Mesenchymal stem cells and oncolytic viruses: joining forces against cancer

The development of oncolytic viruses (OVs) has increased significantly in the past 20 years, with many candidates entering clinical trials and three of them receiving approval for some indications. Recently, OVs have also gathered interest as candidates to use in combination with immunotherapies for cancer due to their immunogenic properties, which include immunogenic cell death and the possibility to carry therapeutic transgenes in their genomes. OVs transform non-immunogenic 'cold' tumors into inflamed immunogenic 'hot' tumors, where immunotherapies show the highest efficacy. However, in monotherapy or in combination with immunotherapy, OVs face numerous challenges that limit their successful application, in particular upon systemic administration, such as liver sequestration, neutralizing interactions in blood, physical barriers to infection, and fast clearance by the immune system. In this regard, the use of mesenchymal stem cells (MSCs) as cells carrier for OV delivery addresses many of these obstacles acting as virus carriers and factories, expressing additional transgenes, and modulating the immune system. Here, I review the current progress of OVs-loaded MSCs in cancer, focusing on their interaction with the immune system, and discuss new strategies to improve their therapeutic efficacy

Engraftment Potential of Adipose Tissue-Derived Human Mesenchymal Stem Cells After Transplantation in the Fetal Rabbit

Due to their favorable intrinsic features, including engraftment, differentiation, and immunomodulatory potential, adult mesenchymal stem cells (MSCs) have been proposed for therapeutic in utero intervention. Further improvement of such attributes for particular diseases might merely be achieved by ex vivo MSC genetic engineering previous to transplantation. Here, we evaluated for the first time the feasibility, biodistribution, long-term engraftment, and transgenic enhanced green fluorescent protein (EGFP) expression of genetically engineered human adipose tissue-derived MSCs (EGFP+-ASCs) after intra-amniotic xenotransplantation at E17 of gestation into our validated pregnant rabbit model. Overall, the procedure was safe (86.4% survival rate; absence of anatomical defects). Stable, low-level engraftment of EGFP+-ASCs was confirmed by assessing the presence of the pWT-EGFP lentiviral provirus in the young transplanted rabbit tissues. Accordingly, similar frequencies of provirus-positive animals were found at both 8 weeks (60%) and 16 weeks (66.7%) after in utero intervention. The presence of EGFP+-ASCs was more frequent in respiratory epithelia (lung and trachea), according to the route of administration. However, we were unable to detect EGFP expression, neither by real-time polymerase chain reaction nor by immunohistochemistry, in the provirus-positive tissues, suggesting EGFP transgene silencing mediated by epigenetic events. Moreover, we noticed lack of both host cellular immune responses against xenogeneic ASCs and humoral immune responses against transgenic EGFP. Therefore, the fetal microchimerism achieved by the EGFP+-ASCs in the young rabbit hosts indicates induction of donor-specific tolerance after fetal rabbit xenotransplantation, which should boost postnatal transplantation for the early treatment/prevention of many devastating congenital disorders

Human Mesenchymal Stem Cells Resolve Airway Inflammation, Hyperreactivity, And Histopathology In A Mouse Model Of Occupational Asthma

Occupational asthma (OA) is characterized by allergic airway inflammation and hyperresponsiveness, leading to progressive airway remodeling and a concomitant decline in lung function. The management of OA remains suboptimal in clinical practice. Thus, establishing effective therapies might overcome the natural history of the disease. We evaluated the ability of human adipose-tissue-derived mesenchymal stem cells (hASCs), either unmodified or engineered to secrete the IL-33 decoy receptor sST2, to attenuate the inflammatory and respiratory symptoms in a previously validated mouse model of OA to ammonium persulfate (AP). Twenty-four hours after a dermal AP sensitization and intranasal challenge regimen, the animals received intravenously 1 x 10(6) cells (either hASCs or hASCs overexpressing sST2) or saline and were analyzed at 1, 3, and 6 days after treatment. The infused hASCs induced an anti-inflammatory and restorative program upon reaching the AP-injured, asthmatic lungs, leading to early reduction of neutrophilic inflammation and total IgE production, preserved alveolar architecture with nearly absent lymphoplasmacytic infiltrates, negligible smooth muscle hyperplasia/hypertrophy in the peribronchiolar areas, and baseline airway hyperreactivity (AHR) to methacholine. Local sST2 overexpression barely increased the substantial efficacy displayed by unmodified hASCs. Thus, hASCs may represent a viable multiaction therapeutic capable to adequately respond to the AP-injured lung environment by resolving inflammation, tissue remodeling, and bronchial hyperresponsiveness typical of OA

Targeting the tumor stroma with an oncolytic adenovirus secreting a fibroblast activation protein-targeted bispecific T-cell engager

BackgroundOncolytic virus (OV)-based therapies have an emerging role in the treatment of solid tumors, involving both direct cell lysis and immunogenic cell death. Nonetheless, tumor-associated stroma limits the efficacy of oncolytic viruses by forming a barrier that blocks efficient viral penetration and spread. The stroma also plays a critical role in progression, immunosuppression and invasiveness of cancer. Fibroblast activation protein- (FAP) is highly overexpressed in cancer-associated fibroblasts (CAFs), the main cellular component of tumor stroma, and in this study we assessed whether arming oncolytic adenovirus (OAd) with a FAP-targeting Bispecific T-cell Engager (FBiTE) could retarget infiltrated lymphocytes towards CAFs, enhancing viral spread and T cell-mediated cytotoxicity against the tumor stroma to improve therapeutic activity.MethodsThe bispecific T-cell Engager against FAP was constructed using an anti-human CD3 single-chain variable fragment (scFv) linked to an anti-murine and human FAP scFv. This FBiTE was inserted in the oncolytic adenovirus ICOVIR15K under the control of the major late promoter, generating the ICO15K-FBiTE. ICO15K-FBiTE replication and potency were assessed in HT1080 and A549 tumor cell lines. The expression of the FBiTE and the activation and proliferation of T cells that induced along with the T cell-mediated cytotoxicity of CAFs were evaluated by flow cytometry in vitro.In vivo, T-cell biodistribution and antitumor efficacy studies were conducted in NOD/scid/IL2rg(-)/(-) (NSG) mice.ResultsFBiTE expression did not decrease the infectivity and replication potency of the armed virus. FBiTE-mediated binding of CD3(+) effector T cells and FAP(+) target cells led to T-cell activation, proliferation, and cytotoxicity of FAP-positive cells in vitro. In vivo, FBiTE expression increased intratumoral accumulation of T cells and decreased the level of FAP, a marker of CAFs, in tumors. The antitumor activity of the FBiTE-armed adenovirus was superior to the parental virus.ConclusionsCombination of viral oncolysis of cancer cells and FBiTE-mediated cytotoxicity of FAP-expressing CAFs might be an effective strategy to overcome a key limitation of oncolytic virotherapy, encouraging its further clinical development

Immune priming using DC- and T cell-targeting gene therapy sensitizes both treated and distant B16 tumors to checkpoint inhibition

Immune checkpoint inhibitors have revolutionized the treatment of metastatic melanoma, but most tumors show resistance. Resistance is connected to a non-T cell inflamed phenotype partially caused by a lack of functional dendritic cells (DCs) that are crucial for T cell priming. Herein, we investigated whether the adenoviral gene vehicle mLOAd703 carrying both DC- and T cell-activating genes can lead to inflammation in a B16-CD46 model and thereby overcome resistance to checkpoint inhibition therapy. B16-CD46 cells were injected subcutaneously in one or both flanks of immuno-competent C57BL/6J mice. mLOAd703 treatments were given intratumorally alone or in combination with intraperitoneal checkpoint inhibition therapy (anti-PD-1, anti-PD-L1, or anti-TIM-3). Tumor, lymph node, spleen, and serum samples were analyzed for the presence of immune cells and cytokines/chemokines. B16-CD46 tumors were non-inflamed and resistant to checkpoint blockade. In contrast, mLOAd703 treatment led to infiltration of the tumor by CD8(+) T cells, natural killer (NK) cells, and CD103(+) DCs, accompanied by a systemic increase of pro-inflammatory cytokines interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-27 (IL-27). This response was even more pronounced after combining the virus with checkpoint therapy, in particular with anti-PD-L1 and anti-TIM-3, leading to further reduced tumor growth in injected lesions. Moreover, anti-PD-L1 combination also facilitated abscopal responses in non-injected lesions

Arming Oncolytic Adenoviruses: Effect of Insertion Site and Splice Acceptor on Transgene Expression and Viral Fitness

Oncolytic adenoviruses (OAds) present limited efficacy in clinics. The insertion of therapeutic transgenes into OAds genomes, known as "arming OAds", has been the main strategy to improve their therapeutic potential. Different approaches were published in the decade of the 2000s, but with few comparisons. Most armed OAds have complete or partial E3 deletions, leading to a shorter half-life in vivo. We generated E3+ OAds using two insertion sites, After-fiber and After-E4, and two different splice acceptors linked to the major late promoter, either the Ad5 protein IIIa acceptor (IIIaSA) or the Ad40 long fiber acceptor (40SA). The highest transgene levels were obtained with the After-fiber location and 40SA. However, the set of codons of the transgene affected viral fitness, highlighting the relevance of transgene codon usage when arming OAds using the major late promoter

Human Menstrual Blood-derived Mesenchymal Stem Cells As Potential Cell Carriers For Oncolytic Adenovirus

Antitumor efficacy of systemically administered oncolytic adenoviruses (OAdv) is limited due to diverse factors such as liver sequestration, neutralizing interactions in blood, elimination by the immune system, and physical barriers in tumors. It is therefore of clinical relevance to improve OAdv bioavailability and tumor delivery. Among the variety of tumor-targeting strategies, the use of stem cells and specifically bone marrow-derived mesenchymal stem cells (BM-MSCs) is of particular interest due to their tumor tropism and immunomodulatory properties. Nonetheless, the invasive methods to obtain these cells, the low number of MSCs present in the bone marrow, and their restricted in vitro expansion represent major obstacles for their use in cancer treatments, pointing out the necessity to identify an alternative source of MSCs. Here, we have evaluated the use of menstrual blood-derived mesenchymal stem cells (MenSCs) as cell carriers for regional delivery of an OAdv in the tumor. Our results indicate that MenSCs can be isolated without invasive methods, they have an increased proliferation rate compared to BM-MSCs, and they can be efficiently infected with different serotype 5-based capsid-modified adenoviruses, leading to viral replication and release. In addition, our in vivo studies confirmed the tumor-homing properties of MenSCs after regional administration

Enhanced antitumor efficacy of an oncolytic adenovirus armed with an EGFR-targeted BiTE using menstrual blood-derived mesenchymal stem cells as carriers

Poor tumor targeting of oncolytic adenoviruses (OAdv) after systemic administration is considered a major limitation for virotherapy of disseminated cancers. The benefit of using mesenchymal stem cells as cell carriers for OAdv tumor targeting is currently evaluated not only in preclinical models but also in clinical trials. In this context, we have previously demonstrated the enhanced antitumor efficacy of OAdv-loaded menstrual blood-derived mesenchymal stem cells (MenSCs). However, although significant, the antitumor efficacy obtained was modest, and we hypothesized that a greater antitumor efficacy could be obtained arming the OAdv with a therapeutic transgene. Here we show that combining MenSCs with ICOVIR15-cBiTE, an OAdv expressing an epidermal growth factor receptor (EGFR)-targeting bispecific T-cell engager (cBiTE), enhances the antitumor efficacy compared to MenSCs loaded with the unarmed virus ICOVIR15. We found that MenSCs properly produce cBiTE after viral infection leading to a greater antitumor potency both in vitro and in vivo. These findings indicate the mutual benefit of combining MenSCs and armed OAdv and support the combination of ICOVIR15-cBiTE and MenSCs as a cancer treatment.We thank CERCA Program/Generalitat de Catalunya for their institutional support. The authors also thank Dolores Ramos and Silvia Torres for their lab technical support and Vanessa Cervera for samples processing. This work was supported by the Asociación Española Contra el Cáncer, BIO2017-897554-C2-1-Rgrant to R. Alemany from the Ministerio de Economía y Competitividad of Spain, Adenonet BIO2015-68990-REDT to R. Alemany from the Ministerio de Economía y Competitividad of Spain, RedADVANCE(CAT) project COMRDI15-1-0013 to R. Alemany from Ris3CAT and 2017SGR449 research grant to RA from the ‘Generalitat de Catalunya’. Cofunded by the European Regional Development Fund, a way to Build Europe to RA

Evidence of Anti-tumoral Efficacy in an Immune Competent Setting with an iRGD-Modified Hyaluronidase-Armed Oncolytic Adenovirus

To enhance adenovirus-mediated oncolysis, different approaches that tackle the selectivity, tumor penetration, and spreading potential of oncolytic adenoviruses have been reported. We have previously demonstrated that insertion of the internalizing Arginine-Glycine-Aspartic (iRGD) tumor-penetrating peptide at the C terminus of the fiber or transgenic expression of a secreted hyaluronidase can improve virus tumor targeting and spreading. Here we report a new oncolytic adenovirus ICOVIR17K-iRGD in which both modifications have been incorporated. In xenografted A549 tumors in nude mice, ICOVIR17K-iRGD shows higher efficacy than the non-iRGD counterpart. To gain insights into the role of the immune system in oncolysis, we have studied ICOVIR17K-iRGD in the tumor isograft mouse model CMT64.6, partially permissive to human adenovirus 5 replication, in immunodeficient or immunocompetent mice. Whereas no efficacy was observed in the immunodeficient setting due to insufficient viral replication, partial efficacy and a polymorphonuclear and CD8+ T cell infiltrate were observed in the immunocompetent mice. The results indicate that the elicitation of a virus-induced anti-tumoral immune response is responsible for the observed partial antitumoral effect

Enhanced antitumor efficacy of oncolytic adenovirus-loaded menstrual blood-derived mesenchymal stem cells in combination with peripheral blood mononuclear cells

Several studies have evaluated the efficacy of using human oncolytic adenovirus-loaded mesenchymal stem cells for cancer treatment. For example, we have described the antitumor efficacy of CELYVIR, autologous bone marrow mesenchymal stem cells infected with the oncolytic adenovirus ICOVIR-5, for treatment of neuroblastoma patients. Results from this clinical trial point out the role of the immune system in the clinical outcome. In this context, a better understanding of the immunophenotypic changes of human mesenchymal stem cells upon adenoviral infection and how these changes affect human autologous or allogeneic peripheral blood mononuclear cells (PBMCs) could guide strategies to improve the antitumor efficacy of infected Mesenchymal Stem Cells (MSCs). In this work, we show how infection by an oncolytic adenovirus (OAdv) induces Toll-like receptor 9 overexpression and activation of the NF-κB pathway in menstrual blood-derived mesenchymal stem cells (MenSCs), leading to a specific cytokine secretion profile. Moreover, a pro-inflammatory environment, mainly mediated by monocyte activation that leads to the activation of both T-cells and natural killer cells (NK cells), is generated when OAdv-loaded MenSCs are co-cultured with allogeneic PBMCs. This combination of allogeneic PBMCs and OAdv-loaded MenSCs enhances antitumor efficacy both in vitro and in vivo, an effect partially mediated monocytes and NK cells. Altogether our results demonstrate not only the importance of the immune system for the oncolytic adenovirus-loaded MSCs antitumor efficacy, but in particular the benefits of using allogeneic MSCs for this therapy