Extracellular vesicles secreted by triple-negative breast cancer stem cells trigger premetastatic niche remodeling and metastatic growth in the lungs.

Tumor secreted extracellular vesicles (EVs) are potent intercellular signaling platforms. They are responsible for the accommodation of the premetastatic niche (PMN) to support cancer cell engraftment and metastatic growth. However, complex cancer cell composition within the tumor increases also the heterogeneity among cancer secreted EVs subsets, a functional diversity that has been poorly explored. This phenomenon is particularly relevant in highly plastic and heterogenous triple-negative breast cancer (TNBC), in which a significant representation of malignant cancer stem cells (CSCs) is displayed. Herein, we selectively isolated and characterized EVs from CSC or differentiated cancer cells (DCC; EVsCSC and EVsDCC , respectively) from the MDA-MB-231 TNBC cell line. Our results showed that EVsCSC and EVsDCC contain distinct bioactive cargos and therefore elicit a differential effect on stromal cells in the TME. Specifically, EVsDCC activated secretory cancer associated fibroblasts (CAFs), triggering IL-6/IL-8 signaling and sustaining CSC phenotype maintenance. Complementarily, EVsCSC promoted the activation of α-SMA+ myofibroblastic CAFs subpopulations and increased the endothelial remodeling, enhancing the invasive potential of TNBC cells in vitro and in vivo. In addition, solely the EVsCSC mediated signaling prompted the transformation of healthy lungs into receptive niches able to support metastatic growth of breast cancer cells.

Selectively Targeting Breast Cancer Stem Cells by 8-Quinolinol and Niclosamide.

Cancer maintenance, metastatic dissemination and drug resistance are sustained by cancer stem cells (CSCs). Triple negative breast cancer (TNBC) is the breast cancer subtype with the highest number of CSCs and the poorest prognosis. Here, we aimed to identify potential drugs targeting CSCs to be further employed in combination with standard chemotherapy in TNBC treatment. The anti-CSC efficacy of up to 17 small drugs was tested in TNBC cell lines using cell viability assays on differentiated cancer cells and CSCs. Then, the effect of 2 selected drugs (8-quinolinol -8Q- and niclosamide -NCS-) in the cancer stemness features were evaluated using mammosphere growth, cell invasion, migration and anchorage-independent growth assays. Changes in the expression of stemness genes after 8Q or NCS treatment were also evaluated. Moreover, the potential synergism of 8Q and NCS with PTX on CSC proliferation and stemness-related signaling pathways was evaluated using TNBC cell lines, CSC-reporter sublines, and CSC-enriched mammospheres. Finally, the efficacy of NCS in combination with PTX was analyzed in vivo using an orthotopic mouse model of MDA-MB-231 cells. Among all tested drug candidates, 8Q and NCS showed remarkable specific anti-CSC activity in terms of CSC viability, migration, invasion and anchorage independent growth reduction in vitro. Moreover, specific 8Q/PTX and NCS/PTX ratios at which both drugs displayed a synergistic effect in different TNBC cell lines were identified. The sole use of PTX increased the relative presence of CSCs in TNBC cells, whereas the combination of 8Q and NCS counteracted this pro-CSC activity of PTX while significantly reducing cell viability. In vivo, the combination of NCS with PTX reduced tumor growth and limited the dissemination of the disease by reducing circulating tumor cells and the incidence of lung metastasis. The combination of 8Q and NCS with PTX at established ratios inhibits both the proliferation of differentiated cancer cells and the viability of CSCs, paving the way for more efficacious TNBC treatments.

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method.

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.

Zileuton™ loaded in polymer micelles effectively reduce breast cancer circulating tumor cells and intratumoral cancer stem cells.

Tumor recurrence, metastatic spread and progressive gain of chemo-resistance of advanced cancers are sustained by the presence of cancer stem cells (CSCs) within the tumor. Targeted therapies with the aim to eradicate these cells are thus highly regarded. However, often the use of new anti-cancer therapies is hampered by pharmacokinetic demands. Drug delivery through nanoparticles has great potential to increase efficacy and reduce toxicity and adverse effects. However, its production has to be based on intelligent design. Likewise, we developed polymeric nanoparticles loaded with Zileuton™, a potent inhibitor of cancer stem cells (CSCs), which was chosen based on high throughput screening. Its great potential for CSCs treatment was subsequently demonstrated in in vitro and in in vivo CSC fluorescent models. Encapsulated Zileuton™ reduces amount of CSCs within the tumor and effectively blocks the circulating tumor cells (CTCs) in the blood stream and metastatic spread.

Improved pharmacokinetic profile of lipophilic anti-cancer drugs using ανß3-targeted polyurethane-polyurea nanoparticles.

Glutathione degradable polyurethane-polyurea nanoparticles (PUUa NP) with a disulfide-rich multiwalled structure and a cyclic RGD peptide as a targeting moiety were synthesized, incorporating a very lipophilic chemotherapeutic drug named Plitidepsin. In vitro studies indicated that encapsulated drug maintained and even improved its cytotoxic activity while in vivo toxicity studies revealed that the maximum tolerated dose (MTD) of Plitidepsin could be increased three-fold after encapsulation. We also found that pharmacokinetic parameters such as maximum concentration (Cmax), area under the curve (AUC) and plasma half-life were significantly improved for Plitidepsin loaded in PUUa NP. Moreover, biodistribution assays in mice showed that RGD-decorated PUUa NP accumulate less in spleen and liver than non-targeted conjugates, suggesting that RGD-decorated nanoparticles avoid sequestration by macrophages from the reticuloendothelial system. Overall, our results indicate that polyurethane-polyurea nanoparticles represent a very valuable nanoplatform for the delivery of lipophilic drugs by improving their toxicological, pharmacokinetic and whole-body biodistribution profiles.

A novel bio-functional material based on mammalian cell aggresomes.

Aggresomes are protein aggregates found in mammalian cells when the intracellular protein degradation machinery is over-titered. Despite that they abound in cells producing recombinant proteins of biomedical and biotechnological interest, the physiological roles of these protein clusters and the functional status of the embedded proteins remain basically unexplored. In this work, we have determined for the first time that, like in bacterial inclusion bodies, deposition of recombinant proteins into aggresomes does not imply functional inactivation. As a model, human α-galactosidase A (GLA) has been expressed in mammalian cells as enzymatically active, mechanically stable aggresomes showing higher thermal stability than the soluble GLA version. Since aggresomes are easily produced and purified, we propose these particles as novel functional biomaterials with potential as carrier-free, self-immobilized catalyzers in biotechnology and biomedicine.

Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A.

Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.

Fluorescent CSC models evidence that targeted nanomedicines improve treatment sensitivity of breast and colon cancer stem cells.

To be able to study the efficacy of targeted nanomedicines in marginal population of highly aggressive cancer stem cells (CSC), we have developed a novel in vitro fluorescent CSC model that allows us to visualize these cells in heterogeneous population and to monitor CSC biological performance after therapy. In this model tdTomato reporter gene is driven by CSC specific (ALDH1A1) promoter and contrary to other similar models, CSC differentiation and un-differentiation processes are not restrained and longitudinal studies are feasible. We used this model for preclinical validation of poly[(d,l-lactide-co-glycolide)-co-PEG] (PLGA-co-PEG) micelles loaded with paclitaxel. Further, active targeting against CD44 and EGFR receptors was validated in breast and colon cancer cell lines. Accordingly, specific active targeting toward surface receptors enhances the performance of nanomedicines and sensitizes CSC to paclitaxel based chemotherapy. FROM THE CLINICAL EDITOR: Many current cancer therapies fail because of the failure to target cancer stem cells. This surviving population soon proliferates and differentiates into more cancer cells. In this interesting article, the authors designed an in vitro cancer stem cell model to study the effects of active targeting using antibody-labeled micelles containing chemotherapeutic agent. This new model should allow future testing of various drug/carrier platforms before the clinical phase.

Prostate tumor OVerexpressed-1 (PTOV1) down-regulates HES1 and HEY1 notch targets genes and promotes prostate cancer progression.

BACKGROUND: PTOV1 is an adaptor protein with functions in diverse processes, including gene transcription and protein translation, whose overexpression is associated with a higher proliferation index and tumor grade in prostate cancer (PC) and other neoplasms. Here we report its interaction with the Notch pathway and its involvement in PC progression. METHODS: Stable PTOV1 knockdown or overexpression were performed by lentiviral transduction. Protein interactions were analyzed by co-immunoprecipitation, pull-down and/or immunofluorescence. Endogenous gene expression was analyzed by real time RT-PCR and/or Western blotting. Exogenous promoter activities were studied by luciferase assays. Gene promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP). In vivo studies were performed in the Drosophila melanogaster wing, the SCID-Beige mouse model, and human prostate cancer tissues and metastasis. The Excel package was used for statistical analysis. RESULTS: Knockdown of PTOV1 in prostate epithelial cells and HaCaT skin keratinocytes caused the upregulation, and overexpression of PTOV1 the downregulation, of the Notch target genes HEY1 and HES1, suggesting that PTOV1 counteracts Notch signaling. Under conditions of inactive Notch signaling, endogenous PTOV1 associated with the HEY1 and HES1 promoters, together with components of the Notch repressor complex. Conversely, expression of active Notch1 provoked the dismissal of PTOV1 from these promoters. The antagonist role of PTOV1 on Notch activity was corroborated in the Drosophila melanogaster wing, where human PTOV1 exacerbated Notch deletion mutant phenotypes and suppressed the effects of constitutively active Notch. PTOV1 was required for optimal in vitro invasiveness and anchorage-independent growth of PC-3 cells, activities counteracted by Notch, and for their efficient growth and metastatic spread in vivo. In prostate tumors, the overexpression of PTOV1 was associated with decreased expression of HEY1 and HES1, and this correlation was significant in metastatic lesions. CONCLUSIONS: High levels of the adaptor protein PTOV1 counteract the transcriptional activity of Notch. Our evidences link the pro-oncogenic and pro-metastatic effects of PTOV1 in prostate cancer to its inhibitory activity on Notch signaling and are supportive of a tumor suppressor role of Notch in prostate cancer progression.

SPARC mediates metastatic cooperation between CSC and non-CSC prostate cancer cell subpopulations.

BACKGROUND: Tumor cell subpopulations can either compete with each other for nutrients and physical space within the tumor niche, or co-operate for enhanced survival, or replicative or metastatic capacities. Recently, we have described co-operative interactions between two clonal subpopulations derived from the PC-3 prostate cancer cell line, in which the invasiveness of a cancer stem cell (CSC)-enriched subpopulation (PC-3M, or M) is enhanced by a non-CSC subpopulation (PC-3S, or S), resulting in their accelerated metastatic dissemination. METHODS: M and S secretomes were compared by SILAC (Stable Isotope Labeling by Aminoacids in Cell Culture). Invasive potential in vitro of M cells was analyzed by Transwell-Matrigel assays. M cells were co-injected with S cells in the dorsal prostate of immunodeficient mice and monitored by bioluminescence for tumor growth and metastatic dissemination. SPARC levels were determined by immunohistochemistry and real-time RT-PCR in tumors and by ELISA in plasma from patients with metastatic or non-metastatic prostate cancer. RESULTS: Comparative secretome analysis yielded 213 proteins differentially secreted between M and S cells. Of these, the protein most abundantly secreted in S relative to M cells was SPARC. Immunodepletion of SPARC inhibited the enhanced invasiveness of M induced by S conditioned medium. Knock down of SPARC in S cells abrogated the capacity of its conditioned medium to enhance the in vitro invasiveness of M cells and compromised their potential to boost the metastatic behavior of M cells in vivo. In most primary human prostate cancer samples, SPARC was expressed in the epithelial tumoral compartment of metastatic cases. CONCLUSIONS: The matricellular protein SPARC, secreted by a prostate cancer clonal tumor cell subpopulation displaying non-CSC properties, is a critical mediator of paracrine effects exerted on a distinct tumor cell subpopulation enriched in CSC. This paracrine interaction results in an enhanced metastatic behavior of the CSC-enriched tumor subpopulation. SPARC is expressed in the neoplastic cells of primary prostate cancer samples from metastatic cases, and could thus constitute a tumor progression biomarker and a therapeutic target in advanced prostate cancer.

A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study.

BACKGROUND: Magnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs. RESULTS: The post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP. CONCLUSION: We have developed a simple, cost-effective relatively high-throughput method for selecting CAs for in vivo experiments. This method requires approximately 800 times less quantity of material than the amount used for in vivo administrations.

Multifunctional nanovesicle-bioactive conjugates prepared by a one-step scalable method using CO2-expanded solvents.

The integration of therapeutic biomolecules, such as proteins and peptides, in nanovesicles is a widely used strategy to improve their stability and efficacy. However, the translation of these promising nanotherapeutics to clinical tests is still challenged by the complexity involved in the preparation of functional nanovesicles and their reproducibility, scalability, and cost production. Here we introduce a simple one-step methodology based on the use of CO2-expanded solvents to prepare multifunctional nanovesicle-bioactive conjugates. We demonstrate high vesicle-to-vesicle homogeneity in terms of size and lamellarity, batch-to-batch consistency, and reproducibility upon scaling-up. Importantly, the procedure is readily amenable to the integration/encapsulation of multiple components into the nanovesicles in a single step and yields sufficient quantities for clinical research. The simplicity, reproducibility, and scalability render this one-step fabrication process ideal for the rapid and low-cost translation of nanomedicine candidates from the bench to the clinic.

3D Bioprinted Tumor-Stroma Models of Triple-Negative Breast Cancer Stem Cells for Preclinical Targeted Therapy Evaluation.

Breast cancer stem cells (CSCs) play a pivotal role in therapy resistance and tumor relapse, emphasizing the need for reliable in vitro models that recapitulate the complexity of the CSC tumor microenvironment to accelerate drug discovery. We present a bioprinted breast CSC tumor-stroma model incorporating triple-negative breast CSCs (TNB-CSCs) and stromal cells (human breast fibroblasts), within a breast-derived decellularized extracellular matrix bioink. Comparison of molecular signatures in this model with different clinical subtypes of bioprinted tumor-stroma models unveils a unique molecular profile for artificial CSC tumor models. We additionally demonstrate that the model can recapitulate the invasive potential of TNB-CSC. Surface-enhanced Raman scattering imaging allowed us to monitor the invasive potential of tumor cells in deep z-axis planes, thereby overcoming the depth-imaging limitations of confocal fluorescence microscopy. As a proof-of-concept application, we conducted high-throughput drug testing analysis to assess the efficacy of CSC-targeted therapy in combination with conventional chemotherapeutic compounds. The results highlight the usefulness of tumor-stroma models as a promising drug-screening platform, providing insights into therapeutic efficacy against CSC populations resistant to conventional therapies.

Interlaboratory comparison of endotoxin contamination assessment of nanomaterials.

Endotoxin contamination is a significant hurdle to the translation of nanomaterials for biomedical applications. Multiple reports now describe that more than one-third of nanomaterials fail early pre-clinical assessment due to levels of endotoxin above regulatory requirements. Additionally, most immunological studies or in vivo studies testing nanomaterials in the literature lack inclusion of this assessment, which may lead to false-positive or false-negative results if high levels of the contaminant are present. The currently approved methods for endotoxin contamination assessment rely on enzymatic activity and wavelength absorbance as their endpoint, and many nanomaterials can interfere with such assays. For this reason, we devised an interlaboratory comparison of endotoxin contamination assessment for a range of nanomaterials to challenge the current international organization for standardization and pharmacopeia standards. Herein, we show that detected endotoxin levels could vary considerably between groups, and, in some instances, nanomaterials could both pass and fail regulatory endotoxin limits for medical devices depending on the group undertaking the assessment, all while passing all quality criteria standards. This work emphasises the requirement for multiple assays to fully assess the endotoxin levels in a nanomaterial and highlights the need for additional assays to be developed in this space.

Human SMC2 protein, a core subunit of human condensin complex, is a novel transcriptional target of the WNT signaling pathway and a new therapeutic target.

Human SMC2 is part of the condensin complex, which is responsible for tightly packaging replicated genomic DNA prior to segregation into daughter cells. Engagement of the WNT signaling pathway is known to have a mitogenic effect on cells, but relatively little is known about WNT interaction with mitotic structural organizer proteins. In this work, we described the novel transcriptional regulation of SMC2 protein by direct binding of the ß-catenin·TCF4 transcription factor to the SMC2 promoter. Furthermore, we identified the precise region in the SMC2 promoter that is required for ß-catenin-mediated promoter activation. Finally, we explored the functional significance of down-regulating SMC2 protein in vivo. Treatment of WNT-activated intestinal tumor cells with SMC2 siRNA significantly reduced cell proliferation in nude mice, compared with untreated controls (p = 0.02). Therefore, we propose that WNT signaling can directly activate SMC2 transcription as a key player in the mitotic cell division machinery. Furthermore, SMC2 represents a new target for oncological therapeutic intervention.

Optimization of [(11)C]raclopride positron emission tomographic rat studies: comparison of methods for image quantification.

The goal of this study was to compare different quantification approaches and reconstruction methods to estimate the binding potential in [11C]raclopride studies in rats. The final aim was to determine if the results obtained with short-acquisition scanning were comparable to the results obtained with long-acquistion (conventional) scanning. We analyzed two rat data sets: a baseline versus a pretreatment study (with cold raclopride) and a young versus an old animal group comparison. The study results support the contention that optimization of [11C]raclopride positron emission tomographic studies in rats by shortening the acquisition time is feasible. In addition, filtered backprojection is recommended as a reconstruction algorithm, although iterative methods may be more sensitive to detect within-group differences.

Solid Lipid Nanoparticles: Multitasking Nano-Carriers for Cancer Treatment.

Despite all the advances seen in recent years, the severe adverse effects and low specificity of conventional chemotherapy are still challenging problems regarding cancer treatment. Nanotechnology has helped to address these questions, making important contributions in the oncological field. The use of nanoparticles has allowed the improvement of the therapeutic index of several conventional drugs and facilitates the tumoral accumulation and intracellular delivery of complex biomolecules, such as genetic material. Among the wide range of nanotechnology-based drug delivery systems (nanoDDS), solid lipid nanoparticles (SLNs) have emerged as promising systems for delivering different types of cargo. Their solid lipid core, at room and body temperature, provides SLNs with higher stability than other formulations. Moreover, SLNs offer other important features, namely the possibility to perform active targeting, sustained and controlled release, and multifunctional therapy. Furthermore, with the possibility to use biocompatible and physiologic materials and easy scale-up and low-cost production methods, SLNs meet the principal requirements of an ideal nanoDDS. The present work aims to summarize the main aspects related to SLNs, including composition, production methods, and administration routes, as well as to show the most recent studies about the use of SLNs for cancer treatment.

Nanoceria as Safe Contrast Agents for X-ray CT Imaging.

Cerium oxide nanoparticles (CeO2NPs) have exceptional catalytic properties, rendering them highly effective in removing excessive reactive oxygen species (ROS) from biological environments, which is crucial in safeguarding these environments against radiation-induced damage. Additionally, the Ce atom's high Z number makes it an ideal candidate for utilisation as an X-ray imaging contrast agent. We herein show how the injection of albumin-stabilised 5 nm CeO2NPs into mice revealed substantial enhancement in X-ray contrast, reaching up to a tenfold increase at significantly lower concentrations than commercial or other proposed contrast agents. Remarkably, these NPs exhibited prolonged residence time within the target organs. Thus, upon injection into the tail vein, they exhibited efficient uptake by the liver and spleen, with 85% of the injected dose (%ID) recovered after 7 days. In the case of intratumoral administration, 99% ID of CeO2NPs remained within the tumour throughout the 7-day observation period, allowing for observation of disease dynamics. Mass spectrometry (ICP-MS) elemental analysis confirmed X-ray CT imaging observations.

Intracellular Delivery of Anti-Kirsten Rat Sarcoma Antibodies Mediated by Polymeric Micelles Exerts Strong In Vitro and In Vivo Anti-Tumorigenic Activity in Kirsten Rat Sarcoma-Mutated Cancers.

The Kirsten rat sarcoma viral oncogene (KRAS) is one of the most well-known proto-oncogenes, frequently mutated in pancreatic and colorectal cancers, among others. We hypothesized that the intracellular delivery of anti-KRAS antibodies (KRAS-Ab) with biodegradable polymeric micelles (PM) would block the overactivation of the KRAS-associated cascades and revert the effect of its mutation. To this end, PM-containing KRAS-Ab (PM-KRAS) were obtained using Pluronic F127. The feasibility of using PM for antibody encapsulation as well as the conformational change of the polymer and its intermolecular interactions with the antibodies was studied, for the first time, using in silico modeling. In vitro, encapsulation of KRAS-Ab allowed their intracellular delivery in different pancreatic and colorectal cancer cell lines. Interestingly, PM-KRAS promoted a high proliferation impairment in regular cultures of KRAS-mutated HCT116 and MIA PaCa-2 cells, whereas the effect was neglectable in non-mutated or KRAS-independent HCT-8 and PANC-1 cancer cells, respectively. Additionally, PM-KRAS induced a remarkable inhibition of the colony formation ability in low-attachment conditions in KRAS-mutated cells. In vivo, when compared with the vehicle, the intravenous administration of PM-KRAS significantly reduced tumor volume growth in HCT116 subcutaneous tumor-bearing mice. Analysis of the KRAS-mediated cascade in cell cultures and tumor samples showed that the effect of PM-KRAS was mediated by a significant reduction of the ERK phosphorylation and a decrease in expression in the stemness-related genes. Altogether, these results unprecedently demonstrate that the delivery of KRAS-Ab mediated by PM can safely and effectively reduce the tumorigenicity and the stemness properties of KRAS-dependent cells, thus bringing up new possibilities to reach undruggable intracellular targets.

The Nanotechnology-Based Approaches against Kirsten Rat Sarcoma-Mutated Cancers.

Kirsten rat sarcoma (KRAS) is a small GTPase which acts as a molecular switch to regulate several cell biological processes including cell survival, proliferation, and differentiation. Alterations in KRAS have been found in 25% of all human cancers, with pancreatic cancer (90%), colorectal cancer (45%), and lung cancer (35%) being the types of cancer with the highest mutation rates. KRAS oncogenic mutations are not only responsible for malignant cell transformation and tumor development but also related to poor prognosis, low survival rate, and resistance to chemotherapy. Although different strategies have been developed to specifically target this oncoprotein over the last few decades, almost all of them have failed, relying on the current therapeutic solutions to target proteins involved in the KRAS pathway using chemical or gene therapy. Nanomedicine can certainly bring a solution for the lack of specificity and effectiveness of anti-KRAS therapy. Therefore, nanoparticles of different natures are being developed to improve the therapeutic index of drugs, genetic material, and/or biomolecules and to allow their delivery specifically into the cells of interest. The present work aims to summarize the most recent advances related to the use of nanotechnology for the development of new therapeutic strategies against KRAS-mutated cancers.