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

The use of therapeutic monoclonal antibodies (mAbs) has revolutionized cancer treatment. The conjugation of mAbs to nanoparticles has been broadly exploited to improve the targeting efficiency of drug nanocarriers taking advantage of high binding efficacy and target selectivity of antibodies for specific cell receptors. However, the therapeutic implications of nanoconjugation have been poorly considered. In this study, half-chain fragments of the anti-EGFR mAb cetuximab were conjugated to colloidal nanoparticles originating stable nanoconjugates that were investigated as surrogates of therapeutic mAbs in triple negative breast cancer (TNBC). Three TNBC cell lines were selected according to EGFR expression, which regulates activation of MAPK/ERK and PI3K/Akt pathways, and to distinctive molecular profiling including KRAS, PTEN, and BRCA1 mutations normally associated with diverse sensitivity to treatment with cetuximab. The molecular mechanisms of action of nanoconjugated half-chain mAb, including cell targeting, interference with downstream signaling pathways, proliferation, cell cycle, and apoptosis, along with triggering of ADCC response, were investigated in detail in sensitive and resistant TNBC cells. We found that half-chain mAb nanoconjugation was able to enhance the therapeutic efficacy and improve the target selectivity against sensitive, but unexpectedly also resistant, TNBC cells. Viability assays and signaling transduction modulation suggested a role of BRCA1 mutation in TNBC resistance to cetuximab alone, whereas its effect could be circumvented using half-chain cetuximab nanoconjugates, suggesting that nanoconjugation not only improved the antibody activity but also exerted different mechanisms of action. Our results provide robust evidence of the potential of half-chain antibody nanoconjugates in the treatment of TNBC, which could offer a new paradigm for therapeutic antibody administration, potentially allowing improved curative efficiency and reduced minimal effective dosages in both sensitive and resistant tumors.

Half-Chain Cetuximab Nanoconjugates Allow Multitarget Therapy of Triple Negative Breast Cancer.

Cancer-associated fibroblasts (CAF) are the most abundant cells of the tumor stroma and they critically influence cancer growth through control of the surrounding tumor microenvironment (TME). CAF-orchestrated reactive stroma, composed of pro-tumorigenic cytokines and growth factors, matrix components, neovessels, and deregulated immune cells, is associated with poor prognosis in multiple carcinomas, including breast cancer. Therefore, beyond cancer cells killing, researchers are currently focusing on TME as strategy to fight breast cancer. In recent years, nanomedicine has provided a number of smart delivery systems based on active targeting of breast CAF and immune-mediated overcome of chemoresistance. Many efforts have been made both to eradicate breast CAF and to reshape their identity and function. Nano-strategies for CAF targeting profoundly contribute to enhance chemosensitivity of breast tumors, enabling access of cytotoxic T-cells and reducing immunosuppressive signals. TME rearrangement also includes reorganization of the extracellular matrix to enhance permeability to chemotherapeutics, and nano-systems for smart coupling of chemo- and immune-therapy, by increasing immunogenicity and stimulating antitumor immunity. The present paper reviews the current state-of-the-art on nano-strategies to target breast CAF and TME. Finally, we consider and discuss future translational perspectives of proposed nano-strategies for clinical application in breast cancer.

Nano-Strategies to Target Breast Cancer-Associated Fibroblasts: Rearranging the Tumor Microenvironment to Achieve Antitumor Efficacy.

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?

Everolimus (Eve) is an immunosuppressive macrolide that is being analyzed in various biological matrices and fluids. Its antitumor activity makes this drug suitable not only for organ transplantation but also for breast cancer treatments. In the attempt to reduce the incidence and severity of its side effects, Eve was loaded in H-ferritin (HFn), a natural biomolecule that is involved in specific cellular uptake pathways. Thus, Eve pre-complexed with Cu(II) and encapsulated in HFn resulted in an Eve nanoformulation, named HEve. The quantification of HEve was performed using a tailored pH-induced procedure to precipitate H-ferritin. This sample preparation was effective enough to reduce the ion suppression effect on the mass spectrometric responses of Eve in electrospray ionization (ESI). The ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-ESI-MS/MS) system operating in positive ionization mode showed to be a versatile technique in achieving more than 77 % recovery of Eve from the cytoplasmic compartment. This simple, selective and sensitive method enabled the quantification of Eve within the linear range of 2.5-100 ng/mL in matrix spiked with the isotope-labeled internal standard, EveD4. This method was validated according to FDA Guidance. The intracellular distribution of HEve and its accumulation at a cytoplasmic level were studied in breast cancer cell lines. As expected, HEve was more effective than free Eve on sensitive (i.e. BT474) and resistant cell lines, as a result of a better penetration into the target subcellular compartment.

Development and validation of a simple and versatile method for the quantification of everolimus loaded in H-ferritin nanocages using UHPLC-MS/MS.

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
Half-Chain Cetuximab Nanoconjugates Allow Multitarget Therapy of Triple Negative Breast Cancer. Bioconjugate chemistry\| PubMed ID: 30350574	2018
Nano-Strategies to Target Breast Cancer-Associated Fibroblasts: Rearranging the Tumor Microenvironment to Achieve Antitumor Efficacy. International journal of molecular sciences\| PubMed ID: 30871158	2019
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
Development and validation of a simple and versatile method for the quantification of everolimus loaded in H-ferritin nanocages using UHPLC-MS/MS. Journal of pharmaceutical and biomedical analysis\| PubMed ID: 32987250	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

Arianna Bonizzi

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