Switchable CAR T cell strategy against osteosarcoma.

Immunotherapy with chimeric antigen receptor T (CAR T) cells has changed the treatment of hematological malignances, but they are still a challenge for solid tumors, including pediatric sarcomas. Here, we report a switchable CAR T cell strategy based on anti-FITC CAR T cells and a switch molecule conjugated with FITC for targeting osteosarcoma (OS) tumors. As a potential target, we analyzed the expression of B7-H3, an immune checkpoint inhibitor, in OS cell lines. In addition, we evaluate the capacity of an anti-B7-H3 monoclonal antibody conjugated with FITC (anti-B7-H3-FITC mAb) to control the antitumor activity of anti-FITC CAR T cells. The effector functions of anti-FITC CAR T cells against OS, measured in vitro by tumor cell killing activity and cytokine production, are dependent on the presence of the anti-B7-H3-FITC mAb switch. Moreover, OS cells stimulate anti-FITC CAR T cells migration. In vivo, anti-B7-H3 mAb penetrates in the tumor and binds 143B OS tumor cells. Furthermore, anti-FITC CAR T cells reach tumor region and exert antitumor effect in an OS NSG mouse model only in the presence of the switch molecule. We demonstrate that anti-B7-H3-FITC mAb redirects the cytotoxic activity of anti-FITC CAR T cells against OS tumors suggesting that switchable CAR T cell platforms might be a plausible strategy against OS.

Toll-like Receptor Signaling-deficient Cells Enhance Antitumor Activity of Cell-based Immunotherapy by Increasing Tumor Homing.

Cancer immunotherapy aims to activate the immune system. Some immunotherapeutic agents can be loaded in carrier cells for delivering to the tumors. However, a challenge with cell-based therapies is the selection of the appropriate cells to produce effective clinical outcomes. We hypothesize that therapies based on cells presenting a natural low proinflammatory profile ("silent cells") in the peripheral blood would result in better antitumor responses by increasing their homing to the tumor site. We studied our hypothesis in an immunotherapy model consisting of mesenchymal stromal cells (MSCs) carrying oncolytic adenoviruses for the treatment of immunocompetent mice. Toll-like receptor signaling-deficient cells (TLR4, TLR9, or MyD88 knockout) were used as "silent cells," while regular MSCs were used as control. Although in vitro migration was similar in regular and knockout carrier cells, in vivo tumor homing of silent cells was significantly higher after systemic administration. This better homing to the tumor site was highly related to the mild immune response triggered by these silent cells in peripheral blood. As a result, the use of silent cells significantly improved the antitumor efficacy of the treatment in comparison with the use of regular MSCs. While cancer immunotherapies generally aim to boost local immune responses in the tumor microenvironment, low systemic inflammation after systemic administration of the treatment may indeed enhance their tumor homing and improve the overall antitumor effect. These findings highlight the importance of selecting appropriate donor cells as therapeutic carriers in cell-based therapies for cancer treatment. Significance: Cells carrying drugs, virus, or other antitumor agents are commonly used for the treatment of cancer. This research shows that silent cells are excellent carriers for immunotherapies, improving tumor homing and enhancing the antitumor effect.

Cellular Heterogeneity and Cooperativity in Glioma Persister Cells Under Temozolomide Treatment.

We have observed a drug-tolerant/persister state in a human glioblastoma (GBM) cell line after exposure to temozolomide, the standard-of-care chemotherapeutic agent for GBM. We used a multicolor lentiviral genetic barcode labeling to follow cell population evolution during temozolomide treatment. We observed no change in the distribution of the different colored populations of cells in persister or resistant cells suggesting that pre-existing minor subpopulations, which would be expected to be restricted to a single color, were not amplified/selected during the response to the drug. We have previously identified four genes (CHI3L1, FAT2, KLK5, and HB-EGF) that were over-expressed during the persister stage. Single-cell analysis of these four genes indicated that they were expressed in different individual cells ruling out the existence of a single persister-specific clone but suggesting rather a global answer. Even so, the transitory silencing of CHI3L1, FAT2, or KLK5 influenced the expression of the other three genes and the survival of U251 cells in absence of temozolomide. Since proteins encoded by the four genes are all localized in the extracellular matrix or interact within the extracellular compartment, we propose that cellular interactions and communications are important during the persister stage before the acquisition of chemo-resistance. Thus, persisters might be a new therapeutically relevant target in GBM.

Combination immunotherapy using G-CSF and oncolytic virotherapy reduces tumor growth in osteosarcoma.

BACKGROUND: Osteosarcoma is the most common malignant solid tumor that affects bones, however, survival rates of patients with relapsed osteosarcoma have not improved in the last 30 years. Oncolytic virotherapy, which uses viruses designed to selectively replicate in cancer cells, has emerged as a promising treatment for solid tumors. Our group uses mesenchymal stem cells (MSCs) to transport oncolytic adenoviruses (OAds) to the tumor site, a therapeutic strategy called Celyvir. This treatment has been already applied in human patients, canine patients and different mouse models. In parallel, previous results have probed that administration of granulocyte-colony stimulating factor (G-CSF) increased immune infiltration in tumors. We then hypothesized that the mobilization of immune cells by G-CSF may increase the antitumor efficacy of Celyvir treatment by increasing the immune infiltration into the tumors. METHODS: In this study, we use a murine version of Celyvir consisting in murine MSCs carrying the murine OAd dlE102-here called OAd-MSCs-in an immunocompetent model of osteosarcoma. We tested the antitumoral efficacy of the combination of OAd-MSCs plus G-CSF. RESULTS: Our results show that treatment with OAd-MSCs or the union of OAd-MSCs with G-CSF (Combination) significantly reduced tumor growth of osteosarcoma in vivo. Moreover, treated tumors presented higher tumor infiltration of immune cells-especially tumor-infiltrating lymphocytes-and reduced T cell exhaustion, which seems to be enhanced in tumors treated with the Combination. The comparison of our results to those obtained from a cohort of pediatric osteosarcoma patients showed that the virotherapy induces immunological changes similar to those observed in patients with good prognosis. CONCLUSIONS: The results open the possibility of using cellular virotherapy for the treatment of bone cancers. Indeed, its combination with G-CSF may be considered for the improvement of the therapy.

AKT and JUN are differentially activated in mesenchymal stem cells after infection with human and canine oncolytic adenoviruses.

There is increasing evidence about the use of oncolytic adenoviruses (Ads) as promising immunotherapy agents. We have previously demonstrated the clinical efficiency of mesenchymal stem cells (MSCs) infected with oncolytic Ads as an antitumoral immunotherapy (called Celyvir) in human and canine patients, using ICOVIR-5 or ICOCAV17 as human and canine oncolytic Ads, respectively. Considering the better clinical outcomes of canine patients, in this study we searched for differences in cellular responses of human and canine MSCs to Ad infection that may help understand the mechanisms leading to higher antitumor immune response. We found that infection of human and canine MSCs with ICOVIR-5 or ICOCAV17 did not activate the NF-κB pathway or the interferon regulatory factors IRF3 and IRF7. However, we observed differences in the profile of cytokines secretion, as infection of canine MSCs with ICOCAV17 resulted in lower secretion of several cytokines. Moreover, we showed that infection of human MSCs with ICOVIR-5 increased the phosphorylation of a number of proteins, including AKT and c-JUN. Finally, we demonstrated that differences in regulation of AKT and c-JUN in human and canine MSCs by ICOVIR-5 or ICOCAV17 are intrinsic to each virus. Our findings suggest that ICOCAV17 induces a more limited host response in canine MSCs, which may be related to a better clinical outcome. This result opens the possibility to develop new human oncolytic Ads with these specific properties. In addition, this improvement could be imitated by selecting specific human MSC on the basis of a limited host response after Ad infection.

Sarcoma treatment in the era of molecular medicine.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.

Cellular Virotherapy Increases Tumor-Infiltrating Lymphocytes (TIL) and Decreases their PD-1+ Subsets in Mouse Immunocompetent Models.

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.

SOX2 Expression and Transcriptional Activity Identifies a Subpopulation of Cancer Stem Cells in Sarcoma with Prognostic Implications.

Stemness in sarcomas is coordinated by the expression of pluripotency factors, like SOX2, in cancer stem cells (CSC). 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. Here, we show that SOX2 depletion dramatically reduced the ability of undifferentiated pleomorphic sarcoma (UPS) cells to form tumorspheres and to initiate tumor growth. Conversely, SOX2 overexpression resulted in increased in vivo tumorigenicity. Moreover, using a reporter system (SORE6) which allows to monitor viable cells expressing SOX2 and/or OCT4, we found that SORE6+ cells were significantly more tumorigenic than the SORE6- subpopulation. In agreement with this findings, SOX2 expression in sarcoma patients was associated to tumor grade, differentiation, invasive potential and lower patient survival. Finally, we studied the effect of a panel of anti-tumor drugs on the SORE6+ cells of the UPS model and patient-derived chondrosarcoma lines. We found that the mithramycin analogue EC-8042 was the most efficient in reducing SORE6+ cells in vitro and in vivo. Overall, this study demonstrates that 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.

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 (OAdv)-loaded mesenchymal stem cells (MSC) for cancer treatment. For example, we have described the antitumor efficacy of CELYVIR, autologous bone marrow-mesenchymal stem cells infected with the OAdv ICOVIR-5, for treatment of patients with neuroblastoma. 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 MSCs upon adenoviral infection and how these changes affect human autologous or allogeneic peripheral blood mononuclear cells (PBMC) could guide strategies to improve the antitumor efficacy of infected MSCs. In this work, we show how infection by an OAdv induces toll-like receptor 9 overexpression and activation of the NFĸB pathway in menstrual blood-derived MSCs, leading to a specific cytokine secretion profile. Moreover, a proinflammatory environment, mainly mediated by monocyte activation that leads to the activation of both T cells and natural killer cells (NK cell), is generated when OAdv-loaded MSCs are cocultured with allogeneic PBMCs. This combination of allogeneic PBMCs and OAdv-loaded MSCs enhances antitumor efficacy both in vitro and in vivo, an effect partially mediated by monocytes and NK cells. Altogether our results demonstrate not only the importance of the immune system for the OAdv-loaded MSCs antitumor efficacy, but in particular the benefits of using allogeneic MSCs for this therapy.

Clonal dynamics in osteosarcoma defined by RGB marking.

Osteosarcoma is a type of bone tumour characterized by considerable levels of phenotypic heterogeneity, aneuploidy, and a high mutational rate. The life expectancy of osteosarcoma patients has not changed during the last three decades and thus much remains to be learned about the disease biology. Here, we employ a RGB-based single-cell tracking system to study the clonal dynamics occurring in a de novo-induced murine osteosarcoma model. We show that osteosarcoma cells present initial polyclonal dynamics, followed by clonal dominance associated with adaptation to the microenvironment. Interestingly, the dominant clones are composed of subclones with a similar tumour generation potential when they are re-implanted in mice. Moreover, individual spontaneous metastases are clonal or oligoclonal, but they have a different cellular origin than the dominant clones present in primary tumours. In summary, we present evidence that osteosarcomagenesis can follow a neutral evolution model, in which different cancer clones coexist and propagate simultaneously.

Antitumor virotherapy using syngeneic or allogeneic mesenchymal stem cell carriers induces systemic immune response and intratumoral leukocyte infiltration in mice.

Oncolytic virotherapy uses oncolytic viruses that selectively replicate in cancer cells. The use of cellular vehicles with migration ability to tumors has been considered to increase their delivery to target sites. Following this approach, the antitumor efficacy of the treatment Celyvir (mesenchymal stem cells infected with the oncolytic adenovirus ICOVIR-5) has been demonstrated in patients with neuroblastoma. However, the better efficacy of syngeneic or allogeneic mesenchymal stem cells as cell carriers and the specific role of the immune system in this therapy are still unknown. In this study we use our virotherapy Celyvir with syngeneic and allogeneic mouse mesenchymal stem cells to determine their antitumor efficacy in a C57BL/6 murine adenocarcinoma model. Adoptive transfer of splenocytes from treated mice to new tumor-bearing mice followed by a secondary adoptive transfer to a third group was performed. Similar reduction of tumor growth and systemic activation of the innate and adaptive immune system was observed in groups treated with syngeneic or allogeneic mesenchymal stem cells loaded with ICOVIR-5. Moreover, a different pattern of infiltration was observed by immunofluorescence in Celyvir-treated groups. While non-treated tumors presented higher density of infiltrating immune cells in the periphery of the tumor, both syngeneic and allogeneic Celyvir-treated groups presented higher infiltration of CD45+ cells in the core of the tumor. Therefore, these results suggest that syngeneic and allogeneic Celyvir induce systemic activation of the immune system, similar antitumor effect and a higher intratumoral infiltration of leukocytes.