Michael P. Plebanek

Update on current and potential nanoparticle cancer therapies

Purpose of review To summarize the most recent preclinical and clinical advancements in therapeutic nano-oncology. Recent findings First-generation nanotherapies are well tolerated in humans and evidence shows that they are efficacious, while at the same time reducing the burden of side-effects. Most of these therapies are not specifically targeted, but take advantage of enhanced passive accumulation within tumors to preferentially deliver chemotherapies that demonstrate off-target toxicities when administered as free drugs. Also, actively targeted nanotherapies are entering the clinical arena and preliminary data are encouraging. Finally, a number of exciting preclinical developments in nanotechnology provide clear evidence that nanotherapies will continue to enter the clinic and will have a significant impact in oncology. Summary A number of intriguing nanoparticle therapies are being tested in preclinical and clinical trials. Nanoparticles with increasing molecular sophistication, specific targeting properties, and unique mechanisms of action will find their way to the clinic. Certainly, nanoparticle-based therapies will be increasingly represented in drug development pipelines, and will continue to provide efficacious and well tolerated drug options for patients with cancer.

Nanoparticle Targeting and Cholesterol Flux Through Scavenger Receptor Type B-1 Inhibits Cellular Exosome Uptake

Exosomes are nanoscale vesicles that mediate intercellular communication. Cellular exosome uptake mechanisms are not well defined partly due to the lack of specific inhibitors of this complex cellular process. Exosome uptake depends on cholesterol-rich membrane microdomains called lipid rafts, and can be blocked by non-specific depletion of plasma membrane cholesterol. Scavenger receptor type B-1 (SR-B1), found in lipid rafts, is a receptor for cholesterol-rich high-density lipoproteins (HDL). We hypothesized that a synthetic nanoparticle mimic of HDL (HDL NP) that binds SR-B1 and removes cholesterol through this receptor would inhibit cellular exosome uptake. In cell models, our data show that HDL NPs bind SR-B1, activate cholesterol efflux, and attenuate the influx of esterified cholesterol. As a result, HDL NP treatment results in decreased dynamics and clustering of SR-B1 contained in lipid rafts and potently inhibits cellular exosome uptake. Thus, SR-B1 and targeted HDL NPs provide a fundamental advance in studying cholesterol-dependent cellular uptake mechanisms.

Pre-metastatic cancer exosomes induce immune surveillance by patrolling monocytes at the metastatic niche

Metastatic cancers produce exosomes that condition pre-metastatic niches in remote microenvironments to favor metastasis. In contrast, here we show that exosomes from poorly metastatic melanoma cells can potently inhibit metastasis to the lung. These “non-metastatic” exosomes stimulate an innate immune response through the expansion of Ly6Clow patrolling monocytes (PMo) in the bone marrow, which then cause cancer cell clearance at the pre-metastatic niche, via the recruitment of NK cells and TRAIL-dependent killing of melanoma cells by macrophages. These events require the induction of the Nr4a1 transcription factor and are dependent on pigment epithelium-derived factor (PEDF) on the outer surface of exosomes. Importantly, exosomes isolated from patients with non-metastatic primary melanomas have a similar ability to suppress lung metastasis. This study thus demonstrates that pre-metastatic tumors produce exosomes, which elicit a broad range of PMo-reliant innate immune responses via trigger(s) of immune surveillance, causing cancer cell clearance at the pre-metastatic niche.Exosomes are extracellular vesicles that can favor tumor development and metastasis. Here, the authors show that cancer exosomes may also exert a suppressive function; in fact, exosomes from non-metastatic melanoma cells can lead to the recruitment of patrolling monocytes, which clear cancer cells at the pre-metastatic niche.

Pathways for Modulating Exosome Lipids Identified By High-Density Lipoprotein-Like Nanoparticle Binding to Scavenger Receptor Type B-1

Exosomes are produced by cells to mediate intercellular communication, and have been shown to perpetuate diseases, including cancer. New tools are needed to understand exosome biology, detect exosomes from specific cell types in complex biological media, and to modify exosomes. Our data demonstrate a cellular pathway whereby membrane-bound scavenger receptor type B-1 (SR-B1) in parent cells becomes incorporated into exosomes. We tailored synthetic HDL-like nanoparticles (HDL NP), high-affinity ligands for SR-B1, to carry a fluorescently labeled phospholipid. Data show SR-B1-dependent transfer of the fluorescent phospholipid from HDL NPs to exosomes. Modified exosomes are stable in serum and can be directly detected using flow cytometry. As proof-of-concept, human serum exosomes were found to express SR-B1, and HDL NPs can be used to label and isolate them. Ultimately, we discovered a natural cellular pathway and nanoparticle-receptor pair that enables exosome modulation, detection, and isolation.

Properties of Native High-Density Lipoproteins Inspire Synthesis of Actively Targeted In Vivo siRNA Delivery Vehicles

Efficient systemic administration of therapeutic short interfering RNA (siRNA) is challenging. High-density lipoproteins (HDL) are natural in vivo RNA delivery vehicles. Specifically, native HDLs: 1) Load single-stranded RNA; 2) Are anionic, which requires charge reconciliation between the RNA and HDL, and 3) Actively target scavenger receptor type B-1 (SR-B1) to deliver RNA. Emphasizing these particular parameters, we employed templated lipoprotein particles (TLP), mimics of spherical HDLs, and self-assembled them with single-stranded complements of, presumably, any highly unmodified siRNA duplex pair after formulation with a cationic lipid. Resulting siRNA templated lipoprotein particles (siRNA-TLP) are anionic and tunable with regard to RNA assembly and function. Data demonstrate that the siRNA-TLPs actively target SR-B1 to potently reduce androgen receptor (AR) and enhancer of zeste homolog 2 (EZH2) proteins in multiple cancer cell lines. Systemic administration of siRNA-TLPs demonstrated no off-target toxicity and significantly reduced the growth of prostate cancer xenografts. Thus, native HDLs inspired the synthesis of a hybrid siRNA delivery vehicle that can modularly load single-stranded RNA complements after charge reconciliation with a cationic lipid, and that function due to active targeting of SR-B1.

Apigenin Inhibits UVB-Induced Skin Carcinogenesis: The Role of Thrombospondin-1 as an Anti-Inflammatory Factor

We have previously demonstrated that apigenin promotes the expression of antiangiogenic protein thrombospondin-1 (TSP1) via a mechanism driven by mRNA-binding protein HuR. Here, we generated a novel mouse model with whole-body THBS-1 gene knockout on SKH-1 genetic background, which allows studies of UVB-induced acute skin damage and carcinogenesis and tests TSP1 involvement in apigenins anticancer effects. Apigenin significantly inhibited UVB-induced carcinogenesis in the wild-type (WT) animals but not in TSP1 KO (TKO) mice, suggesting that TSP1 is a critical component of apigenins chemopreventive function in UVB-induced skin cancer. Importantly, TKO mice presented with the elevated cutaneous inflammation at baseline, which was manifested by increased inflammatory infiltrates (neutrophils and macrophages) and elevated levels of the two key inflammatory cytokines, IL-6 and IL-12. In agreement, maintaining normal TSP1 expression in the UVB-irradiated skin of WT mice using topical apigenin application caused a marked decrease of circulating inflammatory cytokines. Finally, TKO mice showed an altered population dynamics of the bone marrow myeloid progenitor cells (CD11b+), with dramatic expansion of the population of neutrophil progenitors (Ly6ClowLy6Ghigh) compared to the WT control. Our results indicate that the cutaneous tumor suppressor TSP1 is a critical mediator of the in vivo anticancer effect of apigenin in skin, specifically of its anti-inflammatory action.

Scavenger Receptor Type B1 and Lipoprotein Nanoparticle Inhibit Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSC) are innate immune cells that potently inhibit T cells. In cancer, novel therapies aimed to activate T cells can be rendered ineffective due to the activity of MDSCs. Thus, targeted inhibition of MDSCs may greatly enhance T-cell–mediated antitumor immunity, but mechanisms remain obscure. Here we show, for the first time, that scavenger receptor type B-1 (SCARB1), a high-affinity receptor for spherical high-density lipoprotein (HDL), is expressed by MDSCs. Furthermore, we demonstrate that SCARB1 is specifically targeted by synthetic high-density lipoprotein-like nanoparticles (HDL NP), which reduce MDSC activity. Using in vitro T-cell proliferation assays, data show that HDL NPs specifically bind SCARB1 to inhibit MDSC activity. In murine cancer models, HDL NP treatment significantly reduces tumor growth, metastatic tumor burden, and increases survival due to enhanced adaptive immunity. Flow cytometry and IHC demonstrate that HDL NP–mediated suppression of MDSCs increased CD8+ T cells and reduced Treg cells in the metastatic tumor microenvironment. Using transgenic mice lacking SCARB1, in vivo data clearly show that the HDL NPs specifically target this receptor for suppressing MDSCs. Ultimately, our data provide a new mechanism and targeted therapy, HDL NPs, to modulate a critical innate immune cell checkpoint to enhance the immune response to cancer. Mol Cancer Ther; 17(3); 686–97. ©2017 AACR.

Abstract 3673: High-density lipoprotein-like nanoparticles target SR-B1 and inhibit the cellular uptake of melanoma-cell derived exosomes

Exosomes play a crucial role in the progression of cancer through the transport of a variety molecular cargo, including proteins, lipids, and nucleic acids, to and from cells as a means of intercellular communication. Unraveling mechanisms of exosome-cell interactions may open avenues for studying cellular communication and lead to new therapies. Cellular exosome uptake depends on cholesterol-rich membrane microdomains called lipid rafts. Non-specific depletion of lipid raft cholesterol reduces cellular exosome uptake; however, to our knowledge, no targeted mechanism of inhibiting cellular exosome uptake has been reported. Scavenger receptor type B-1 (SR-B1) localizes to lipid rafts, and is a high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL). SR-B1 is an intriguing therapeutic target because it is upregulated in many different cancers due to the high need for cholesterol of rapidly dividing cancer cells. Therefore, we hypothesized that specific targeting of SR-B1 and modulation of cholesterol flux through this receptor with biomimetic HDL-like nanoparticles (HDL NPs) would disrupt cellular exosome uptake. As a model, we explored exosomes derived from melanoma cells as they have been shown to promote angiogenesis and immunosuppression both crucial events in melanoma progression. Melanoma exosomes have also been shown to actively prepare metastatic sites, creating a suitable microenvironment allowing for the development of metastasis. Because of this, targeting exosomes and intercellular signaling could be beneficial for the treatment of metastatic melanoma. Using a variety of techniques including confocal microscopy, flow cytometry and automated image analysis, data demonstrate that HDL NPs specifically target SR-B1 in lipid rafts in melanoma cells and modulate cholesterol flux through this receptor. This leads to a clustering of SR-B1 at the cell membrane and potent inhibition of the cellular uptake of melanoma cell-derived exosomes. Citation Format: Michael P. Plebanek, Alexandre Matov, Kannan Mautharasan, Jesse Gatlin, C. Shad Thaxton. High-density lipoprotein-like nanoparticles target SR-B1 and inhibit the cellular uptake of melanoma-cell derived exosomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2015-3673