Towards Protein-Based Viral Mimetics for Cancer Therapies

High resistance and recurrence rates, along with elevated drug clearance, compel the use of maximum tolerated drug doses in cancer therapy, resulting in high-grade toxicities and limited clinical applicability. Promoting active drug accumulation in tumor tissues would minimize such issues and improve therapeutic outcomes. A new class of therapeutic drugs suitable for the task has emerged based on the concept of virus-mimetic nanocarriers, or 'artificial viruses.' Among the spectrum of materials under exploration in nanocarrier research, proteins offer unparalleled structural and functional versatility for designing viruslike molecular vehicles. By exhibiting 'smart' functions and biomimetic traits, protein-based nanocarriers will be a step ahead of the conventional drug-protein conjugates already in the clinics in ensuring efficient delivery of passenger antitumor drugs

uPA/uPAR and SERPINE1 in head and neck cancer: role in tumor resistance, metastasis, prognosis and therapy

There is strong evidence supporting the role of the plasminogen activator system in head and neck squamous cell carcinoma (HNSCC), particularly of its uPA (urokinase plasminogen activator) / uPAR (urokinase plasminogen activator receptor) and SERPINE1 components. Overexpression of uPA/uPAR and SERPINE1 enhances tumor cell migration and invasion and plays a key role in metastasis development, conferring poor prognosis. The apparent paradox of uPA/uPAR and its inhibitor SERPINE1 producing similar effects is solved by the identification of SERPINE1 activated signaling pathways independent of uPA inhibition. Both uPA/uPAR and SERPINE1 are directly linked to the induction of epithelial-to-mesenchymal transition, the acquisition of stem cell properties and resistance to antitumor agents. The aim of this review is to provide insight on the deregulation of these proteins in all these processes. We also summarize their potential value as prognostic biomarkers or potential drug targets in HNSCC patients. Concomitant overexpression of uPA/uPAR and SERPINE1 is associated with a higher risk of metastasis and could be used to identify patients that would benefit from an adjuvant treatment. In the future, the specific inhibitors of uPA/uPAR and SERPINE1, which are still under development, could be used to design new therapeutic strategies in HNSCCs

All-in-one biofabrication and loading of recombinant vaults in human cells

Altres ajuts: Fundación Mutua Madrileña (FMMA) through project 'Targeted therapy for selective elimination of metastatic stem cells CXCR4+ in endometrial cancer' (AP1666942017)Altres ajuts: Asociación Española Contra el Cancer (AECC) through project 'Development of an antitumor protein delivery system into ovarian cancer cells using the subcellular vault' (IDEAS18038BENI)One of the most promising approaches in the drug delivery field is the use of naturally occurring self-assembling protein nanoparticles, such as virus-like particles, bacterial microcompartments or vault ribonucleoprotein particles as drug delivery systems (DDSs). Among them, eukaryotic vaults show a promising future due to their structural features, in vitro stability and non-immunogenicity. Recombinant vaults are routinely produced in insect cells and purified through several ultracentrifugations, both tedious and time-consuming processes. As an alternative, this work proposes a new approach and protocols for the production of recombinant vaults in human cells by transient gene expression of a His-tagged version of the major vault protein (MVP-H6), the development of new affinity-based purification processes for such recombinant vaults, and the all-in-one biofabrication and encapsulation of a cargo recombinant protein within such vaults by their co-expression in human cells. Protocols proposed here allow the easy and straightforward biofabrication and purification of engineered vaults loaded with virtually any INT-tagged cargo protein, in very short times, paving the way to faster and easier engineering and production of better and more efficient DDS

Industrial By-Products As a Novel Circular Source of Biocompatible Extracellular Vesicles

Extracellular vesicles (EVs) constitute an intricate system of molecular exchange that has recently gained tremendous interest. However, sustainable sources of safe biological EVs remain scarce and elusive. This study explores and defines the use of food industry by-products (BP) as a circular source of safe biocompatible EVs. Averaged diameter and molecular compositions indicate a large yield of exosomes and high abundancy of membrane lipids with signaling capacity in these vesicles. Complex proteomes mimicking those circulating in human blood plasma are also identified. Furthermore, BP-EVs do not show relevant cytotoxicity and display excellent oral and intravenous bioavailability together with specific organ targeting capacity. Collectively, it is believed that the novel findings reported here will open substantial venues for the use of BP as an optimal source of biocompatible nanovesicles in manifold applications of the biotechnological and biomedical fields.The authors sincerely thank Gemma Plaza, oenologist at the Castell del Remei winery in Penelles, Lleida, Spain and Juan Carlos Blanco, production manager at Mahou San Miguel in Alovera, Madrid, Spain for their kind and altruistic help on the obtention of their respective industry by-product samples. The authors also thank Dr. Hector Peinado and his research group at the National Center for Oncology Research (CNIO) in Madrid (Spain) for their support on the morphometric characterization of BP-EVs. Support for this work was provided by the Research and Education Council of the Community of Madrid, Spain (2018-T1/ BIO-10633), Ministry of Science and Innovation, Spain (PID2020- 114885RB-C21) and a FIS project by Carlos III Institute of Health (ISCIII), Spain (PI20/00623). A.S. acknowledges a grant from the Talento Program 2018 of the Community of Madrid. X.G.-P. acknowledges grants from Sara Borrell postdoctoral program (CD19/00243) and Miguel Servet tenure track program (CP21/00096) of the Instituto de Salud Carlos III (ISCIII, Spain), respectively awarded on the 2019 and 2021 calls under ISCIII-Health Strategy Actions [These grants are cofunded with European Union Funds (ISCIII Miguel Servet Program 2021 is cofunded by Fondo Social Europeo Plus, FSE+)]. M.V.C. acknowledges a Miguel Servet program contract (CPII20/00007). C.L.’s Ph.D. was funded by the Regional Ministry of Science, Universities and Innovation of the Community of Madrid and the European Social Fund for the recruitment of predoctoral researchers (PEJD-2019-PRE/BIO-16475). IRBLLEIDA and X.G.-P. are co-funded by CERCA Program/Generalitat de Catalunya

A novel orally available inhibitor of focal adhesion signaling increases survival in a xenograft model of diffuse large B-cell lymphoma with central nervous system involvement

Central nervous system dissemination is a relatively uncommon but almost always fatal complication in diffuse large B-cell lymphoma patients. Optimal therapy for central nervous involvement in this malignancy has not been established. In this paper, we aimed to evaluate the therapeutic effect of E7123, a celecoxib derivative that inhibits focal adhesion signaling, in a novel xenograft model of diffuse large B-cell lymphoma with central nervous system involvement. Cells obtained after disaggregation of HT subcutaneous tumors (HT-SC cells) were intravenously injected in NOD/SCID mice. These mice received oral vehicle or 75 mg/kg of E7123 daily until they were euthanized for weight loss or signs of sickness. The antitumor effect of E7123 was validated in an independent experiment using a bioluminescent mouse model. Intravenously injected HT-SC cells showed higher take rate and higher central nervous system tropism (associated with increased expression of beta 1-integrin and p130Cas proteins) than HT cells. The oral administration of E7123 significantly increased survival time in 2 independent experiments using mice injected with unmodified or bioluminescent HT-SC cells. We have developed a new xenograft model of diffuse large B-cell lymphoma with central nervous system involvement that can be used in the pre-clinical evaluation of new drugs for this malignancy. E7123 is a new, well-tolerated and orally available therapeutic agent that merits further investigation since it may improve current management of diffuse large B-cell lymphoma patients with central nervous system involvement

Efficient bioactive oligonucleotide-protein conjugation for cell-targeted cancer therapy

Oligonucleotide-protein conjugates have important applications in biomedicine. Simple and efficient methods are described for the preparation of these conjugates. Specifically, we describe a new method in which a bifunctional linker is attached to thiol-oligonucleotide to generate a reactive intermediate that is used to link to the protein. Having similar conjugation efficacy compared with the classical method in which the bifunctional linker is attached first to the protein, this new approach produces significantly more active conjugates with higher batch to batch reproducibility. In a second approach, direct conjugation is proposed using oligonucleotides carrying carboxyl groups. These methodologies have been applied to prepare nanoconjugates of an engineered nanoparticle protein carrying a T22 peptide with affinity for the CXCR4 chemokine receptor and oligomers of the antiproliferative nucleotide 2'-deoxy-5-fluorouridine in a very efficient way. The protocols have potential uses for the functionalization of proteins, amino-containing polymers or amino-lipids in order to produce complex therapeutic nucleic acid delivery system

CXCR7 expression in diffuse large B-cell lymphoma identifies a subgroup of CXCR4+ patients with good prognosis

© 2018 Moreno et al.The CXCR4/CXCL12 axis has been extensively associated with different types of cancer correlating with higher aggressiveness and metastasis. In diffuse large B-cell lymphoma (DLBCL), the expression of the chemokine receptor CXCR4 is involved in the dissemination of malignant B cells and is a marker of poor prognosis. CXCR7 is a chemokine receptor that binds to the same ligand as CXCR4 and regulates de CXCR4-CXCL12 axis. These findings together with the report of CXCR7 prognostic value in several tumor types, led us to evaluate the expression of CXCR7 in diffuse large B-cell lymphoma biopsies. Here, we describe that CXCR7 receptor is an independent prognostic factor that associates with good clinical outcome. Moreover, the expression of CXCR7 associates with increased survival in CXCR4+ but not in CXCR4- DLBCL patients. Thus, the combined immunohistochemical evaluation of both CXCR7 and CXCR4 expression in DLBCL biopsies may improve their prognostic value as single markers. Finally, we show that CXCR7 overexpression in vitro is able to diminish DLBCL cell survival and increase their sensitivity to antitumor drugs. Hence, further studies on the CXCR7 receptor may establish its role in DLBCL and the molecular mechanisms that modulate CXCR4 activity.This work was supported by Instituto de Salud Carlos III (co-founding from FEDER) [ FIS PI11/00872, RD12/0036/0071, FIS PI14/00450 to J.S., FIS PI15/00378 to R.M., PIE15/00028 to R.M. and J.S., RD12/0036/0069, PS09/01382 to M.G.D, PI15/01393 to M.A, CD13/00074 to V.P.]; Centro de Investigación Biomédica en Red (CIBER) [CB06/01/1031 and Nanomets3 to R.M. and CB16/12/00233 to M.G.D.]; Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) [2014-SGR-1041, 2014PROD0005 to R.M and 2014-SGR-1281 to J.S]; Fundació La Marató TV3 [416/C/2013-2030 to R.M, 100830/31/32 to J.S and M.G.D.]; Josep Carreras Leukaemia Research Institute [P/AG 2014 to R.M.]; the Health Council of Castilla y León (BIO/SA56/13 and BIO/SA78/15 to M.A.); Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) [2017FI_B00680 to A.F.]

Antitumoral effect of maintained neutrophilia induced by rhG-CSF in a murine model of pancreatic cancer

Although the protumoral functions of polymorphonuclear neutrophils are well known, some now-forgotten studies report antitumoral roles for these cells. The present work examines the antitumoral effect of maintained neutrophilia induced via the injection of recombinant human granulocyte colony stimulating factor (rhG-CSF, 100 μg/kg/day) in a Panc-1 subcutaneous xenograft murine model of pancreatic cancer. This treatment was compared with gemcitabine administration (120 mg/kg every two days) and a saline control (n = 6–7 mice per group). Compared to the controls, both the rhG-CSF- and gemcitabine-treated mice showed significantly suppressed tumor growth by day 4 (p < 0.001 and p = 0.013 respectively). From a mean starting volume of 106.9 ± 3.1 mm3 for all treatment groups, the final mean tumor volumes reached were 282.0 ± 30.7 mm3 for the rhG-CSF-treated mice, 202.6 ± 18.1 mm3 for the gemcitabine-treated mice and 519.4 ± 62.9 mm3 for the control mice (p < 0.004 and p < 0.01, respectively, vs. control). The rhG-CSF-treated tumors showed higher percentage necrosis than those treated with gemcitabine (37.4 ± 4.6 vs. 7.5 ± 3.0; p < 0.001). This is the first report of a clear anti-tumoral effect of rhG-CSF when used in monotherapy against pancreatic cancer. Since rhG-CSF administration is known to be associated with very few adverse events, it may offer an attractive alternative in the clinical treatment of pancreatic cancer

Collaborative membrane activity and receptor-dependent tumor cell targeting for precise nanoparticle delivery in CXCR4+ colorectal cancer

Altres ajuts: EU COST Action CA 17140. AV received an ICREA ACADEMIA award.By the appropriate selection of functional peptides and proper accommodation sites, we have generated a set of multifunctional proteins that combine selectivity for CXCR4 cell binding and relevant endosomal escape capabilities linked to the viral peptide HA2. In particular, the construct T22-GFP-HA2-H6 forms nanoparticles that upon administration in mouse models of human, CXCR4 colorectal cancer, accumulates in primary tumor at levels significantly higher than the parental T22-GFP-H6 HA2-lacking version. The in vivo application of a CXCR4 antagonist has confirmed the prevalence of the CXCR4 tumor tissue selectivity over unspecific cell penetration, upon systemic administration of the material. Such specificity is combined with improved endosomal escape, what overall results in a precise and highly efficient tumor biodistribution. These data strongly support the functional recruitment as a convenient approach to generate protein materials for clinical applications. More precisely, they also support the unexpected concept that enhancing the unspecific membrane activity of a protein material does not necessarily compromise, but it can even improve, the selective cell targeting offered by an accompanying functional module. Statement of Significance: We have shown here that the combination of cell-penetrating and tumor cell-targeting peptides dramatically enhances precise tumor accumulation of protein-only nanoparticles intended for selective drug delivery, in mouse models of human colorectal cancer. This fact is a step forward for the rational design of multifunctional protein nanomaterials for improved cancer therapies

Nanostructures for delivery of therapeutic oligonucleotides

Fluoropyrimidines, such as 5-fluorouracil (5-FU) and related prodrugs are considered one of the most-successful agents in the treatment of colorectal cancer, yet poor specificity and tumor cell resistance remain major limiting bottlenecks. In this work, we exploited the ability of two DNA nanostructures, DNA tetrahedron (Td) and rectangle DNA origami, to incorporate 5-fluoro-2¿-deoxyuridine (FdUn) oligomers. DNA nanostructures bearing FdUn revealed to be able to circumvent 5-FU low sensitivity of colorectal drug-resistant cancer cells. Both DNA nanostructures attained comparable cytotoxic effect yet Td displays higher antiproliferative action. DNA nanoscaffolds functionalized with FdUn exhibited an enhanced cytotoxicity and higher ability to trigger apoptosis in colorectal cancer cells relative to conventional 5-FU and FdU drugs, especially having cholesterol as internalization helper. In addition, we will describe the preparation of a protein nanoparticle carrying FdUn oligomers with high affinity for cancer stem cells preventing the formation of metastasis in mice. The present work shows that DNA and protein nanoparticles are privileged carriers for delivering fluoropyrimidines, opening new avenues to the development of promising therapeutics for cancer treatment