The immune response is a fundamental process in the treatment of solid tumors. Here, we present a protocol for implanting diffuse midline glioma cells in the brain of immune-competent mice and ...characterizing the different immune populations in the tumor microenvironment in a flow cytometry panel. We describe steps for processing of brain tumors, isolating the immune cells, and subsequent staining with antibodies for flow cytometry. We then detail procedures for implementing the gating strategy.
For complete details on the use and execution of this protocol, please refer to Ausejo-Mauleon et al.1
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•Development of an orthotopic pontine diffuse midline glioma model•Implantable guide-screw system for injection of tumor cells and drugs into the brain•Steps described for isolating immune cells from orthotopic DMG mouse model•Characterization of the brain immune populations with a single flow cytometry panel
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The immune response is a fundamental process in the treatment of solid tumors. Here, we present a protocol for implanting diffuse midline glioma cells in the brain of immune-competent mice and characterizing the different immune populations in the tumor microenvironment in a flow cytometry panel. We describe steps for processing of brain tumors, isolating the immune cells, and subsequent staining with antibodies for flow cytometry. We then detail procedures for implementing the gating strategy.
...paediatric patients have been treated with therapeutic regimens based on those of adults, which have failed. ...TIM-3 appears to be a therapy in patients to modulate both the myeloid cells and the ...T cells that exist in the tumour (Figure 1). ...TIM-3 blockade is presented as an ideal treatment to be combined with other immunotherapies that increase T-cell infiltration due to the activation of myeloid cells in the tumour microenvironment and new infiltrating T cells. TIM-3 blockade appears as a new therapy capable of inducing profound proinflammatory changes in the DIPG myeloid immune populations, leading to the activation of T cells in the tumour microenvironment of these patients. ...these pre-clinical data offer strong support for initiating a clinical trial with an anti-TIM-3 antibody for the treatment of DIPG as monotherapy or even with other already proven treatments, such as oncolytic viruses or CAR-T cells.
BackgroundGlioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising ...strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma.MethodsThe in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF.ResultsDelta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge.ConclusionsIn summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors.
Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors, and patient survival has not changed despite many therapeutic efforts, emphasizing the urgent need for effective ...treatments. Here, we evaluated the anti-DIPG effect of the oncolytic adenovirus Delta-24-ACT, which was engineered to express the costimulatory ligand 4-1BBL to potentiate the antitumor immune response of the virus. Delta-24-ACT induced the expression of functional 4-1BBL on the membranes of infected DIPG cells, which enhanced the costimulation of CD8+ T lymphocytes. In vivo, Delta-24-ACT treatment of murine DIPG orthotopic tumors significantly improved the survival of treated mice, leading to long-term survivors that developed immunological memory against these tumors. In addition, Delta-24-ACT was safe and caused no local or systemic toxicity. Mechanistic studies showed that Delta-24-ACT modulated the tumor-immune content, not only increasing the number, but also improving the functionality of immune cells. All of these data highlight the safety and potential therapeutic benefit of Delta-24-ACT the treatment of patients with DIPG.
BackgroundDiffuse Midline Gliomas (DMG) are aggressive pediatric brain tumors that arise in the brainstem of children between 5–10 years old. DMGs are the leading cause of pediatric death caused by a ...brain tumor, with a median survival of only 9 months.1 2 We have previously shown that the administration of the oncolytic adenovirus Delta-24-RGD is safe and lead to an increase in long-term survivors in murine models.3 4 In order to further increase the antitumor effect of Delta-24-RGD by boosting the immune response, we have constructed a new adenovirus, Delta-24-ACT, which incorporates the 4-1BBL (CD137L) into its backbone. 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells and NK cells and the generation and maintenance of memory CD8+ T cells, among other functions.5 6 MethodsMurine and human DMG cell lines were used. 4-1BBL expression was assessed in infected cells by flow cytometry and immunofluorescence. Viral protein expression was measured by western blot, viral replication was analyzed using a method based on hexon detection and the oncolytic effect by MTS assay. For in vivo experiments, cells were injected in the pons of mice using a screw-guided system.7 A single administration of the adenovirus was injected intratumorally using the same procedure. The tumor immune populations were analyzed by flow cytometry.ResultsWe first confirmed by flow cytometry that DMG cells infected with Delta-24-ACT expressed 4-1BBL in their membrane in a dose-dependent manner. Afterwards, we analyzed the oncolytic effect of Delta-24-ACT in vitro. Delta-24-ACT was able to express viral early and late proteins in murine and human DMG cell lines and to replicate efficiently in human cells. In addition, the virus caused cell death in a dose-dependent manner. In vivo, Delta-24-ACT administration demonstrated to be safe and to produce a significant survival benefit in murine DMG models, obtaining 30–50% of long-term survivors depending on the model. More importantly, Delta-24-ACT generated immune memory, as long-term survivors were disease-free after cell rechallenge. On the other hand, we analyzed immune infiltration 7 or 10 days after the viral administration into the tumor and observed a significant increase of tumor infiltration in treated mice, which showed an activated state.ConclusionsDelta-24-ACT administration into DMG murine tumor models significantly increases the recruitment and activation of immune cells, which leads to long term survivors and immunological memory.ReferencesCooney T, Lane A, Bartels U, Bouffet E, Goldman S, Leary S, Foreman NK, Packer RJ, Broniscer A, Minturn JE, Shih C, Chintagumpala M, Hassall T, Gottardo NG, Dholaria H, Hoffman L, Chaney B, Baugh J, Doughman R, Leach JL, Jones BV, Fouladi M, Warren KE, Monje M. Contemporary survival endpoints: an International diffuse Intrinsic pontine glioma registry study. Neuro Oncol 2017;19(9):1279–1280.Grasso CS, Tang Y, Truffaux N, Berlow NE, Liu L, Debily MA, Quist MJ, Davis LE, Huang EC, Woo PJ, Ponnuswami A, Chen S, Johung TB, Sun W, Kogiso M, Du Y, Qi L, Huang Y, Hütt-Cabezas M, Warren KE, Le Dret L, Meltzer PS, Mao H, Quezado M, van Vuurden DG, Abraham J, Fouladi M, Svalina MN, Wang N, Hawkins C, Nazarian J, Alonso MM, Raabe EH, Hulleman E, Spellman PT, Li XN, Keller C, Pal R, Grill J, Monje M. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nat Med 2015;21(6):555–9.Martínez-Vélez N, Garcia-Moure M, Marigil M, González-Huarriz M, Puigdelloses M, Gallego Pérez-Larraya J, Zalacaín M, Marrodán L, Varela-Guruceaga M, Laspidea V, Aristu JJ, Ramos LI, Tejada-Solís S, Díez-Valle R, Jones C, Mackay A, Martínez-Climent JA, García-Barchino MJ, Raabe E, Monje M, Becher OJ, Junier MP, El-Habr EA, Chneiweiss H, Aldave G, Jiang H, Fueyo J, Patiño-García A, Gomez-Manzano C, Alonso MM. The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models. Nat Commun 2019;10(1):2235.Garcia-Moure M, Gonzalez-Huarriz M, Labiano S, Guruceaga E, Bandres E, Zalacain M, Marrodan L, de Andrea C, Villalba M, Martinez-Velez N, Laspidea V, Puigdelloses M, Gallego Perez-Larraya J, Iñigo-Marco I, Stripecke R, Chan JA, Raabe EH, Kool M, Gomez-Manzano C, Fueyo J, Patiño-García A, Alonso MM. Delta-24-RGD, an oncolytic adenovirus, increases survival and promotes proinflamatory immune landscape remodeling in models of AT/RT and CNS-PNET. Clin Cancer Res 2021;27(6):1807–1820.Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1):49–57.Yonezawa A, Dutt S, Chester C, Kim J, Kohrt HE. Boosting cancer immunotherapy with anti-CD137 antibody therapy. Clin Cancer Res 2015;21(14):3113–20.Marigil M, Martinez-Velez N, Domínguez PD, Idoate MA, Xipell E, Patiño-García A, Gonzalez-Huarriz M, García-Moure M, Junier MP, Chneiweiss H, El-Habr E, Diez-Valle R, Tejada-Solís S, Alonso MM. Development of a DIPG orthotopic model in mice using an implantable guide-screw system. PLoS One 2017;12(1):e0170501.
The outcomes of metastatic and nonresponder pediatric osteosarcoma patients are very poor and have not improved in the last 30 years. These tumors harbor a highly immunosuppressive environment, ...making existing immunotherapies ineffective. Here, we evaluated the use of Semliki Forest virus (SFV) vectors expressing galectin-3 (Gal3) inhibitors as therapeutic tools, since both the inhibition of Gal3, which is involved in immunosuppression and metastasis, and virotherapy based on SFV have been demonstrated to reduce tumor progression in different tumor models.
In vitro
, inhibitors based on the Gal3 amino-terminal domain alone (Gal3-N) or fused to a Gal3 peptide inhibitor (Gal3-N-C12) were able to block the binding of Gal3 to the surface of activated T cells.
In vivo
, SFV expressing Gal3-N-C12 induced strong antitumor responses in orthotopic K7M2 and MOS-J osteosarcoma tumors, leading to complete regressions in 47% and 30% of mice, respectively. Pulmonary metastases were also reduced in K7M2 tumor-bearing mice after treatment with SFV-Gal3-N-C12. Both the antitumor and antimetastatic responses were dependent on modulation of the immune system, primarily including an increase in tumor-infiltrating lymphocytes and a reduction in the immunosuppressive environment inside tumors. Our results demonstrated that SFV-Gal3-N-C12 could constitute a potential therapeutic agent for osteosarcoma patients expressing Gal3.
A single intratumoral administration of a Semliki Forest virus vector expressing a galectin-3 inhibitor (SFV-Gal3-N-C12) reduced tumor growth and metastasis development, generating long-term survivors, in orthotopic osteosarcoma mouse models. SFV-Gal3-N-C12 decreased the tumor immunosuppressive environment, increasing tumor-specific CD8
+
T cells, which were more resilient to becoming exhausted.
Abstract
With a 2-year survival less than 20%, Diffuse Intrinsic Pontine Glioma (DIPG) is the principal cause of pediatric death. Despite recent advances in the current treatments, the outcome for ...children with DIPGs remains dismal. Since the approval of T-VEC for melanoma by the FDA, oncolytic adenoviruses have emerged as a promising therapeutic strategy for brain tumors. Thus, our group launched the first world clinical trial phase I with the oncolytic adenovirus Delta-24-RGD (DNX-2401 in the clinic) for newly diagnosed DIPG (NCT03178032), which has shown safety and feasibility. Despite DNX-2401 increases the recruitment of T cells into the tumor, they usually become inactive due to the non-responsive tumor microenvironment evidencing the urgent need to improve this strategy focusing on the generation of effective long-term immune responses. Therefore, we decided to combine the Delta-24-RGD with the targeting of the costimulatory molecule CD40 in immunocompetent mice bearing orthotopic DIPG. The activation of the CD40 receptor, which is expressed by antigen presenting cells (APC) such as microglia, macrophages, and dendritic cells, is known to increase antigen presentation and enable T-cell priming and activation. Here, we observed that in addition to Delta-24-RGD anti-tumor effects, the stimulation of CD40 (using an agonistic antibody) on the tumor APCs results in a remodeling of the tumor immune compartment towards a proinflammatory scenario and a more efficient T-cell infiltration. Of importance, the combination therapy extends survival of treated mice as compared to single treatments or non-treated mice. In addition, we observe a complete regression of tumors in more than 40% of treated mice and the development of long-term anti-tumor immunity. We believe that these results provide a translational breakthrough in the treatment of these lethal tumors and open the door for a future innovative clinical trial.
Abstract
Pediatric diffuse midline gliomas-H3-K27M-mutant are aggressive brain tumours that arise during childhood. Despite new advances in genomic knowledge and the significant number of clinical ...trial testing new targeted therapies, patient outcome is still insufficient. Cancer immunotherapy is opening new therapeutic options representing a hope for this orphan disease. Aptamers are single-stranded nucleic acid ligands design to achieve a remarkable affinity and specificity to their targets, comparable to antibodies. TIM-3, is a potential immune checkpoint target, typically involved in T-cell exhaustion. Recent studies showed that TIM-3 is also expressed in tumour and glial cells and it plays an important role in brain tumour responses mediated by myeloid cells. In this work, we examined the anti-tumour effect of an aptamer against TIM-3 alone or in combination with radiotherapy. Of importance, we tested TIM-3 aptamer in a murine glioma and DIPG model, where we not observed any toxicity. TIM-3 administration increased overall survival but was unable to control the disease. Of importance, TIM-3 combination with radiotherapy improved the overall survival of treated mice when compared with single treatments leading to 50% of long-term survivors. TIM-3 aptamer administration increase T-infiltration in the tumour site compared to non-treated or library control. Mechanistic studies performed on day 16 showed an increase in CD8 effector cells, a decrease in T-regulators Foxp3+ cells and an increase in IFN-gamma expression suggesting the triggering of an antitumor-immune response. Rechallenge experiments demonstrated immune memory in the long-term responders that led to reject tumour re-implantation, confirming that TIM-3 aptamer treatment in combination with RT elicits specific antitumor immunity in mouse glioma models. These results suggest that immuno-therapies approaches in combination with radiotherapy would be worth exploring in the treatment of deadly DMG-H3K27-Mutant tumours.
Abstract
Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic ...virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models. In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory. Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.
Abstract
Despite the advances in our understanding of pediatric diffuse midline gliomas (DMG) they remain the leading cause of pediatric death caused by cancer. Our group has demonstrated that the ...administration of Delta-24-RGD in DIPG models is safe and therapeutically efficacious. An on-going clinical trial with this virus has proven to be safe for these patients. To further improve the anti-tumoral response of the virus, we armed Delta-24-RGD with 4-1BBL (Delta-24-ACT). 41BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. Here, we showed that in vitro Delta-24-ACT can infect and express 4-1BBL in murine and human DIPG cell lines. Importantly, 4-1BBL expression in DIPG cell lines was able to activate lymphocytes and increase their IFN-gamma production. In addition, Delta-24-ACT triggered immunogenic cell death in DIPG cell lines, as shown by the release of DAMPs such as ATP, HMGB1, Hsp90a and calreticulin translocation. Delta-24-ACT administration in orthotopic DIPG models was well tolerated and safe. We confirmed the expression of the ligand within the tumor. Moreover, using flow cytometry and multispectral immunohistochemistry we observed profound changes in the tumor microenvironment with an increase in the T-cell populations and a remodeling of the myeloid component before and after treatment. Functional studies showed no differences in lymphocytes isolated from mice treated splenocytes but uncovered TILS that showed a significantly increased in the production of IFN-gamma. Delta-24-ACT treatment of mice bearing orthotopic DIPG murine tumors resulted in a significant increase in the median survival and led to free of disease long-term survivors. In summary, Delta-24-ACT is a virus that builds in our clinical experience with Delta-24-RGD in DIPG patients going a step further to boost the antitumor effect of viral therapy while maintaining a safe profile.
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