Differential Cytopathology and Kinetics of Measles Oncolysis in Two Primary B-cell Malignancies Provides Mechanistic Insights
Authors: Bella Patel, Aditi Dey, Ehsan Ghorani, Shaji Kumar, Yogeshkumar Malam, Lena Rai, Andrew J Steele, Jennifer Thomson, R Gitendra Wickremasinghe, Yu Zhang, Anna Z Castleton, Adele K Fielding
Journal: Molecular Therapy (2011)

Clinical trials using vaccine measles virus (MV) as anticancer therapy are already underway. We compared the oncolytic potential of MV in two B-cell malignancies; adult acute lymphoblastic leukemia (ALL, an aggressive leukemia) and chronic lymphocytic leukemia (CLL, an indolent leukemia overexpressing Bcl-2) using patient-derived material. In vitro, distinct cytopathological effects were observed between MV-infected primary ALL and CLL cells, with large multinucleated syncytia forming in ALL cultures compared to minimal cell-to-cell fusion in infected CLL cells. Cell viability and immunoblotting studies confirmed rapid cell death in MV-infected ALL cultures and slower MV oncolysis of CLL cells. In cell lines, overexpression of Bcl-2 diminished MV-induced cell death providing a possible mechanism for the slower kinetic of MV oncolysis in CLL. In vivo, intratumoral MV treatment of established subcutaneous ALL xenografts had striking antitumor activity leading to complete resolution of all tumors. The antitumor activity of MV was also evident in disseminated ALL xenograft models. In summary, both ALL and CLL are targets for MV-mediated lysis albeit with different kinetics. The marked sensitivity of both primary ALL cells and ALL xenografts to MV oncolysis highlights the tremendous potential of MV as a novel replicating-virus therapy for adult ALL. Clinical trials using vaccine measles virus (MV) as anticancer therapy are already underway. We compared the oncolytic potential of MV in two B-cell malignancies; adult acute lymphoblastic leukemia (ALL, an aggressive leukemia) and chronic lymphocytic leukemia (CLL, an indolent leukemia overexpressing Bcl-2) using patient-derived material. In vitro, distinct cytopathological effects were observed between MV-infected primary ALL and CLL cells, with large multinucleated syncytia forming in ALL cultures compared to minimal cell-to-cell fusion in infected CLL cells. Cell viability and immunoblotting studies confirmed rapid cell death in MV-infected ALL cultures and slower MV oncolysis of CLL cells. In cell lines, overexpression of Bcl-2 diminished MV-induced cell death providing a possible mechanism for the slower kinetic of MV oncolysis in CLL. In vivo, intratumoral MV treatment of established subcutaneous ALL xenografts had striking antitumor activity leading to complete resolution of all tumors. The antitumor activity of MV was also evident in disseminated ALL xenograft models. In summary, both ALL and CLL are targets for MV-mediated lysis albeit with different kinetics. The marked sensitivity of both primary ALL cells and ALL xenografts to MV oncolysis highlights the tremendous potential of MV as a novel replicating-virus therapy for adult ALL. IntroductionViruses that conditionally replicate and lyse transformed cells are promising agents for cancer therapy. Live attenuated measles virus (MV) derived from the Edmonston-B vaccine strain is particularly attractive as an oncolytic agent for human tumors.1Fielding AK Measles as a potential oncolytic virus.Rev Med Virol. 2005; 15: 135-142Crossref PubMed Scopus (34) Google Scholar A long safety record derived from four decades of application as a vaccine against measles disease together with a viral genome that is readily engineered are attributes that favor its use for virotherapy. The clinical relevance of MV as a cancer therapeutic is further strengthened by a phase 1 trial which reported no dose limiting toxicities with this approach.2Galanis E Hartmann LC Cliby WA Long HJ Peethambaram PP Barrette BA et al.Phase I trial of intraperitoneal administration of an oncolytic measles virus strain engineered to express carcinoembryonic antigen for recurrent ovarian cancer.Cancer Res. 2010; 70: 875-882Crossref PubMed Scopus (217) Google ScholarThe vaccine strain of MV is a negative strand RNA virus which enters cells predominantly through the CD46 receptor3Yanagi Y The cellular receptor for measles virus–elusive no more.Rev Med Virol. 2001; 11: 149-156Crossref PubMed Scopus (19) Google Scholar and has been previously shown to have specific cytotoxic activity in transformed cells from several tumor types.4Peng KW TenEyck CJ Galanis E Kalli KR Hartmann LC Russell SJ Intraperitoneal therapy of ovarian cancer using an engineered measles virus.Cancer Res. 2002; 62: 4656-4662PubMed Google Scholar,5Phuong LK Allen C Peng KW Giannini C Greiner S TenEyck CJ et al.Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblastoma multiforme.Cancer Res. 2003; 63: 2462-2469PubMed Google Scholar,6Msaouel P Iankov ID Allen C Morris JC von Messling V Cattaneo R et al.Engineered measles virus as a novel oncolytic therapy against prostate cancer.Prostate. 2009; 69: 82-91Crossref PubMed Scopus (79) Google Scholar The most susceptible target cells are arguably B-cell malignancies.7Peng KW Ahmann GJ Pham L Greipp PR Cattaneo R Russell SJ Systemic therapy of myeloma xenografts by an attenuated measles virus.Blood. 2001; 98: 2002-2007Crossref PubMed Scopus (159) Google Scholar,8Grote D Russell SJ Cornu TI Cattaneo R Vile R Poland GA et al.Live attenuated measles virus induces regression of human lymphoma xenografts in immunodeficient mice.Blood. 2001; 97: 3746-3754Crossref PubMed Scopus (212) Google Scholar By contrast minimal cytopathic effects are seen in MV-infected normal peripheral blood lymphocytes.7Peng KW Ahmann GJ Pham L Greipp PR Cattaneo R Russell SJ Systemic therapy of myeloma xenografts by an attenuated measles virus.Blood. 2001; 98: 2002-2007Crossref PubMed Scopus (159) Google Scholar The efficiency of MV entry into neoplastic B-lymphocytes (targets relatively refractory to transduction by conventional vectors) is further highlighted by reports of high transduction rates in leukemic B-cells by lentiviral vectors pseudotyped with MV envelope proteins.9Frecha C Costa C Lévy C Nègre D Russell SJ Maisner A et al.Efficient and stable transduction of resting B lymphocytes and primary chronic lymphocyte leukemia cells using measles virus gp displaying lentiviral vectors.Blood. 2009; 114: 3173-3180Crossref PubMed Scopus (70) Google ScholarThe mode of action of MV oncolysis is not clear. CD46 overexpression on tumor cells is implicated in the selective lysis of malignant cells.10Anderson BD Nakamura T Russell SJ Peng KW High CD46 receptor density determines preferential killing of tumor cells by oncolytic measles virus.Cancer Res. 2004; 64: 4919-4926Crossref PubMed Scopus (245) Google Scholar In respect of how MV kills tumor cells, expression of the MV envelope glycoproteins, hemaglutinin (MV-H) and fusion (MV-F) in infected cells results in considerable cell-to-cell fusion which mediates a potent cytopathic effect (CPE) in vitro. By contrast, syncytia are rarely observed in excised MV-sensitive human tumor xenografts, hence there is debate over the necessity for multinucleated cell formation as a prelude to MV-induced death.8Grote D Russell SJ Cornu TI Cattaneo R Vile R Poland GA et al.Live attenuated measles virus induces regression of human lymphoma xenografts in immunodeficient mice.Blood. 2001; 97: 3746-3754Crossref PubMed Scopus (212) Google Scholar,11Grote D Cattaneo R Fielding AK Neutrophils contribute to the measles virus-induced antitumor effect: enhancement by granulocyte macrophage colony-stimulating factor expression.Cancer Res. 2003; 63: 6463-6468PubMed Google Scholar Furthermore, most work has been carried out using cell lines, which may not be broadly representative of MV infection response in primary malignancies.In this study, we have investigated the utility of MV oncolysis in two different B-cell malignancies, in both of which the need for novel agents is pressing.Outcome for adults with acute lymphoblastic leukemia (ALL), a rapidly progressive acute malignancy—is poor with <40% of patients surviving long term.12Goldstone AH Richards SM Lazarus HM Tallman MS Buck G Fielding AK et al.In adults with standard-risk acute lymphoblastic leukemia, the greatest benefit is achieved from a matched sibling allogeneic transplantation in first complete remission, and an autologous transplantation is less effective than conventional consolidation/maintenance chemotherapy in all patients: final results of the International ALL Trial (MRC UKALL XII/ECOG E2993).Blood. 2008; 111: 1827-1833Crossref PubMed Scopus (573) Google Scholar Chronic lymphocytic leukemia (CLL), although a much more indolent disease, is generally considered incurable.13Chiorazzi N Rai KR Ferrarini M Chronic lymphocytic leukemia.N Engl J Med. 2005; 352: 804-815Crossref PubMed Scopus (1313) Google Scholar To get the most realistic insights possible, we aimed to use primary patient material for as much of our study as was feasible. In particular, we wished to determine the possible differences in the outcome of MV infection in two B-cell malignancies with very different clinical behaviors. CLL is a malignancy which overexpresses Bcl-2, a protein which confers a block to apoptosis. The key feature of CLL is the accumulation of mature and relatively indolent clonal B-cells whereas in ALL the malignant cells are rapidly proliferative cells derived from stem cells or cells early in the lymphoid differentiation pathway.14Swerdlow SH Campo E Harris NL Jaffe ES Pileri SA Stein H et al.WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC, Lyon, France2008Google Scholar We were interested in determining the relationship between cell killing, MV replication, protein expression, and syncytia formation in these two distinct B-cell malignancies, with the overall aims of gaining mechanistic insight into mechanisms of oncolysis and of determining in which B-cell leukemia to best focus future efforts at translating MV oncolysis into the clinic.ResultsMV-induced cytopathology is distinct in primary ALL and CLLTo confirm susceptibility of primary ALL (n = 6) and CLL (n = 5) cells to MV infection, we demonstrated cell surface expression of CD46 by fluorescence-activated cell sorting (data not shown). We infected primary ALL (n = 5) and CLL (n = 3) cells with MV expressing green fluorescent protein (MV-GFP) at a multiplicity of infection of 1.0 and compared CPE of the virus in culture. Photomicrographs of representative experiments are shown in Figure 1. Primary ALL specimens typically showed characteristic MV-induced cytopathic changes—including multinucleated syncytium formation which began very early postinfection and peaked at day 3, with rapid death thereafter. By contrast, the MV-GFP infected CLL cultures showed little or no evidence of cell-to-cell fusion even by day 6 postinfection. Despite a relative lack of syncytia in infected CLL cultures, cytopathic effects did eventually become apparent although only at later stages of infection when the vast majority of cells exhibited cell shrinkage and surface membrane contraction compared to mostly normal cytomorphological appearances in uninfected cultures at this time.To compare the extent of MV-induced cell death in the infected ALL and CLL cultures we assessed MTT (CLL, n = 5) and MTS (ALL, n = 6) reduction in infected specimens and corresponding uninfected controls. The ability of viable cells to reduce tetrazolium to formazan in these assays is directly proportional to the number of living cells in culture. Raji B-cells which are highly susceptible to MV-oncolysis8Grote D Russell SJ Cornu TI Cattaneo R Vile R Poland GA et al.Live attenuated measles virus induces regression of human lymphoma xenografts in immunodeficient mice.Blood. 2001; 97: 3746-3754Crossref PubMed Scopus (212) Google Scholar were used as a positive control. Figure 2a,b show the ratio of formazan product in infected cells:uninfected control cells. Surprisingly, bioreduction of MTT/MTS was always greater in infected primary cultures indicating an apparent enhancement of cell viability in MV-infected ALL (Figure 2a) and CLL (Figure 2b) cells. By contrast, rapid cell death ensued in MV-infected Raji cells as expected.Figure 2MTS and MTT assays demonstrate enhanced production of formazan by measles virus (MV)-infected primary acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) cells. (a,b) show results of MTS/MTT testing following MV infection (multiplicity of infection 1.0) of primary ALL (n = 6) and CLL (n = 5) specimens, respectively, compared to a Raji cell line positive control. Datapoints represent the ratio of mean MTT/MTS absorbance in infected cells:uninfected control cells for each patient specimen. A ratio of <1 indicates reduced formazan production compared to control whereas a ratio of >1 indicates enhanced formazan production compared to control. MTT testing in Raji cells from three independent experiments are shown. Horizontal bars represent the means of independent experiments in each tumor group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)These unexpected results led us to assay cell viability postinfection by alternative methods: flow cytometric quantification of TO-PRO staining in ALL cells (n = 5) and direct counting of nonviable cells after Trypan blue staining in CLL specimens (n = 3). To exclude the influence of spontaneous cell death of primary cells in vitro, rates of "MV-specific cell death" were calculated and results are shown in Figure 3. Although a clear enhancement of cell death in both MV-infected ALL (Figure 3a) and CLL (Figure 3b) cultures was seen at all timepoints, the kinetics of this response appeared distinct with cell death ensuing rapidly in infected ALL cells (MV-specific cell death at day 1: 54.87 ± 10.37%; compared to 31.01 ± 13.06% enhancement of cell death in CLL cells at day 2. (P = 0.14).Figure 3Cell viability following measles virus expressing green fluorescent protein (MV-GFP) infection of primary acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) cells. The percentage of MV-specific cell death is shown. (a) Primary ALL (n = 5) and (b) primary CLL (n = 3). Data represent the mean ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A difference in the rapidity of killing between MV-infected ALL and CLL cells was also apparent when apoptosis induction in these targets was examined. Cell lysates from primary ALL and CLL cells infected with MV-GFP were immunoblotted to assess cleavage of endogenous poly (ADP-ribose) polymerase cleavage at serial timepoints. Evidence of poly (ADP-ribose) polymerase cleavage was detected as early as day 1 postinfection in infected ALL specimens (Figure 4a) whereas in CLL samples significant poly (ADP-ribose) polymerase cleavage could only be detected at 6 (Figure 4b) and 4 days after infection. Taken together, these data suggest a slower induction of MV-mediated apoptotic cell death in CLL targets compared to human ALL tumors.Figure 4Enhanced poly (ADP-ribose) polymerase (PARP) cleavage in primary acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) cells infected with measles virus expressing green fluorescent protein (MV-GFP). Immunoblotting for PARP cleavage (a) ALL cells. (b) CLL cells. Results for MV-GFP infected (+) or uninfected (−) cells are shown at various times postinfection. The data are representative of three and two experiments, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MV infection and replication is equivalent in primary ALL and CLL cellsTo determine whether differences in viral genome replication and/or protein expression could be responsible for the difference in CPE and speed of killing between MV-infected ALL and CLL cells, we quantified viral genome replication in MV-infected primary cell cultures by relative-quantitative PCR. Figure 5a,b shows a 5–6 log increase in MV nucleocapsid (MV-N) RNA occurring over 6 days postinfection in specimens from both malignancies, confirming that differential viral genome replication rates were not responsible for the different cytopathology or timescale of cell death between ALL and CLL.Figure 5Measles virus nucleocapsid (MV-N) RNA and proteins are produced in abundance after infection of primary acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) cells. Relative-quantitative PCR expression of MV-N mRNA in MV-green fluorescent protein (GFP) infected patient specimens (a) ALL (n = 3) (b) CLL (n = 3). The MV-N mRNA level is expressed relative to the expression of the housekeeping gene GAPDH. Data represent the mean values ± SEM. Representative immunoblots of lysates from MV-GFP infected (+) or uninfected (−) probed for MV-N, MV-fusion (F), and MV-hemaglutinin (H) at various days postinfection in (c) ALL (MV-N and MV-H only) and (d) CLL cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next assessed expression of MV proteins, in infected CLL (MV-H, MV-N, and MV-F) and ALL cells (MV-H and MV-N only) by western blotting. Figure 5c,d show representative blots for ALL and CLL, respectively, and demonstrates abundant expression of MV-N and the envelope glycoprotein MV-H in both CLL and ALL cells after infection. MV-F protein expression was additionally sought in infected CLL cells which exhibited minimal cell-to-cell fusion. MV-F expression was confirmed in all cases indicating that lack of cell-to-cell fusion in these cells was not due to a failure of viral envelope protein expression. To assess whether MV production was responsible for the distinct antitumor kinetic of MV in our primary B-cell models, lysates and supernatants from MV-infected CLL (n = 6) and ALL cells (n = 2) were titrated by 50% end point dilution assay (TCID50) at serial timepoints (data not shown). In both cell types, <5 × 103 plaque forming unit (pfu)/ml of cell associated virus was detected at the day 3 postinfection timepoint alone. No virus was detected in the supernatant.Overexpression of Bcl-2 reduces cell death in MV-infected cellsElevated Bcl-2 expression is a major determinant of drug resistance in CLL15Buggins AG Pepper CJ The role of Bcl-2 family proteins in chronic lymphocytic leukaemia.Leuk Res. 2010; 34: 837-842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar but not in ALL.16Uckun FM Yang Z Sather H Steinherz P Nachman J Bostrom B et al.Cellular expression of antiapoptotic BCL-2 oncoprotein in newly diagnosed childhood acute lymphoblastic leukemia: a Children's Cancer Group Study.Blood. 1997; 89: 3769-3777PubMed Google Scholar,17Coustan-Smith E Kitanaka A Pui CH McNinch L Evans WE Raimondi SC et al.Clinical relevance of BCL-2 overexpression in childhood acute lymphoblastic leukemia.Blood. 1996; 87: 1140-1146Crossref PubMed Google Scholar We thus hypothesized that overexpression of Bcl-2 diminishes MV-induced oncolysis in primary CLL. To investigate this hypothesis, we examined cell survival and activated caspase-3 activity in MV-infected cell lines overexpressing Bcl-2 compared with unmanipulated counterparts. Figure 6b shows MTS absorbance at day 3 post MV infection in Raji, Daudi, and Jurkat cells. In all three cell lines cell viability was greater in Bcl-2 overexpressing cell lines compared to unmanipulated controls. A timecourse experiment in Jurkat cells, where the difference was least pronounced, confirmed that this effect could be sustained up to 11 days postinfection (Figure 6c). Caspase-3 activity, quantified by enzyme-linked immunosorbent assay (Quantikine; R&D Systems, Abingdon, UK), was also consistently lower for Bcl-2 overexpressing cell lines compared to unmanipulated cell controls (Figure 6d). Thus, in all cell lines, overexpression of Bcl-2 conferred enhanced survival and reduction in apoptosis. Taken together, these data suggest that the ability of MV to lyse cells can be compromised by Bcl-2 overexpression which taken in the context of established Bcl-2 associated therapy resistance in human CLL provides a possible explanation for the slower speed of MV-mediated oncolysis in primary CLL cells.Figure 6Overexpression of Bcl-2 blunts an oncolytic response to measles virus (MV). Cell lines engineered to overexpress Bcl-2 were infected with MV (multiplicity of infection 1.0). (a) Western blot staining of Bcl-2 in Raji, Daudi, and Jurkat cell lines after retroviral transduction. The DoHH2 Bcl-2 expressing cell line is included as a positive control. (b) Cell viability as measured by MTS absorbance values carried out at day 3 post MV infection in Daudi, Raji, and Jurkat cells. Black shaded bars represent unmanipulated cells, gray bars represent Bcl-2 overexpressing cells. (c) Percentage cell viability in MV-infected Jurkat and Jurkat Bcl-2 expressing cell lines over an 11 day time course experiment. Data represent the mean ± SEM of four independent experiments. (d) Activated caspase-3 activity as determined by enzyme-linked immunosorbent assay at days 3 and 6 post infection in Daudi and Raji cells. Data show the means of one experiment performed in duplicate. Black shaded bars represent unmanipulated cells, gray bars represent Bcl-2 overexpressing cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Established Nalm-6 pre-B ALL xenografts regress after MV treatmentNext, we investigated whether the in vitro findings translated to in vivo antitumor activity. We studied specifically ALL since human xenotransplantation models for this disease are available. First, we tested the in vitro oncolytic efficacy of MV against four different B-precursor ALL-derived cell lines, REH, SEM, 697 and Nalm-6, all of which were readily killed (Figure 7a) by classic MV-induced cell-to-cell fusion with Nalm-6 being the slowest to respond. The less sensitive Nalm-6 cells was selected for in-vivo studies. A one-step MV growth curve on Nalm-6 cells is shown in Figure 7b. Mice bearing subcutaneous Nalm-6 tumors were treated with 10 intratumoral injections of 1 × 106 pfu of MV or UV inactivated MV (UV-MV). Median tumor volumes in the MV (215.7 mm3) and UV-MV (269.5 mm3) treated groups prior to the start of injections were not significantly different. As shown in Figure 8a, MV had striking antitumor activity. Complete resolution of all established subcutaneous ALL tumors was observed in 11/12 MV-treated mice by day 44 with the single remaining tumor fully regressing by day 62. In contrast, all UV-MV treated tumors progressed. The difference in tumor growth between the MV-treated and UV-MV control groups was highly statistically significant (P < 0.0001).Figure 7Measles virus (MV) oncolysis in acute lymphoblastic leukemia (ALL) cell lines. (a) In order to determine which ALL cell line to use for the murine model, four B-ALL cell lines REH (closed triangles), SEM (closed squares), 697 (closed inverted triangles) and Nalm-6 (closed diamonds) were infected with MV-green fluorescent protein (GFP) and viability determined daily by Trypan blue estimation. The Y axis denotes percent viability normalized to control uninfected cells. (b) One-step growth curve showing the titer of cell-associated virus after MV infection of Nalm-6 cells over time.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Measles virus (MV) oncolysis in a Nalm-6 subcutaneous model of acute lymphoblastic leukemia (ALL). (a) Tumor volumes in mm3 after intratumoral injection of MV (closed squares) or UV-MV(closed circles) into Nalm-6 ALL xenografts. (b) Kaplan–Meier survival curves of mice bearing disseminated Nalm-6 xenografts treated intravenously with a total dose of 6 × 106 plaque forming unit (pfu) of MV (broken line) or UV-MV control (solid line). Data represent the results from three independent experiments (n = 3–6 per group). Brackets with arrows indicate the period of MV/UV-MV administration.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since all MV-treated subcutaneous xenografts had completely regressed, three further mice with large, established Nalm-6 tumors were injected daily with 1 × 106 pfu MV for three consecutive days and tumors harvested at days 1, 2, and 5 after the 3rd injection in order to obtain evidence of in vivo viral replication. MV was recovered after Vero co-culture of washed tumor (but not from the wash supernatant) from tumors harvested at days 1 and 2 but not at day 5, confirming ongoing viral replication early after MV therapy in these Nalm-6 xenografts. Interestingly, in contrast to the CPE of MV-infected Nalm-6 cells in vitro, syncytia could not be identified in any of the harvested xenograft tumors.Next, we evaluated the efficacy of intravenous administration of MV in a disseminated model of ALL. Severe combined immunodeficient mice injected intravenously with Nalm-6 cells received 1 × 106 pfu of MV or UV-MV control over a 4-week period and observed for signs of illness. Five of 10 mice receiving replication competent MV survived beyond 100 days whereas all mice receiving UV-MV died by 67 days. Fluorescence-activated cell sorting analysis of bone marrow of UV-MV treated mice demonstrated 50–100% of human CD19+/CD10+ cells, confirming death by leukemia. The median survival was 54 days in UV-MV controls and 114 days in the MV group. (P < 0.0001). A Kaplan–Meier analysis showing overall survival of the two groups is shown in Figure 8b. Interestingly, there was no evidence of Nalm-6 leukemia in bone marrows of the five surviving mice at experiment termination, 123 days after therapy, indicating a complete remission rate of 42% for MV-treatment in this model of disseminated ALL (data not shown).DiscussionOur interest in development of MV as a therapy for B-cell leukemias led us to investigate oncolysis in primary patient-derived material to gain as much mechanistic information as possible. We found a number of differences between the replication and CPE of oncolytic MV in primary cells as compared to cell lines. An interesting and unexpected finding was the limited utility of MTT/MTS assay in determining MV-mediated cell death in primary ALL and CLL cells. There was a consistent enhancement of formazan production in MV-infected primary malignant B-cells compared to uninfected controls. Clearly, MV infection did not increase the number of viable primary cells in the culture (data not shown), instead it is likely that initial infection enhanced mitochondrial reductase activity, enabling infected cells to more effectively reduce MTT or MTS relative to "unactivated" controls. Hence, on a pragmatic note, we suggest that assays of cell death based on mitochondrial reductase activity might be misleading in the situation of MV infection of primary cells.The heterogeneity in the extent of cell-to-cell fusion after MV infection in primary cells was unexpected. Variability of cell-to-cell fusion between different ALL samples was noted but the almost complete absence of syncytia in infected CLL cells despite clear evidence of infection and replication was unexpected. To date, published data on MV therapy in a variety of tumors consistently report substantial induction of fusion in tumor targets, at least in vitro4Peng KW TenEyck CJ Galanis E Kalli KR Hartmann LC Russell SJ Intraperitoneal therapy of ovarian cancer using an engineered measles virus.Cancer Res. 2002; 62: 4656-4662PubMed Google Scholar,5Phuong LK Allen C Peng KW Giannini C Greiner S TenEyck CJ et al.Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblastoma multiforme.Cancer Res. 2003; 63: 2462-2469PubMed Google Scholar,6Msaouel P Iankov ID Allen C Morris JC von Messling V Cattaneo R et al.Engineered measles virus as a novel oncolytic therapy against prostate cancer.Prostate. 2009; 69: 82-91Crossref PubMed Scopus (79) Google Scholar,7Peng KW Ahmann GJ Pham L Greipp PR Cattaneo R Russell SJ Systemic therapy of myeloma xenografts by an attenuated measles virus.Blood. 2001; 98: 2002-2007Crossref PubMed Scopus (159) Google Scholar,8Grote D Russell SJ Cornu TI Cattaneo R Vile R Poland GA et al.Live attenuated measles virus induces regression of human lymphoma xenografts in immunodeficient mice.Blood. 2001; 97: 3746-3754Crossref PubMed Scopus (212) Google Scholar,18Allen C Paraskevakou G Liu C Iankov ID Msaouel P Zollman P et al.Oncolytic measles virus strains in the treatment of gliomas.Expert Opin Biol Ther. 2008; 8: 213-220Crossref PubMed Scopus (56) Google Scholar and this has been regarded as a potential mechanism of MV-induced oncolysis. However, cell lines—as opposed to primary material—were the subject of study in all except one of these reports. Our own data show substantial differences in the levels of syncytia formation between cell lines and primary cells—Nalm-6, SEM, 697, and REH all readily formed very large multi