Transporte de adenovirus oncolítico canino mediado por células tumorales para el tratamiento de osteosarcoma canino

El osteosarcoma es el tumor óseo más frecuente que afecta al perro. En veterinaria, las opciones para su manejo terapéutico son limitadas y el pronóstico para estos pacientes es pobre. Los adenovirus oncolíticos son una atractiva herramienta de tratamiento experimental ya que se pueden replicar y extender dentro del tumor para inducir directamente la destrucción del tejido tumoral. Sin embargo, el mayor impedimento de la administración sistémica de los adenovirus oncolíticos es la presencia de anticuerpos neutralizantes preformados. Éste obstáculo puede ser resuelto administrando el adenovirus oncolítico dentro de una célula transportadora capaz de ocultar los antígenos virales durante el transporte sistémico. Las células tumorales tienen tropismo tumoral, lo que permite su utilización como potenciales vehículos de transporte. En este trabajo, se estudió la capacidad de las células tumorales caninas infectadas con adenovirus oncolítico in vitro y administradas sistémicamente para escapar de las defensas antivirales, alcanzar el sitio tumoral y, subsecuentemente, de liberar la progenie viral para infectar a la célula tumoral diana en un modelo xenoinjertado de osteosarcoma canino. Los estudios in vivo utilizando células tumorales transportadoras demostraron el eficiente transporte sistémico de adenovirus oncolítico al tumor, incluso en presencia de anticuerpos anti-adenovirus. Al contrario, la presencia de anticuerpos anti-adenovirus reducieron el transporte del virus desnudo al tumor. La administración intratumoral y sistémica de las células transportadoras infectadas con un adenovirus canino de replicación selectiva (OCCAV) mostró una disminución significativa en el crecimiento del tumor comparado con la inyección del virus desnudo en presencia de altos títulos de anticuerpos neutralizantes. La inmunosupresión con ciclofosfamida no afectó la actividad terapéutica de las células tumorales infectadas con OCCAV, pero resultó en un beneficio terapéutico respecto al virus desnudo. Por otro lado, la administración intratumoral y sistémica de células tumorales infectadas con OCCAV en dos perros con osteosarcoma fue bien tolerada. En conjunto, estos hallazgos soportan la idea de la administración por vía sistémica del sistema celular infectado con virus oncolítico para el tratamiento del osteosarcoma canino.Osteosarcoma is the most frequent bone tumor affecting dogs. Veterinary options for its therapeutic management are limited and the prognosis for patients is poor. Oncolytic adenoviruses constitute an attractive experimental treatment tool since they are able to replicate and extend within the tumor to directly induce tumoral tissue destruction. However, the greatest hindrance for the systemic administration of oncolytic adenoviruses is the presence of preformed neutralizing antibodies. This obstacle can be surmounted by administering the virus inside a transporter cell which can hide the viral antigens during systemic transport. Malignant cells display tumor tropism, which allows for their use as potential transport vehicles. In this work, we studied the ability of canine malignant cells infected in vitro with oncolytic adenoviruses and systemically administered to escape from antiviral defences, reach the site of the tumor and subsequently release the viral progeny to infect target tumor cells in a xenografted canine osteosarcoma model. In vivo studies using transporter malignant cells showed efficient systemic transport of oncolytic adenovirus to the tumor, even in the presence of anti-adenovirus antibodies. On the contrary, the presence of anti-adenoviral antibodies reduced the transport of naked viral particles to the tumor. Intratumoral and systemic administration of transporter cells infected with a selective replication canine adenovirus (OCCAV) showed a significant decrease in tumor growth as compared to the injection of naked viral particles in the presence of high titers of neutralizing antibodies. Immunosuppression with cyclophosphamide did not affect the therapeutic activity of malignant cells infected with OCCAV, but resulted in therapeutic benefit in the case of naked viral particles. Furthermore, intratumoral and systemic administration of malignant cells infected with OCCAV in two dogs with osteosarcoma was well tolerated. Altogether, these findings support the concept of systemic administration of oncolytic virus-infected cells for the treatment of canine osteosarcoma

Camouflage strategies for therapeutic exosomes evasion from phagocytosis

Background: Even though exosome-based therapy has been shown to be able to control the progression of different pathologies, the data revealed by pharmacokinetic studies warn of the low residence time of exogenous exosomes in circulation that can hinder the clinical translation of therapeutic exosomes. The macrophages related to the organs of the mononuclear phagocytic system are responsible primarily for the rapid clearance and retention of exosomes, which strongly limits the amount of exosomal particles available to reach the target tissue, accumulate in it and release with high efficiency its therapeutic cargo in acceptor target cells to exert the desired biological effect. Aim of review: Endowing exosomes with surface modifications to evade the immune system is a plausible strategy to contribute to the suppression of exosomal clearance and increase the efficiency of their targeted content delivery. Here, we summarize the current evidence about the mechanisms underlying the recognition and sequestration of therapeutic exosomes by phagocytic cells. Also, we propose different strategies to generate 'invisible' exosomes for the immune system, through the incorporation of different anti-phagocytic molecules on the exosomes’ surface that allow increasing the circulating half-life of therapeutic exosomes with the purpose to increase their bioavailability to reach the target tissue, transfer their therapeutic molecular cargo and improve their efficacy profile. Key scientific concepts of review: Macrophage-mediated phagocytosis are the main responsible behind the short half-life in circulation of systemically injected exosomes, hindering their therapeutic effect. Exosomes ‘Camouflage Cloak’ strategy using antiphagocytic molecules can contribute to the inhibition of exosomal clearance, hence, increasing the on-target effect. Some candidate molecules that could exert an antiphagocytic role are CD47, CD24, CD44, CD31, β2M, PD-L1, App1, and DHMEQ. Pre- and post-isolation methods for exosome engineering are compatible with the loading of therapeutic cargo and the expression of antiphagocytic surface molecules

Osteosarcoma cells as carriers to allow antitumor activity of canine oncolytic adenovirus in the presence of neutralizing antibodies

Osteosarcoma (OSA) is the most common bone tumor affecting the dog. The veterinary options for therapeutic management of OSA are limited and prognosis for such patients is poor. Oncolytic adenoviruses are attractive tools for experimental therapeutics as they can replicate and spread within tumors to directly induce tumor destruction. However, a major impediment to systemic oncolytic adenoviruses injection is the presence of pre-existing neutralizing antibodies (Nabs). In this study, we investigated the effect of a replication-selective canine adenovirus (OCCAV) to treat OSA in the presence of Nabs and the use of canine OSA cells as carrier vehicles for evading Nabs. Our systemic biodistribution data indicated that canine tumor cells could successfully reach the tumor site and deliver OCCAV to tumor cells in an immunized mice model. Furthermore, the use of carrier cells also reduced adenovirus uptake by the liver. Importantly, OCCAV alone was not effective to control tumor growth in a pre-immunized xenograft mouse model. On the contrary, systemic antitumoral activity of carrier-cell OCCAV was evident even in the presence of circulating antibodies, which is a relevant result from a clinical point of view. These findings are of direct translational relevance for the future design of canine clinical trials

Transporte de adenovirus oncolítico canino mediado por células tumorales para el tratamiento de osteosarcoma canino

El osteosarcoma es el tumor óseo más frecuente que afecta al perro. En veterinaria, las opciones para su manejo terapéutico son limitadas y el pronóstico para estos pacientes es pobre. Los adenovirus oncolíticos son una atractiva herramienta de tratamiento experimental ya que se pueden replicar y extender dentro del tumor para inducir directamente la destrucción del tejido tumoral. Sin embargo, el mayor impedimento de la administración sistémica de los adenovirus oncolíticos es la presencia de anticuerpos neutralizantes preformados. Éste obstáculo puede ser resuelto administrando el adenovirus oncolítico dentro de una célula transportadora capaz de ocultar los antígenos virales durante el transporte sistémico. Las células tumorales tienen tropismo tumoral, lo que permite su utilización como potenciales vehículos de transporte. En este trabajo, se estudió la capacidad de las células tumorales caninas infectadas con adenovirus oncolítico in vitro y administradas sistémicamente para escapar de las defensas antivirales, alcanzar el sitio tumoral y, subsecuentemente, de liberar la progenie viral para infectar a la célula tumoral diana en un modelo xenoinjertado de osteosarcoma canino. Los estudios in vivo utilizando células tumorales transportadoras demostraron el eficiente transporte sistémico de adenovirus oncolítico al tumor, incluso en presencia de anticuerpos anti-adenovirus. Al contrario, la presencia de anticuerpos anti-adenovirus reducieron el transporte del virus desnudo al tumor. La administración intratumoral y sistémica de las células transportadoras infectadas con un adenovirus canino de replicación selectiva (OCCAV) mostró una disminución significativa en el crecimiento del tumor comparado con la inyección del virus desnudo en presencia de altos títulos de anticuerpos neutralizantes. La inmunosupresión con ciclofosfamida no afectó la actividad terapéutica de las células tumorales infectadas con OCCAV, pero resultó en un beneficio terapéutico respecto al virus desnudo. Por otro lado, la administración intratumoral y sistémica de células tumorales infectadas con OCCAV en dos perros con osteosarcoma fue bien tolerada. En conjunto, estos hallazgos soportan la idea de la administración por vía sistémica del sistema celular infectado con virus oncolítico para el tratamiento del osteosarcoma canino.Osteosarcoma is the most frequent bone tumor affecting dogs. Veterinary options for its therapeutic management are limited and the prognosis for patients is poor. Oncolytic adenoviruses constitute an attractive experimental treatment tool since they are able to replicate and extend within the tumor to directly induce tumoral tissue destruction. However, the greatest hindrance for the systemic administration of oncolytic adenoviruses is the presence of preformed neutralizing antibodies. This obstacle can be surmounted by administering the virus inside a transporter cell which can hide the viral antigens during systemic transport. Malignant cells display tumor tropism, which allows for their use as potential transport vehicles. In this work, we studied the ability of canine malignant cells infected in vitro with oncolytic adenoviruses and systemically administered to escape from antiviral defences, reach the site of the tumor and subsequently release the viral progeny to infect target tumor cells in a xenografted canine osteosarcoma model. In vivo studies using transporter malignant cells showed efficient systemic transport of oncolytic adenovirus to the tumor, even in the presence of anti-adenovirus antibodies. On the contrary, the presence of anti-adenoviral antibodies reduced the transport of naked viral particles to the tumor. Intratumoral and systemic administration of transporter cells infected with a selective replication canine adenovirus (OCCAV) showed a significant decrease in tumor growth as compared to the injection of naked viral particles in the presence of high titers of neutralizing antibodies. Immunosuppression with cyclophosphamide did not affect the therapeutic activity of malignant cells infected with OCCAV, but resulted in therapeutic benefit in the case of naked viral particles. Furthermore, intratumoral and systemic administration of malignant cells infected with OCCAV in two dogs with osteosarcoma was well tolerated. Altogether, these findings support the concept of systemic administration of oncolytic virus-infected cells for the treatment of canine osteosarcoma

Turning adversity into opportunity: Small extracellular vesicles as nanocarriers for tumor‐associated macrophages re‐education

Abstract Currently, small extracellular vesicles (sEV) as a nanoscale drug delivery system, are undergoing biotechnological scaling and clinical validation. Nonetheless, preclinical pharmacokinetic studies revealed that sEV are predominantly uptaken by macrophages. Although this “sEV‐macrophage” propensity represents a disadvantage in terms of sEV targeting and their bioavailability as nanocarriers, it also represents a strategic advantage for those therapies that involve macrophages. Such is the case of tumor‐associated macrophages (TAMs), which can reprogram/repolarize their predominantly immunosuppressive and tumor‐supportive phenotype toward an immunostimulatory and anti‐tumor phenotype using sEV as nanocarriers of TAMs reprogramming molecules. In this design, sEV represents an advantageous delivery system, providing precision to the therapy by simultaneously matching their tropism to the therapeutic cell target. Here, we review the current knowledge of the role of TAMs in the tumoral microenvironment and the effect generated by the reprogramming of these phagocytic cells fate using sEV. Finally, we discuss how these vesicles can be engineered by different bioengineering techniques to improve their therapeutic cargo loading and preferential uptake by TAMs

The Reparative Abilities of Menstrual Stem Cells Modulate the Wound Matrix Signals and Improve Cutaneous Regeneration

Considerable advances have been made toward understanding the cellular and molecular mechanism of wound healing, however, treatments for chronic wounds remain elusive. Emerging concepts utilizing mesenchymal stem cells (MSCs) from umbilical cord, adipose tissue and bone marrow have shown therapeutical advantages for wound healing. Based on this positive outcome, efforts to determine the optimal sources for MSCs are required in order to improve their migratory, angiogenic, immunomodulatory, and reparative abilities. An alternative source suitable for repetitive, non-invasive collection of MSCs is from the menstrual fluid (MenSCs), displaying a major practical advantage over other sources. This study aims to compare the biological functions and the transcriptomic pattern of MenSCs with umbilical cord MSCs in conditions resembling the wound microenvironment. Consequently, we correlate the specific gene expression signature from MenSCs with changes of the wound matrix signals in vivo. The direct comparison revealed a superior clonogenic and migratory potential of MenSCs as well as a beneficial effect of their secretome on human dermal fibroblast migration in vitro. Furthermore, MenSCs showed increased immunomodulatory properties, inhibiting T-cell proliferation in co-culture. We further, investigated the expression of selected genes involved in wound repair (growth factors, cytokines, chemokines, AMPs, MMPs) and found considerably higher expression levels in MenSCs (ANGPT1 1.5-fold; PDGFA 1.8-fold; PDGFB 791-fold; MMP3 21.6-fold; ELN 13.4-fold; and MMP10 9.2-fold). This difference became more pronounced under a pro-inflammatory stimulation, resembling wound bed conditions. Locally applied in a murine excisional wound splinting model, MenSCs showed a significantly improved wound closure after 14 days, as well as enhanced neovascularization, compared to the untreated group. Interestingly, analysis of excised wound tissue revealed a significantly higher expression of VEGF (1.42-fold) among other factors, translating an important conversion of the matrix signals in the wound site. Furthermore, histological analysis of the wound tissue from MenSCs-treated group displayed a more mature robust vascular network and a genuinely higher collagen content confirming the pro-angiogenic and reparative effect of MenSCs treatment. In conclusion, the superior clonogenicity, immunosuppressive and migration potential in combination with specific paracrine signature of MenSCs, resulted in an enhanced wound healing and cutaneous regeneration process

Additional file 2: Figure S2. of Combination therapy of menstrual derived mesenchymal stem cells and antibiotics ameliorates survival in sepsis

Experimental design for the in vivo studies. Diagram of the experimental design of (a) survival studies and (b) therapeutic effects studies of MenSC administration in mice with CLP-induced sepsis. AB antibiotics, CFU colony-forming unit, CLP cecal ligation and puncture, CM conditioned medium, H&E hematoxylin and eosin, MenSCs menstrual derived mesenchymal stem cells. (PDF 370 kb

Additional file 3: Figure S3. of Combination therapy of menstrual derived mesenchymal stem cells and antibiotics ameliorates survival in sepsis

Effect of MenSCs treatment in mice with polymicrobial sepsis. Serum was isolated 24 hours after sepsis induction and administration of different treatments with AB or MenSCs or both (sham, n = 3; saline, n = 5; AB, n = 2–4; MenSCs, n = 4; MenSCs + AB, n = 4) to determine the concentrations of alkaline phosphatase (ALP) (left panel) and albumin (right panel). Dot plots represent individual values, horizontal bars represent mean values, and vertical bars represent standard error values. **P ≤ 0.01. AB antibiotics, MenSCs menstrual derived mesenchymal stem cells, ns not significant. (PDF 180 kb

Additional file 4: Figure S4. of Combination therapy of menstrual derived mesenchymal stem cells and antibiotics ameliorates survival in sepsis

Effect of MenSCs treatment on CD4+ and CD8+ lymphocytes in mice with polymicrobial sepsis. Blood samples were obtained at different time points after induction of sepsis and administration of various treatments to determine specific lymphocyte subsets using flow cytometry (sham, n = 3–5; saline, n = 3–10; AB, n = 3–6; MenSCs, n = 3–6; MenSCs + AB, n = 3–5). Graphs show the percentages of CD45+ CD3+ CD4+ and CD45+ CD3+ CD8+ lymphocytes and the ratios of CD4+/CD8+ at (a) 24 hours and (b) 40 hours in untreated and treated mice. Dot plots represent individual values, horizontal bars represent mean values, and vertical bars represent standard error values. *P ≤ 0.05. AB antibiotics, MenSCs menstrual derived mesenchymal stem cells, ns not significant. (PDF 238 kb

Additional file 1: Figure S1. of Combination therapy of menstrual derived mesenchymal stem cells and antibiotics ameliorates survival in sepsis

Characterization of MenSCs and BMSCs. Stem cells isolated from the menstrual fluid and bone marrow were characterized according to International Society for Cellular Therapy guidelines. a MenSCs and BMSCs showed stem cell-like immunophenotypic markers. Fluorescence-activated cell sorting profile of representative cell samples. Blue and orange filled histograms denote the fluorescent profile of the indicated antigens in MenSCs and BMSCs, respectively; red histograms correspond to isotype-matched controls. b MenSCs and BMSCs displayed mesodermal differentiation. Tri-lineage differentiation of representative MenSCs and BMSC samples. Cells were cultured with adipogenic, osteogenic, and chondrogenic induction media for 14–21 days and then stained with Oil Red O, Alizarin Red, and Safranin O staining, respectively. Scale bar: 200 μm. BMSCs bone marrow-derived mesenchymal stem cells,MenSCs menstrual derived mesenchymal stem cells. (PDF 489 kb