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Oncolytic virotherapy uses viruses designed to selectively replicate in cancer cells. An alternative to intratumoral administration is to use mesenchymal stem cells (MSCs) to transport the oncolytic viruses to the tumor site. Following this strategy, our group has already applied this treatment to children and adults in a human clinical trial and a veterinary trial, with good clinical responses and excellent safety profiles. However, the development of immunocompetent cancer mouse models is still necessary for the study and improvement of oncolytic viroimmunotherapies. Here we have studied the antitumor efficacy, immune response, and mechanism of action of a complete murine version of our cellular virotherapy in mouse models of renal adenocarcinoma and melanoma. We used mouse MSCs infected with the mouse oncolytic adenovirus dlE102 (OAd-MSCs). In both models, treatment with OAd-MSCs significantly reduced tumor volumes by 50% and induced a pro-inflammatory tumor microenvironment. Furthermore, treated mice harboring renal adenocarcinoma and melanoma tumors presented increased infiltration of tumor-associated macrophages (TAMs), natural killer cells, and tumor-infiltrating lymphocytes (TILs). Treated mice also presented lower percentage of TILs expressing programmed cell death protein 1 (PD-1)—the major regulator of T cell exhaustion. In conclusion, treatment with OAd-MSCs significantly reduced tumor volume and induced changes in tumor-infiltrating populations of melanoma and renal cancer.

Background: Stemness in sarcomas is coordinated by the expression of pluripotency factors, like SOX2, in cancer stem cell (CSC) subpopulations. The role of SOX2 in tumor initiation and progression has been well characterized in osteosarcoma. However, the pro-tumorigenic features of SOX2 have been scarcely investigated in other sarcoma subtypes. Methods: shRNA and cDNA lentiviral constructions were used to deplete and overexpress SOX2 in a model of undifferentiated pleomorphic sarcoma (UPS). In addition, we introduced a reporter system (SORE6) which allows to detect and isolate viable cells expressing SOX2 and/or OCT4 in the UPS model and patient-derived chondrosarcoma lines. Functional CSC characterization included tumorsphere formation, in vivo xenograft growth assays, and pharmacologic testing of the effects of doxorubicin, placlitaxel, trabectedin or the mithramycin analogue EC8042. Immunohistochemical analysis of a cohort of 88 formalin-fixed and paraffin-embedded sarcoma samples was performed to correlate SOX2 expression with clinical parameters. Findings: SOX2 depletion dramatically reduced the ability of sarcoma cells to form tumorspheres and to initiate tumor growth in vivo. Conversely, SOX2 overexpression resulted in increased in vivo tumorigenicity. In sarcoma patients, SOX2 expression was associated to advanced disease stages, aggressive phenotypes and lower survival. Finally, SORE6+ cells were significantly more tumorigenic than the SORE6- population and EC-8042 effectively targeted SORE6+ cells in vitro and in vivo. Interpretation: SOX2 is a pro-tumorigenic factor with prognostic potential in sarcoma. Moreover, SORE6 transcriptional activity is a bona fide CSC marker in sarcoma and constitutes an excellent biomarker for evaluating the efficacy of anti-tumor treatments on CSC subpopulations. Funding Statement: This work was supported by the Agencia Estatal de Investigacion (AEI) [MINECO/Fondo Europeo de Desarrollo Regional (FEDER) (SAF-2016-75286-R to R.R.), ISC III/FEDER (Miguel Servet Program CPII16/00049 to R.R., Sara Borrell Program CD16/00103 to S.T.M. and PI16/00280 and PI19/00560 to J.M.G-P) and Consorcio CIBERONC CB16/12/00390)] and the Plan de Ciencia Tecnologia e Innovacion del Principado de Asturias/FEDER (IDI/2018/155) to J.P.R. PharmaMar and EntreChem also provided support. Finally, the authors acknowledge the Principado de Asturias BioBank (PT17/0015/0023), financed jointly by Servicio de Salud del Principado de Asturias, Instituto de Salud Carlos III and Fundacion Bancaria Cajastur and integrated in the Spanish National Biobanks Network, for its collaboration. Declaration of Interests: The authors declare they have no competing interests. Ethics Approval Statement: All experimental protocols have been performed in accordance with institutional review board guidelines and were approved by the Institutional Ethics Committee of the Principado de Asturias. All samples from human origin were obtained upon signed informed consent. All experimental protocols were carried out in accordance with the institutional guidelines of the University of Oviedo and were approved by the Animal Research Ethical Committee of the University of Oviedo prior to the study.

Osteosarcoma (OS) is a highly aggressive tumor characterized by malignant cells producing pathologic bone; the disease presents a natural tendency to metastasize. Genetic studies indicate that the OS genome is extremely complex, presenting signs of macro-evolution, and linear and branched patterns of clonal development. However, those studies were based on the phylogenetic reconstruction of next-generation sequencing (NGS) data, which present important limitations. Thus, testing clonal evolution in experimental models could be useful for validating this hypothesis. In the present study, lentiviral LeGO-vectors were employed to generate colorimetric red, green, blue (RGB)-marking in murine, canine, and human OS. With this strategy, we studied tumor heterogeneity and the clonal dynamics occurring in vivo in immunodeficient NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ (NSG) mice. Based on colorimetric label, tumor clonal composition was analyzed by confocal microscopy, flow cytometry, and different types of supervised and unsupervised clonal analyses. With this approach, we observed a consistent reduction in the clonal composition of RGB-marked tumors and identified evident clonal selection at the first passage in immunodeficient mice. Furthermore, we also demonstrated that OS could follow a neutral model of growth, where the disease is defined by the coexistence of different tumor sub-clones. Our study demonstrates the importance of rigorous testing of the selective forces in commonly used experimental models, This research was funded by Fondo de Investigaciones Sanitarias (FIS: PI11/00377, PI14CIII/00005 and PI17CIII/00013 to J.G.-C., FIS: CP11/00206 to A.A., FIS: PI18/006146 to A.P.- G. and RTICC: RD12/0036/0027 to J.G.-C.), the Madrid Regional Government (P2010/BMD-2420 and B2017/BMD-3692 to J.G.-C.), the Asociación Pablo Ugarte, and AFANION grant support