isolation-primary-cancer-associated-fibroblasts-from-syngeneic-murine

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

Università di Milano

Everolimus (Eve) is an FDA approved drug that inhibits mammalian target of rapamycin (mTOR). It is employed in breast cancer treatment even if its responsiveness is controversial. In an attempt to increase Eve effectiveness, we have developed a novel Eve nanoformulation exploiting H-ferritin nanocages (HEve) to improve its subcellular delivery. We took advantage of the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1). Breast cancer cells overexpressing TfR-1 were successfully recognized by H-Ferritin, displaying quick nanocage internalization. HEve has been tested and compared to Eve for in vitro efficacy in sensitive and resistant breast cancer cells. Nanoformulated Eve induced remarkable antiproliferative activity in vitro, making even resistant cell lines sensitive to Eve. Moreover, the antiproliferative activity of HEve is fully in accordance with cytotoxicity observed by cell death assay. Furthermore, the significant increase in anticancer efficacy displayed in HEve-treated samples is due to the improved drug accumulation, as demonstrated by UHPLC-MS/MS quantifications. Our findings suggest that optimizing Eve subcellular delivery, thanks to nanoformulation, determines its improved antitumor activity in a panel of Eve-sensitive or resistant breast cancer cell lines.

Everolimus Nanoformulation in Biological Nanoparticles Increases Drug Responsiveness in Resistant and Low-Responsive Breast Cancer Cell Lines.

Indocyanine green (ICG) is a Food and Drug Administration-approved near-infrared fluorescent dye, employed as an imaging agent for different clinical applications due to its attractive physicochemical properties, high sensitivity, and safety. However, free ICG suffers from some drawbacks, such as relatively short circulation half-life, concentration-dependent aggregation, and rapid clearance from the body, which would confine its feasible application in oncology. Here, we aim to discuss encapsulation of ICG within a nanoparticle formulation as a strategy to overcome some of its current limitations and to enlarge its possible applications in cancer diagnosis and treatment. Our purpose is to provide a short but exhaustive overview of clinical outcomes that these nanocomposites would provide, discussing opportunities, limitations, and possible impacts with regard to the main clinical needs in oncology.

Indocyanine Green Nanoparticles: Are They Compelling for Cancer Treatment?

Protein nanocages represent an emerging candidate among nanoscaled delivery systems. Indeed, they display unique features that proved to be very interesting from the nanotechnological point of view such as uniform structure, stability in biological fluids, suitability for surface modification to insert targeting moieties and loading with different drugs and dyes. However, one of the main concerns regards the production as recombinant proteins in , which leads to a product with high endotoxin contamination, resulting in nanocage immunogenicity and pyrogenicity. Indeed, a main challenge in the development of protein-based nanoparticles is finding effective procedures to remove endotoxins without affecting protein stability, since every intravenous injectable formulation that should be assessed in preclinical and clinical phase studies should display endotoxins concentration below the admitted limit of 5 EU/kg. Different strategies could be employed to achieve such a result, either by using affinity chromatography or detergents. However, these strategies are not applicable to protein nanocages as such and require implementations. Here we propose a combined protocol to remove bacterial endotoxins from nanocages of human H-ferritin, which is one of the most studied and most promising protein-based drug delivery systems. This protocol couples the affinity purification with the Endotrap HD resin to a treatment with Triton X-114. Exploiting this protocol, we were able to obtain excellent levels of purity maintaining good protein recovery rates, without affecting nanocage interactions with target cells. Indeed, binding assay and confocal microscopy experiments confirm that purified H-ferritin retains its capability to specifically recognize cancer cells. This procedure allowed to obtain injectable formulations, which is preliminary to move to a clinical trial.

Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin.

Cancer-associated fibroblasts (CAFs) are key actors in regulating cancer progression. They promote tumor growth, metastasis formation, and induce drug resistance. For these reasons, they are emerging as potential therapeutic targets. Here, with the aim of developing CAF-targeted drug delivery agents, we functionalized H-ferritin (HFn) nanocages with fibroblast activation protein (FAP) antibody fragments. Functionalized nanocages (HFn-FAP) have significantly higher binding with FAP CAFs than with FAP cancer cells. We loaded HFn-FAP with navitoclax (Nav), an experimental Bcl-2 inhibitor pro-apoptotic drug, whose clinical development is limited by its strong hydrophobicity and toxicity. We showed that Nav is efficiently loaded into HFn (HNav), maintaining its mechanism of action. Incubating Nav-loaded functionalized nanocages (HNav-FAP) with FAP cells, we found significantly higher cytotoxicity as compared to non-functionalized HNav. This was correlated with a significantly higher drug release only in FAP cells, confirming the specific targeting ability of functionalized HFn. Finally, we showed that HFn-FAP is able to reach the tumor and to target CAFs in a mouse syngeneic model of triple negative breast cancer after intravenous administration. Our data show that HNav-FAP could be a promising tool to enhance specific drug delivery into CAFs, thus opening new therapeutic possibilities focused on tumor microenvironment.

Selective Targeting of Cancer-Associated Fibroblasts by Engineered H-Ferritin Nanocages Loaded with Navitoclax.

Indocyanine green (ICG) is a near infrared fluorescent tracer used in image-guided surgery to assist surgeons during resection. Despite appearing as a very promising tool for surgical oncology, its employment in this area is limited to lymph node mapping or to laparoscopic surgery, as it lacks tumor targeting specificity. Recently, a nanoformulation of this dye has been proposed with the aim toward tumor targeting specificity in order to expand its employment in surgical oncology. This nanosystem is constituted by 24 monomers of H-Ferritin (HFn), which self-assemble into a spherical cage structure enclosing the indocyanine green fluorescent tracer. These HFn nanocages were demonstrated to display tumor homing due to the specific interaction between the HFn nanocage and transferrin receptor 1, which is overexpressed in most tumor tissues. Here, we provide an ex vivo detailed comparison between the biodistribution of this nanotracer and free ICG, combining the results obtained with the Karl Storz endoscope that is currently used in clinical practice and the quantification of the ICG signal derived from the fluorescence imaging system IVIS Lumina II. These insights demonstrate the suitability of this novel HFn-based nanosystem in fluorescence-guided oncological surgery.

Tumor Accumulation and Off-Target Biodistribution of an Indocyanine-Green Fluorescent Nanotracer: An Ex Vivo Study on an Orthotopic Murine Model of Breast Cancer.

Article	Year
Everolimus Nanoformulation in Biological Nanoparticles Increases Drug Responsiveness in Resistant and Low-Responsive Breast Cancer Cell Lines. Pharmaceutics\| PubMed ID: 31382388	2019
Indocyanine Green Nanoparticles: Are They Compelling for Cancer Treatment? Frontiers in chemistry\| PubMed ID: 32766203	2020
Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin. Pharmaceutics\| PubMed ID: 33562060	2021
Selective Targeting of Cancer-Associated Fibroblasts by Engineered H-Ferritin Nanocages Loaded with Navitoclax. Cells\| PubMed ID: 33562504	2021
Tumor Accumulation and Off-Target Biodistribution of an Indocyanine-Green Fluorescent Nanotracer: An Ex Vivo Study on an Orthotopic Murine Model of Breast Cancer. International journal of molecular sciences\| PubMed ID: 33562574	2021

Leopoldo Sitia

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