Differential pattern of susceptibility of sarcoma cell lines to MeV-SCD-mediated oncolysis being modulated by addition of the prodrug 5-FC.
To investigate the susceptibility of sarcoma cells to MeV-mediated oncolysis, we infected a panel of eight sarcoma cell lines with MeV-SCD at an MOI of 1. Primary resistance to virotherapy was defined as a remaining cell mass of >50% relative to mock-infected controls (i.e., incubation with medium only) at 96 hpi. As a result, five cell lines were found to be susceptible to oncolysis by MeV-SCD, with remaining cell masses ranging from 3 to 40%, whereas three cell lines exhibited primary resistance (remaining cell masses between 72 and 95%) (). To differentiate between inhibition of cell proliferation (as determined by SRB assays ) and cell lysis, we additionally measured a parameter indicating direct cell lysis, i.e., MeV-mediated oncolysis. For this purpose, the release of lactate dehydrogenase (LDH) was determined in all 8 sarcoma cell lines 96 hpi at an MOI of 1 (LDH release in mock-infected/uninfected cells was used as the baseline control). Whereas in the oncolysis-resistant cell lines values of LDH release only ranged from 11 to 18% (CCS, SCOS, and SRH) (, right), values of LDH release were found to vary between 48 and 67% in the susceptible cell lines (BR, ST, A673, ZF, and HT1080) (, left). These data indicate that the reduction in cell mass compared to that of mock-infected cells as measured by SRB assay is predominantly due to cell lysis/oncolysis. Inhibition of cell proliferation seems to play a minor role at best.
Fig 1 Susceptibility of sarcoma cell lines to MeV-mediated oncolysis. Sarcoma cell lines were infected with MeV-SCD at an MOI of 1 without addition of the prodrug 5-FC. (A) At 96 hpi, the remaining cell mass was determined by SRB assay. Primary resistance was (more ...)
Cells next were infected with MeV-SCD at MOIs of 0.01, 0.1, and 1 (susceptible cell lines) () as well as 0.1 and 1 (resistant cell lines) (). At 3 hpi, different concentrations of the prodrug 5-FC (10−4 to 100 mM) were added, followed by assessment of tumor cell viability again at 96 hpi. In the susceptible cell lines, high 5-FC concentrations (1 mM) had already strongly enhanced the oncolytic effect of MeV-SCD at a low MOI of 0.01 or 0.1 (). Interestingly, addition of 5-FC also enhanced the oncolytic effect of MeV-SCD in two otherwise resistant cell lines (SRH and CCS), resulting in a reduction of the remnant cell mass from 71 to 36% for SRH cells (MOI of 1; 1 mM 5-FC) and from 94 to 21% for CCS cells (MOI of 1; 1 mM 5-FC). In contrast, SCOS cells still were found to be highly resistant even when infection with MeV-SCD was combined with a treatment of 1 mM 5-FC at an MOI of 1 (), exhibiting a remnant cell mass of 60%, while that of cells without 5-FC treatment was 82%.
Fig 2 Effect of the addition of the prodrug 5-FC on MeV-SCD-mediated oncolysis. Sarcoma cell lines were infected with MeV-SCD at MOIs of 0.01, 0.1, and 1 (A) or at MOIs of 0.1 and 1 (B) or were mock infected and then treated with increasing concentrations of (more ...) Expression levels of known measles virus receptors in our sarcoma cell panel.
To get insight into the mechanisms of the observed differential susceptibilities to MeV-mediated oncolysis, we first confirmed the expression of the measles vaccine virus receptor CD46 by flow cytometry in cell lines that were susceptible or resistant to MeV-mediated oncolysis (). Calculation of mean fluorescence indices (MFI) (35
) revealed higher expression levels in the susceptible cell lines (A673, ZF, HT1080, ST, and BR) than in resistant cell lines (CCS, SRH, and SCOS). Interestingly, none of the 8 cell lines of our sarcoma panel was found to express either of the other two known MeV receptors, SLAM and nectin-4 (data not shown).
Fig 3 Receptor expression and primary infection rates. (A) Sarcoma cell lines were stained with anti-CD46 antibody or an isotype control. Expression was analyzed by flow cytometry. MFI indicates the ratios of the arithmetic means of the CD46 staining/isotype (more ...) Primary infection rates in sarcoma cells.
Primary infection rates were determined next. For this purpose, all eight sarcoma cell lines were infected with a GFP marker gene encoding MeV vector (MeV-GFP) at MOIs of 0.1, 1, and 10 or were mock infected, followed by quantification of GFP expression at 24 hpi by flow cytometry ( and ). At an MOI of 0.1, primary infection rates in the susceptible cell lines ranged from 5.6 to 11.1% (average, 8.54%) () and from 0.9 to 2.9% in the resistant cell lines (average, 1.7%) (). At an MOI of 1, primary infection rates in the susceptible cell lines varied between 37.3 and 64.4% (average, 49%) () and in the resistant cell lines between 4.4 and 23.6% (average, 12.2%) (). At an MOI of 10, all susceptible cell lines showed very high primary infection rates, between 86 and 96% (average, 90.9%) (); in contrast, resistant cell lines displayed much lower rates, between 31.3 and 66.1% (average, 45.9%) (). In summary, susceptible cell lines were found to be infected more efficiently than resistant ones.
Viral replication in sarcoma cell lines.
To determine whether resistance to MeV-mediated oncolysis is due to an inhibition of viral replication, we next performed viral growth curve assays starting with a fixed inoculation dosage of MeV-SCD at an MOI of 0.03 (i.e., 3,000 infectious viral particles per 105 sarcoma cells per well) for all cell lines of our sarcoma cell panel and observed the following patterns (). Three susceptible cell lines (BR, ST, and ZF) showed a continuous increase in virus yield over time, reaching titers in the range of 105 to 106 fluorescent focus-forming units per ml (FFU/ml). In contrast, A673 cells had already reached a plateau at 48 hpi. Furthermore, in HT1080 cells, titers reached a peak at 48 hpi (105 FFU/ml) and then were found to slowly decline in virus production (). In contrast, in the resistant cell lines there was either no relevant virus production at all (SCOS cells), a transient increase in viral titers (SRH cells) reaching a maximum of 1.3 × 103 FFU/ml (supernatant) and 2.2 × 103 FFU/ml (lysate), or a very slow and weak viral replication, ending in titers below 103 FFU/ml (CCS cells) (). Dashed horizontal lines in indicate a threshold level of 104 FFU/ml, which is not reached in oncolysis-resistant cell lines, being indicative of this quite insufficient production of infectious progeny virus particles in all oncolysis-resistant cell lines.
Fig 4 Replication of MeV-SCD in sarcoma cell lines. Sarcoma cell lines that were susceptible (A) or resistant (B) to MeV-based oncolysis were infected with an identical/fixed dose of MeV-SCD (MOI of 0.03). Supernatants (black lines) and cells (gray dotted lines) (more ...)
To further investigate to what extent these results are influenced by differences in virus entry between the different cell lines, we thought it helpful to examine virus growth kinetics for a subset of our sarcoma cell lines after infection with virus titers that result in similar rates of primary infection. For this purpose, viral growth curve assays were repeated with different MOIs yielding quite similar primary infection rates, ranging from 7.7 to 18.2% (), as indicated by the percentage of GFP-positive cells at 24 hpi. In detail, cell lines susceptible to oncolysis and viral growth (HT1080, ST, and ZF) were infected at an MOI of 0.5, whereas cell lines displaying reduced viral growth were infected at an MOI of 1 (SRH) and 2 (SCOS) (). In the resistant cell lines SRH and SCOS, viral replication was found to be transient, resulting in peak titers of only 104 FFU/ml at 48 hpi (). In susceptible ZF cells, viral titers of 1.4 × 106 FFU/ml were reached at 96 hpi in the supernatant and at 72 hpi in the cell lysate. In susceptible HT1080 cells, a peak titer of 4.5 × 105 FFU/ml was reached in the supernatant at 48 hpi, whereas in the cell lysate there was a continuous increase in viral titers up to 1.6 × 106 at 96 hpi. In susceptible ST cells, viral replication reached a maximum at 2.4 × 105 in the supernatant and at 1.6 × 105 in the cell lysate at 72 hpi.
Fig 5 Differences in viral replication between sarcoma cell lines that were susceptible or resistant to MeV-based oncolysis are not primarily due to differences in virus entry. (A) In order to yield similar rates of primary infection, a subset of sarcoma cell (more ...)
Taken together, these data indicate that differences in viral replication between oncolysis-resistant and -susceptible sarcoma cell lines are not primarily due to differences in virus entry but rather are caused subsequently by an inhibition of replication. Thus, growth curves revealed a profound inhibition of viral replication in the resistant cell lines.
Induction of pathogen receptor and ISG expression upon MeV-SCD infection.
Naturally, viral infections lead to the activation of cytoplasmic receptors MDA5 and RIG-I, triggering expression of IFN-β and of interferon-stimulated genes (ISG). To further elucidate the molecular mechanisms of resistance to MeV-SCD-mediated virotherapy, expression levels of both intracellular pathogen receptors as well as ISGs were analyzed by quantitative RT-PCR (qRT-PCR). For this purpose, sarcoma cell lines were infected with MeV-SCD at an MOI of 1 or were mock infected. At 6 hpi, total RNA was isolated and qRT-PCR was performed using primers specific for TLR3, MDA5, RIG-I, IFN-β, and IFIT1, representing a prototypic ISG (). As a result, no clear correlation between virotherapy resistance patterns and expression levels of TLR3, MDA5, and IFN-β were detectable; however, a strong induction of RIG-I expression upon infection was found in resistant cell lines SRH (16.8-fold) and SCOS (14.9-fold), whereas the resistant cell line CCS showed a weaker induction of only 4.3-fold ( and ). In contrast, in the susceptible cell lines the induction of RIG-I expression was found to be 3.1-fold (for HT1080 cells) or less ( and ). Also, expression of IFIT1 was strongly induced upon infection with MeV-SCD in resistant cell lines SRH (12.9-fold) and SCOS (13.9-fold), whereas the induction in CCS cells again was much weaker (3.1-fold) ( and ). In contrast, susceptible cell lines exhibited only weak induction rates of 2-fold (for A673 cells) and 1.5-fold (for ST cells) or less with only one exception: HT1080 cells displayed a 16.3-fold induction of IFIT1 expression.
Fig 6 Induction of TLR3, RIG-I, MDA5, IFN-β, and IFIT1 in sarcoma cell lines. Sarcoma cell lines were mock infected or infected with MeV-SCD at an MOI of 1. At 6 hpi, RNA was isolated and qRT-PCR was performed. Values were normalized to the housekeeping (more ...)
mRNA expression levels of sarcoma cells either mock infected or infected with MeV-SCD (MOI of 1) and harvested at 6 hpi
In addition, we also compared the relative mRNA expression levels after grouping the results of mock-infected and MeV-SCD-infected susceptible versus resistant sarcoma cell lines (). Mean values for expression of IFN-β in mock-infected cell lines were 0.08 (susceptible) and 0.39 (resistant), respectively, whereas at 6 hpi with MeV-SCD (MOI of 1), mean values of 0.34 (susceptible) and 5.62 (resistant) were reached. For IFIT1, there was an 11-fold difference in basal expression between susceptible and resistant cell lines (0.06 versus 0.66). At 6 hpi, the difference was 35-fold and therefore even more prominent, reaching mean values of 0.17 in susceptible cell lines and 5.96 in resistant cell lines. For RIG-I, there were minor differences in mock-infected cells (0.57 versus 0.83). At 6 hpi, mean expression values were 1.11 (susceptible cell lines) and 9.21 (resistant cell lines). For MDA5, mock-infected resistant cell lines again displayed higher relative mRNA expression levels than susceptible cell lines (1.87 versus 0.26). Upon infection, mean values rose to 0.52 in susceptible cell lines and 11.87 in resistant cell lines. For TLR3, levels increased from 0.13 to 0.5 in susceptible and from 0.53 to 1.88 in resistant cell lines.
These data suggest that resistance to virotherapy is at least in part due to elevated levels of cytoplasmic pathogen receptors and ISGs. Resistant cell lines in general display higher basal mRNA expression levels of pathogen receptors and ISGs and also show higher induction rates upon infection with MeV-SCD.
Secretion of IFN-β upon MeV-SCD infection.
We observed quite low baseline levels of IFN-β secretion into cell culture supernatants, which after infection with MeV-SCD (MOI of 1) were found to increase in only two out of three oncolysis-resistant cell lines (, right, SCOS and SRH). A maximum IFN secretion of 1,200 pg/ml was reached in SRH cells at 48 hpi and of 1,800 pg/ml in SCOS cells at 36 hpi. In contrast, no IFN-β was detectable in the supernatants of oncolysis-resistant CCS cells. When looking at the patterns of MeV-SCD-based IFN induction in the oncolysis-susceptible cell lines (, left), two out of five cell lines displayed little (BR) or no (ZF) induction of IFN-β release. In contrast, A673, HT1080, and ST cell lines exhibited patterns of IFN-β release similar to those of the two oncolysis-resistant cell lines SCOS and SRH, reaching maxima of 1,900 pg/ml (HT1080) at 48 hpi, 2,000 pg/ml (ST) at 48 hpi, and 2,200 pg/ml (A673) at 36 hpi, respectively. These data are in line with the data obtained by qRT-PCR showing no clear correlation between IFN levels and resistance to oncolysis.
Fig 7 Secretion of IFN-β in sarcoma cell lines. Sarcoma cell lines were mock infected or infected with MeV-SCD at an MOI of 1. Supernatants were collected at the indicated time points. IFN-β in the supernatants was determined by ELISA. Means (more ...) Resistant cell lines exhibit a strong persistent activation of IFN signaling.
Binding of IFN-β to its cognate receptor leads to activation of the Jak-Stat signaling pathway, resulting in the phosphorylation of Stat1 and Stat2 and formation of trimeric complexes together with IRF-9, which then activates transcription of ISGs. In order to investigate a potential modulation of Stat1 phosphorylation and IFIT1 protein expression in the course of MeV-mediated virotherapy, all sarcoma cell lines were infected with MeV-SCD at an MOI of 1. At 24, 48, and 72 hpi, cells were lysed and immunoblotting was performed (). As a result, Stat1 constitutively was found to be weakly phosphorylated in resistant cell lines SRH and CCS (, upper); at 24 hpi, phosphorylation was detectable in all resistant cell lines (CCS, SCOS, and SRH), which persisted up to 72 hpi. In contrast, in the susceptible sarcoma cell lines, different patterns were found. In ZF cells, there was no phosphorylation of Stat1 at all. In ST and A673 cells, a transient phosphorylation at 24 hpi was detectable. In HT1080 cells, Stat1 was found to be transiently phosphorylated at 24 and 48 hpi. In BR cells, Stat1 phosphorylation only took place in a delayed manner at 72 hpi. Due to a lack of viable cells at 72 hpi, no immunoblotting could be performed for ZF cells at this late time point.
Fig 8 Phosphorylation of Stat1 and expression of IFIT1 in sarcoma cell lines in the course of MeV-SCD infection. Sarcoma cell lines were infected with MeV-SCD (MOI of 1) or were mock infected. At the indicated time points cells were lysed. Mock-infected cells (more ...)
Concerning expression of prototypic ISGs, IFIT1 was found to be constitutively expressed in the resistant SRH and CCS cells (). Upon infection with MeV-SCD, IFIT1 expression was found to be increased in the resistant cell lines, persisting for up to 72 hpi. No IFIT1 expression was detectable in ZF cells, correlating with the missing Stat1 phosphorylation. In BR cells, IFIT1 expression was found only in a delayed manner at 72 hpi. In ST cells, expression was transiently detectable only at 48 hpi. In A673 cells, IFIT1 expression appeared first at 48 hpi. The only susceptible cell line which showed strong and persistent expression of IFIT1 was HT1080. These findings support our data obtained by qRT-PCR, indicating that differences in the innate immune defense account for differences in the susceptibility of sarcoma cell lines to MeV-mediated oncolysis.
Exogenous stimulation with IFN-β results in activation of the Jak-Stat signaling pathway and ISG expression.
To find out whether susceptible cell lines had a general defect in IFN signaling, all sarcoma cells were stimulated with IFN-β for different time periods, followed by immunoblot analysis of the activation of Stat1 and expression of IFIT1. In all sarcoma cell lines, phosphorylation of Stat1 was detectable after 30 min and persisted for up to 2 h (). IFIT1 expression also was found in all sarcoma cell lines after 24 h (), indicating that exogenous stimulation with IFN-β resulted in both activation of the Jak-Stat signaling pathway and expression of ISGs independently of patterns of susceptibility or resistance to MeV-mediated oncolysis. Therefore, differences accounting for differential susceptibility supposedly are located further upstream.
Fig 9 IFN-β induces Stat1 phosphorylation and IFIT1 expression in sarcoma cell lines. (A) Cells were stimulated with IFN-β for the indicated time periods or left untreated. Immunoblotting was performed to detect the expression of phosphorylated (more ...) Effect of pretreatment with IFN-β on susceptibility to MeV-mediated oncolysis and viral replication.
Since resistant cell lines showed a strong and persistent activation of the IFN signaling pathway, we investigated whether pretreatment of susceptible cell lines with IFN-β rendered them more resistant to MeV-mediated oncolysis. Susceptible cell lines were pretreated for 20 h with IFN-β (1,000 U/ml) and then infected with MeV-SCD at MOIs of 0.01, 0.1, 1, and 10. At 96 hpi, the remaining tumor cell mass was determined (). In BR and ZF cells, no protective effect of IFN pretreatment was found. In A673 and HT1080 cells, pretreatment with IFN-β increased cell survival at an MOI of 1 from to 17 to 73% and 5 to 18%, respectively. In ST cells, the effect was moderate, with an increase in viability from 23 to 52%.
Fig 10 Effects of prestimulation with IFN-β on MeV-mediated oncolysis and viral replication. (A) Cells were prestimulated with IFN-β (1,000 U/ml) for 20 h (white bars) or were left unstimulated (black bars), followed by infection with MeV-SCD (more ...)
To determine if increased survival paralleled inhibition in viral replication, growth curve assays were performed in the presence or absence of IFN-β pretreatment (). Consistent with the missing effect on cell survival, IFN-β pretreatment did not influence viral replication in BR and ZF cells. In A673 and HT1080 cells, viral replication was delayed but reached similar titers at a later time point, i.e., at 96 hpi. In ST cells, replication was also delayed in the course of IFN-β pretreatment, and viral titers were still lower at 96 hpi. Interestingly, primary infection rates (measured using MeV-GFP) were also influenced by IFN-β pretreatment as determined by FACS analysis at 24 hpi (). At an MOI of 1, pretreatment with IFN-β led to a reduction of the primary infection rates in cell lines A673 (from 64 to 27%) and ST (from 34 to 12%). In HT1080 cells, a moderate reduction from 24 to 16% was observed. In cell lines ZF and BR, only a minor reduction from 41 to 35% and 48 to 36%, respectively, was found. Thus, pretreatment with exogenous IFN-β was found to be able to protect sarcoma cell lines to different extents from MeV-mediated oncolysis. This protection was demonstrated to go along with an inhibition of viral replication and reduced rates of primary infection.
Fig 11 Primary infection rates in the absence or presence of IFN-β. Sarcoma cell lines were pretreated for 20 h with IFN-β (1,000 U/ml) or left untreated, followed by infection with MeV-GFP at MOIs of 0.1, 1, and 10 or mock infection. For determination (more ...) Suppression of IFIT1 expression confers susceptibility to MeV-mediated oncolysis in SRH cells but not in SCOS and CCS cells.
Since IFIT1 was shown to bind to 5′-triphosphorylated RNA, which arises during the life cycle of many negative-stranded RNA viruses, thereby inhibiting viral replication, we investigated if knockdown of IFIT1 using two different siRNAs and a combination of both renders resistant cells more susceptible to MeV-mediated oncolysis. SRH cells, constitutively expressing IFIT1 (, lanes 1 and 3), were transfected with IFIT1 siRNAs or control siRNA or were mock transfected, and 24 h later they were infected with MeV-SCD at MOIs 1 and 10. As a result, immunoblots revealed an almost complete knockdown of IFIT1 expression (, lanes 5 to 8). On this basis of significant suppression of IFIT1, SRH cell mass was determined at 72 hpi (). At an MOI of 1, SRH cell mass was found to be reduced to 73% in mock-transfected cells and to 83% in control siRNA-transfected cells. In SRH cells with IFIT1 knockdown, the remnant cell mass was lowered to 62, 66, and 61%, respectively. At an MOI of 10, the oncolytic effect observed in the course of IFIT1 knockdown was even more prominent, resulting in remnant cell masses at 72 hpi of 25, 26, and 21%, respectively, compared to 50 and 57% in mock- and control siRNA-transfected cells. These data suggest that the constitutive expression of IFIT1 observed in SRH cells plays a key role in conferring resistance to MeV-mediated oncolysis in these cells.
Fig 12 Influence of IFIT1 knockdown in sarcoma cells on resistance to MeV-mediated oncolysis. Sarcoma cells were transfected with control siRNA (si control), two different IFIT1 siRNAs (siIFIT1 #2 and siIFIT1 #6), or a combination of both (siIFIT1 #2 + 6). Twenty-four (more ...)
In order to determine the contribution of IFIT1 to oncolysis resistance in other oncolysis-resistant cell lines, we examined CCS and SCOS cells. In contrast to SRH cells, both CCS and SCOS cells were found to exhibit no constitutive IFIT1 protein expression (, lanes 1 and 3, and F, lanes 1 and 3). Whereas transfections of CCS and SCOS cells with IFIT1 siRNAs again resulted in a nearly complete suppression of MeV-induced IFIT1 expression (, lanes 6 and 8, and F, lanes 6 and 8), as was the case in SRH cells, this did not result in any significant reduction of the cell mass of either CCS or SCOS tumor cells, even when performing infections at an MOI of 10 ( and ). Thus, in the absence of a relevant constitutive expression of IFIT1, knockdown of MeV-induced IFIT1 expression (, lanes 6 and 8, and F, lanes 6 and 8) does not result in a reversion of resistance to MeV-based oncolysis, again pointing out the heterogeneity of oncolysis resistance mechanisms in sarcoma cells.
Increased MOI as well as the addition of 5-FC are potent means to overcome primary resistance toward MeV-mediated oncolysis.
To investigate if increasing the MOI in combination with application of the prodrug 5-FC constitutes a suitable regimen to overcome primary resistance to MeV-mediated oncolysis, resistant sarcoma cell lines SRH, SCOS, and CCS were infected at an MOI of 10 (). At 3 hpi, the prodrug 5-FC was added (1 mM), and tumor cell viability was determined at 96 hpi. In CCS cells, viability was reduced to 50% at an MOI of 10 (, upper). Addition of 1 mM 5-FC further decreased the remnant cell mass to a very low level of only 8%. In SRH cells, infection with MeV-SCD at an MOI of 10 without and with 1 mM 5-FC resulted in a remaining cell mass of 32 and 11%, respectively (, middle). In SCOS cells, neither increasing the MOI up to 10 nor addition of 5-FC was able to decrease cell viability (82 and 77% remaining cell mass, respectively) (, lower). Of note, this phenomenon is explained at least in part by finding a lack of sensitivity to 5-FU of this specific cell line (data not shown). To investigate whether this reduction in cell mass at an MOI of 10 was primarily due to cell lysis or to an inhibition of cell proliferation, we also measured the release of LDH in the course of MeV-mediated oncolysis at an MOI of 10 (). Interestingly, MeV-SCD-induced oncolysis of resistant sarcoma cells at an MOI of 10 led to an LDH release of 32 (SCOS), 55 (SRH), and 59% (CCS), which was much higher than the results obtained at an MOI of 1 (). These data again indicate that the reduction in cell mass at 96 hpi obtained with MeV-SCD is mainly caused by cell lysis irrespective of employment of an MOI of 1 or 10.
Fig 13 Effect of increased MOI together with addition of prodrug on the survival of resistant sarcoma cell lines. (A) Resistant sarcoma cell lines were infected with MeV-SCD at an MOI of 10 or were mock infected. The prodrug 5-FC was added 3 hpi. Cell viability (more ...)
In summary, raising the MOI up to 10 together with the application of 5-FC constitutes a regimen that is able to overcome primary resistance to MeV-mediated oncolysis in two out of three primarily resistant cell lines.