Computational Design of Novel Cyclic Peptides Endowed with Autophagy-Inhibiting Activity on Cancer Cell Lines.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

Priming with oncolytic adenovirus followed by anti-PD-1 and paclitaxel treatment leads to improved anti-cancer efficacy in the 3D TNBC model.

Triple-negative breast cancer (TNBC) is considered one of the most incurable malignancies due to its clinical characteristics, including high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Therefore, it remains a critical unmet medical need. On the other hand, poor delivery efficiency continues to reduce the efficacy of anti-cancer therapeutics developed against solid tumours using various strategies, such as genetically engineered oncolytic vectors used as nanocarriers. The study was designed to evaluate the anti-tumour efficacy of a novel combinatorial therapy based on oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with an anti-PD-1 (pembrolizumab) and paclitaxel (PTX). Here, we first tested the antineoplastic effect in two-dimensional (2D) and three-dimensional (3D) breast cancer models in MDA-MB-231, MDA-MB-468 and MCF-7 cells. Then, to further evaluate the efficacy of combinatorial therapy, including immunological aspects, we established a three-dimensional (3D) co-culture model based on MDA-MB-231 cells with peripheral blood mononuclear cells (PBMCs) to create an integrated system that more closely mimics the complexity of the tumour microenvironment and interacts with the immune system. Treatment with OV as a priming agent, followed by pembrolizumab and then paclitaxel, was the most effective in reducing the tumour volume in TNBC co-cultured spheroids. Further, T-cell phenotyping analyses revealed significantly increased infiltration of CD8+, CD4+ T and Tregs cells. Moreover, the observed anti-tumour effects positively correlated with the level of CD4+ T cell infiltrates, suggesting the development of anti-cancer immunity. Our study demonstrated that combining different immunotherapeutic agents (virus, pembrolizumab) with PTX reduced the tumour volume of the TNBC co-cultured spheroids compared to relevant controls. Importantly, sequential administration of the investigational agents (priming with the vector) further enhanced the anti-cancer efficacy in 3D culture over other groups tested. Taken together, these results support further evaluation of the virus in combination with anti-PD-1 and PTX for the treatment of triple-negative breast cancer patients. Importantly, further studies with in vivo models should be conducted to better understand the translational aspects of tested therapy.

Structural basis for specific inhibition of salicylate synthase from Mycobacterium abscessus.

Blocking iron uptake and metabolism has been emerging as a promising therapeutic strategy for the development of novel antimicrobial compounds. Like all mycobacteria, M. abscessus (Mab) has evolved several countermeasures to scavenge iron from host carrier proteins, including the production of siderophores, which play a crucial role in these processes. In this study, we solved, for the first time, the crystal structure of Mab-SaS, the first enzyme involved in the biosynthesis of siderophores. Moreover, we screened a small, focused library and identified a compound exhibiting a potent inhibitory effect against Mab-SaS (IC50 ≈ 2 µM). Its binding mode was investigated by means of Induced Fit Docking simulations, performed on the crystal structure presented herein. Furthermore, cytotoxicity data and pharmacokinetic predictions revealed the safety and drug-likeness of this class of compounds. Finally, the crystallographic data were used to optimize the model for future virtual screening campaigns. Taken together, the findings of our study pave the way for the identification of potent Mab-SaS inhibitors, based on both established and unexplored chemotypes.

Novel combinatorial therapy of oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with anti PD-1 exhibits enhanced anti-cancer efficacy through promotion of intratumoral T-cell infiltration and modulation of tumour microenvironment in mesothelioma mouse model.

Introduction: Malignant mesothelioma is a rare and aggressive form of cancer. Despite improvements in cancer treatment, there are still no curative treatment modalities for advanced stage of the malignancy. The aim of this study was to evaluate the anti-tumor efficacy of a novel combinatorial therapy combining AdV5/3-D24-ICOSL-CD40L, an oncolytic vector, with an anti-PD-1 monoclonal antibody. Methods: The efficacy of the vector was confirmed in vitro in three mesothelioma cell lines - H226, Mero-82, and MSTO-211H, and subsequently the antineoplastic properties in combination with anti-PD-1 was evaluated in xenograft H226 mesothelioma BALB/c and humanized NSG mouse models. Results and discussion: Anticancer efficacy was attributed to reduced tumour volume and increased infiltration of tumour infiltrating lymphocytes, including activated cytotoxic T-cells (GrB+CD8+). Additionally, a correlation between tumour volume and activated CD8+ tumour infiltrating lymphocytes was observed. These findings were confirmed by transcriptomic analysis carried out on resected human tumour tissue, which also revealed upregulation of CD83 and CRTAM, as well as several chemokines (CXCL3, CXCL9, CXCL11) in the tumour microenvironment. Furthermore, according to observations, the combinatorial therapy had the strongest effect on reducing mesothelin and MUC16 levels. Gene set enrichment analysis suggested that the combinatorial therapy induced changes to the expression of genes belonging to the "adaptive immune response" gene ontology category. Combinatorial therapy with oncolytic adenovirus with checkpoint inhibitors may improve anticancer efficacy and survival by targeted cancer cell destruction and triggering of immunogenic cell death. Obtained results support further assessment of the AdV5/3-D24-ICOSL-CD40L in combination with checkpoint inhibitors as a novel therapeutic perspective for mesothelioma treatment.

In vitro immune evaluation of adenoviral vector-based platform for infectious diseases.

New prophylactic vaccine platforms are imperative to combat respiratory infections. The efficacy of T and B memory cell-mediated protection, generated through the adenoviral vector, was tested to assess the effectiveness of the new adenoviral-based platforms for infectious diseases. A combination of adenovirus AdV1 (adjuvant), armed with costimulatory ligands (ICOSL and CD40L), and rRBD (antigen: recombinant nonglycosylated spike protein rRBD) was used to promote the differentiation of T and B lymphocytes. Adenovirus AdV2 (adjuvant), without ligands, in combination with rRBD, served as a control. In vitro T-cell responses to the AdV1+rRBD combination revealed that CD8+ platform-specific T-cells increased (37.2 ± 0.7% vs. 23.1 ± 2.1%), and T-cells acted against SARS-CoV-2 via CD8+TEMRA (50.0 ± 1.3% vs. 36.0 ± 3.2%). Memory B cells were induced after treatment with either AdV1+rRBD (84.1 ± 0.8% vs. 82.3 ± 0.4%) or rRBD (94.6 ± 0.3% vs. 82.3 ± 0.4%). Class-switching from IgM and IgD to isotype IgG following induction with rRBD+Ab was observed. RNA-seq profiling identified gene expression patterns related to T helper cell differentiation that protect against pathogens. The analysis determined signaling pathways controlling the induction of protective immunity, including the MAPK cascade, adipocytokine, cAMP, TNF, and Toll-like receptor signaling pathway. The AdV1+rRBD formulation induced IL-6, IL-8, and TNF. RNA-seq of the VERO E6 cell line showed differences in the apoptosis gene expression stimulated with the platforms vs. mock. In conclusion, AdV1+rRBD effectively generates T and B memory cell-mediated protection, presenting promising results in producing CD8+ platform-specific T cells and isotype-switched IgG memory B cells. The platform induces protective immunity by controlling the Th1, Th2, and Th17 cell differentiation gene expression patterns. Further studies are required to confirm its effectiveness.

From Immunosuppression to Immunomodulation - Turning Cold Tumours into Hot.

Cancer cells employ various mechanisms to evade and suppress anti-cancer immune responses generating a "cold" immunosuppressive tumour microenvironment. Oncolytic viruses are a promising tool to convert tumour immunosuppression to immunomodulation and improve the efficacy of cancer treatment. Emerging preclinical and clinical findings confirm that oncolytic viruses act in a multimodal scheme, triggering lyses, immunogenic cell death and finally inducing anti-cancer immune responses. In this paper, we tested the local administration of a novel oncolytic adenovirus AdV-D24-ICOSL-CD40L expressing co-stimulatory molecules ICOSL and CD40L to induce the production of tumour infiltrating lymphocytes to the site of injection. Subsequently, in immunocompetent mouse models, we studied possible correlation between tumour infiltrates and anti-cancer efficacy. Described results showed that the delivery of oncolytic viruses encoding immunomodulatory transgenes in combination with anti-PD1 resulted in synergistic inhibition of both melanoma and mesothelioma tumours. Importantly anti-cancer effect positively correlated with cytotoxic CD8+ tumour-infiltrating lymphocytes exerting a central role in the tumour volume control thus generating beneficial outcomes that will undoubtedly provide new insights into possible future treatment strategies to combat cancer. Altogether our findings highlight the importance of oncolytic vectors able to modulate anti-cancer immune responses that can correlate with efficacy in solid malignancies.

Novel Insights Into Mesothelioma Therapy: Emerging Avenues and Future Prospects.

Malignant mesothelioma is a rare and aggressive cancer that develops in the thin layer surrounding the mesothelium and is mainly caused by asbestos exposure. Despite improvements in patient prognosis with conventional cancer treatments, such as surgery, chemotherapy, and radiotherapy, there are still no curative treatment modalities for advanced disease. In recent years, new therapeutic avenues have been explored. Improved understanding of the mechanisms underlying the dynamic tumor interaction with the immune system has led to the development of immunotherapeutic approaches. Numerous recent clinical trials have shown a desire to develop more effective treatments that can be used to fight against the disease. Immune checkpoint inhibitors, oncolytic adenoviruses, and their combination represent a promising strategy that can be used to synergistically overcome immunosuppression in the mesothelioma tumor microenvironment. This review provides a synthesized overview of the current state of knowledge on new therapeutic options for mesothelioma with a focus on the results of clinical trials conducted in the field.

Focused Design of Novel Cyclic Peptides Endowed with GABARAP-Inhibiting Activity.

(1) Background: Disfunctions in autophagy machinery have been identified in various conditions, including neurodegenerative diseases, cancer, and inflammation. Among mammalian autophagy proteins, the Atg8 family member GABARAP has been shown to be greatly involved in the autophagy process of prostate cancer cells, supporting the idea that GABARAP inhibitors could be valuable tools to fight the progression of tumors. (2) Methods: In this paper, starting from the X-ray crystal structure of GABARAP in a complex with an AnkirinB-LIR domain, we identify two new peptides by applying in silico drug design techniques. The two ligands are synthesized, biophysically assayed, and biologically evaluated to ascertain their potential anticancer profile. (3) Results: Two cyclic peptides (WC8 and WC10) displayed promising biological activity, high conformational stability (due to the presence of disulfide bridges), and Kd values in the low micromolar range. The anticancer assays, performed on PC-3 cells, proved that both peptides exhibit antiproliferative effects comparable to those of peptide K1, a known GABARAP inhibitor. (4) Conclusions: WC8 and WC10 can be considered new GABARAP inhibitors to be employed as pharmacological tools or even templates for the rational design of new small molecules.

The Multifaceted Roles of Mast Cells in Immune Homeostasis, Infections and Cancers.

Mast cells (MCs) play important roles in normal immune responses and pathological states. The location of MCs on the boundaries between tissues and the external environment, including gut mucosal surfaces, lungs, skin, and around blood vessels, suggests a multitude of immunological functions. Thus, MCs are pivotal for host defense against different antigens, including allergens and microbial pathogens. MCs can produce and respond to physiological mediators and chemokines to modulate inflammation. As long-lived, tissue-resident cells, MCs indeed mediate acute inflammatory responses such as those evident in allergic reactions. Furthermore, MCs participate in innate and adaptive immune responses to bacteria, viruses, fungi, and parasites. The control of MC activation or stabilization is a powerful tool in regulating tissue homeostasis and pathogen clearance. Moreover, MCs contribute to maintaining the homeostatic equilibrium between host and resident microbiota, and they engage in crosstalk between the resident and recruited hematopoietic cells. In this review, we provide a comprehensive overview of the functions of MCs in health and disease. Further, we discuss how mouse models of MC deficiency have become useful tools for establishing MCs as a potential cellular target for treating inflammatory disorders.

MUG Mel3 Cell Lines Reflect Heterogeneity in Melanoma and Represent a Robust Model for Melanoma in Pregnancy.

Melanomas are aggressive tumors with a high metastatic potential and an increasing incidence rate. They are known for their heterogeneity and propensity to easily develop therapy-resistance. Nowadays they are one of the most common cancers diagnosed during pregnancy. Due to the difficulty in balancing maternal needs and foetal safety, melanoma is challenging to treat. The aim of this study was to provide a potential model system for the study of melanoma in pregnancy and to illustrate melanoma heterogeneity. For this purpose, a pigmented and a non-pigmented section of a lymph node metastasis from a pregnant patient were cultured under different conditions and characterized in detail. All four culture conditions exhibited different phenotypic, genotypic as well as tumorigenic properties, and resulted in four newly established melanoma cell lines. To address treatment issues, especially in pregnant patients, the effect of synthetic human lactoferricin-derived peptides was tested successfully. These new BRAF-mutated MUG Mel3 cell lines represent a valuable model in melanoma heterogeneity and melanoma pregnancy research. Furthermore, treatment with anti-tumor peptides offers an alternative to conventionally used therapeutic options-especially during pregnancy.

Polymer Coated Oncolytic Adenovirus to Selectively Target Hepatocellular Carcinoma Cells.

Despite significant advances in chemotherapy, the overall prognosis of hepatocellular carcinoma (HCC) remains extremely poor. HCC targeting strategies were combined with the tumor cell cytotoxicity of oncolytic viruses (OVs) to develop a more efficient and selective therapeutic system. OVs were coated with a polygalactosyl-b-agmatyl diblock copolymer (Gal32-b-Agm29), with high affinity for the asialoglycoprotein receptor (ASGPR) expressed on the liver cell surface, exploiting the electrostatic interaction of the positively charged agmatine block with the negatively charged adenoviral capsid surface. The polymer coating altered the viral particle diameter (from 192 to 287 nm) and zeta-potential (from -24.7 to 23.3 mV) while hiding the peculiar icosahedral symmetrical OV structure, as observed by TEM. Coated OVs showed high potential therapeutic value on the human hepatoma cell line HepG2 (cytotoxicity of 72.4% ± 4.96), expressing a high level of ASGPRs, while a lower effect was attained with ASPGR-negative A549 cell line (cytotoxicity of 54.4% ± 1.59). Conversely, naked OVs showed very similar effects in both tested cell lines. Gal32-b-Agm29 OV coating enhanced the infectivity and immunogenic cell death program in HepG2 cells as compared to the naked OV. This strategy provides a rationale for future studies utilizing oncolytic viruses complexed with polymers toward effective treatment of hepatocellular carcinoma.

PEG-polyaminoacid based micelles for controlled release of doxorubicin: Rational design, safety and efficacy study.

A library of amphiphilic monomethoxypolyethylene glycol (mPEG) terminating polyaminoacid co-polymers able to self-assemble into colloidal systems was screened for the delivery and controlled release of doxorubicin (Doxo). mPEG-Glu/Leu random co-polymers were generated by Ring Opening Polymerization from 5 kDa mPEG-NH2 macroinitiator using 16:0:1, 8:8:1, 6:10:1, 4:12:1 γ-benzyl glutamic acid carboxy anhydride monomer/leucine N-carboxy anhydride monomer/PEG molar ratios. Glutamic acid was selected for chemical conjugation of Doxo, while leucine units were introduced in the composition of the polyaminoacid block as spacer between adjacent glutamic repeating units to minimize the steric hindrance that could impede the Doxo conjugation and to promote the polymer self-assembly by virtue of the aminoacid hydrophobicity. The benzyl ester protecting the γ-carboxyl group of glutamic acid was quantitatively displaced with hydrazine to yield mPEG5kDa-b-(hydGlum-r-Leun). Doxo was conjugated to the diblock co-polymers through pH-sensitive hydrazone bond. The Doxo derivatized co-polymers obtained with a 16:0:1, 8:8:1, 6:10:1 Glu/Leu/PEG ratios self-assembled into 30-40 nm spherical nanoparticles with neutral zeta-potential and CMC in the range of 4-7 µM. At pH 5.5, mimicking endosome environment, the carriers containing leucine showed a faster Doxo release than at pH 7.4, mimicking the blood conditions. Doxo-loaded colloidal formulations showed a dose dependent cytotoxicity on two cancer cell lines, CT26 murine colorectal carcinoma and 4T1 murine mammary carcinoma with IC50 slightly higher than those of free Doxo. The carrier assembled with the polymer containing 6:10:1 hydGlu/Leu/PEG molar ratio {mPEG5kDa-b-[(Doxo-hydGlu)6-r-Leu10]} was selected for subsequent in vitro and in vivo investigations. Confocal imaging on CT26 cell line showed that intracellular fate of the carrier involves a lysosomal trafficking pathway. The intratumor or intravenous injection to CT26 and 4T1 subcutaneous tumor bearing mice yielded higher antitumor activity compared to free Doxo. Furthermore, mPEG5kDa-b-[(Doxo-hydGlu)6-r-Leu10] displayed a better safety profile when compared to commercially available Caelyx®.

Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model.

Malignant melanoma, an aggressive form of skin cancer, has a low five-year survival rate in patients with advanced disease. Immunotherapy represents a promising approach to improve survival rates among patients at advanced stage. Herein, the aim of the study was to design and produce, by using engineering tools, a novel oncolytic adenovirus AdV-D24- inducible co-stimulator ligand (ICOSL)-CD40L expressing potent co-stimulatory molecules enhancing clinical efficacy through the modulation of anti-cancer immune responses. Firstly, we demonstrated the vector's identity and genetic stability by restriction enzyme assay and sequencing, then, by performing in vitro and in vivo pre-clinical studies we explored the anti-cancer efficacy of the virus alone or in combination with anti PD-1 inhibitor in human melanoma cell lines, i.e., MUG Mel-1 and MUG Mel-2, and in immunocompetent C57BL/6 melanoma B16V mouse model. We showed that both monotherapy and combination approaches exhibit enhanced anti-cancer ability and immunogenic cell death in in vitro settings. Furthermore, AdV-D24-ICOSL-CD40L combined with anti PD-1 revealed a fall in tumor volume and 100% survival in in vivo context, thus suggesting enhanced efficacy and survival via complementary anti-cancer properties of those agents in melanoma therapy. Collectively, the novel oncolytic vector AdV-D24-ICOSL-CD40L alone or in combination with anticancer drugs, such as check point inhibitors, may open novel therapeutic perspectives for the treatment of melanoma.

Circulating extracellular vesicles release oncogenic miR-424 in experimental models and patients with aggressive prostate cancer.

Extracellular vesicles (EVs) are relevant means for transferring signals across cells and facilitate propagation of oncogenic stimuli promoting disease evolution and metastatic spread in cancer patients. Here, we investigated the release of miR-424 in circulating small EVs or exosomes from prostate cancer patients and assessed the functional implications in multiple experimental models. We found higher frequency of circulating miR-424 positive EVs in patients with metastatic prostate cancer compared to patients with primary tumors and BPH. Release of miR-424 in small EVs was enhanced in cell lines (LNCaPabl), transgenic mice (Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG) and patient-derived xenograft (PDX) models of aggressive disease. EVs containing miR-424 promoted stem-like traits and tumor-initiating properties in normal prostate epithelial cells while enhanced tumorigenesis in transformed prostate epithelial cells. Intravenous administration of miR-424 positive EVs to mice, mimicking blood circulation, promoted miR-424 transfer and tumor growth in xenograft models. Circulating miR-424 positive EVs from patients with aggressive primary and metastatic tumors induced stem-like features when supplemented to prostate epithelial cells. This study establishes that EVs-mediated transfer of miR-424 across heterogeneous cell populations is an important mechanism of tumor self-sustenance, disease recurrence and progression. These findings might indicate novel approaches for the management and therapy of prostate cancer.

Transplantation of autologous extracellular vesicles for cancer-specific targeting.

Nano- and microsized extracellular vesicles (EVs) are naturally occurring cargo-bearing packages of regulatory macromolecules, and recent studies are increasingly showing that EVs are responsible for physiological intercellular communication. Nanoparticles encapsulating anti-tumor theranostics represent an attractive "exosome-interfering" strategy for cancer therapy. Methods: Herein, by labeling plasma-derived EVs with indocyanine green (ICG) and following their biodistribution by in vivo and ex vivo imaging, we demonstrate the existence of nanoparticles with a highly selective cancer tropism in the blood of colorectal cancer (CRC) patients but not in that of healthy volunteers. Results: In CRC patient-derived xenograft (PDX) mouse models, we show that transplanted EVs recognize tumors from the cognate nanoparticle-generating individual, suggesting the theranostic potential of autologous EVs encapsulating tumor-interfering molecules. In large canine breeds bearing spontaneous malignant skin and breast tumors, the same autologous EV transplantation protocol shows comparable safety and efficacy profiles. Conclusions: Our data show the existence of an untapped resource of intercellular communication present in the blood of cancer patients, which represents an efficient and highly biocompatible way to deliver molecules directly to the tumor with great precision. The novel EV-interfering approach proposed by our study may become a new research direction in the complex interplay of modern personalized cancer therapy.

Cancer-derived EVs show tropism for tissues at early stage of neoplastic transformation.

From the past decade, extracellular vesicles (EVs) have attracted considerable attention as tools for the selective delivery of anti-neoplastic drugs to cancer tissues. Compared to other nanoparticles, EVs display interesting unique features including immune compatibility, low toxicity and the ability to encapsulate a large variety of small- and macro-molecules. However, in virtually all studies, investigations on EVs have been focused on fully transformed cancers: the possibility to apply EV technology also to early-stage tumors has never been explored. Methods: Herein, we studied the ability of cancer-derived EVs to recognize and deliver their cargo also to incipient cancers. To this purpose, EV biodistribution was studied in MMTV-NeuT genetically modified mice during early mammary transformation, in fully developed breast tumors and in the normal gland of wild type syngeneic mice. EVs were loaded with indocyanine green (ICG), a near-infrared (NIR) dye together with oncolytic viruses and i.v. injected in mice. The nanoparticle biodistribution was assayed by in vivo and ex vivo optical imaging (detecting the ICG) and semiquantitative real-time PCR (measuring the adenoviral genome) in different tissues. Results: Our results demonstrate the ability of cancer-derived EVs to recognize early-stage neoplastic tissues opening the possibility to selectively deliver theranostics also for tumor prevention. Conclusions: Taken together our study demonstrates the ability of EVs to recognize and deliver diagnostic and therapeutic agents not only to fully transformed tissues but also to early stage tumors. These findings pave the way for the synthesis of "universal" EVs-based formulation for targeted cancer therapy.

Glabrescione B delivery by self-assembling micelles efficiently inhibits tumor growth in preclinical models of Hedgehog-dependent medulloblastoma.

Aberrant activation of the Hedgehog (Hh) pathway leads to the development of several tumors, including medulloblastoma (MB), the most common pediatric brain malignancy. Hh inhibitors acting on GLI1, the final effector of Hh signaling, offer a valuable opportunity to overcome the pitfalls of the existing therapies to treat Hh-driven cancers. In this study, the toxicity, delivery, biodistribution, and anticancer efficacy of Glabrescione B (GlaB), a selective GLI1 inhibitor, were investigated in preclinical models of Hh-dependent MB. To overcome its poor water solubility, GlaB was formulated with a self-assembling amphiphilic polymer forming micelles, called mPEG5kDa-cholane. mPEG5kDa-cholane/GlaB showed high drug loading and stability, low cytotoxicity, and long permanence in the bloodstream. We found that mPEG5kDa-cholane efficiently enhanced the solubility of GlaB, thus avoiding the use of organic solvents. mPEG5kDa-cholane/GlaB possesses favorable pharmacokinetics and negligible toxicity. Remarkably, GlaB encapsulated in mPEG5kDa-cholane micelles was delivered through the blood-brain barrier and drastically inhibited tumor growth in both allograft and orthotopic models of Hh-dependent MB. Our findings reveal that mPEG5kDa-cholane/GlaB is a good candidate for the treatment of Hh-driven tumors and provide relevant implications for the translation of GlaB into clinical practice.

Thermosensitive "Smart" Surfaces for Biorecognition Based Cell Adhesion and Controlled Detachment.

The biorecognition-based control of attachment/detachment of MCF-7 cancer cells from polymer-coated surfaces is demonstrated. A glass surface is coated with a thermoresponsive statistical copolymer of poly(N-isopropylacrylamide-co-acrylamide) [p(NIPAm-co-Am)], which is end-capped with the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide, and the hydrophilic polymer poly(ethylene glycol) (PEG). Below the lower critical solution temperature (LCST) of p(NIPAm-co-Am) (38 °C), the copolymers are in the extended conformation, allowing for accessibility of the GRGDS peptides to membrane-associated integrins thus enabling cell attachment. Above the LCST, the p(NIPAm-co-Am) polymers collapse into globular conformations, resulting in the shielding of the GRGDS peptides into the PEG brush with consequent inaccessibility to cell-surface integrins, causing cell detachment. The surface coating is carried out by a multi-step procedure that included: glass surface amination with 3-aminopropyltriethoxysilane; reaction of mPEG5kDa -N-hydroxysuccinimide (NHS) and p(NIPam-co-Am)15.1kDa -bis-NHS with the surface aminopropyl groups and conjugation of GRGDS to the carboxylic acid termini of p(NIPam-co-Am)15.1kDa -COOH. A range of spectrophotometric, surface, and microscopy assays confirmed the identity of the polymer-coated substrates. Competition studies prove that MCF-7 cancer cells are attached via peptide recognition at the coated surfaces according to the mPEG5kDa /p(NIPam-co-Am)15.1kDa -GRGDS molar ratio. These data suggest the system can be exploited to modulate cell integrin/GRGDS binding for controlled cell capture and release.

Doxorubicin liposomes cell penetration enhancement and its potential drawbacks for the tumor targeting efficiency.

The clinical efficacy of the PEGylated doxorubicin liposomes (PLD) is limited by low tumor accumulation and limited intra-tumoral disposition. Decoration with the cell penetration enhancers (CPEs) can increase the PLD permeability via the biological barriers, however at the expense of enhanced distribution to the non-target organs and tissues, and may interfere with their tumor accumulation and with the resulting anti-cancer effects. We investigated the effect of the surface CPE agent tetraArg-[G-1]-distearoyl glycerol (DAG-Arg4) on the systemic and intra-tumoral accumulation of PLD, using a 4 T1-Luc murine orthotopic model of breast cancer, using several analytical approaches. CPE-decorated liposomes undergo efficient in vitro endocytosis, and delivered doxorubicin to the cell nuclei. In vivo, they had lower tumor and spleen accumulation, similar liver accumulation, and higher lung accumulation, as compared to those of the PLD. Despite the lower tumor accumulation, CPE-decorated liposomes induced more prominent in vivo anti-cancer effects, as compared to the PLD, apparently ascribable to the higher intra-tumoral permeability mediated by the CPE surface residues. Overall, liposomes decoration with the CPE residues had mostly beneficial effects on their systemic and intra-tumoral disposition. The mechanisms of the CPE-mediated effects on the liposome disposition should be further assessed with additional experimental models using robust analytical methods with high spatial resolution.

From Conventional Therapies to Immunotherapy: Melanoma Treatment in Review.

In this review, we discuss the use of oncolytic viruses and checkpoint inhibitors in cancer immunotherapy in melanoma, with a particular focus on combinatory therapies. Oncolytic viruses are promising and novel anti-cancer agents, currently under investigation in many clinical trials both as monotherapy and in combination with other therapeutics. They have shown the ability to exhibit synergistic anticancer activity with checkpoint inhibitors, chemotherapy, radiotherapy. A coupling between oncolytic viruses and checkpoint inhibitors is a well-accepted strategy for future cancer therapies. However, eradicating advanced cancers and tailoring the immune response for complete tumor clearance is an ongoing problem. Despite current advances in cancer research, monotherapy has shown limited efficacy against solid tumors. Therefore, current improvements in virus targeting, genetic modification, enhanced immunogenicity, improved oncolytic properties and combination strategies have a potential to widen the applications of immuno-oncology (IO) in cancer treatment. Here, we summarize the strategy of combinatory therapy with an oncolytic vector to combat melanoma and highlight the need to optimize current practices and improve clinical outcomes.