(575f) Enhancing Macrophage Immunotherapy Via Supramolecular Nanoparticles for Dual Inhibition of CSF1R and MAPK Pathways

Introduction: Among the numerous immune interactions in the tumor microenvironment (TME), tumor-associated macrophage (TAM)- cancer cell interactions have shown to play a critical role in modulating the TME to an immune-suppressed state, promoting accelerated tumor growth, survival, and metastatic spread. These TAMs are predominantly polarized to a pro-tumorigenic M2 phenotype through macrophage colony-stimulating factor 1 (MCSF), which binds to the colony-stimulating factor 1 receptor (CSF1R). This MCSF-CSF1R interaction causes phosphorylation of CSF1R, phosphorylating and activating downstream signaling pathways including mitogen-activated protein kinase (MAPK) pathway leading to proliferation and functional activity of M2 TAMs. Therapeutic inhibition of CSF1R and MAPK signaling could effectively repolarize TAMs from M2 to an anti-tumorigenic M1 phenotype; however, this is challenging. Systemic administration of certain therapeutics unfortunately does not allow for targeted treatment to the TME, and have been known to impact essential organs such as the liver, kidneys, and spleen. Utilizing nanoparticle vectors, therapeutics can be packaged and delivered to certain sites within the body based on their biophysical and chemical characteristics, avoiding unwanted systemic or off-target toxicities. To address the possibility of TAM repolarization, we engineered dual-kinase inhibiting supramolecular nanoparticles (DSN) that can cause both concurrent and sustained inhibition of CSF1R and MAPK signaling pathways. TAMs will internalize and degrade the DSN system upon reaching the TME, releasing the CSF1R and MAPK inhibitors (BLZ-945; BLZ and selumetinib; SEL respectively) to cause vertical pathway inhibition and repolarization to the M1 phenotype (Figure 1A) resulting in tumor regression.

Methods: This DSN system incorporates four building blocks: two kinase signaling inhibitors and two biocompatible phospholipids. This lipid-based nanoparticle system was synthesized using the biocompatible phospholipids phosphatidylcholine and DSPE-PEG₂₀₀₀-amine. The BLZ and SEL were conjugated with cholesterol through an ester bond to overcome low loading and increase stability. Through a lipid film-hydration technique which initiates self-assembly through hydrophilic-hydrophobic interactions, both lipids and cholesterol-tagged inhibitors form this repolarization-capable supramolecule in optimized ratios. Next, we performed a physiochemical characterization of the DSN system, including size and stability using dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM) analysis. Upon confirmation of stable size and drug loading, in vitro assessments were conducted to effectively evaluate the efficacy of this system. Utilizing fluorescence microscopy, internalization comparisons between free drug and DSNs were conducted. Further, flow cytometry was conducted in order to assess the cytotoxicity to and repolarization of RAW264.7 macrophages, in addition to western blot for insight into inhibition of phosphorylated CSF1R and MAPK pathways. We investigated the DSNs ability to accumulate into a highly-aggressive 4T1 model through IVIS near-infrared particle tracking and fluorescence microscopy. Finally, tumor regression studies due to macrophage-mediated anti-tumorigenic activity via repolarization were conducted and analyzed ex vivo through flow cytometry, western blot, and fluorescence microscopy.

Results: We have shown that we can successfully synthesize tandem-loaded DSNs using cholesterol-tagged inhibitors and biocompatible phospholipids. The DLS and cryo-TEM analysis shows spherical liposome structures averaging ~150 nm in diameter (Figure 1B). This system obtained low drug-release while in PBS, but exhibited a burst-release mechanism when incubated in macrophage lysate which indicates drug-release activity possible within the TAMs. We showed that DNS achieved significantly higher internalization in comparison to free drugs, all while exhibiting significantly lower cell death as analyzed using Annexin V. Repolarization of M2-converted macrophages over multiple time points shows sustained inhibition via biomarker analysis and western blot compared to free-inhibitors, single-inhibitor supramolecular nanoparticles (SNPs), and tandem delivery of BLZ and SEL SNP. Significant increases in the M1:M2 macrophage ratio (CD80:CD206) were seen at 24 hours and continued into 48 hours (Figure 1C), in addition to reductions in phosphorylated-CSF1R and phosphorylated-ERK1/2 over time. The increased repolarization demonstrates that having both inhibitors within the same system being delivered to a single cell is more effective than single-drug treatments. Upon confirmation of repolarization capabilities, we i.v. injected DSNs in sub-optimal doses into aggressive 4T1-bearing BALB/c mice. Suboptimal administration of this system was utilized to elucidate the impact of synergistic delivery of BLZ and SEL in tandem in comparison to single-inhibitor administration. NIR dye-tagged DSN trafficked to the TME showing increased fluorescence intensity to the tumor site over 24 hours (Figure 1D) while having minimal impact on other major organs. The DSN treatment significantly outperformed control groups of free-inhibitors, single-inhibitor SNPs, and the combination of single-inhibitor SNPs, exhibiting significant tumor regression (Figure 1E). Finally, biomarker and western blot analysis was conducted on resected tumors, indicating that mice treated with DSNs had significantly inhibited phosphorylated CSF1R and ERK1/2, along with increased M1-correlated biomarkers.

Conclusions: This study shows that treatment of macrophages with synergistic CSF1R- and MAPK-inhibiting drugs via self-assembling supramolecules can allow for more potent repolarization of macrophages, increasing tumor regression capabilities. This platform and proof-of-concept study opens the door for tandem delivery of repolarization-capable molecules to macrophages. As the discovery of more M1 and M2 TAM-related pathways increase, the ability for more platforms to incorporate vertical or parallel inhibition increase with it. Efficacy was shown in suboptimal dosing also give rise to lower dosing regimens due to increased efficacy, warding off treatment-based side effects and allowing for highly effective immunotherapeutic treatment.

References: Brouillard A. et al. “Dual inhibition of CSF1R and MAPK pathways using supramolecular nanoparticles enhances macrophage immunotherapy.” Biomaterials, 2020, 227, 119559.

Figure 1. (A) Schematic shows that the higher concentration of MCSF in the tumor microenvironment results in the polarization of macrophages to immunosuppressive M2 phenotype by binding to its CSF1R receptor resulting in the activation of downstream signaling pathways such as the MAPK pathway. Concurrent and sustained inhibition of CSF1R and MAPK pathways in M2 TAMs using dual-inhibitor loaded supramolecular nanoparticles (DSNs) results in efficient repolarization to pro-inflammatory M1 phenotype, resulting in an enhanced anti-tumor effect. (B) Representative high-resolution cryo-TEM image of DSNs. (C) Quantification of flow cytometry data demonstrating a time-dependent relationship between the relative expression of M1 (CD11b⁺, CD80⁺) compared to M2 (CD11b⁺, CD206⁺) macrophages. (D) Representative NIR fluorescence images of 4T1 tumor bearing mice at different time points after NIR-dye tagged DSN injection (tumor locations indicated by black circles) (E) Relative tumor growth profiles of 4T1 tumor-bearing mice after multi-dose treatments (injections indicated with white arrows).