miR-203 drives breast cancer cell differentiation.

A hallmark of many malignant tumors is dedifferentiated (immature) cells bearing slight or no resemblance to the normal cells from which the cancer originated. Tumor dedifferentiated cells exhibit a higher capacity to survive to chemo and radiotherapies and have the ability to incite tumor relapse. Inducing cancer cell differentiation would abolish their self-renewal and invasive capacity and could be combined with the current standard of care, especially in poorly differentiated and aggressive tumors (with worst prognosis). However, differentiation therapy is still in its early stages and the intrinsic complexity of solid tumor heterogeneity demands innovative approaches in order to be efficiently translated into the clinic. We demonstrate here that microRNA 203, a potent driver of differentiation in pluripotent stem cells (ESCs and iPSCs), promotes the differentiation of mammary gland tumor cells. Combining mouse in vivo approaches and both mouse and human-derived tridimensional organoid cultures, we report that miR-203 influences the self-renewal capacity, plasticity and differentiation potential of breast cancer cells and prevents tumor cell growth in vivo. Our work sheds light on differentiation-based antitumor therapies and offers miR-203 as a promising tool for directly confronting the tumor-maintaining and regeneration capability of cancer cells.

Whole-body imaging of lymphovascular niches identifies pre-metastatic roles of midkine.

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches. Injectable lymphatic tracers have been developed, but their limited diffusion precludes whole-body imaging at visceral sites. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive 'lymphoreporter' because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a 'MetAlert' discovery platform for drivers and inhibitors of metastasis.

Clickable Albumin Nanoparticles for Pretargeted Drug Delivery toward PD-L1 Overexpressing Tumors in Combination Immunotherapy.

We present a simple methodology to design a pretargeted drug delivery system, based on clickable anti-programmed death ligand 1 (anti-PD-L1) antibodies (Abs) and clickable bovine serum albumin (BSA) nanoparticles (NPs). Pretargeted drug delivery is based on the decoupling of a targeting moiety and a drug-delivering vector which can then react in vivo after separate injections. This may be key to achieve active targeting of drug-delivering NPs toward cancerous tissue. In pretargeted approaches, drug-delivering NPs were observed to accumulate in a higher amount in the targeted tissue due to shielding-related enhanced blood circulation and size-related enhanced tissue penetration. In this work, BSA NPs were produced using the solvent precipitation methodology that renders colloidally stable NPs, which were subsequently functionalized with a clickable moiety based on chlorosydnone (Cl-Syd). Those reactive groups are able to specifically react with dibenzocyclooctyne (DBCO) groups in a click-type fashion, reaching second-order reaction rate constants as high as 1.9 M-1·s-1, which makes this reaction highly suitable for in vivo applications. The presence of reactive Cl-Syd was demonstrated by reacting the functionalized NPs with a DBCO-modified sulfo-cyanine-5 dye. With this reaction, it was possible to infer the number of reactive moieties per NPs. Finally, and with the aim of demonstrating the suitability of this system to be used in pretargeted strategies, functionalized fluorescent NPs were used to label H358 cells with a clickable anti-PD-L1 Ab, applying the reaction between Cl-Syd and DBCO as corresponding clickable groups. The results of these experiments demonstrate the bio-orthogonality of the system to perform the reaction in vitro, in a period as short as 15 min.

Role of Activator Protein-1 Complex on the Phenotype of Human Osteosarcomas Generated from Mesenchymal Stem Cells.

Osteosarcoma (OS) is a highly aggressive bone tumor that usually arises intramedullary at the extremities of long bones. Due to the fact that the peak of incidence is in the growth spurt of adolescence, the specific anatomical location, and the heterogeneity of cells, it is believed that osteosarcomagenesis is a process associated with bone development. Different studies in murine models showed that the tumor-initiating cell in OS could be an uncommitted mesenchymal stem cell (MSC) developing in a specific bone microenvironment. However, only a few studies have reported transgene-induced human MSCs transformation and mostly obtained undifferentiated sarcomas. In our study, we demonstrate that activator protein 1 family members induce osteosarcomagenesis in immortalized hMSC. c-JUN or c-JUN/c-FOS overexpression act as tumorigenic factors generating OS with fibroblastic or pleomorphic osteoblastic phenotypes, respectively. Stem Cells 2018;36:1487-1500.

K-RasV14I recapitulates Noonan syndrome in mice.

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-Ras(V14I), a recurrent KRAS mutation in NS patients. K-Ras(V14I)-mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-Ras(V14I)-mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome.

Bone environment is essential for osteosarcoma development from transformed mesenchymal stem cells.

The cellular microenvironment plays a relevant role in cancer development. We have reported that mesenchymal stromal/stem cells (MSCs) deficient for p53 alone or together with RB (p53(-/-)RB(-/-)) originate leiomyosarcoma after subcutaneous (s.c.) inoculation. Here, we show that intrabone or periosteal inoculation of p53(-/-) or p53(-/-)RB(-/-) bone marrow- or adipose tissue-derived MSCs originated metastatic osteoblastic osteosarcoma (OS). To assess the contribution of bone environment factors to OS development, we analyzed the effect of the osteoinductive factor bone morphogenetic protein-2 (BMP-2) and calcified substrates on p53(-/-)RB(-/-) MSCs. We show that BMP-2 upregulates the expression of osteogenic markers in a WNT signaling-dependent manner. In addition, the s.c. coinfusion of p53(-/-)RB(-/-) MSCs together with BMP-2 resulted in appearance of tumoral osteoid areas. Likewise, when p53(-/-)RB(-/-) MSCs were inoculated embedded in a calcified ceramic scaffold composed of hydroxyapatite and tricalciumphosphate (HA/TCP), tumoral bone formation was observed in the surroundings of the HA/TCP scaffold. Moreover, the addition of BMP-2 to the ceramic/MSC implants further increased the tumoral osteoid matrix. Together, these data indicate that bone microenvironment signals are essential to drive OS development.

Constitutive activation of B-Raf in the mouse germ line provides a model for human cardio-facio-cutaneous syndrome.

RASopathies are a class of developmental syndromes that result from congenital mutations in key elements of the RAS/RAF/MEK signaling pathway. A well-recognized RASopathy is the cardio-facio-cutaneous (CFC) syndrome characterized by a distinctive facial appearance, heart defects, and mental retardation. Clinically diagnosed CFC patients carry germ-line mutations in four different genes, B-RAF, MEK1, MEK2, and K-RAS. B-RAF is by far the most commonly mutated locus, displaying mutations that most often result in constitutive activation of the B-RAF kinase. Here, we describe a mouse model for CFC generated by germ-line expression of a B-RafLSLV600E allele. This targeted allele allows low levels of expression of B-RafV600E, a constitutively active B-Raf kinase first identified in human melanoma. B-Raf+/LSLV600E mice are viable and display several of the characteristic features observed in CFC patients, including reduced life span, small size, facial dysmorphism, cardiomegaly, and epileptic seizures. These mice also show up-regulation of specific catecholamines and cataracts, two features detected in a low percentage of CFC patients. In addition, B-Raf+/LSLV600E mice develop neuroendocrine tumors, a pathology not observed in CFC patients. These mice may provide a means of better understanding the pathophysiology of at least some of the clinical features present in CFC patients. Moreover, they may serve as a tool to evaluate the potential therapeutic efficacy of B-RAF inhibitors and establish the precise window at which they could be effective against this congenital syndrome.

Design and validation of novel flow cytometry panels to analyze a comprehensive range of peripheral immune cells in mice.

The use of flow cytometry in mice is constrained by several factors, including the limited availability of mouse-specific antibodies and the need to work with small volumes of peripheral blood. This is particularly challenging for longitudinal studies, as serial blood samples should not exceed 10% of the total blood volume in mice. To address this, we have developed two novel flow cytometry panels designed to extensively analyze immune cell populations in mice during longitudinal studies, using only 50 µL of peripheral blood per panel. Additionally, a third panel has been designed to conduct a more detailed analysis of cytotoxic and inhibitory markers at the end point. These panels have been validated on a lipopolysaccharide (LPS)-induced lung inflammation model. Two experiments were conducted to 1) validate the panels' sensitivity to immune challenges (n=12) and 2) to assess intrinsic variability of measurements (n=5). In both experiments, we collected 50 µL of peripheral blood for each cytometry panel from the maxillary venous sinus. All antibodies were titrated to identify the optimal concentration that maximized the signal from the positive population while minimizing the signal from the negative population. Samples were processed within 1 hour of collection using a MACSQuant Analyzer 16 cytometer. Our results demonstrate that these immunological panels are sensitive enough to detect changes in peripheral blood after LPS induction. Moreover, our findings help determine the sample size needed based on the immune population variability. In conclusion, the panels we have designed enable a comprehensive analysis of the murine immune system with a low blood volume requirement, enabling the measure of both absolute values and relative percentages effectively. This approach provides a robust platform for longitudinal studies in mice and can be used to uncover significant insights into immune responses.

Combined supplementation with hesperidin, phytosterols and curcumin decreases adiposity and improves metabolic health in ovariectomized rats.

In recent years many women have looked for alternative therapies to address menopause. Hesperidin, phytosterols and curcumin are bioactive compounds that can ameliorate some cardiovascular risk factors associated with menopause, although there are no data concerning the effects of their combined supplementation. We used ovariectomized (OVX) rats, a postmenopausal model with oestrogen deficiency, to evaluate whether supplementation with a multi-ingredient (MI) including hesperidin, phytosterols and curcumin for 57 days would display beneficial effects against fat mass accretion and metabolic disturbances associated with menopause. Twenty OVX rats were orally supplemented with either MI (OVX-MI) or vehicle (OVX). Furthermore, 10 OVX rats orally received the vehicle along with subcutaneous injections of 17ß-oestradiol biweekly (OVX-E2), whereas 10 rats were sham operated and received oral and injected vehicles (control group; SH). MI supplementation partly counteracted the fat mass accretion observed in OVX animals, which was evidenced by decreased total fat mass, adiposity index, the weight of retroperitoneal, inguinal and mesenteric white adipose tissue (MWAT) depots and MWAT adipocyte hypertrophy. These effects were accompanied by a significant decrease in the circulating levels of leptin and the mRNA levels of the fatty acid uptake-related genes Lpl and Cd36 in MWAT. These results were very similar to those observed in OVX-E2 animals. OVX-MI rats also displayed a higher lean body mass, lean/fat mass ratio, adiponectin-to-leptin ratio and insulin sensitivity than their OVX counterparts. Our findings can pave the way for using this MI formulation as an alternative therapy to manage obesity and to improve the cardiometabolic health of menopausal women.

A cocktail of histidine, carnosine, cysteine and serine reduces adiposity and improves metabolic health and adipose tissue immunometabolic function in ovariectomized rats.

Many women have sought alternative therapies to address menopause. Recently, a multi-ingredient supplement (MIS) containing L-histidine, L-carnosine, L-serine, and L-cysteine has been shown to be effective at ameliorating hepatic steatosis (HS) in ovariectomized (OVX) rats, a postmenopausal oestrogen deficiency model. Considering that HS frequently accompanies obesity, which often occurs during menopause, we aimed to investigate the effects of this MIS for 8 weeks in OVX rats. Twenty OVX rats were orally supplemented with either MIS (OVX-MIS) or vehicle (OVX). Ten OVX rats received vehicle orally along with subcutaneous injections of 17ß-oestradiol (OVX-E2), whereas 10 rats underwent a sham operation and received oral and injected vehicles (control group). MIS consumption partly counteracted the fat mass accretion observed in OVX animals, leading to decreased total fat mass, adiposity index and retroperitoneal white adipose tissue (RWAT) adipocyte hypertrophy. OVX-MIS rats also displayed increased lean mass and lean/fat ratio, suggesting a healthier body composition, similar to the results reported for OVX-E2 animals. MIS consumption decreased the circulating levels of the proinflammatory marker CRP, the total cholesterol-to-HDL-cholesterol ratio and the leptin-to-adiponectin ratio, a biomarker of diabetes risk and metabolic syndrome. RWAT transcriptomics indicated that MIS favourably regulated genes involved in adipocyte structure and morphology, cell fate determination and differentiation, glucose/insulin homeostasis, inflammation, response to stress and oxidative phosphorylation, which may be mechanisms underlying the beneficial effects described for OVX-MIS rats. Our results pave the way for using this MIS formulation to improve the body composition and immunometabolic health of menopausal women.

Radioimmunotheragnosis in Cancer Research.

The combination of immunoPET-where an antibody (Ab) is labeled with an isotope for PET imaging-and radioimmunotherapy (RIT), using the same antibody with a therapeutic isotope, offers significant advantages in cancer management. ImmunoPET allows non-invasive imaging of antigen expression, which aids in patient selection for subsequent radioimmunotherapy. It also facilitates the assessment of tumor response to therapy, allowing for treatment adjustments if necessary. In addition, immunoPET provides critical pharmacokinetic data, including antibody biodistribution and clearance rates, which are essential for dosimetry calculations and treatment protocol optimization. There are still challenges to overcome. Identifying appropriate target antigens that are selectively expressed on cancer cells while minimally expressed on normal tissues remains a major hurdle to reduce off-target toxicity. In addition, it is critical to optimize the pharmacokinetics of radiolabeled antibodies to maximize tumor uptake and minimize normal tissue uptake, particularly in vital organs such as the liver and kidney. This approach offers the potential for targeted and personalized cancer therapy with reduced systemic toxicity by exploiting the specificity of monoclonal antibodies and the cytotoxic effects of radiation. However, further research is needed to address remaining challenges and to optimize these technologies for clinical use.

Target engagement of an anti-MT1-MMP antibody for triple-negative breast cancer PET imaging and beta therapy.

PURPOSE: Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that lacks effective diagnostic and therapeutic options. Membrane type 1 matrix metalloproteinase (MT1-MMP) is an attractive biomarker for improving patient selection. This study aimed to develop a theranostic tool using a highly tumour-selective anti-MT1-MMP antibody (LEM2/15) radiolabelled with 89Zr for PET and 177Lu for therapy in a TNBC murine model. METHODS: The LEM2/15 antibody and IgG isotype control were radiolabelled with 89Zr. PET imaging was performed in a TNBC orthotopic mouse model at 1, 2, 4, and 7 days after administration. Tissue biodistribution and pharmacokinetic parameters were analysed and Patlak linearisation was used to calculate the influx rate of irreversible uptake. The TNBC mice were treated with [177Lu]Lu-DOTA-LEM2/15 (single- or 3-dose regimen) or saline. Efficacy of [177Lu]Lu-DOTA-LEM2/15 was evaluated as tumour growth and DNA damage (γH2AX) in MDA 231-BrM2-831 tumours. RESULTS: At 7 days post-injection, PET uptake in tumour xenografts revealed a 1.6-fold and 2.4-fold higher tumour-to-blood ratio for [89Zr]Zr-Df-LEM2/15 in the non-blocked group compared to the blocked and IgG isotype control groups, respectively. Specific uptake of LEM2/15 in TBNC tumours mediated by MT1-MMP-binding was demonstrated by the Patlak linearisation method, providing insights into the potential efficacy of LEM2/15-based treatments. A similar uptake was found for [89Zr]Zr-Df-LEM2/15 and [177Lu]Lu-DOTA-LEM2/15 in tumours 7 days post-injection (6.80 ± 1.31 vs. 5.61 ± 0.66 %ID/g). Tumour doubling time was longer in the [177Lu]Lu-DOTA-LEM2/15 3-dose regimen treated group compared to the control (50 vs. 17 days, respectively). The percentage of cells with γH2AX-foci was higher in tumours treated with [177Lu]Lu-DOTA-LEM2/15 3-dose regimen compared to tumours non-treated or treated with [177Lu]Lu-DOTA-LEM2/15 single-dose (12 % vs. 4-5 %). CONCLUSIONS: The results showed that the 89Zr/177Lu-labelled anti-MT1-MMP mAb (LEM2/15) pair facilitated immune-PET imaging and reduced tumour growth in a preclinical TNBC xenograft model.

Kras oncogene ablation prevents resistance in advanced lung adenocarcinomas.

KRASG12C inhibitors have revolutionized the clinical management of patients with KRASG12C-mutant lung adenocarcinoma. However, patient exposure to these inhibitors leads to the rapid onset of resistance. In this study, we have used genetically engineered mice to compare the therapeutic efficacy and the emergence of tumor resistance between genetic ablation of mutant Kras expression and pharmacological inhibition of oncogenic KRAS activity. Whereas Kras ablation induces massive tumor regression and prevents the appearance of resistant cells in vivo, treatment of KrasG12C/Trp53-driven lung adenocarcinomas with sotorasib, a selective KRASG12C inhibitor, caused a limited antitumor response similar to that observed in the clinic, including the rapid onset of resistance. Unlike in human tumors, we did not observe mutations in components of the RAS-signaling pathways. Instead, sotorasib-resistant tumors displayed amplification of the mutant Kras allele and activation of xenobiotic metabolism pathways, suggesting that reduction of the on-target activity of KRASG12C inhibitors is the main mechanism responsible for the onset of resistance. In sum, our results suggest that resistance to KRAS inhibitors could be prevented by achieving a more robust inhibition of KRAS signaling mimicking the results obtained upon Kras ablation.

Rapid identification of ETP-46992, orally bioavailable PI3K inhibitor, selective versus mTOR.

Phosphoinositide-3-kinases (PI3K) are a family of lipid kinases mediating numerous cell processes such as proliferation, migration and differentiation. PI3K is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the rapid identification of ETP-46992, within 2-aminocarbonyl imidazo [1,2-a] pyrazine series, with suitable pharmacokinetic (PK) properties that allows the establishment of mechanism of action and efficacy in vivo studies. ETP-46992 showed tumor growth inhibition in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation and in tumor xenograft models with PI3K pathway deregulated (BT474).

Identification of ETP-46321, a potent and orally bioavailable PI3K α, δ inhibitor.

Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the optimization of imidazo [1,2-a] pyrazines, which allow us to identify compound 14 (ETP-46321), with potent biochemical and cellular activity and good pharmacokinetic properties (PK) after oral dosing. ETP-46321 PK/PD studies showed time dependent downregulation of AKT(Ser473) phosphorylation, which correlates with compound levels in tumor tissue and demonstrating to be efficacious in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation. Treatment with ETP-46321 resulted in significant tumor growth inhibition.

ImmunoPET in oncology.

Due to increase of immunotherapy in oncology, it is essential to have a biological characterization of tumors. Knowing which antigens are expressed both on the surface of the tumor cell and at tumor microenvironment in order to predict the tretment response different therapeutic antibodies, has become a need. ImmunoPET is a non-invasive diagnostic imaging tool that combines the high specificity of antibodies against antigens with the high sensitivity, resolution and quantification capacity of PET imaging. With ImmunoPET we obtain a virtual biopsy of tumors, it has a big present and future in preclinical-clinical research, being already a reality in predicting and monitoring the response to treatments with monoclonal antibodies, allowing a selection of patients and therapies reaching a personalized medicine contributing to improve clinical decisions.

Genetic interaction between PLK1 and downstream MCPH proteins in the control of centrosome asymmetry and cell fate during neural progenitor division.

Alteration of centrosome function and dynamics results in major defects during chromosome segregation and is associated with primary autosomal microcephaly (MCPH). Despite the knowledge accumulated in the last few years, why some centrosomal defects specifically affect neural progenitors is not clear. We describe here that the centrosomal kinase PLK1 controls centrosome asymmetry and cell fate in neural progenitors during development. Gain- or loss-of-function mutations in Plk1, as well as deficiencies in the MCPH genes Cdk5rap2 (MCPH3) and Cep135 (MCPH8), lead to abnormal asymmetry in the centrosomes carrying the mother and daughter centriole in neural progenitors. However, whereas loss of MCPH proteins leads to increased centrosome asymmetry and microcephaly, deficient PLK1 activity results in reduced asymmetry and increased expansion of neural progenitors and cortical growth during mid-gestation. The combination of PLK1 and MCPH mutations results in increased microcephaly accompanied by more aggressive centrosomal and mitotic abnormalities. In addition to highlighting the delicate balance in the level and activity of centrosomal regulators, these data suggest that human PLK1, which maps to 16p12.1, may contribute to the neurodevelopmental defects associated with 16p11.2-p12.2 microdeletions and microduplications in children with developmental delay and dysmorphic features.

Development of anti-membrane type 1-matrix metalloproteinase nanobodies as immunoPET probes for triple negative breast cancer imaging.

Triple-negative breast cancer (TNBC) is characterized by aggressiveness and high rates of metastasis. The identification of relevant biomarkers is crucial to improve outcomes for TNBC patients. Membrane type 1-matrix metalloproteinase (MT1-MMP) could be a good candidate because its expression has been reported to correlate with tumor malignancy, progression and metastasis. Moreover, single-domain variable regions (VHHs or Nanobodies) derived from camelid heavy-chain-only antibodies have demonstrated improvements in tissue penetration and blood clearance, important characteristics for cancer imaging. Here, we have developed a nanobody-based PET imaging strategy for TNBC detection that targets MT1-MMP. A llama-derived library was screened against the catalytic domain of MT1-MMP and a panel of specific nanobodies were identified. After a deep characterization, two nanobodies were selected to be labeled with gallium-68 (68Ga). ImmunoPET imaging with both ([68Ga]Ga-NOTA-3TPA14 and [68Ga]Ga-NOTA-3CMP75) in a TNBC mouse model showed precise tumor-targeting capacity in vivo with high signal-to-background ratios. (68Ga)Ga-NOTA-3CMP75 exhibited higher tumor uptake compared to (68Ga)Ga-NOTA-3TPA14. Furthermore, imaging data correlated perfectly with the immunohistochemistry staining results. In conclusion, we found a promising candidate for nanobody-based PET imaging to be further investigated as a diagnostic tool in TNBC.

Combination of injectable multiple growth factor-releasing scaffolds and cell therapy as an advanced modality to enhance tissue neovascularization.

OBJECTIVE: Vasculogenic progenitor cell therapy for ischemic diseases bears great potential but still requires further optimization for justifying its clinical application. Here, we investigated the effects of in vivo tissue engineering by combining vasculogenic progenitors with injectable scaffolds releasing controlled amounts of proangiogenic growth factors. METHODS AND RESULTS: We produced biodegradable, injectable polylactic coglycolic acid-based scaffolds releasing single factors or combinations of vascular endothelial growth factor, hepatocyte growth factor, and angiopoietin-1. Dual and triple combinations of scaffold-released growth factors were superior to single release. In murine hindlimb ischemia models, scaffolds releasing dual (vascular endothelial growth factor and hepatocyte growth factor) or triple combinations improved effects of cord blood-derived vasculogenic progenitors. Increased migration, homing, and incorporation of vasculogenic progenitors into the vasculature augmented capillary density, translating into improved blood perfusion. Most importantly, scaffold-released triple combinations including the vessel stabilizer angiopoietin-1 enhanced the number of perivascular smooth muscle actin(+) vascular smooth muscle cells, indicating more efficient vessel stabilization. CONCLUSIONS: Vasculogenic progenitor cell therapy is significantly enhanced by in vivo tissue engineering providing a proangiogenic and provasculogenic growth factor-enriched microenvironment. Therefore, combined use of scaffold-released growth factors and cell therapy improves neovascularization in ischemic diseases and may translate into more pronounced clinical effects.

Osteoporosis Remission and New Bone Formation with Mesoporous Silica Nanoparticles.

Nanotechnology changed the concept of treatment for a variety of diseases, producing a huge impact regarding drug and gene delivery. Among the different targeted diseases, osteoporosis has devastating clinical and economic consequences. Since current osteoporosis treatments present several side effects, new treatment approaches are needed. Recently, the application of small interfering RNA (siRNA) has become a promising alternative. Wnt/ß-catenin signaling pathway controls bone development and formation. This pathway is negatively regulated by sclerostin, which knock-down through siRNA application would potentially promote bone formation. However, the major bottleneck for siRNA-based treatments is the necessity of a delivery vector, bringing nanotechnology as a potential solution. Among the available nanocarriers, mesoporous silica nanoparticles (MSNs) have attracted great attention for intracellular delivery of siRNAs. The mesoporous structure of MSNs permits the delivery of siRNAs together with another biomolecule, achieving a combination therapy. Here, the effectiveness of a new potential osteoporosis treatment based on MSNs is evaluated. The proposed system is effective in delivering SOST siRNA and osteostatin through systemic injection to bone tissue. The nanoparticle administration produced an increase expression of osteogenic related genes improving the bone microarchitecture. The treated osteoporotic mice recovered values of a healthy situation approaching to osteoporosis remission.