Therapeutic Evaluation of Palbociclib and Its Compatibility with Other Chemotherapies in Primary and Recurrent Nasopharyngeal Carcinoma

Abstract Background: Recent genomic analyses revealed that druggable molecule targets could only be detected in around 6% of nasopharyngeal carcinoma (NPC) patients. Yet, an addiction to dysregulated CDK4/6-cyclinD1 signalling pathway is an essential event in the pathogenesis of NPC. Using our newly established xenografts and cell lines derived from primary, recurrent and metastatic NPC, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemodrugs in treating NPC.Methods: The efficacy of single treatment of palbociclib on NPC models was first evaluated, followed by concurrent treatment with cisplatin or suberanilohydroxamic acid (SAHA). RNA sequencing was used to profile the related pathways in governing the drug response. Palbociclib-resistant NPC cell lines were also established to demonstrate if cisplatin could be used as a second-line treatment once the cells developed resistance to palbociclib. The efficacy of palbociclib treatment on cisplatin-resistant NPC cells was also examined. Results: Palbociclib single drug treatment was confirmed to have a cell cycle arresting effect of NPC cells in G1 phase in vitro. It also had a significant inhibitory effect in all the 6 NPC tumor models in vivo, with a substantial reduction in total tumor volume and proliferation marker Ki-67. Concurrent use of palbociclib dampened the cytotoxic effect of cisplatin in NPC cells in vitro. Notably, combination of palbociclib with SAHA resulted in synergistic cell death of NPC both in vitro and in vivo. Autophagy-associated cell death was found to be involved in the enhanced tumor growth inhibitory effect in the combined palbociclib+SAHA treatment. NPC cell lines trained to sustain growth in high dose of palbociclib and cisplatin remained sensitive in subsequent treatment of cisplatin or palbociclib respectively.Conclusions: This study provides essential evidences to position palbociclib as an alternative therapeutic option to NPC treatment, and to aware the effective administrative timing of palbociclib with other chemodrugs. The findings give the basis for planning of the first-in-human clinical trials of palbociclib regimens in NPC patients.

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Therapeutic evaluation of palbociclib and its compatibility with other chemotherapies for primary and recurrent nasopharyngeal carcinoma

10.21203/rs.3.rs-36929/v2 ◽

2020 ◽

Author(s):

zhichao xue ◽

Vivian Wai Yan Lui ◽

Yongshu Li ◽

Jia Lin ◽

Chanping You ◽

...

Keyword(s):

Cell Death ◽

Nasopharyngeal Carcinoma ◽

Cell Lines ◽

Chemotherapeutic Drugs ◽

Induce Cell Cycle Arrest ◽

Ki 67 ◽

Concurrent Treatment ◽

Xenograft Models

Abstract Background: Recent genomic analyses revealed that druggable molecule targets were detectable in approximately 6% of patients with nasopharyngeal carcinoma (NPC). However, a dependency on dysregulated CDK4/6–cyclinD1 pathway signaling is an essential event in the pathogenesis of NPC. In this study, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemotherapeutic drugs for the treatment of NPC by using newly established xenograft models and cell lines derived from primary, recurrent, and metastatic NPC. Methods: We evaluated the efficacies of palbociclib monotherapy and concurrent treatment with palbociclib and cisplatin or suberanilohydroxamic acid (SAHA) in NPC cell lines and xenograft models. RNA sequencing was then used to profile the drug response–related pathways. Palbociclib-resistant NPC cell lines were established to determine the potential use of cisplatin as a second-line treatment after the development of palbociclib resistance. We further examined the efficacy of palbociclib treatment against cisplatin-resistant NPC cells. Results: In NPC cells, palbociclib monotherapy was confirmed to induce cell cycle arrest in the G1 phase in vitro . Palbociclib monotherapy also had significant inhibitory effects in all six tested NPC tumor models in vivo , as indicated by substantial reductions in the total tumor volumes and in Ki-67 proliferation marker expression. In NPC cells, concurrent palbociclib treatment mitigated the cytotoxic effect of cisplatin in vitro . Notably, concurrent treatment with palbociclib and SAHA synergistically promoted NPC cell death both in vitro and in vivo . This combination also further inhibited tumor growth by inducing autophagy-associated cell death. NPC cell lines with induced palbociclib or cisplatin resistance remained sensitive to treatment with cisplatin or palbociclib, respectively. Conclusions: Our study findings provide essential support for the use of palbociclib as an alternative therapy for NPC and increase awareness of the effective timing of palbociclib administration with other chemotherapeutic drugs. Our results provide a foundation for the design of first-in-human clinical trials of palbociclib regimens in patients with NPC.

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Therapeutic evaluation of palbociclib and its compatibility with other chemotherapies for primary and recurrent nasopharyngeal carcinoma

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01763-z ◽

2020 ◽

Vol 39 (1) ◽

Author(s):

Zhichao Xue ◽

Vivian Wai Yan Lui ◽

Yongshu Li ◽

Lin Jia ◽

Chanping You ◽

...

Keyword(s):

Cell Death ◽

Nasopharyngeal Carcinoma ◽

Cell Lines ◽

Chemotherapeutic Drugs ◽

Induce Cell Cycle Arrest ◽

Ki 67 ◽

Concurrent Treatment ◽

Xenograft Models

Abstract Background Recent genomic analyses revealed that druggable molecule targets were only detectable in approximately 6% of patients with nasopharyngeal carcinoma (NPC). However, a dependency on dysregulated CDK4/6–cyclinD1 pathway signaling is an essential event in the pathogenesis of NPC. In this study, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemotherapeutic drugs for the treatment of NPC by using newly established xenograft models and cell lines derived from primary, recurrent, and metastatic NPC. Methods We evaluated the efficacies of palbociclib monotherapy and concurrent treatment with palbociclib and cisplatin or suberanilohydroxamic acid (SAHA) in NPC cell lines and xenograft models. RNA sequencing was then used to profile the drug response–related pathways. Palbociclib-resistant NPC cell lines were established to determine the potential use of cisplatin as a second-line treatment after the development of palbociclib resistance. We further examined the efficacy of palbociclib treatment against cisplatin-resistant NPC cells. Results In NPC cells, palbociclib monotherapy was confirmed to induce cell cycle arrest in the G1 phase in vitro. Palbociclib monotherapy also had significant inhibitory effects in all six tested NPC tumor models in vivo, as indicated by substantial reductions in the total tumor volumes and in Ki-67 proliferation marker expression. In NPC cells, concurrent palbociclib treatment mitigated the cytotoxic effect of cisplatin in vitro. Notably, concurrent treatment with palbociclib and SAHA synergistically promoted NPC cell death both in vitro and in vivo. This combination also further inhibited tumor growth by inducing autophagy-associated cell death. NPC cell lines with induced palbociclib or cisplatin resistance remained sensitive to treatment with cisplatin or palbociclib, respectively. Conclusions Our study findings provide essential support for the use of palbociclib as an alternative therapy for NPC and increase awareness of the effective timing of palbociclib administration with other chemotherapeutic drugs. Our results provide a foundation for the design of first-in-human clinical trials of palbociclib regimens in patients with NPC.

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The Histone Deacetylase Inhibitor Panobinostat (LBH-589) Exerts Anti-Leukaemic Activity in a MLL-Rearranged ALL Xenograft Mouse Model

Blood ◽

10.1182/blood.v124.21.3709.3709 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3709-3709

Author(s):

Patricia Garrido Castro ◽

Eddy HJ Van Roon ◽

Sandra S Mimoso Pinhancos ◽

Pauline Schneider ◽

Mark JB Kerstjens ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Histone Modification ◽

Histone Deacetylase ◽

Cell Lines ◽

Disease Onset ◽

High Dose ◽

Nsg Mice

Abstract BACKGROUND: Infant acute lymphoblastic leukaemia (ALL) is a rare but aggressive malignancy, mainly presenting with chromosomal rearrangements of the MLL (Mixed Lineage Leukaemia) gene locus on 11q23. The majority of these MLL rearrangements involve the translocation partners AF4, AF9 or ENL within the translocation events t(4;11)(q21;q23), t(9;11)(p22;q23) and t(11;19)(q23;p13.3), respectively. The resulting fusion genes, MLL-AF4, MLL-AF9 and MLL-ENL, code for chimeric transcription regulators acting as strong oncogenic drivers, rewriting the epigenetic landscape of the cell and profoundly altering gene expression. Consequently, these cytogenetic lesions define an ALL subtype both biologically and clinically distinct from other subtypes, strongly associated with drug resistance to first-line chemotherapeutics, high relapse rates and a dismal prognosis. Hence, novel treatment strategies which specifically target the underlying molecular pathobiology of this disease are urgently needed. AIMS: Previously, our group performed extensive patient cohort profiling on both transcript and epigenetic level in order to understand the molecular events underlying the disease, and identified histone deacetylase inhibitors (HDACi) as effective therapeutic drugs both in silico and in vitro. The aim of the current study was to elucidate potential molecular mechanisms by which the candidate HDACi Panobinostat is able to target MLL-rearranged ALL (MLLr-ALL) cells, and to confirm its efficacy in vivo using pre-clinical MLLr-ALL xenograft mouse models able to recapitulate the disease phenotype observed in humans. METHODS: Immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were injected intrafemurally with a MLL-AF4+ B-ALL cell line (SEM) genetically modified to express a luciferase reporter. These mice were subsequently either treated with low-dose (1mg/kg) or high-dose (5mg/kg) Panobinostat using a continuous 5-day-on-2-day-off regimen for a period of up to 12 weeks, or they were assigned to a control group and left untreated. Disease onset and progression was monitored using in vivo bioluminescence imaging, and systemic human ALL cell infiltration was determined by multi-colour flow cytometry and histochemistry. In addition, molecular changes induced by Panobinostat exposure in MLLr-ALL and non-MLLr-ALL cell lines were assessed in vitro using immunoblotting and cell death assays. RESULTS: High-dose Panobinostat resulted in a significantly and substantially delayed MLLr-ALL disease onset and progression in NSG mice when compared to controls; this was accompanied by a reduction of the systemic disease burden, as evidenced by significantly lower whole-body luminescence signals and substantially decreased splenomegaly. Furthermore, immunohistochemical and flow cytometric data showed hypocellularity and increased cell death in the BM of xenografted NSG mice treated with Panobinostat when compared to untreated control xenografts. This finding correlated well with in vitro results, where exposure with 5 nM Panobinostat induced cell death in MLLr-ALL cells, but not in non-MLLr ALL cells, as determined by both ANNEXINV/7AAD flow cytometry assays and immunoblotting. In addition, on a molecular level, in vitro exposure with Panobinostat induced histone H3 hyperacetylation in all leukaemic cell lines, but did not affect other histone modification marks investigated such as, i.e., histone H3K4 methylation or histone H3K79 methylation. A notable exception was observed in MLLr-ALL cell lines, where Panobinostat exposure correlated with a reduction in histone H2B ubiquitination, a histone modification recently reported to be pivotal for MLLr leukaemogenesis. Concomitantly, Panobinostat - or more generally - HDACi-mediated loss of H2B ubiquitination might play a role in the observed sensitivity of MLLr-ALL cell towards this drug class. CONCLUSIONS: Both the in vivo and the molecular in vitro results show the HDACi Panobinostat to have promising therapeutic potential against MLLr-ALL. Currently, we are investigating Panobinostat in combination with other epigenetic drugs in xenograft models with primary MLLr-ALL patient material in order to consolidate these observations, and to confirm HDACi as a novel powerful treatment strategy in MLLr-ALL. Disclosures No relevant conflicts of interest to declare.

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LncRNA SNHG15 contributes to doxorubicin resistance of osteosarcoma cells through targeting the miR-381-3p/GFRA1 axis

Open Life Sciences ◽

10.1515/biol-2020-0086 ◽

2020 ◽

Vol 15 (1) ◽

pp. 871-883

Author(s):

Jinshan Zhang ◽

Dan Rao ◽

Haibo Ma ◽

Defeng Kong ◽

Xiaoming Xu ◽

...

Keyword(s):

Cell Lines ◽

Noncoding Rna ◽

Xenograft Model ◽

Inhibitory Effect ◽

Cell Counting ◽

Luciferase Reporter ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells

AbstractBackgroundOsteosarcoma is a common primary malignant bone cancer. Long noncoding RNA small nucleolar RNA host gene 15 (SNHG15) has been reported to play an oncogenic role in many cancers. Nevertheless, the role of SNHG15 in the doxorubicin (DXR) resistance of osteosarcoma cells has not been fully addressed.MethodsCell Counting Kit-8 assay was conducted to measure the half-maximal inhibitory concentration value of DXR in osteosarcoma cells. Western blotting was carried out to examine the levels of autophagy-related proteins and GDNF family receptor alpha-1 (GFRA1). Quantitative reverse transcription-polymerase chain reaction was performed to determine the levels of SNHG15, miR-381-3p, and GFRA1. The proliferation of osteosarcoma cells was measured by MTT assay. The binding sites between miR-381-3p and SNHG15 or GFRA1 were predicted by Starbase bioinformatics software, and the interaction was confirmed by dual-luciferase reporter assay. Murine xenograft model was established to validate the function of SNHG15 in vivo.ResultsAutophagy inhibitor 3-methyladenine sensitized DXR-resistant osteosarcoma cell lines to DXR. SNHG15 was upregulated in DXR-resistant osteosarcoma tissues and cell lines. SNHG15 knockdown inhibited the proliferation, DXR resistance, and autophagy of osteosarcoma cells. MiR-381-3p was a direct target of SNHG15, and GFRA1 bound to miR-381-3p in osteosarcoma cells. SNHG15 contributed to DXR resistance through the miR-381-3p/GFRA1 axis in vitro. SNHG15 depletion contributed to the inhibitory effect of DXR on osteosarcoma tumor growth through the miR-381-3p/GFRA1 axis in vivo.ConclusionsSNHG15 enhanced the DXR resistance of osteosarcoma cells through elevating the autophagy via targeting the miR-381-3p/GFRA1 axis. Restoration of miR-381-3p expression might be an underlying therapeutic strategy to overcome the DXR resistance of osteosarcoma.

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TERT Promoter Revertant Mutation Inhibits Melanoma Growth through Intrinsic Apoptosis

Biology ◽

10.3390/biology11010141 ◽

2022 ◽

Vol 11 (1) ◽

pp. 141

Author(s):

Yanbing Wang ◽

Yiwu Chen ◽

Chang Li ◽

Zhiwei Xiao ◽

Hongming Yuan ◽

...

Keyword(s):

Therapeutic Option ◽

B Cell Lymphoma ◽

Inhibitory Effect ◽

Migration And Invasion ◽

Catalytic Core ◽

Tert Promoter ◽

Human Telomerase ◽

Mutant Cells

Human telomerase is a specialized DNA polymerase whose catalytic core includes both TERT and human telomerase RNA (hTR). Telomerase in humans, which is silent in most somatic cells, is activated to maintain the telomere length (TEL) in various types of cancer cells, including melanoma. In the vast majority of tumor cells, the TERT promoter is mutated to promote proliferation and inhibit apoptosis. Here, we exploited NG-ABEmax to revert TERT -146 T to -146 C in melanoma, and successfully obtained TERT promoter revertant mutant cells. These TERT revertant mutant cells exhibited significant growth inhibition both in vitro and in vivo. Moreover, A375−146C/C cells exhibited telomere shortening and the downregulation of TERT at both the transcription and protein levels, and migration and invasion were inhibited. In addition, TERT promoter revertant mutation abrogated the inhibitory effect of mutant TERT on apoptosis via B-cell lymphoma 2 (Blc-2), ultimately leading to cell death. Collectively, the results of our work demonstrate that reverting mutations in the TERT promoter is a potential therapeutic option for melanoma.

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Anticancer Activity of Cynomorium coccineum

Cancers ◽

10.3390/cancers10100354 ◽

2018 ◽

Vol 10 (10) ◽

pp. 354 ◽

Cited By ~ 7

Author(s):

Mouna Sdiri ◽

Xiangmin Li ◽

William Du ◽

Safia El-Bok ◽

Yi-Zhen Xie ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Anticancer Activity ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Cell Cycle Progression ◽

Cycle Progression

The extensive applications of Cynomorium species and their rich bioactive secondary metabolites have inspired many pharmacological investigations. Previous research has been conducted to examine the biological activities and numerous interesting pharmaceutical activities have been reported. However, the antitumor activities of these species are unclear. To understand the potential anticancer activity, we screened Cynomorium coccineum and Cynomorium songaricum using three different extracts of each species. In this study, the selected extracts were evaluated for their ability to decrease survival rates of five different cancer cell lines. We compared the cytotoxicity of the three different extracts to the anticancer drug vinblastine and one of the most well-known medicinal mushrooms Amaurederma rude. We found that the water and alcohol extracts of C. coccineum at the very low concentrations possessed very high capacity in decreasing the cancer cells viability with a potential inhibition of tumorigenesis. Based on these primitive data, we subsequently tested the ethanol and the water extracts of C. coccineum, respectively in in vitro and in vivo assays. Cell cycle progression and induction of programmed cell death were investigated at both biological and molecular levels to understand the mechanism of the antitumor inhibitory action of the C. coccineum. The in vitro experiments showed that the treated cancer cells formed fewer and smaller colonies than the untreated cells. Cell cycle progression was inhibited, and the ethanol extract of C. coccineum at a low concentration induced accumulation of cells in the G1 phase. We also found that the C. coccineum’s extracts suppressed viability of two murine cancer cell lines. In the in vivo experiments, we injected mice with murine cancer cell line B16, followed by peritoneal injection of the water extract. The treatment prolonged mouse survival significantly. The tumors grew at a slower rate than the control. Down-regulation of c-myc expression appeared to be associated with these effects. Further investigation showed that treatment with C. coccineum induced the overexpression of the tumor suppressor Foxo3 and other molecules involved in inducing autophagy. These results showed that the C. coccineum extract exerts its antiproliferative activity through the induction of cell death pathway. Thus, the Cynomorium plants appear to be a promising source of new antineoplastic compounds.

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IMMU-14. ONCOLYTIC VIRUS EXPRESSING A POSITIVE IMMUNE CHECKPOINT MODULATOR AS A THERAPEUTIC APPROACH FOR DIPG

Neuro-Oncology ◽

10.1093/neuonc/noz175.508 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi122-vi122

Author(s):

Virginia Laspidea ◽

Montse Puigdelloses ◽

Ignacio Iñigo-Marco ◽

Marc Garcia-Moure ◽

Iker Ausejo ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Cell Death ◽

Cell Lines ◽

Oncolytic Virus ◽

Murine Models ◽

Antitumoral Effect ◽

Infected Cells

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.

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Iron Causes Lipid Oxidation and Inhibits Proteasome Function in Multiple Myeloma Cells: A Proof of Concept for Novel Combination Therapies

Cancers ◽

10.3390/cancers12040970 ◽

2020 ◽

Vol 12 (4) ◽

pp. 970 ◽

Cited By ~ 3

Author(s):

Jessica Bordini ◽

Federica Morisi ◽

Fulvia Cerruti ◽

Paolo Cascio ◽

Clara Camaschella ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Iron Toxicity ◽

Ammonium Citrate ◽

Ferric Ammonium Citrate ◽

Ferric Carboxymaltose ◽

High Dose ◽

Iron Loading

Adaptation to import iron for proliferation makes cancer cells potentially sensitive to iron toxicity. Iron loading impairs multiple myeloma (MM) cell proliferation and increases the efficacy of the proteasome inhibitor bortezomib. Here, we defined the mechanisms of iron toxicity in MM.1S, U266, H929, and OPM-2 MM cell lines, and validated this strategy in preclinical studies using Vk*MYC mice as MM model. High-dose ferric ammonium citrate triggered cell death in all cell lines tested, increasing malondialdehyde levels, the by-product of lipid peroxidation and index of ferroptosis. In addition, iron exposure caused dose-dependent accumulation of polyubiquitinated proteins in highly iron-sensitive MM.1S and H929 cells, suggesting that proteasome workload contributes to iron sensitivity. Accordingly, high iron concentrations inhibited the proteasomal chymotrypsin-like activity of 26S particles and of MM cellular extracts in vitro. In all MM cells, bortezomib-iron combination induced persistent lipid damage, exacerbated bortezomib-induced polyubiquitinated proteins accumulation, and triggered cell death more efficiently than individual treatments. In Vk*MYC mice, addition of iron dextran or ferric carboxymaltose to the bortezomib-melphalan-prednisone (VMP) regimen increased the therapeutic response and prolonged remission without causing evident toxicity. We conclude that iron loading interferes both with redox and protein homeostasis, a property that can be exploited to design novel combination strategies including iron supplementation, to increase the efficacy of current MM therapies.

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P06.01 Delta24-ACT oncolytic adenovirus as a therapeutic approach for DIPG

Neuro-Oncology ◽

10.1093/neuonc/noz126.123 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii36-iii36

Author(s):

V Laspidea ◽

M Puigdelloses ◽

M García-Moure ◽

I Iñigo-Marco ◽

J Gallego ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Cell Lines ◽

In Vivo Studies ◽

Immunogenic Cell Death ◽

Murine Models ◽

Antitumoral Effect ◽

Infected Cells

Abstract BACKGROUND 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. MATERIALS AND METHODS We use the NP53 and XFM murine DIPG cell lines. Flow cytometry was used to assess cell infectivity and ligand expression. We analyzed viral replication using a method based in hexon detection, and viral cytotoxic effect using the MTS assay. For immunogenic cell death analysis, we measured ATP secretion by a luminometric assay and calreticulin location by flow cytometry and immunofluorescence. For in vivo studies, cells and virus were injected in the pons of the mice, using the screw-guided system. RESULTS 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. CONCLUSIONS 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.

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