Possible misinterpretation of the mode of action of therapeutic antibodies in vitro: homotypic adhesion and flow cytometry result in artefactual direct cell death

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

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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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Combination of the glycoengineered Type II CD20 antibody obinutuzumab (GA101) and The novel Bcl-2 selective Inhibitor GDC-0199 Results in superior In Vitro and In Vivo Anti-tumor activity in models Of B-Cell Malignancies

Blood ◽

10.1182/blood.v122.21.4412.4412 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4412-4412 ◽

Cited By ~ 4

Author(s):

Deepak Sampath ◽

Sylvia Herter ◽

Frank Herting ◽

Ellen Ingalla ◽

Michelle Nannini ◽

...

Keyword(s):

Cell Death ◽

Single Agent ◽

Employment Equity ◽

Direct Cell ◽

Xenograft Models ◽

Equity Ownership ◽

Anti Cd20 ◽

Agent Treatment

Introduction Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody induces a high level of direct cell death. As a result of glycoengineering, GA101 has increased affinity for FcgRIIIa on effector cells resulting in enhanced direct cell death and ADCC induction. GA101 is currently in pivotal clinical trials in CLL, indolent NHL and DLCBL. ABT-199 (GDC-0199) is a novel, orally bioavailable, selective Bcl-2 inhibitor that induces robust apoptosis in preclinical models of hematological malignancies and is currently in clinical trials for CLL, NHL and MM. Based on their complementary mechanisms of action involving increased apoptosis (GDC-0199) or direct cell death (GA101) the combination of anti-CD20 therapy with a Bcl-2 inhibitor has the potential for greater efficacy in treating B lymphoid malignancies. Experimental Methods The combination of GA101 or rituximab with GDC-0199 was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (AxV/Pi positivity) on WSU-DLCL2, SU-DHL4 DLBCL and Z138 MCL cells by FACS and the impact of Bcl-2 inhibition on ADCC induction. In vivo efficacy of the combination of GA101 or rituximab and GDC-0199 was evaluated in SU-DHL4 and Z138 xenograft models. Results GA101 and rituximab enhanced cell death induction when combined with GDC-0199 in SU-DHL4, WSU-DLCL2 and Z138 cell lines. When combined at optimal doses an additive effect of the two drugs was observed. GDC-0199 did not negatively impact the capability of GA101 or rituximab to induce NK-cell mediated ADCC. Combination of GDC-0199 and GA101 induced a greater than additive anti-tumor effects in the SU-DHL4 and Z138 xenograft models resulting in tumor regressions and delay in tumor regrowth when compared to monotherapy. Moreover, continued single-agent treatment with GDC-0199 after combination with GA101 resulted in sustained in vivo efficacy in the SU-DHL4 model. Conclusions Our data demonstrate that the combination of GA101 with GDC-0199 results in enhanced cell death and robust anti-tumor efficacy in xenograft models representing NHL sub-types that is comparable to the combination of rituximab with GDC-0199. In addition, single-agent treatment with GDC-0199 following combination with GA101 sustains efficacy in vivo suggesting a potential benefit in continued maintenance therapy with GDC-0199. Collectively the preclinical data presented here supports clinical investigation of GA101 and GDC-0199 combination therapy, which is currently in a phase Ib clinical trial (clinical trial.gov identifier NCT01685892). Disclosures: Sampath: Genentech: Employment, Equity Ownership. Herter:Roche: Employment. Herting:Roche: Employment. Ingalla:Genentech: Employment. Nannini:Genentech: Employment. Bacac:Roche: Employment. Fairbrother:Genentech: Employment, Equity Ownership. Klein:Roche Glycart AG: Employment.

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Highlighting curcumin-induced crosstalk between autophagy and apoptosis: A biochemical approach coupling impedancemetry, imaging, and flow cytometry

10.1101/827279 ◽

2019 ◽

Author(s):

Francisco José Sala de Oyanguren ◽

Nathan E. Rainey ◽

Aoula Moustapha ◽

Ana Saric ◽

Franck Sureau ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Er Stress ◽

Calcium Release ◽

Curcuma Longa ◽

Cancer Cell Growth ◽

Unfolded Protein ◽

The Status ◽

Type Ii Cell

Curcumin, a major active component of turmeric (Curcuma longa, L.), is known to have various effects on both healthy and cancerous tissues. In vitro studies suggest that curcumin inhibits cancer cell growth by activating apoptosis, but the mechanism underlying the anticancer effects of curcumin is still unclear. Since there is a consensus about endoplasmic reticulum (ER) stress being involved in the cytotoxicity of many natural compounds, we investigated by Amnis®Imaging flow cytometry the mechanistic aspects of curcumin’s destabilization of the ER, but also the status of the lysosomal compartment involved in curcumin-associated apoptosis. Curcumin induces ER stress thereby causing an unfolded protein response (UPR) and calcium release which destabilize the mitochondrial compartment and induce apoptosis. These events are also associated with secondary lysosomal membrane permeabilization and activation of caspase-8, mediated by activation of cathepsins and calpains. We previously showed that sequence lead to the generation of truncated tBid and disruption of mitochondrial homeostasis. These two pathways of different intensities and momentum converge towards an amplification of cell death that still needs to be studied in more detail. It has been suggested that it may be possible to exploit autophagy for cancer therapy. There is a complex interplay involving early autophagy as soon as mitochondria produce superoxide anions and hydrogen peroxide. Treatments with 10 µM to 20 µM curcumin induce autophagosome formation, while only early events of cell death are detectable.In the present study, curcumin-induced autophagy failed to rescue all cells since most cells underwent type II cell death following initial autophagic processes. However, a small number of cells blocked in the cell cycle escaped and were rescued to give rise to a novel proliferation phase.

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FTY720 Has Potent Anti-Leukemic Effects on Acute Lymphoblastic Leukemia Cells and Results In Caspase Independent Cell Death

Blood ◽

10.1182/blood.v116.21.3260.3260 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3260-3260

Author(s):

Craig T. Wallington-Beddoe ◽

John Hewson ◽

Kenneth Francis Bradstock ◽

Linda J. Bendall

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Cell Lines ◽

Western Blotting ◽

Caspase 3 ◽

Lymphoblastic Leukemia ◽

Parp Cleavage ◽

Oxygen Species ◽

Ic50 Values

Abstract Abstract 3260 Introduction: Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, which usually responds to chemotherapy. Long-term survival in adults is poor with most developing disease relapse, whilst Ph+ ALL has a particularly poor prognosis. FTY720 is an immunosuppressive drug that has recently demonstrated efficacy in phase 3 trials of relapsing/remitting multiple sclerosis. FTY720 also appears promising in a number of malignancies with the proposed mechanism being the reactivation of PP2A, a protein serine/threonine phosphatase whose activity may be reduced in malignant cells. Here we report findings of in vitro testing of FTY720 on Ph+ and negative ALL cell lines and primary patient samples, describing mechanisms of cell death. Methods: ALL cell lines and primary patient samples were treated with 1 nM - 100 μM FTY720 for 24 hours. Viability was measured by flow cytometry using propidium iodide and annexin V staining. Cellular proliferation was measured by 3H-thymidine incorporation. Flow cytometry and western blotting were used to measure caspase 3 activation whilst western blotting was used to assess caspase 3, PARP cleavage and LC3II formation. Electron microscopy permitted a detailed examination of cell ultra-structure and confocal microscopy with lysosensor blue staining enabled visualisation of acidic vacuoles. Reactive oxygen species generation was assessed by flow cytometry using the cell permeable dye carboxy-H2DCFDA. Results: FTY720 produced a profound reduction in proliferation and viability of Ph+ (ALL1 cells) and Ph− (REH, NALM6 and LK63 cells) cell lines and patient samples (n=7) in the low micromolar range. IC50 values for loss of viability at 24 hours ranged from 5.3 μM for ALL1 to 7.9 μM for LK63. The IC50 values for proliferation at 24 hours were 1.4 μM for ALL1 and 3.5 μM for REH. Caspase 3 activation was observed only at very low levels by flow cytometry whilst both caspase 3 and PARP cleavage were not detected by western blotting. Inhibition of caspases by ZVAD-FMK failed to rescue ALL cells from FTY720 induced cell death, demonstrating a caspase independent cell death mechanism. Light microscopy revealed prominent cytoplasmic vacuolation, and electron microscopy showed features consistent with autophagy and necrosis. Western blotting demonstrated strong LC3II bands and confocal microscopy, using lysosensor blue, revealed prominent acidic vacuolation, all confirming the induction of autophagy. Reactive oxygen species were generated in response to FTY720 treatment and partial reversal of this by N-acetyl-cysteine produced a concomitant increase in cell viability. PP2A inhibition with okadaic acid failed to rescue cells from FTY720-induced cell death. Conclusion: FTY720 is a highly active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase independent mechanism of cell death with the occurrence of autophagy and necrosis. PP2A activation is not solely responsible for leukemic cell death. Data on the in vivo effects of FTY720 on ALL cells in NOD-SCID mice will be presented. Disclosures: Bendall: Genzyme: Honoraria.

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The Anti-CD20 Monoclonal Antibody GA101 Displays More Robust Anti-Tumor Activity Versus Rituximab In Waldenstrom's Macroglobulinemia (WM)

Blood ◽

10.1182/blood.v116.21.4904.4904 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4904-4904 ◽

Cited By ~ 2

Author(s):

Guang Yang ◽

Christina Hanzis ◽

Sigitas Verselis ◽

Lian Xu ◽

Zachary Hunter ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Death ◽

B Cell ◽

Nk Cells ◽

Healthy Donor ◽

B Cell Depletion ◽

Adcc Activity ◽

Direct Cell ◽

Anti Cd20

Abstract Abstract 4904 Background: Rituximab is an IgG class CD20-directed monoclonal antibody used in the treatment of B-cell malignancies, including WM. We and others have previously demonstrated dependence for IgG class therapeutic antibodies on polymorphisms at FcγRIIIA-158. Approximately half of WM patients express V/V or V/F, and the remainder half express F/F at this polymorphic locus. Patients with WM expressing FcγRIIIA-158 V/V or V/F show improved rituximab single agent activity, as well as attainment of deeper responses (VGPR or CR) with combination Rituximab therapy. GA101 is a novel humanized anti-CD20 antibody with a glyco-engineered Fc domain that exhibits increased Fcg receptor binding and ADCC activity. Methods: In this study, we examined the in vitro activity of GA101 and Rituximab against WM cells, and also examined the activity of these antibodies in context of FcγRIIIA-158 polymorphisms. ADCC activity for GA101 and Rituximab was assessed using genotyped healthy donor derived NK cells against BCWM.1 WM cells, as well autologous NK cells against the patient's own lymphoplasmacytic cells. In vitro B-cell depletion and direct cell death induction assays were also performed. Results: We observed significantly greater ADCC activity against WM cells for GA101 versus Rituximab in both healthy donor, as well as autologous NK cell assays. GA101 mediated ADCC activity was particularly more robust versus Rituximab in patients expressing FcγRIIIA-158 F/F versus V/V or V/F (Figure 1). In addition, GA101 induced significant direct cell death against WM lymphoplasmacytic cells, as well as in vitro B-cell depletion assays in comparison to Rituximab, which exhibited little direct cell death induction activity. Nuclear translocation of apoptosis inducing factor (AIF) was observed following GA101 by immunofluorescence microscopy. Conclusions: GA101 is associated with enhanced ADCC activity relative to Rituximab by NK cells, particularly for those subjects expressing FcγRIIIA-158 F/F. In addition, GA101 demonstrated direct cell death in WM lymphoplasmacytic cells through an AIF mediated caspase-independent pathway. These studies provide the framework for the investigation of GA101 in WM, and suggest particular benefit for those patients who express FcγRIIIA-158 F/F. Disclosures: No relevant conflicts of interest to declare.

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Flow cytometry evaluation of HeLa S3 cell death induced by gamma-radiation

Jugoslovenska medicinska biohemija ◽

10.2298/jmh0402135n ◽

2004 ◽

Vol 23 (2) ◽

pp. 135-142 ◽

Cited By ~ 3

Author(s):

Ana Niciforovic ◽

Bozidarka Zaric ◽

Aleksandra Dakic ◽

Nevena Tisma ◽

Marija Radojcic

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Radiation Dose ◽

Gamma Radiation ◽

Fluorescent Microscopy ◽

Annexin V ◽

Hela S3 ◽

Induced Apoptosis ◽

60Co Gamma Radiation

In this study we followed the effects of radiation on human uterin cervix HeLa S3 cells viability, morphology and DNA structure 2-96 hours after treatment with 2-10 Gy from 60Co gamma radiation source. Staining of cells with Annexin V-FITC and propidium iodide showed very low degree of radiation-induced apoptosis. The prevailing form of HeLa S3 cell death according to flow-cytometry, DNA fragmentation and fluorescent microscopy, was necrosis. The gamma-radiation dose necessary to induce 50% of necrosis (termed DD50) was twice higher compared to dose that induced 50% inhibition of cell proliferation (LD50). These in vitro data suggested, that the increase in radiation dose might eradicate tumor cells, rather than just control their proliferation and growth.

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Characterization of the CD47/SIRP-Alpha System in Patients with Immune Thrombocytopenia.

Blood ◽

10.1182/blood.v114.22.1309.1309 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1309-1309

Author(s):

Lucia Catani ◽

Daria Sollazzo ◽

Francesca Ricci ◽

Nicola Polverelli ◽

Francesca Palandri ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Immune Thrombocytopenia ◽

Conflicts Of Interest ◽

Regulatory Protein ◽

Platelet Apoptosis ◽

Cell Death Pathway ◽

Macrophage Phagocytosis

Abstract Abstract 1309 Poster Board I-331 Introduction The CD47 antigen is a transmembrane glycoprotein ubiquitously expressed on hematopoietic and non-hematopoietic cells. It serves as a ligand for SIRP-alpha (signal regulatory protein-alpha) receptor and as a receptor for Thrombospondin, acting, respectively, as antagonistic to phagocyte activity and as regulator of apoptosis. Based on mouse studies, a novel mechanism of platelet destruction involving the CD47/SIRP-alpha system has been recently proposed in immune thrombocytopenia. This mechanism suggests that platelet homeostasis is regulated by platelet expression of CD47 and that interaction between platelet CD47 and macrophage SIRP-alpha receptor is important in regulating platelet macrophage phagocytosis. However, the role of this system in platelet uptake/phagocytosis by dendritic cells (DCs) has never been investigated in immune thrombocytopenia (ITP) in humans. Therefore, our purpose was to evaluate whether alterations of the CD47/SIRP-alpha system may have a role in the pathogenesis of ITP in humans. Patients and methods Twenty five ITP patients were studied. We phenotypically characterized apoptosis (Annexin-V FITC staining) and the expression of CD47 on platelets and SIRP-alpha on CD14-derived and circulating DCs by flow cytometry. To determine whether platelet apoptosis was due to activation of CD47-cell death pathway, in parallel experiments, we assessed the in vitro sensitivity of platelets to antibody CD47 ligation. In addition, to investigate the role of CD47/SIRP-alpha system on platelet phagocytic capacity of CD14-derived DCs, immature DCs were coincubated with PKH26-labelled platelets in the presence or absence of antibodies against CD47 and SIRP-alpha. The percentage of ingested platelets was then evaluated by flow cytometry. Results We demonstrate that in ITP: 1) CD47 expression is not altered in freshly isolated platelets; 2) after in vitro aging, platelet apoptosis is increased as compared with the normal counterparts; 3) CD47 expression is unchanged in apoptotic platelets; by contrast, it increases in normal platelets; 4) the increased platelet apoptosis is not due to the activation of the CD47-induced cell death pathway; 5) despite low level expression of SIRP-alpha in CD14-derived DCs and in circulating DCs, the CD47/ SIRP-alpha system does not play a central role for in vitro platelet phagocytosis of DCs, since blockage of SIRP-alpha on DCs or CD47 on platelets by specific antibodies failed to modify phagocytosis. Conclusions In conclusion, we demonstrate that in ITP platelet CD47 expression does not play a role in the pathogenesis of the disease. We also show that, in ITP patients, higher platelet apoptosis is not due to different CD47-induced cell death susceptibility. Whether this is due to the fact that CD47 expressed on apoptotic platelets from ITP patients may have a peculiar conformation, avoiding the delivery of cell death signal, remains open question. Furthermore, the platelet uptake/phagocytosis by DCs is not significantly influenced by the CD47/ SIRP-alpha system. Supported in part by BolognaAIL (Italian association against Leukemia, Bologna section) Disclosures No relevant conflicts of interest to declare.

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The Antimalarial Agent Artesunate Overcomes Bortezomib Resistance in Myeloma Cell Lines Through Non-Caspase Mediated Apoptosis

Blood ◽

10.1182/blood.v120.21.4015.4015 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4015-4015

Author(s):

Xenofon Papanikolaou ◽

Ruslana Tytarenko ◽

Tarun K. Garg ◽

Sarah K. Johnson ◽

Erming Tian ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Lines ◽

Mode Of Action ◽

Nuclear Translocation ◽

Proteasome Inhibition ◽

Cross Resistance ◽

Dependent Manner ◽

Caspase Activation ◽

Rpmi 8226

Abstract Abstract 4015 During terminal differentiation of a B-lymphocyte to a mature plasma cell, the protein load increases considerably while the proteasome capacity decreases. The fact that plasma cells operate close to the limit of their proteasomal capacity is being exploited therapeutically and proteasome inhibition has become one of the pillars of myeloma treatment. The importance of the proteasome in Multiple Myeloma (MM) has also been established in prognostic terms (1). De novo or acquired resistance to proteasome inhibitors (PI) confers poor prognosis in MM. In vitro bortezomib resistance is accompanied by cross-resistance to other known PIs, thus highlighting (2), the need for treatment alternatives. Based on reports that various antimalarial drugs exhibit in vitro anti-MM activity through either α-ring proteasome inhibition (mefloquine) or aggresome formation inhibition (quinine), we examined whether Artesunate (ART), a new highly effective antimalarial drug with a known excellent safety profile, could overcome resistance to PI's currently in clinical use. Bortezomib (BZ)-resistant sublines of the BZ naïve and sensitive JJN3 and U266 cell lines were developed by exposure to progressively increasing BZ concentrations. Eventually the JJN3BR and U266BR sublines were developed with a 20- and 25-fold IC50 increase, respectively, compared to their BZ naïve counterparts. ART was able to inhibit viability in clinically achievable dosages and in a time-dependent manner in IL-6-independent and -dependent MM cell lines (Table 1). JJN3BR and U266BR showed no evidence of cross-resistance compared to their parental cell lines. The RPMI 8226 cell line known to exhibit one of the highest BZ IC50s among MM cell lines proved to be among the most sensitive to ART. The in-vitro antiMM effectiveness of ART was evident also in primary CD-138+ MM cells from six (6) patients with Relapsed/Refractory MM (Table 2). ART in-vitro anti MM activity was also verified by Annexin-V and 7-AAD flow cytometry for its ability to induce both early and late apoptosis. To elucidate ART's mode of action, we measured the sequence and level of caspase activation through the luminescence assay Caspase-Glo. ART exposure initiated the extrinsinc pathway of apoptosis with a brisk increase in activated caspase-8 levels and effector caspase3/7 levels within 3h, followed by caspase 9 activation at 6h. However, actual apoptosis by ART was not accompanied by a marked caspase activity especially when compared to other potent anti-MM agents. The pan-caspase inhibitor Z-VAD-MFK effectively inhibited caspase activation by ART but failed to inhibit ART's effect on viability, as at least 70% of ART's effect was retained in JJN3, U266 and RPMI 8226 cells. Similar results were obtained in flow cytometry-based apoptosis assays. Immunoblotting of the mitochondrial derived non-caspase mediating apoptosis factors AIF, EndoG and HtrA2 in ART treated JJN3 cells revealed that ART-mediated apoptosis was related to the cytoplasmic and subsequently nuclear translocation of AIF. Conclusion: ART shows marked anti-MM activity and overcomes BZ resistance in vitro. Its benign side effect profile support its potential for myeloma therapy. Most anti-cancer agents induce apoptosis in a caspase-dependent manner. ART's mode of action is mainly caspase-independent, indicating that its role in overcoming drug resistance, could also extend to other anti-MM agents. Experiments aimed at further elucidating the mechanism of action of ART and the evaluation of drug combinations including ART are currently under way. Disclosures: Tian: University of Arkansas for Medical Sciences: Employment, under pending process, under pending process Patents & Royalties.

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Cell death induced by cytotoxic CD8+T cells is immunogenic and primes caspase-3–dependent spread immunity against endogenous tumor antigens

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-000528 ◽

2020 ◽

Vol 8 (1) ◽

pp. e000528 ◽

Cited By ~ 6

Author(s):

Paula Jaime-Sanchez ◽

Iratxe Uranga-Murillo ◽

Nacho Aguilo ◽

Sofia C Khouili ◽

Maykel A Arias ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

T Cell ◽

Cancer Cells ◽

Caspase 3 ◽

Tumor Antigens ◽

Tumor Development ◽

Dominant Negative Mutant

BackgroundElimination of cancer cells by some stimuli like chemotherapy and radiotherapy activates anticancer immunity after the generation of damage‐associated molecular patterns, a process recently named immunogenic cell death (ICD). Despite the recent advances in cancer immunotherapy, very little is known about the immunological consequences of cell death activated by cytotoxic CD8+T (Tc) cells on cancer cells, that is, if Tc cells induce ICD on cancer cells and the molecular mechanisms involved.MethodsICD induced by Tc cells on EL4 cells was analyzed in tumor by vaccinating mice with EL4 cells killedin vitroorin vivoby Ag-specific Tc cells. EL4 cells and mutants thereof overexpressing Bcl-XLor a dominant negative mutant of caspase-3 and wild-type mice, as well as mice depleted of Tc cells and mice deficient in perforin, TLR4 and BATF3 were used.Ex vivocytotoxicity of spleen cells from immunized mice was analyzed by flow cytometry. Expression of ICD signals (calreticulin, HMGB1 and interleukin (IL)-1β) was analyzed by flow cytometry and ELISA.ResultsMice immunized with EL4.gp33 cells killed in vitro or in vivo by gp33-specific Tc cells were protected from parental EL4 tumor development. This result was confirmed in vivo by using ovalbumin (OVA) as another surrogate antigen. Perforin and TLR4 and BATF3-dependent type 1 conventional dendritic cells (cDC1s) were required for protection against tumor development, indicating cross-priming of Tc cells against endogenous EL4 tumor antigens. Tc cells induced ICD signals in EL4 cells. Notably, ICD of EL4 cells was dependent on caspase-3 activity, with reduced antitumor immunity generated by caspase-3–deficient EL4 cells. In contrast, overexpression of Bcl-XLin EL4 cells had no effect on induction of Tc cell antitumor response and protection.ConclusionsElimination of tumor cells by Ag-specific Tc cells is immunogenic and protects against tumor development by generating new Tc cells against EL4 endogenous antigens. This finding helps to explain the enhanced efficacy of T cell-dependent immunotherapy and provide a molecular basis to explain the epitope spread phenomenon observed during vaccination and chimeric antigen receptor (CAR)-T cell therapy. In addition, they suggest that caspase-3 activity in the tumor may be used as a biomarker to predict cancer recurrence during T cell-dependent immunotherapies.

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