Nanoparticle pre-treatment for enhancing the survival and activation of pulmonary macrophage transplant

Abstract

Despite recent clinical successes of chimeric antigen receptor T cell therapies in treating liquid cancers, many lingering challenges stand in the way of therapeutic translation to broader types of malignancies. Macrophages have been proposed as alternatives to T cells given macrophages' advantages in promoting tumor infiltration, acquiring diverse antigens, and possessing the ability to continuously stimulate adaptive responses. However, the poor survival of macrophages upon transplantation in addition to transient anti-tumor phenotypical states have been major obstacles standing in the way of macrophage-based cell therapies. Given recent discoveries of nanoparticle strategies in improving macrophage survival and promoting phenotype retention, we herein report the ability to extend the survival and phenotype of macrophage transplants in murine lungs via pre-treatment with nanoparticles of varying degradation rates. Macrophages pre-treated with 100 µg/ml dose of poly(ethylene glycol) diacrylate nanoparticle formulations improve pulmonary macrophage transplant survival over untreated cells beyond 7 days, where degradable nanoparticle formulations result in over a 50% increase in retention of transplanted cell counts relative to untreated cells. Furthermore, pre-treated macrophages more efficiently retain an imposed pro-inflammatory-like polarization state following transplantation out to 7 days compared to macrophages pre-treated with a classical pro-inflammatory stimulus, interferon-gamma, where CD86 costimulatory molecule expression is greater than 150% higher in pre-treated macrophage transplants compared to untreated counterparts. These findings provide an avenue for a major improvement in the lifespan and efficacy of macrophage-based cell therapies and have broader implications to other phagocyte-based cellular therapeutics and administration routes.

Keywords: Cell therapy; Macrophages; Nanoparticles; Polarization; Pulmonary transplant; Survival.

Fig. 1

Pulmonary macrophage transplant (PMT) studies dosing schedule. BMMs were isolated from B6.SJL-Ptprc^a Pepc^b/BoyJ mice. In parallel, host C57BL/6 J mice were prepared for transplants by three daily orotracheal instillations of clodronate liposomes on Days − 4 through − 2. Transplants were performed on Day 0 and flow cytometric analysis on lung digests was performed on Days 3 and 7 for identification of CD45.1 + transplant survival and phenotypical analysis. Treatment groups are shown along with color legend for subsequent figures

Fig. 2

Survival of CD45.1 + BMMs on A Day 3 and B Day 7 following PMT in whole lung digests. *p < 0.05 and ***p < 0.001; ns is not significant (compared to untreated transplant, UT) using Dunnett’s multiple comparisons test (one-way ANOVA) (N = 5 mice; representative results from duplicate experiments). Bars represent the mean and error bars represent SEM

Fig. 3

H&E histological analysis (4 × magnification) of lungs at Days 1 and 3 from mice receiving no transplant, untreated transplant, 0% NP-treated transplant, 20% NP-treated transplant, and IFN-$\gamma$-treated transplant. Scale bar 100 µm

Fig. 4

Detection of CD45.1 + transplant cells in lung sections with immunohistochemistry at Day 3 from mice receiving untreated transplant, 0% NP-treated transplant, 20% NP-treated transplant, and IFN-$\gamma$-treated transplant (20 × magnification). Scale bar 100 µm. Insets show 40 × magnification demonstrating transplanted CD45.1 cells in each group. Arrows indicate the presence of CD45.1 + transplant cells

Fig. 5

Representative lysosomal tracking and imaging at 20 × magnification with LysoBrite™ Green of lung digest cells on Day 3 post-transplant of BMMs treated with 100 µg/ml of 0% and 20% NPs, 25 ng/ml IFN-$\gamma$, or untreated BMMs. Scale bar 100 µm. Phase contrast (PC). Images are representative of three biological replicates; results representative of duplicate experiments

Fig. 6

Representative TUNEL apoptosis imaging analysis at 20 × magnification of lung digest cells on Day 3 post-transplant of BMMs treated with 100 µg/ml of 0% and 20% NPs, 25 ng/ml IFN-$\gamma$, or untreated BMMs. Scale bar 100 µm. Phase contrast (PC). Images are representative of three biological replicates; results representative of duplicate experiments

Fig. 7

Expression of representative CD86 activation marker of CD45.1 + BMMs treated with 100 µg/ml of 0% and 20% NPs, 25 ng/ml IFN-$\gamma$, or untreated BMMs for 24 h. A, D Pre-transplant (Day 0). B, E Day 3 PMT. C, F Day 7 PMT. Top panel: Representative flow cytometric histograms of CD86 expression of CD45.1 + BMMs. Bottom panel: CD86 median fluorescence intensity of CD45.1 + BMMs. *p < 0.05, **p < 0.01, and ****p < 0.0001; ns is not significant using Tukey’s multiple comparisons tests as part of a one-way ANOVA (N = 3–5 mice; results representative of duplicate experiments). Error bars represent SEM

Fig. 8

Expression of representative MHCII activation marker of CD45.1 + BMMs treated with 100 µg/ml of 0% and 20% NPs, 25 ng/ml IFN-$\gamma$, or untreated BMMs for 24 h. A, D Pre-transplant (Day 0). B, E Day 3 PMT. C, F Day 7 PMT. Top panel: Representative flow cytometric histograms of MHCII expression of CD45.1 + BMMs. Bottom panel: MHCII median fluorescence intensity of CD45.1 + BMMs. ****p < 0.0001; ns is not significant using Tukey’s multiple comparisons tests as part of a one-way ANOVA (N = 3–5 mice; results representative of duplicate experiments). Error bars represent SEM