticle clearance, and suggest that further consideration of immune function is required for success in preclinical and clinical nanoparticle studies.

Keywords: immunology; intravital; nanoparticle; orthotopic; pharmacokinetic.

Figure 1

Circulation of intravenously injected PRINT hydrogels is reduced in tumor-bearing mice. (A) Still images from intravital microscopy in naïve and tumor-bearing mice depict lower initial fluorescence and faster clearance in tumor-bearing mice compared to naïve mice (scale-bar = 50 μm). (B) Particle fluorescence in blood over time and exposure (inset) expressed as area-under-the-curve reveals a tumor-induced pharmacokinetic modulation. (****p < 0.0001; one-way ANOVA).

Figure 2

Tumor presence alters nanoparticle circulation and accumulation in organs with salient immune cell activity. The plasma profile (A) and pharmacokinetic parameters (B) of particles were significantly altered by the presence of a tumor, including exposure (AUC), clearance rate from circulation (CL), and volume of distribution (V_d). The behavior of free cisplatin, however, remained unaffected (C). Additionally, there was a significant increase in initial sequestration of particles in both liver (D) and spleen (E) in tumor-bearing mice compared to naïve mice. Measured as platinum (Pt) content via inductively coupled plasma mass spectroscopy (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA).

Figure 3

Serum collected from tumor-bearing mice induced an increase in ex vivo macrophage activity compared to serum collected from naïve mice. PRINT hydrogel nanoparticle association with ex vivo macrophages incubated with serum from naïve and tumor-bearing mice (****p < 0.0001; one-way ANOVA).

Figure 4

Tumor presence alters immune cell interactions with particles. Immune cell distribution of PRINT hydrogel nanoparticles in blood (A), lung (B), spleen (C), and liver (D). Significant increases in association and MFI were seen for several populations, including macrophages and dendritic cells in the lung and liver. MFI = median fluorescence intensity (*p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA).

Figure 5

Presence of A549 tumors skewed macrophage populations from M1 to M2-like. Representative flow cytometry histograms (A) of CD206 expression in macrophages of the liver show an increase in M2-like phenotype in tumor-bearing mice. A significant increase in the population of liver and spleen M2-like macrophages (B) was observed in tumor-bearing mice compared to naïve mice (**p < 0.01, ****p < 0.0001; two-way ANOVA).

Figure 6

M1 and M2-like macrophages have higher affinity for nanoparticles in tumor-bearing mice. Macrophage subset association of PRINT hydrogel nanoparticles by flow cytometry in lung (A), spleen (B), and liver (C) of tumor-bearing and naïve mice. Differences in particle association and MFI were revealed: significant increases in the same macrophage subset between naïve and tumor-bearing mice, and also between different macrophage subsets within the same mouse model. MFI = median fluorescence intensity (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; two-way ANOVA).