Iuliana Matei
email: iuliana.matei@malix.univ-paris1.fr.

Peter Howitt

Mathilde Maurel

Sandra Poncet

Prices and output co-movements: an empirical investigation for the CEECs

Keywords: European monetary integration, co-movements, AR models, CEECs Cet article étudie Integration monétaire européenne, co-mouvements, modèles AR, PECO JEL Classification : C22, E30, F33, F42, F47

, we evaluate the size and the persistence of prices and outputs shocks between each CEECs and euro zone. Results will contribute to the debate around the participation of the new members to the EMU.

Introduction

From 1st January 2007, the European Union counts 27 member states. Ten CEECs (Bulgaria, Czech Republic, Estonia, Hungary, Latvia, Lithuania, Poland, Romania, Slovak Republic and Slovenia) along with Cyprus and Malta have joined the 15 older EU members. Following the largest expansion in its history, 27 countries must now strive to respect the acquis communautaire 1 especially in terms of single currency project and to integrate their national structures and their citizens on a scale never seen before.

This recent enlargement of EU raises essential questions particularly around the participation of the new members to the EMU. Due to their progress during a still unfinished transition and convergence process, the new EU states have as objective to join euro zone for some reasons: this entry could guarantee them lower interest rate, a certain monetary stability and a more easily access to foreign founds. Contrary to the case of Denmark and the United Kingdom in 1999, they can not beneficiate of the opt-out clause. Consequently, they have to prepare their accession to Euroland as soon as possible in agreement principally with the Maastricht criteria. The accession to euro zone and its effects became consequently a crucial key issue for their future.

The entry into a monetary union involves both costs and benefits for member countries. The costs of the adoption of a common currency are generally established according to two criterions:

(i) institutional criterion (Maastricht convergence criteria) and (ii) criteria derived from the theory of optimal currency areas (OCA) initiated by [START_REF] Mundell | A theory of Optimum Currency Areas[END_REF], [START_REF] Mckinnon | Optimum Currency Areas[END_REF] and Kenen (1969). If the Maastricht treaty privileges criterions of nominal convergence to evaluate the practical feasibility of the euro area and the risks weighing on the independence of the European Central Bank, the OCA criterions emphasize structural criterions such as those related to the benefits of a monetary union: two countries or areas will profit from a monetary union if they are characterized by a great similarity of the business cycles, have strong commercial linkages and a mechanism to mitigate the negative effects of the asymmetrical shocks. It is worth noting that the most commonly identified cost in the theoretical and empirical literature is the loss of monetary policy as national stabilization tool and the most cited benefit of a monetary union is the increase in trade and foreign investments. Beyond these various structural criteria of OCA theory which can guide in the choice of the exchange rate regimes, an important role plays the 1 The acquis communautaire 1 means the same EU laws and rules that apply to the older members. nature of the shocks affecting a monetary area. In the case of the symmetrical shocks affecting the economies, the fixed exchange rate regimes are preferable compared to their flexibility. In the case of asymmetrical shocks, the opposite situation has to be taken into account: a more flexibility of the exchange rate regimes. But, perfectly symmetrical shocks don't exist. Because of this raison, the most part of the literature focuses on the degree of shocks symmetries rather than the nature of the shocks. Hence, the principal idea of this approach is that costs of a monetary union decrease proportionally with the degree of symmetry of the shocks (i.e, the business cycles should be synchronized when the currencies are anchored to the euro because of the existence of a common monetary policy).

The empirical literature on the nature and the degree of the shock's symmetries exploit usually descriptive statistics, econometric estimates or stochastic simulations. The descriptive statistics focuses on the correlations between the representatives economic variables such as the GDP growth, variability of cyclical components of industrial production etc. and consider that a strongly positive correlation of the cyclical components implies the existence of the shocks mainly symmetrical. These methods ignore the structural changes and don't distinguish between the shocks and the answer to the shocks. Concerning the econometric estimates, the majority of studies uses as econometric method a structural VAR model developed by [START_REF] Blanchard | The Dynamic Effects of Aggregate Demand and Supply Disturbances[END_REF] and Bayoumi andEichengreen (1993, 1996) to study both the supply and demand shocks affecting the EU members and their degree of (a)symmetry. Finally, the stochastic simulations are based on an econometric model in which the residuals are compared to macroeconomic shocks.

These studies estimate a model drawing randomly from the shocks in the estimated distributions and compute then the variance of the different representative variables (production, prices) entering in these functions [START_REF] Masson | Evaluating the EMS and EMU: Some issues[END_REF]. Other methods could be used to analyze the nature of shocks and the degree of shocks asymmetries such as the co-movements features for representative economic variables [START_REF] Alesina | Optimal currency areas[END_REF].

The focus of this paper is to analyze the characteristics of production and prices co-movements between each new EU member states and euro zone using partially the methodology developed by [START_REF] Alesina | Optimal currency areas[END_REF]. To our best knowledge, it was never tempted to provide an investigation of the co-movements in the case of the new EU member's states. According to [START_REF] Alesina | Optimal currency areas[END_REF] approach, a positive response of the co-movements to the monetary union would lead to a higher degree of consensus around a common monetary policy without involving imbalances between participants and to lower costs generated by the loss of the monetary autonomy. A negative response of the co-movements would have the opposite effect: it would generate divergent fluctuations and in this case, the exchange rate policies will be crucial to support the economic activity. Remarkably little attention has been paid to methods for measuring the co-movements, principally, in the case of new member states. In this sense, [START_REF] Alesina | Optimal currency areas[END_REF] approach exploit one of the various sensitivity indices -the conditional variance -to measure the co-movements. From the methodological point of view, the purpose of this paper is to examine the co-movements by exploiting recently new class of socalled variance-based sensitivity indices: the conditional variance and, respectively, the unconditional variance. The two concepts are very different, as illustrated for example in studies of inflation, in which the unconditional variance simply measures inflation variability, while the conditional forecast error variance measures inflation uncertainty (see, for example, [START_REF] Ball | Inflation and Uncertainty at Short and Long Horizons[END_REF]. In its basic form, the link between these two concepts is highlighted by the law of total variance and can be expressed by " varY -E[varY|X] " as sensitivity index which is the "unconditional variance minus expected conditional variance of Y given parameter X" and may be interpreted as the "amount of the variance of Y that is expected to be eliminated if the true value of parameter X will become known". These methodological contributions lead to results suggesting that price co-movements are higher than production co-movements and that, generally, they are closed to those of the former EU member states. Our contribution is twofold.

Firstly, we concentrate our attention on the recent experience of the new EU member states which are interesting examples to evaluate the prices and outputs co-movements in the ex-ante conditions (i.e., before entering into EU): countries characterized by the heterogeneity of the exchange rate regimes, recently integrated into EU and candidates for the adoption of the euro.

Secondly, from the methodological point of view, we provide an additional test of the [START_REF] Alesina | Optimal currency areas[END_REF] approach to study the co-movements. More precisely, in the general framework of the autoregressive models, we carried out this analysis using both sensitivity indices: the conditional variance and the unconditional variance to point out business cycles synchronization.

The remainder of the paper is as follows. The next section reviews the literature on the costs and the benefits of a monetary union focusing on the new EU member's case. Section 3 present some stylized facts related to the shocks symmetries. Section 4 details the empirical methodology and data sources. Section 5 reports the main results of our empirical model and discuss them. Section

Literature review

The empirical literature around the costs and the benefits of a monetary union is relatively recent.

We can distinguish two large approaches: the first investigating the occurrence of the asymmetric shocks between the old and the new members of EU and the second, focusing on the effects of a monetary union on the trade linkages. To have a coherent outline, we propose to present briefly a review of the empirical literature on the costs and the benefits of a monetary union in the CEECs context with a particular focus on the research about the shocks (a)symmetries.

Concerning the first approach, the recent studies on the shocks (a)symmetries between the old and the new EU member states become numerous after the adoption of the Maastricht treaty which maked credible the idea of the European Monetary Integration. The most part of these studies use as econometric method a structural vector autoregressive model (VAR model) developped initially by [START_REF] Blanchard | The Dynamic Effects of Aggregate Demand and Supply Disturbances[END_REF] and Bayoumi andEichengreen (1993, 1996) in order to analyse the supply and demand shocks affecting the EU countries and their degree of asymmetry. [START_REF] Frenkel | How symmetric are the shock Adjustment Dynamics Between the Euro Area and the Central and Eastern European Countries?[END_REF] and Horvath and Ratfai (2004) show that neither the correlation of the supply shocks or demand shocks make it possible to conclude in favor of the convergence for European countries. Fidrmuc and Korhonen ( 2001) highlight a certain heterogeneity concerning the correlation of the supply shocks between the EU and CEECs. The correlation of the demand shocks appears significant for Estonia and Hungary, while for the other CEECs, the results of the estimates seem to be non-significant. Babetski, [START_REF] Boone | Symmetry and asymmetry of Supply and demand Shocks in the European Union: a Dynamic Analysis[END_REF][START_REF] Babetskii | Exchange Rate Regimes and Supply Shocks Asymmetry: the case of the Accession Countries[END_REF] widen the analysis of the supply and demand shocks by measuring the correlation in time starting from the methodology of [START_REF] Boone | Symmetry and asymmetry of Supply and demand Shocks in the European Union: a Dynamic Analysis[END_REF]. Their results show a process in course of convergence of the demande shocks between the EU and its new members while the supply shocks tend to diverge because of the transition process and the Balassa-Samuelson effect.

In the general framework of the structural VAR, [START_REF] Frenkel | How symmetric are the shock Adjustment Dynamics Between the Euro Area and the Central and Eastern European Countries?[END_REF] identify and compare demand and supply shocks between euro area countries and CEECs. Compared to Babetski, [START_REF] Boone | Symmetry and asymmetry of Supply and demand Shocks in the European Union: a Dynamic Analysis[END_REF][START_REF] Babetskii | Exchange Rate Regimes and Supply Shocks Asymmetry: the case of the Accession Countries[END_REF] which use data since 1990 (corresponding to falls of the productions during the transition process to market economy), [START_REF] Frenkel | How symmetric are the shock Adjustment Dynamics Between the Euro Area and the Central and Eastern European Countries?[END_REF] use more recent data (1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000)(2001) and thus, less affected by the structural shocks. Their analysis shows that there are still differences in the shocks and in the process of adjustment to shocks between the euro area and CEECs. But, they find also some similarities with euro area countries for several individual CEECs. [START_REF] Boone | L'ancrage de l'Europe Centrale et Orientale à l'Union Européenne[END_REF] study the date for which it would be o p t i m a l f o r C E E C s t o f o r m a m o n e t a r y u n i o n w i t h G e r m a n y o r w i t h E U . U s i n g t h e methodology of Reichlin andForni (1997) andFuss (1997) and using data over the period 1991-1997 for four CEECs, they find that the correlations of industrial production and unemployment cycles in the CEECs and the EU, point towards a deeper integration of the CEECs with Germany than with the EU. They also emphasise that pegging the currencies is a good policy option for CEECs. [START_REF] Alesina | Optimal currency areas[END_REF] study the comouvements of prices and outputs for various countries compared to the euro, the dollar and the yen and identify the "natural" OCA for these three monetary anchors. They use data on inflation, trade and prices and output co-movements over the period 1960 -1997. Their results show that there exist monetary areas well defined by the dollar and the euro -as potential monetary anchors, but not defined by the yen. They highlight that pegging the currencies to euro and dollar increases the bilateral commercial linkages and the price co-movements and note that both prices and output co-movements don't appear systematically corelated. In the same vain with Frankel and Rose (1998) approach, they argue that co-movements in "ex-ante" conditions could under-estimate the potential benefits of integration into a monetary union.

Another group of papers focus on the links between exhange rate regimes and the performances in terms of inflation in the case of EU countries. [START_REF] Borghijs | Exchange Rates in Central Europe : A Blessing or a Curse?[END_REF] analyze the costs and the benefit generated by the loss of the monetary autonomy which depends on the nature of the asymmetrical shocks striking an economy and on the capacity of the exchange rate regimes to absorb these shocks. Using a structural VAR model for five CEECs (Czech Republic, Hungary, Poland, Slovakia and Slovenia), their results suggest that the exchange rate chanel seems to be more important as propagator of the monetary and financial shocks than as an effective absorber of the real shocks. [START_REF] Coricelli | Exchange Rate Pass-Through in Candidate Countries[END_REF] analyze the link between the exchange rate regimes and the performances in terms of inflation for four CEECs: Czech Republic, Hungary, Poland and Slovenia and find that a fast adoption of the euro could provide a more effective framework to reduce inflation. In the same line, [START_REF] Goldfajn | The pass-through from depreciation to inflation : a panel study[END_REF] analyze the relation between the depreciation of the exchange rate and the inflation for 71 countries between 1980-1998 while [START_REF] Darvas | Exchange Rate Pass-Through and Real Exchange Rate in EU Candidate Countries[END_REF] analyze the same problematic over the period 1993 -2000, but, in the case of four CEECs (Czech Republic, Hungary, Poland and Slovenia).

The second approach focus on the effects of the monetary union on the trade. Recent papers have questioned on this problematic such as: [START_REF] Artis | International Business Cycles and the ERM : Is there a European Bussiness Cycle?[END_REF], Frankel andRose (2002, 1996), [START_REF] Rose | One Money, One Market: Estimating the Effect of Common Currencies on Trade[END_REF], [START_REF] Engel | Currency Unions and International Integration[END_REF], [START_REF] Alesina | Optimal currency areas[END_REF], [START_REF] Glick | Contagion and Trade : Why are Currency Crisis Regional ?[END_REF]Rose (2001), Flandrau and[START_REF] Engel | Currency Unions and International Integration[END_REF] etc.. [START_REF] Artis | International Business Cycles and the ERM : Is there a European Bussiness Cycle?[END_REF] show that during the 80s and 90s, parallel to a better integration in Europe, the degree of correlation of the business cycles economies increased. [START_REF] Frankel | An Estimate of the Effect of Currency Unions on Trade and Output[END_REF] estimate the effects of the monetary unions on the trade and the production by using economic and geographical data for 200 countries. Their results reveal that the positive effects of the monetary unions on the macroeconomic performances of each country are rather due to the trade likages than to an adoption of a common monetary policy or to other types of macroeconomic influence. [START_REF] Frankel | Currency Crashes in Emerging Markets : An Empirical Treatement[END_REF] also analyze the impact of the intensity of the commercial links on the bilateral economic activity and conclude that a more intense commercial link is strongly associated with a high correlation of the economic activities. These authors highlight a negative correlation between the degree of (a) symmetry of the shocks and the trade integration. Finally, a large empirical literature has investigated the shocks persistence in macroeconomics.

This analysis becomes popular in macroeconomics with [START_REF] Nelson | Trends and random walks in macroeconomic time S[END_REF] insights about the stochastic trends. More precisely, they demonstrate that the real gross domestic product follows a unit root model. Other developments have succeeded around this problematic: [START_REF] Cochrane | How big is the random walk in GNP?[END_REF], Rudebusch [1993], Macaro [2007] which argue that the presence of a unit root in the real gross domestic product is uncertain. A much smaller literature (Babetski, [START_REF] Boone | Symmetry and asymmetry of Supply and demand Shocks in the European Union: a Dynamic Analysis[END_REF][START_REF] Babetskii | Exchange Rate Regimes and Supply Shocks Asymmetry: the case of the Accession Countries[END_REF], [START_REF] Boone | L'ancrage de l'Europe Centrale et Orientale à l'Union Européenne[END_REF]) led to a long and fruitful interlude in the development of ideas about economic fluctuations and about methods to be used for the analysis of these fluctuations in the CEECs context.

Some stylized facts

A manner to study the degree of (a) symmetry of the shocks is to compare the business cycles between CEECs and euro zone or EU. Our purpose in this section is to analyze some properties of the business cycles for two important macroeconomic variables. The variables considered are the industrial production (as proxy of GDP) and the consumer price index. In appendix, the tables from 8 to 12 expose the business cycles correlation between EU countries and euro zone.

The results are presented at the same time over the whole period 1994-2004 and on the subperiods (1994-1998 and 1999-2004). It should be noted that the series were broken up using the Hodrick Prescott filter to extract the trend and the cycle. Concerning the industrial production, we note that the business cycles of the CEECs appear more or less correlated with that of the euro area. Compared to the other CEECs, only Slovenia is correlated with the euro area at the same time on the sub-periods and on the whole period. Its business cycle is correlated more with euro area cycle for the sub-period 1999-2004 than on the whole period because of the transition process to the market economy (0.51 for 1999-2004 and 0.41 for 1994-1998). This observation underlines the advantages or the disadvantages related to the possibility to enter into euro zone.

These results incline the balance rather in favour of an integration in the euro zone for Slovenia.

C o n c e r n i n g t h e p r i c e s e v o l u t i o n s , t h e C E E C s f l u c t u a t i o n s a r e v e r y c o r r e l a t e d ( e x c e p t f o r

Slovakia) with those of the euro area particularly on 1999-2004. This fact could be justified by the price stability objective or targeting inflation strategy to join the EU in 2004. We observe that the CEECs correlations of business cycles with the euro area are different between countries: from 0.73 for Estonia or 0.70 for Slovenia to 0.01 for Slovakia on the sub-period 1999-2004. It will be interesting to see if the same results are obtained if we use the co-movement approach.

Methodology and data sources

The modern economies are closely dependent through the trade, the monetary and financial flows and the investments and consequently, events occurring in a country can have effects on other countries. The recent developments on the international transmission of the fluctuations concentrate at the same time on connections between these various countries and on the impacts that those could have on the transmission of the fluctuations. In the academic research, this aspect is studied in terms of volatility and co-movements in the macroeconomic series between country or regions. Even though they cannot claim to be precise, econometric models to estimate co-movements offer quantitative information which merits considerable attention and answer questions about the possible cyclical features of the evolution of economies to which they apply.

Methodology

We estimate a model which combines insights from Alesina, Barro and Tenreyro [2002, ABT], [START_REF] Cochrane | How big is the random walk in GNP?[END_REF] and [START_REF] Ball | Inflation and Uncertainty at Short and Long Horizons[END_REF]. As evidenced by ABT [2002] approach, the price equation corresponding to a pair of countries (i,j) is given by: ln P it /P jt = f 0 + f 1 ln P i,t-1 /P j,t-1 + f 2 ln P i,t-2 /P j,t-2 + ε tij .

(1)

where P it /P jt represent the price level in country i (i.e., one of the CEECs) relative to that in the euro zone noted with j. For each pair of countries, (i, j), we compute then the second order autoregression presented above on quarterly data using the consumer price index. The estimated residual ê t, i, j measures the relative prices that can not be predictable from the two prior values of relative prices. The co-movement of prices VP ij is calculated using the root-mean-squared error: VPij = √(1/T-3)∑(êijt)2 where T = the number of observations which is 37.

(2)

We note that the co-movements are defined using the conditional variance (var x t |x t-1 Λ x t-2 ) which take into account the passed values of our series x t (where x t = ln Pit/Pjt). We can observe that the lower VP ij , the greater the co-movement of prices between countries i and j and therefore, the costs of a monetary union should be lower than the benefits. It is worth noting that, if both series are affected by symmetric shocks, the conditional variance of the ratio of the series should be near to zero.

The same method is applied to compute a measure of output co-movement (VY ij ) using the similar concept: the conditional variance. For each pair of countries, (i, j), the value VY ij comes from the estimated residuals from the second order autoregression on quarterly data for industrial production:

ln (Y it /Y jt ) = g 0 + g 1 ln (Y i,t-1 /Y j,t-1 ) + g 2 ln (Y i,t-2 /Y j,t-2 ) + u tij .
(3)

In the same vain, the estimated residuals û tij measure the outputs that can not be predictable from their two prior values. Using the root-mean-squared error, we compute analogously the output co-movement VY ij :

VY ij = √(1/T-2)∑( û ijt ) 2
where T = 37 (4)

The conditional variance constitutes one of the various indices of sensitivity (also called indices of "uncertainty importance") used by ABT [2002] approach to measure the co-movements. In this paper, we contribute to the extent of co-movements by using another variance index such as the unconditional variance. The unconditional variance can be defined as the variance that don't take into account the passed values of our series x t = lnP it /P jt and provides a best prediction on the autoregressive process. If the unconditional variance refers to the variability of the macroeconomic series, the conditional variance concerns principally their predictability.

According to the law of variance decomposition, the difference between unconditional variance (i.e, the variance of changes in inflation for example) and the conditional variance (i.e, the variance of unanticipated changes in inflation) offer the complementary part of the total variance and may be interpreted as the "amount of the variance of x that is expected to be eliminated

given the past values of x 2 . This strategy supposes to compute the co-movements using the results obtained before. The price co-movement takes then the following form (see appendix):

VPij (1 -Φ2) (5) VP*ij = (1 + Φ2) (1 -Φ2 -Φ1 ) (1 -Φ2 + Φ1)
where VP = var(εtij), VP*ij = var(xt), xt = lnPit/Pjt and the denominator different from zero.

Finally, the production co-movement obtained using the unconditional variance is given by the following expression:

VY ij (1 -Φ 2 ) (6) VY* ij = (1 + Φ 2 ) (1 -Φ 2 -Φ 1 ) (1 -Φ 2 + Φ 1 )
where VY = var(ε tij ), VY* ij = var(x t ), x t = ln Y it /Y jt and the denominator different from zero.

Data sources

Data on production and prices come from the IFS -International Financial Statistics and Eurostat. Combining both sources, we form a panel of countries with quarterly data on outputs and prices from 1995:01 to 2004:01 (i.e, before their accession into EU). For production, we use industrial production index and for prices, the consumer price index with 2000=100. To motivate our own stochastic model, we perform a preliminary analysis of CEECs using data for 1994:01-2004:04. We compute then simple measures to analyze the correlation of business cycles using industrial production index and the consumer price index. .

Results

This section presents our main empirical findings. We estimate our models and look at the relation between the co-movements obtained with both methods. We analyze these relations across countries: for some former EU members and new EU members states. The study of shocks persistence suppose to test the existence of unit roots in the macroeconomic series using different unit roots procedures such us: Augmented Dickey Fuller test (ADF test, 1981) and the Kwiatkowski, Philips, Schmidt and Shin (KPSS test, 1992). The KPSS test (1992) which has countries with very high inflation is similar to the relation in moderate-inflation countries, though somewhat stronger.

stationarity under its null is used for confirming or cancelling ADF tests results and is performed with a Bartlett kernel where the bandwidth parameter is selected using the Newey and West (1994) automatic parameter method. Both procedures are applied to check whether they fit the data. Our estimates indicate that output and prices series follow an AR (2) model. Thus, series are non-stationary in level and have unit roots. A large part of the existing empirical literature on the EU countries has indicated that the euro zone series are less volatile than the national series. The results reported below on the relative size of shocks between CEECs and euro zone will be useful to fit this kind of comparisons.

The second column of tables 1 and 2 shows the results on co-movements using the conditional variance using the ABT method. The third column presents the co-movement using the unconditional variance. The last method has the advantage that it takes into account the volatility of the permanent component (the trend) which is propriety of all autocorrelations. This issue is important because the conventional procedures (ADF procedure) focus usually on the first correlations to fit the short dynamic of the macroeconomic series and sometimes there is a risk of spurious regressions. But, what is important is that the residuals from the regressions (1) and

(3) capture the unanticipated changes in relative prices or in the output differentials between the new members and the former members (i.e, the part not predictable from the two prior members of each equation noted above). By computing the co-movements using the conditional variance and the unconditional variance, we measure the relation between variability and the uncertainty of relative prices or output differentials. As one can observe, the results suggest that the uncertainty-variability distinction is not so important in all computations.

Concerning the new EU member states, the results of the table 1 and 2 show that the comovements of prices are higher than the co-movements of outputs and that they are not systematically correlated. In the other hand, the co-movements of prices of the CEECs are lower than the co-movements of prices of the former EU and euro zone. The smallest co-movement of price is that of the Slovak Republic (0.131 in both cases of the variance indices) follow by the comovement of the Czech Republic (0.0083 or 0.0087). The price's co-movements of Estonia (0.0069) and Slovenia (0.0062) are closer to those of the former EU member's states which are between 0.0030 and 0.0048. The prices co-movements for the Poland (0.0073 or 0.0084) and Hungary (0.0071 or 0.0084) are much closer in terms of the conditional variance. For these countries, the difference between both variance indices is larger than in the case of the other CEECs of our sample. The Tables 3 and4 present the results of our estimations in the case of the former EU countries.

These results are closed to those obtained by ABT [2002] for some EU members between 1960 and 1997 (see tables 5 and 6 in appendix) using annual data. We observe that the output co-movements for CEECs are closed to the co-movements of some countries of euro zone like: Germany, Belgium or Finland. These results are similar to those obtained by ABT [2002] for Norway (0.0210) or Cyprus (0.0227). Again the co-movement of production of the Slovak Republic (0.0305 or 0.0307) followed by the production co-movements of the Czech Republic (0.0277 or 0.0280) are smaller than the other CEECs co-movements which suggest higher costs for abandoning the monetary policy. In other words, their output comovements imply that there are still differences in the level of production with euro zone because of the transition process or the Balassa Samuelson effect. It should be noted that the existence of the conditional convergence in terms of output between CEECs and euro zone should imply that the new member states should beneficiate of the same economic and political context which suggest a capacity to reform the institutions independently of the level of richness.

In contrast, the concept of the unconditional convergence imply that the convergence between regions or countries should be favoured by cultural homogeneity, capital and employment mobility, specific budgetary redistribution to each country etc.

Concerning the prices co-movements, we can argue that they are higher than the output comovements and tend to be closed to those of EU_15 countries. We have also to emphasize that both types of co-movements (output and prices co-movements) aren't systematically correlated which is in line with the ABT results for some European countries for the period 1960-1997.

Conclusions

The focus on this article was double: to provide new estimates about the co-movements of prices and output between CEECs and euro zone before the entry of these countries into EU using two methods and to compare them afterwards with the results obtained for the former members of EU between 1960 and 1997 by ABT researchers. Based on this analysis, we find in the same line with ABT research that the co-movements of prices are higher than the co-movements of production in the CEECs case and they are not systematically correlated. The co-movements of production are much closed to the production co-movements of some Euro zone members which involve lower costs to accession. On the other hand, the prices co-movements of CEECs are lower than the prices co-movements of the former members of EU. It should be interesting to study in a future empirical research the characteristics of the co-movements after the entry of CEECs in EU in order to see if our results would underestimate the potential benefits from joining a currency union (as emphasized also by [START_REF] Frankel | Currency Crashes in Emerging Markets : An Empirical Treatement[END_REF] and ABT ( 2002)).

Appendix 2. The co-movements computed with un-conditional variance

The co-movements computed using the un-conditional variance has the following form:

VP (1 -Φ 2 )

VP* ij = (1)

(1 + Φ 2 ) (1 -Φ 2 -Φ 1 ) (1 -Φ 2 + Φ 1 )

Where VP = var(εtij), VP*ij = var(xt) and xt = ln Pit/Pjt.

Let's demonstrate this relation. We consider the following equation:

X t -f 1 X t-1 -f 2 X t-2 = e t Þ X t = f 1 X t-1 + f 2 X t-2 + e t |. X t -τ
Þ X t X t -τ = f 1 X t-1 X t -τ + f 2 X t-2 X t -τ + e t X t -τ Þ γ τ = E(X t X t -τ ) = E(f 1 X t-1 X t -τ ) + E(f 2 X t-2 X t -τ ) + E(e t X t -τ ).

(2)

Þ γ τ = E(X t X t -τ ) = f 1 E(X t-1 X t -τ ) + f 2 E(X t-2 X t -τ ) + E(e t X t -τ ).

(3)

For τ=0, the relation (3) became: γ 0 = f 1 E(X t-1 X t ) + f 2 E(X t-2 X t ) + E(e t X t ).

Þ γ 0 = f 1 γ 1 + f 2 γ 2 + σ 2 ε Þ σ 2 x =f 1 γ 1 + f 2 γ 2 + σ 2 ε (4)

For τ=1, the relation (3) can be written as follows :

γ 1 = E(X t X t-1 )= f 1 E(X t-1 X t-1 ) + f 2 E(X t-2 X t-1 ) + E(e t X t-1 ).

But, E(e t X t-1 ) = 0.

Þ γ 1 = f 1 σ 2 x + f 2 γ 1 (5) Þ γ 1 -f 2 γ 1 = f 1 σ 2 x Þ γ 1 (1-f 2 ) = f 1 σ 2 x Þ γ 1 = (f 1 σ 2 x )/(1-f 2 ) (6) with 1-f 2 ≠ 0 Þ f 2 ≠ 1.
For τ = 2, the relation (3) becomes:

Þ γ 2 = E(X t X t-2 )= f 1 E(X t-1 X t-2 ) + f 2 E(X t-2 X t-2 ) + E(e t X t-2 ), avec E(e t X t-2 ) = 0, Þ γ 2 = f 1 γ 1 + f 2 σ 2 x (7). If we replaced in (4) the value γ 2 obtained in (7), we have that:

σ 2 x =f 1 γ 1 + f 2 γ 2 + σ 2 ε Þ σ 2 x = f 1 γ 1 + f 2 (f 1 γ 1 + f 2 σ 2 x ) + σ 2 ε Þ Þ σ 2 x = f 1 γ 1 + f 2 f 1 γ 1 + f 2 2 σ 2 x + σ 2 ε Þ σ 2 x -f 2 2 σ 2 x = f 1 γ 1 + f 2 f 1 γ 1 + σ 2 ε Þ Þ σ 2 x (1-f 2 2 ) = f 1 γ 1 + f 2 f 1 γ 1 + σ 2 ε Þ σ 2 x (1-f 2 2 ) = γ 1 (f 1 + f 2 f 1 ) + σ 2 ε Þ σ 2 x (1-f 2 2 ) = [f 1 σ 2 x /(1-f 2 )] (f 1 + f 2 f 1 ) + σ 2 ε Þ σ 2 x (1-f 2 2 ) -[σ 2 x f 2 1 (1 + f 2 )]/(1-f 2 ) = σ 2 ε Þ σ 2 x (1-f 2 2 ) -[f 2 1 (1 + f 2 )/(1-f 2 )]} = σ 2 ε , with 1-f 2 ≠ 0 Þ f 2 ≠ 1.
Finally, we obtain the relation (8) that is, the relation (1) written in a different manner.

σ 2 ε (1-f 2 ) Þ σ 2 x = (8)
(1 + Φ 1 ) (1 -Φ 2 -Φ 1 ) (1 -Φ 2 +Φ 1 )

where VP = var(ε tij ) = σ 2 ε , VP* ij = var(x t ) = σ 2

x and x t = ln P it /P jt .

Table 1 .

 1 Co-movements of relative prices for six CEECs

			Co-movements of prices
	Countries	Conditional Variance	Non-conditional Variance
	Czech Rep.	0.0083	0.0087
	Estonia	0.0069	0.0070
	Hungary	0.0071	0.0084
	Poland	0.0073	0.0076
	Slovak Rep.	0.0131	0.0131
	Slovenia	0.0062	0.0062

Table 2 .

 2 Co-movements of production for six CEECs

		Co-movements of production
	Countries	Conditional Variance	Non-conditional Variance
	Czech Rep.	0.0277	0.0280
	Estonia	0.0279	0.0289
	Hungary	0.0207	0.0208
	Poland	0.0260	0.0266
	Slovak Rep.	0.0305	0.0307
	Slovenia	0.0203	0.0236

Table 3 .

 3 Co-movements of prices for some former EU members

			Co-movements of prices
	Countries	Conditional Variance	Non-conditional Variance
	Austria	0.0048	0.0053
	Belgium	0.0028	0.0035
	Finland	0.0042	0.0042
	France	0.0032	0.0032
	Germany	0.0030	0.0030
	Spain	0.0046	0.0050
	United Kingdom	0.0046	0.0049

Table 4 .

 4 Co-movements of production for some former EU members

		Co-movements of production
	Countries	Conditional Variance	Non-conditional Variance
	Austria	0.0175	0.0200
	Belgium	0.0189	0.0267
	Finland	0.0226	0.0252
	France	0.0167	0.0213
	Germany	0.0226	0.0342
	Spain	0.0158	0.0239
	United Kingdom	0.0157	0.0190

Table 8 : Correlation of output business cycles on the period 1994:01-2004 :04

 8

	Note : Author Calculations

Table 9 : Correlation of output business cycles on the period 1999:01-2004 :04

 9

	Note : Author Calculations

Table 10 : Correlation of output business cycles on the period 1994:01-1998 :12

 10

	Note : Author Calculations

Table 11 : Correlation of output business cycles on the period 1994:01-2004:04

 11

	Note : Author Calculations

Table 12 : Correlation of output business cycles on the period 1999:01-2004 :04

 12

	Note : Author Calculations

Table 13 : Correlation of output business cycles on the period 1994 :01-1998 :12

 13

	Note : Author Calculations

Several findings in the literature[START_REF] Ball | Inflation and Uncertainty at Short and Long Horizons[END_REF] highlight that the inflation-uncertainty relation across countries differs from the relation over time in a given country. Across countries, short-term as well as long-term uncertainty rises with average inflation. Finally, the inflation-uncertainty relation in

Appendix 1: The values of co-movements obtained by ABT method for 1960-1997. 0


Jérémy Béguin
email: jeremy.beguin@vet-alfort.fr

Johann Foloppe

Christelle Maurey

Eve Laloy

Julie Hortelano

Virginie Nourtier

Christelle Pichon

Sandrine Cochin

Pascale Cordier

Hélène Huet

Eric Quemeneur

Bernard Klonjkowski

Philippe Erbs
email: erbs@transgene.fr

Boulevard Gonthier

Preclinical Evaluation of the Oncolytic Vaccinia Virus TG6002 by Translational Research on Canine Breast Cancer

Oncolytic virotherapy is a promising therapeutic approach for the treatment of cancer. TG6002 is a recombinant oncolytic vaccinia virus deleted in the thymidine kinase and ribonucleotide reductase genes and armed with the suicide gene FCU1, which encodes a bifunctional chimeric protein that efficiently catalyzes the direct conversion of the nontoxic 5-fluorocytosine into the toxic metabolite 5-fluorouracil. In translational research, canine tumors and especially mammary cancers are relevant surrogates for human cancers and can be used as preclinical models. Here, we report that TG6002 is able to replicate in canine tumor cell lines and is oncolytic in such cells cultured in 2D or 3D as well as canine mammary tumor explants. Furthermore, intratumoral injections of TG6002 lead to inhibition of the proliferation of canine tumor cells grafted into mice. 5-fluorocytosine treatment of mice significantly improves the anti-tumoral activity of TG6002 infection, a finding that can be correlated with its conversion into 5-fluorouracil within infected fresh canine tumor biopsies. In conclusion, our study suggests that TG6002 associated with 5-fluorocytosine is a promising therapy for human and canine cancers.

INTRODUCTION

Cancer is one of the leading causes of death for both humans and pet animals. Current treatment strategies involve surgery, radiation, chemotherapy, and immunotherapy. Existing therapies often fail to cure cancer; therefore, the development of novel ones is needed. Oncolytic virotherapy is one of the promising approaches. Indeed, a genetically engineered herpes simplex virus (Imlygic) has been approved by the European Medicines Agency and the U.S. Food and Drug Administration for the local treatment of unresectable melanoma. [START_REF] Andtbacka | Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma[END_REF] Oncolytic viruses are naturally occurring or engineered viruses that selectively infect and replicate in cancer cells, inducing oncolysis and antitumoral immune responses while sparing healthy tissue. Targeting of these viruses to cancer cells is based on natural tropism and the deletion of viral genes required for replication in non-dividing cells. A wide array of viruses has been studied in preclinical and clinical studies, including adenovirus, vaccinia virus (VACV), herpes virus, vesicular stomatitis virus, and measles. [START_REF] Miest | New viruses for cancer therapy: meeting clinical needs[END_REF][START_REF] Pol | Trial Watch-Oncolytic viruses and cancer therapy[END_REF] VACV belongs to the Poxviridae family, which comprises large, double-stranded DNA viruses that replicate in the cell cytoplasm. Its ability to multiply, lyse host cells, evade immune responses, and spread across a broad range of mammalian hosts makes it a valid candidate for oncolytic virotherapy. [START_REF] Haddad | Genetically Engineered Vaccinia Viruses As Agents for Cancer Treatment, Imaging, and Transgene Delivery[END_REF] Moreover, an intrinsic tumor tropism has been reported for VACV. [START_REF] Mcfadden | Poxvirus tropism[END_REF] In addition, VACV has a large genome that can host transgenes to increase its oncolytic potency. [START_REF] Haddad | Genetically Engineered Vaccinia Viruses As Agents for Cancer Treatment, Imaging, and Transgene Delivery[END_REF] VACV also displays biosafety due to the absence of DNA integration and the availability of effective antiviral agents. [START_REF] Shen | Fighting cancer with vaccinia virus: teaching new tricks to an old dog[END_REF] To be used as an oncolytic agent, genomic modifications are necessary to selectively target tumor cells and restrict virus propagation to the tumor. VACV can be easily engineered, and numerous viral genes have been deleted to generate a safe and efficient oncolytic vector. [START_REF] Guo | Vaccinia virus-mediated cancer immunotherapy: cancer vaccines and oncolytics[END_REF] One of the outstanding candidates developed so far is Pexa-Vec (JX-594) a thymidine-kinase (TK)deleted Wyeth strain of VACV expressing the granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine to activate immune cells at the tumor site. [START_REF] Kim | Systemic armed oncolytic and immunologic therapy for cancer with JX-594, a targeted poxvirus expressing GM-CSF[END_REF][START_REF] Breitbach | Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans[END_REF] Another highly attenuated oncolytic VACV, designated TG6002, has been developed. [START_REF] Foloppe | The Enhanced Tumor Specificity of TG6002, an Armed Oncolytic Vaccinia Virus Deleted in Two Genes Involved in Nucleotide Metabolism[END_REF] TG6002 is a Copenhagen strain VACV deleted in two genes: TK (J2R) and a subunit of the ribonucleotide reductase (RR; I4L). TG6002 has demonstrated strong tumor selectivity and retained a full capacity to replicate and lyse human cancer cell lines. [START_REF] Foloppe | The Enhanced Tumor Specificity of TG6002, an Armed Oncolytic Vaccinia Virus Deleted in Two Genes Involved in Nucleotide Metabolism[END_REF] TK/RR-deleted VACV replication depends on the cellular pool of TK and RR and, thus, on the expression of cellular TK and RR, which is known to be overexpressed in tumor cells. The double deletion leads to an improved safety profile as a result of very high attenuation in healthy tissues. [START_REF] Foloppe | The Enhanced Tumor Specificity of TG6002, an Armed Oncolytic Vaccinia Virus Deleted in Two Genes Involved in Nucleotide Metabolism[END_REF] TG6002 is armed with the suicide gene FCU1, which encodes a bifunctional chimeric protein that catalyzes the conversion of nontoxic 5-fluorocytosine (5-FC) into toxic metabolites 5-fluorouracil (5-FU) and 5-fluorouridine monophosphate. [START_REF] Erbs | In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene[END_REF] The expression of the FCU1 gene by the virus allows targeted chemotherapy within the tumor. [START_REF] Foloppe | The Enhanced Tumor Specificity of TG6002, an Armed Oncolytic Vaccinia Virus Deleted in Two Genes Involved in Nucleotide Metabolism[END_REF] Much of the research on human diseases relies on animal models. Due to several advantages, the mouse is the most frequently used model. Indeed, technologies are available that allow the modulation of the expression of mouse genes in the entire organism or in selected tissues. [START_REF] Gondo | Next-generation gene targeting in the mouse for functional genomics[END_REF] A major limitation of the mouse model is that tumors are grafted or induced and do not arise spontaneously as in humans or dogs. Furthermore, unlike for human cancers for which disease is polygenic, cancers induced in mouse models involve one or only a few genes. For these reasons, mouse models are not excellent models of human cancers. Spontaneous canine cancers, such as mammary cancers, appear to be more relevant models of human cancers. Canine mammary tumors are naturally occurring cancers that have several features in common with human breast cancer. [START_REF] Gray | Naturally-Occurring Canine Mammary Tumors as a Translational Model for Human Breast Cancer[END_REF] Indeed, clinical presentation, hormonal etiology, environment, histological features, molecular profiles (steroid hormone receptors, proliferation markers, epidermal growth factor, p53 suppressor gene mutations, cyclooxygenases or metalloproteinases), and response and resistance to therapy are quite similar. [START_REF] Visan | In vitro comparative models for canine and human breast cancers[END_REF][START_REF] Donnay | Comparison of estrogen and progesterone receptor expression in normal and tumor mammary tissues from dogs[END_REF][START_REF] Ahern | Expression of the oncogene c-erbB-2 in canine mammary cancers and tumor-derived cell lines[END_REF][START_REF] Kumaraguruparan | Of humans and canines: Immunohistochemical analysis of PCNA, Bcl-2, p53, cytokeratin and ER in mammary tumours[END_REF][START_REF] Sleeckx | Canine mammary tumours, an overview[END_REF][START_REF] Shinoda | Significance of ERa, HER2, and CAV1 expression and molecular subtype classification to canine mammary gland tumor[END_REF] Canine mammary tumors account for almost 50% of all canine neoplasms. Thus, the increased prevalence facilitates timely completion of clinical studies. [START_REF] Moe | Population-based incidence of mammary tumours in some dog breeds[END_REF] Thereby, the evaluation of the potency of oncolytic viruses on spontaneous canine cancers should be valuable for assessing their potential benefit in human medicine.

The first objective of this study was to examine the in vitro susceptibility and rate of replication of double-deleted DI4LDJ2R VACV on 2D and 3D canine tumor models. The second objective was to evaluate the effects of TG6002 in a subcutaneous canine mammary carcinoma xenograft model. Finally, we assessed the susceptibility and oncolytic potency of TG6002 on canine tumor explants.

RESULTS

Susceptibility of Human and Canine Tumor Cell Lines

To evaluate the relative ability of double-deleted VACV to enter and produce infectious progeny in canine tumor cells, we performed a replication assay in a canine mammary carcinoma cell line (REM134) and a canine tumor cell line (A72) in parallel with two well-known human cancer cell lines, HeLa and MDA-MB-231. [START_REF] Else | The characteristics of a canine mammary carcinoma cell line, REM 134[END_REF][START_REF] Binn | Establishment of a canine cell line: derivation, characterization, and viral spectrum[END_REF] As shown in Figure 1A, the A72 cell line produced the highest virus yield with an amplification factor of 2 Â 10 7 after infection at a multiplicity of infection (MOI) of 10 À5 ; 20 times more than the amplification factor in HeLa, 500 times more than in MDA-MB-231, and 1,000 times more than in REM134. The results were equivalent at a MOI of 10 À4 . The REM134 cell line was the least productive for VACV replication but with an amplification factor similar to that obtained in MDA-MB-231. The canine cell lines used in this assay were able to amplify double-deleted VACV as efficiently as human tumor cell lines. These results filled a first requirement in considering the value of recombinant oncolytic VACV in canine cancer. We decided to pursue studies with the less permissive cell line (REM134) to validate the ability of TG6002 to be used as a therapy in veterinary medicine.

VACV Infects, Replicates, and Induces Lysis in REM134 Cells

In Vitro

The overall permissiveness of REM134 cells for VACV was determined using a DI4LDJ2RVACV expressing GFP (VVTG17990) and measuring the percentage of GFP-positive cells at early time points after infection. REM134 cells were susceptible to VACV infection and showed a high transduction efficiency (greater than 90%) at both MOIs of 10 À1 and 1 (Figure 1B). To evaluate the kinetics of TG6002 replication in canine tumor cell lines, we measured the virus yields produced after 24, 48, and 72 h of infection in REM134 cells at a MOI of 10 À3 . A constant increase in yield was observed over the course of the experiment to reach, 72 h post-infection, an amplification factor between 4,000 and 5,000 (Figure 1C). The REM134 cancer cell line was also used in a cell-killing assay to assess the anti-tumor potency of TG6002 at various MOIs. TG6002 was able to kill canine tumor cells in a dose-dependent manner (Figure 1D). Five days post-infection, with TG6002 used at MOIs of 10 À5 and 10 À4 , cell lysis was low. More than 95% of tumor cells were killed at a MOI of 10 À2 , and more than 80% lethality was detected at a lower MOI of 10 À3 , indicating the ability of TG6002 to destroy canine tumor cells (Figure 1D).

TG6002/5-FC Cell Killing by Combination of Prodrug Activation with Viral Oncolysis

Prior to the evaluation of the oncolytic effect of TG6002 in the presence of the prodrug 5-FC, the sensitivity of REM134 to 5-FU was examined at different concentrations to ensure the ability of the chemotherapeutic agent to induce toxicity in the tumor canine cell line. Concentrations of 1 mM and 10 mM 5-FU led to a 90% reduction of cell viability, and a half-maximum inhibitory concentration (IC 50 ) of the order of 0.5 mM was determined, demonstrating 5-FU efficacy on this canine tumor cell line (data not shown). We next compared the anti-proliferative activity of TG6002 alone or combined with 5-FC treatment (Figure 1E). REM134 cells were infected with a suboptimal dose of TG6002 (MOI of 10 À4 ), inducing only 10% of cytotoxicity. 5-FC, added in the culture medium 48 h post-infection, largely killed REM134 cells infected by TG6002 in a prodrug dose-dependent manner. In the presence of 10 mM and 100 mM 5-FC, cell viability was reduced by 40% and 80%, respectively, compared to uninfected cells treated with the same amount of 5-FC (Figure 1E). These results indicate that TG6002 with 5-FC acquired an enhanced in vitro anti-tumor activity in the canine tumor cell line REM134, thanks to the conversion of the 5-FC to the cytotoxic agent 5-FU.

Evaluation of TG6002 in Canine Tumor Spheroids

We investigated the therapeutic potential of TG6002 in a 3D culture system. First, we generated spheroids from the canine mammary carcinoma cell line REM134 by using ultra-low attachment plates. After confirmation of spheroid formation by microscopic visualization at day 4, TG6002 was added into the spheroid cultures, and the kinetics of viral multiplication were determined over a 4-day period (Figure 2A). The data showed that a significant amount of virus was obtained with an amplification factor of around 10,000 in 3 days (Figure 2A). In addition, we determined cell viability in the 3D culture system using the CellTiter-Blue Cell Viability Assay and found that TG6002 induced cytotoxicity in REM134 spheroids in a dosedependent manner (Figure 2B). Moreover, the results indicated that spheroids treated with 10 2 PFUs, 10 3 PFUs, and 10 4 PFUs of TG6002 and 5-FC showed a significant decrease of viability compared with the spheroids treated only with TG6002 (p < 0.05) (Figure 2B).

Immunohistochemical Analyses of Canine Tumor Xenografts

In mice, subcutaneous REM134 tumors are organized into glandular structures with islets of tumor cells surrounded by connective tissue (data not shown). CD31 staining confirmed the vascularization of the xenograft (Figures 3A1, 3A4, and3A7). Expression of GFP was observed in the REM134 tumors 2 weeks after intratumoral (10 6 PFUs) or intravenous (10 7 PFUs) injection of VVTG17990 (Figures 3A4 and3A7, respectively). For both intratumoral and intravenous protocols with VVTG17990, immunohistochemical analyses using cleaved caspase-3 immunostaining confirmed areas of apoptosis in tumor tissues (Figures 3A6 and3A9, respectively). Furthermore, VACV immunostaining (Figures 3A5 and3A8) was observed in the same location as caspase immunostaining, confirming that lysis and apoptosis were induced by VACV. For both intratumoral and intravenous injections, VACV was detected in numerous sites within the tumor, showing that VACV could replicate and invade the tumor (Figures 3A5 and3A8, respectively).

Antitumor Effect of TG6002/5-FC in a Canine Xenograft Tumor Model

Nude mice bearing established REM134 xenografts were treated with intratumoral (10 6 plaque-forming units [PFUs]) or intravenous (10 7 PFUs) injections of TG6002. The animals were then treated with per os administration of water or 5-FC for 3 weeks. As shown in Figure 3B, the administration of 5-FC alone had no effect on REM134 tumor growth. Intravenous injections of TG6002 resulted in a slight inhibition of tumor growth compared with the control groups (no treatment or 5-FC alone). In contrast, the 5-FC treatment significantly improved TG6002 antitumor activity when compared to TG6002 alone (p < 0.05), indicating that the FCU1/5-FC approach compensated for the lack of potent oncolytic activity of TG6002 injected intravenously in this model (Figure 3B). In this canine xenograft tumor model, TG6002 injected intratumorally, without 5-FC treatment, induced a significant antitumor effect, with a reduction of 50% of tumor volume, as compared to the control groups (p < 0.05) (Figure 3B). The administration of 5-FC enhanced the antitumor activity of TG6002 injected intratumorally, resulting in a strong inhibition of tumor growth as compared to the control groups (p < 0.05), with a tumor volume reduction greater than 70%. For mice treated intratumorally with TG6002, from day 35 after tumor implantation, there was a significant difference in tumor size between the groups with and without 5-FC administration (p < 0.05). Thus, our in vivo results suggest that TG6002 with 5-FC can effectively inhibit the growth of canine tumor in a subcutaneous xenograft mice model.

Evaluation of Canine Neoplastic Explants

Three canine grade I 23 complex tubular mammary adenocarcinomas with a Ki67 of 3.48%, 6.26%, or 7.02% were used in this study to assess susceptibility to VACV and oncolytic potency of TG6002 with 5-FC. Tumor samples were obtained from dogs undergoing therapeutic surgical excision, at the National Veterinary School of Alfort, with pet owner consent. Fresh biopsy samples of canine mammary tumors were collected immediately after surgery. To assess the susceptibility of canine mammary tumors to VACV, one sample of each tumor was infected with VVTG17990. GFP fluorescence was observed at the periphery of infected explants after 48 h (Figures 4A2, 4A6, and4A10), and infection progressed toward the center until the fourth day for all samples (Figures 4A4, 4A8, and4A12). Immunohistochemical analyses confirmed the infection by VVTG17990 (Figure 4B).

The level of necrosis was measured on three uninfected samples to evaluate the effect of culture conditions. The percentage of spontaneous necrosis was between 25% and 50% for one biopsy and between 50% and 75% for two biopsies (Figure 5A2). A percentage of necrosis between 25% and 75% was estimated for three canine mammary explants with 5-FU (data not shown).

One sample of 6.26% and one sample of 7.02% Ki67 grade I complex tubular mammary adenocarcinoma were infected, respectively, with 10 6 PFUs or 10 7 PFUs of TG6002 for 6 days. After infection with TG6002 and 5-FC, necrosis was more extensive as compared to the control sample (Figures 5A3 and5A4). Necrosis was estimated to represent 75%-100% of biopsies, regardless of the dosage of TG6002 associated with administration of 5-FC (Figures 5A3 and5A4).

To evaluate FCU1 activity in the infected biopsies, and knowing that 5-FC and 5-FU are diffusible across cells membranes, [START_REF] Erbs | In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene[END_REF] we measured the concentration of 5-FC and 5-FU in the culture supernatants. Analysis of the cell culture supernatants by high-performance liquid chromatography revealed a decrease of 5-FC and an increase in the amount of 5-FU in the extracellular medium of canine mammary tumor explants infected by TG6002 with 5-FC (Figure 5B). This result indicates that TG6002 infects tumor biopsies and produces large amounts of FCU1 that are able to catalyze 5-FC into 5-FU. As expected, the production of 5-FU in the supernatants was dependent on the dose of virus with, 5 days post-infection, 50% and 90% of 5-FC deaminated into 5-FU at 10 6 PFUs and 10 7 PFUs, respectively (Figure 5B). In contrast, no 5-FU was detected in the extracellular medium of non-infected biopsies (Figure 5B). Taken together, our data indicate that TG6002 can infect canine mammary tumor explants inducing expression of functional FCU1 protein.

DISCUSSION

Breast cancer is a major concern in human oncology. It represents the most prevalent cancer and the leading cause of cancer death in women worldwide. [START_REF] Ferlay | Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012[END_REF] Indeed, in 2012, approximately 2 million women were diagnosed with breast cancer. [START_REF] Desantis | International Variation in Female Breast Cancer Incidence and Mortality Rates[END_REF] As a result, the development of new treatments for breast cancer are needed. As in human oncology, canine mammary cancer remains the most common canine cancer, with an estimated annual incidence of 182 per 100,000 female dogs. [START_REF] Merlo | Cancer incidence in pet dogs: findings of the Animal Tumor Registry of Genoa, Italy[END_REF] There is some evidence to suggest that neutering bitches before the age of 2.5 years is associated with a significant reduction in the risk of malignant mammary tumors. [START_REF] Beauvais | The effect of neutering on the risk of mammary tumours in dogs-a systematic review[END_REF] Moreover, this risk may be reduced further by neutering before the first estrous cycle. [START_REF] Beauvais | The effect of neutering on the risk of mammary tumours in dogs-a systematic review[END_REF] A study established that bitches spayed before any estrous cycles had approximately 0.5% of the mammary cancer risk, those that had only one estrous cycle had 8%, and dogs that had two or more estrous cycles before neutering had 26%. [START_REF] Schneider | Factors influencing canine mammary cancer development and postsurgical survival[END_REF] Comparative oncological studies have successfully used canine mammary tumors as a suitable model for human breast cancer research. [START_REF] Vail | Spontaneously occurring tumors of companion animals as models for human cancer[END_REF][START_REF] Porrello | Oncology of companion animals as a model for humans. an overview of tumor histotypes[END_REF][START_REF] Uva | Comparative expression pathway analysis of human and canine mammary tumors[END_REF][START_REF] Rivera | Molecular biological aspects on canine and human mammary tumors[END_REF][START_REF] Pinho | Canine tumors: a spontaneous animal model of human carcinogenesis[END_REF][START_REF] Abdelmegeed | Canine mammary tumors as a model for human disease[END_REF] Human and dog cancers share clinical and etiological similarities such as the age of onset, the spontaneous nature of the tumor, the hormonal etiology, and the course of the disease. [START_REF] Donnay | Comparison of estrogen and progesterone receptor expression in normal and tumor mammary tissues from dogs[END_REF][START_REF] Bernstein | Epidemiology of endocrine-related risk factors for breast cancer[END_REF] Furthermore, many studies have reported significant similarities regarding the expression of molecular markers such as the overexpression of steroid hormone receptors, proliferation markers, epidermal growth factor, p53 suppressor gene mutations, cyclooxygenases, or metalloproteinases. [START_REF] Visan | In vitro comparative models for canine and human breast cancers[END_REF] In addition, similar histological patterns between the two species have been described for mammary cancers. [START_REF] Ahern | Expression of the oncogene c-erbB-2 in canine mammary cancers and tumor-derived cell lines[END_REF][START_REF] Kumaraguruparan | Of humans and canines: Immunohistochemical analysis of PCNA, Bcl-2, p53, cytokeratin and ER in mammary tumours[END_REF][START_REF] Sleeckx | Canine mammary tumours, an overview[END_REF][START_REF] Shinoda | Significance of ERa, HER2, and CAV1 expression and molecular subtype classification to canine mammary gland tumor[END_REF] To improve the relevance of our experimental models, a canine mammary carcinoma cell line and fresh canine mammary tumor biopsies from dogs with spontaneous mammary carcinoma were used. Previous studies have reported a significant inhibition of tumor growth in xenograft models after administration of oncolytic VACV expressing FCU1 with oral administration of 5-FC. [START_REF] Foloppe | The Enhanced Tumor Specificity of TG6002, an Armed Oncolytic Vaccinia Virus Deleted in Two Genes Involved in Nucleotide Metabolism[END_REF][START_REF] Foloppe | Targeted delivery of a suicide gene to human colorectal tumors by a conditionally replicating vaccinia virus[END_REF][START_REF] Fend | Oncolytic virotherapy with an armed vaccinia virus in an orthotopic model of renal carcinoma is associated with modification of the tumor microenvironment[END_REF] In our study, a significant reduction of tumor growth was observed after intratumoral treatment with TG6002 associated with oral 5-FC administration. This emphasizes the relevance of the suicide gene FCU1 to improve treatment response. Even though TG6002 is able to target tumors after intravenous administration, better results were noticed after intratumoral administration. Thus, this route of administration may be a better option in cancer treatment. Prior studies have demonstrated that the double-deleted VACV may also reduce tumor load at sites distant from its local administration, [START_REF] Fend | Immune Checkpoint Blockade, Immunogenic Chemotherapy or IFN-a Blockade Boost the Local and Abscopal Effects of Oncolytic Virotherapy[END_REF] a finding that remains to be investigated in the case of canine tumors. The use of cancer cell lines as experimental models is a common procedure in cancer research. Cell lines are generally cultured in 2D conditions. However, discrepancies with real-life situations may occur, particularly because of variability in cell morphologies, the inability to communicate with other cells, and failure to facilitate metabolic functions. A growing number of publications report the advantages of 3D culture compared to 2D culture. Cell cultures maintained in 3D conditions exhibit morphologic and proliferative features that are closer to the natural state. [START_REF] Vinci | Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation[END_REF] Indeed, spheroids can mimic the tumor microenvironment with high oxygen and nutrition supply at the rim and accumulation of metabolic end products in the core. This can better model neoplasia, with a proliferative periphery and quiescent or necrotic center. Moreover, cells cultured in 3D conditions exhibit cell interactions that can influence the oncolytic activity of the virus and the bystander effect induced by the suicide gene. The kind and extent of tumor cell interactions like gap junctions exert an impact on suicide-gene-armed virotherapy. [START_REF] Finocchiaro | Herpes simplex virus thymidine kinase/ganciclovir system in multicellular tumor spheroids[END_REF] Due to different forms of tumor cell interactions, multiple cell layers, and drug distribution, the bystander effect can differ between 2D and 3D cell cultures. [START_REF] Kostova | An armed, YB-1-dependent oncolytic adenovirus as a candidate for a combinatorial anti-glioma approach of virotherapy, suicide gene therapy and chemotherapeutic treatment[END_REF] Indeed, the spread of 5-FU by diffusion into neighboring uninfected tumor cells is more accurately characterized in 3D culture. Furthermore, cells cultured in 3D conditions appear to be less susceptible to drugs. [START_REF] Loessner | Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells[END_REF][START_REF] Millerot-Serrurot | 3D collagen type I matrix inhibits the antimigratory effect of doxorubicin[END_REF] Thus, the demonstration of a treatment response in 3D cultures can be more relevant and predictive of drug effectiveness. Our results highlight an efficacy of TG6002 combined with 5-FC in spheroids enhancing the demonstration of their oncolytic potency.

Cell lines and xenograft models constitute conventional models to establish efficacy of treatment in oncology. Because of differential gene expression and function in immortalized cells, data obtained do not often match those observed in in vivo studies. [START_REF] Borrell | How accurate are cancer cell lines?[END_REF][START_REF] Weinstein | Drug discovery: Cell lines battle cancer[END_REF] Even if mouse xenograft models are more relevant for pharmacodynamic studies than in vitro systems, several limitations are well described. [START_REF] Day | Preclinical mouse cancer models: a maze of opportunities and challenges[END_REF] Indeed, about 11% of anti-neoplastic therapies that demonstrate efficacy in murine models have been approved for human use. [START_REF] Garden | Companion animals in comparative oncology: One Medicine in action[END_REF] In order to improve translational relevance, oncolytic potency was also investigated in canine biopsies from naturally occurring cancers.

The ex vivo tumor biopsy culture is a very useful experimental tool that provides an alternative approach to evaluate oncolytic potency of drugs. Tumor biopsies maintained ex vivo retain the heterogeneous cell composition, the stromal architecture, and the extracellular matrix of a solid tumor. [START_REF] Kloker | Three-dimensional tumor cell cultures employed in virotherapy research[END_REF] Tissue explants harbor immune cells; in organotypic tissue, these cells have been shown to be able to generate an inflammatory environment and even an antiviral response. [START_REF] Wu | Precision-cut human liver slice cultures as an immunological platform[END_REF] Many experimental systems and culture media that preserve neoplastic features have been developed for the long-term maintenance of tumor explants. [START_REF] Dame | Human colon tissue in organ culture: preservation of normal and neoplastic characteristics[END_REF] Depending on tissue and techniques, the viability of biopsy explants is reported to be several days.

Transduction of VACV was daily assessed by fluorescence microscopy. The increase of fluorescence could reflect the tumor transduction of VACV. This has been confirmed by immunohistochemistry. Little information is available on fresh canine tumor biopsies. Autio et al. [START_REF] Autio | Anti-tumour activity of oncolytic Western Reserve vaccinia viruses in canine tumour cell lines, xenografts, and fresh tumour biopsies[END_REF] assessed the transduction of an oncolytic Western Reserve VACV in a mammary carcinoma, an osteosarcoma, and a grade II mast cell tumor. Assessment of tumor transduction has been more frequently evaluated in human organotypic slices or tissue explants. [START_REF] Duntsch | Recombinant vesicular stomatitis virus vectors as oncolytic agents in the treatment of high-grade gliomas in an organotypic brain tissue slice-glioma coculture model[END_REF][START_REF] Stoff-Khalili | Preclinical evaluation of transcriptional targeting strategies for carcinoma of the breast in a tissue slice model system[END_REF][START_REF] Pennington | The utility of a tissue slice model system to determine breast cancer infectivity by oncolytic adenoviruses[END_REF][START_REF] Potts | Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti-tumor immunity with superior safety in bladder cancer models[END_REF] Most primary tumors are difficult to maintain ex vivo, which is why cell lines are typically preferred in laboratory research. As tumor lysis is expected with the treatment, pre-analytical requirements were needed. Necrosis of samples can be related to the site of sampling, the culture conditions, the oncolytic potency of TG6002, and the production of cytotoxic 5-FU. To avoid necrosis bias induced by sampling, peripheral parts of the tumor were selected. To evaluate necrosis induced by culture conditions and, thus, provide a better evaluation of the lytic potency of TG6002, uninfected samples were cultured in the same conditions. The percentage of necrosis was about 75% to 100% for explants treated with TG6002 and lower for uninfected biopsies. This model suffers from several drawbacks. [START_REF] Udden | The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies[END_REF] First, explants do not grow and proliferate like monolayer cell cultures; therefore, they cannot be expanded. Second, even if additional growth factors are added in culture medium, the culture conditions and the absence of efficient vascularization induce spontaneous cell death in the explants. Third, explants are not able to mount an adaptive immune response. The antitumoral activity of the adaptive immune response cannot be evaluated in this model.

In conclusion, this study demonstrates that TG6002 is able to infect and replicate in canine tumor cell lines and is oncolytic in both cell lines, xenograft models, and canine mammary adenocarcinoma samples. These results suggest that an oncolytic VACV expressing the FCU1 gene may offer an effective treatment in canine mammary tumors. As a first step, evaluations of safety and viral shedding will be performed on healthy dogs before the use of TG6002 in pet dogs. Then TG6002 will be evaluated in dogs with canine breast cancer or other epithelial tumors such as digestive carcinoma, urothelial carcinoma, or head and neck carcinoma. As canine mammary adenocarcinomas are a valuable model for human breast cancer, TG6002 could be a promising therapeutic alternative.

MATERIALS AND METHODS

Cell Culture

Human cervical cancer cell line HeLa, human mammary breast adenocarcinoma cell line MDA-MB-231, canine fibroblast cancer cell line A72, and Vero (African green monkey kidney) cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Canine mammary carcinoma cell line REM134 was obtained from the European Collection of Authenticated Cell Cultures (ECACC: Salisbury, UK). HeLa and A72 cell lines were grown in Iscove's Modified Dulbecco's medium supplemented with 20% fetal bovine serum (FBS). [START_REF] Else | The characteristics of a canine mammary carcinoma cell line, REM 134[END_REF][START_REF] Binn | Establishment of a canine cell line: derivation, characterization, and viral spectrum[END_REF] REM 134 and MDA-MB-231 were cultivated in Eagle's minimal essential medium supplemented with 10% FBS. Vero cells, used for virus titration, were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Primary chicken embryonic fibroblasts (CEFs) were used for recombination, amplification, and production of viral vectors. CEF cells were prepared as previously described and maintained in Eagle-based medium supplemented with 5% FBS. [START_REF] Erbs | Modified vaccinia virus Ankara as a vector for suicide gene therapy[END_REF]

Generation of Recombinant VACVs

All oncolytic VACVs were derived from the Copenhagen strain and are deleted in thymidine kinase (J2R) and in the large subunit of RR (I4L) genes.

TG6002 expressing the fusion gene FCU1 (DI4LDJ2R/FCU1 VACV) under the control of the p11K7.5 promoter was constructed and characterized previously. [START_REF] Foloppe | The Enhanced Tumor Specificity of TG6002, an Armed Oncolytic Vaccinia Virus Deleted in Two Genes Involved in Nucleotide Metabolism[END_REF] The same methods were used to generate the double-deleted VACV expressing GFP (DI4LDJ2R/GFP VACV), designated VVTG17990, by homologous recombination between the previously described DJ2R/GFP VACV and the pDI4L shuttle plasmid containing the selection cassette encoding the guanine phosphoribosyltransferase surrounded by the flanking sequences of VACV I4L gene. [START_REF] Foloppe | Targeted delivery of a suicide gene to human colorectal tumors by a conditionally replicating vaccinia virus[END_REF] TG6002 and VVTG17990 were amplified in CEF and purified, and virus stocks were titrated on CEFs by plaque assay.

Infection Efficiency on Adherent Cell Cultures

To determine the in vitro infection efficiency of double-deleted VACV, 3 Â 10 5 REM134 cells were plated in six-well culture plates and infected with VVTG17990 at MOIs of 10 À3 , 10 À2 , 10 À1 , and 1. Quantitative evaluation of fluorescence was performed 24 h after infection by flow cytometry (BD FACSCanto IITM, BD Biosciences, San Jose, CA, USA). Prior to flow cytometry, single-cell suspensions were fixed with 4% paraformaldehyde (PFA).

Virus Titration Adherent Cell Cultures

HeLa, MDA-MB-231, A72, and REM134 cells were seeded at 3 Â 10 5 cells per well in six-well plates and infected with VVTG17990 at a MOI of 10 À5 or 10 À4 . Four days after infection, cells and culture medium were collected and submitted to a quick freeze-thaw cycle and sonication to release intra-cellular viral particles. For viral replication kinetics, 3 Â 10 5 REM134 cells were plated in six-well culture plates and infected with TG6002 at 300 PFUs per well (MOI of 10 À3 ). At 1, 2, and 3 days post-infection, the virus titers were determined by plaque assay on Vero cells.

Spheroid Cultures

REM134 cells were seeded at 500 cells per well in 96-well ultra-lowattachment plates (Corning, Corning, NY, USA) and allowed to form spheroids for 96 h. Spheroids were then infected with TG6002 at 100 PFUs per well. At 1, 2, 3, and 4 days post-infection, spheroids and supernatant were harvested together and submitted to a quick freezethaw cycle and sonication. Viral titers were determined on Vero cells by plaque assay.

Cell Viability of Infected/Prodrug-Converting Cells REM134 cells were seeded as described earlier for both adherent and spheroid cultures. Adherent cells were infected with TG6002 at MOIs of 10 À5 , 10 À4 , 10 À3 , and 10 À2 . Five days post-infection, cell viability was determined by trypan blue exclusion, using a Vi-CELL Cell Counter (Beckman Coulter, Brea, CA, USA). For sensitivity to 5-FC, adherent REM134 cells were infected with TG6002 at a MOI of 10 À4 . Two days after infection, the cells were exposed to various concentrations of 5-FC for 3 days before determination of cell viability by trypan blue exclusion. To assay cell viability in the 3D model, REM134 spheroids were infected at 10, 10 2 , 10 3 , and 10 4 PFUs per well. Two days after infection, 5-FC (at a final concentration of 1 mM) was added, and cell viability was determined 8 days later by a fluorescent assay (CellTiter-Blue, Promega, Madison, WI, USA) using an automated microplate.

Mouse Experiments

All animal protocols were carried out according to standard operating procedures of the Federation of European Laboratory Animal Science Associations and have been approved by the French Research and Education Ministry. Swiss nude mice were obtained from Charles River Laboratories (Saint-Germain-Nuelles, France). Animals used in the studies were uniform in age (6 weeks) and body weight (20-23 g).

Subcutaneous Tumor Model

For immunohistochemical analysis and evaluation of the therapeutic activity of VACVs in the canine xenograft model, 5 Â 10 6 REM134 cells were injected subcutaneously into the flank of the mice. When tumors reached a volume of 100-150 mm 3 , the mice were randomized in a blinded manner and treated with the indicated vectors.

Immunohistochemical Analyses of Xenograft Tumors

Immunohistochemical analyses were performed on REM134 tumor xenografts resected from mice injected once with VVTG17990 intratumorally at 10 6 PFUs or intravenously at 10 7 PFUs. Tumors were harvested 2 weeks after virus injection and fixed in 4% PFA for 1 h and overnight in 30% sucrose and OCT (coating medium for frozen tissue sections) embedded. To evaluate VACV infection, vascularization, and apoptosis of the tumor, samples were sectioned and stained using the following primary antibodies: rat anti-CD31 monoclonal antibody MC13.3 (dilution 1/500) (557355, BD Biosciences, San Jose, CA, USA), rabbit anti-VACV polyclonal antibody (dilution 1/ 1,000) (B65101R, Meridian Life Science, Memphis, TN, USA), and rabbit anti-cleaved caspase-3 polyclonal antibody (dilution 1/200) (9661, Cell Signaling Technology, Leiden, the Netherlands). In addition to the immunohistochemistry staining, slides were counterstained with DAPI to label nuclear DNA, and direct detection of GFP expression under fluorescence microscopy on tissue section was done.

In Vivo Antitumoral Activity of TG6002 in the REM134 Subcutaneous Model Mice were treated three times intratumorally or intravenously (days 0, 2, and 4) with TG6002 at a dose of 10 6 PFUs or 10 7 PFUs, respectively. Seven days after viral infection, 5-FC was given by oral gavage at 100 mg/kg twice daily for 3 weeks. Tumors were measured, and mice were weighed twice weekly for 25 days.

Canine Mammary Tumor Explants

The study was approved by the Ethics Committee of the National Veterinary School of Alfort (2017-12-01), and all owners had signed the informed consent form. Samples of canine mammary adenocarcinomas were collected immediately after therapeutic surgical excision for three dogs. Canine mammary adenocarcinomas were characterized by Ki67 evaluation. Ki67 antibodies were used on resection specimens. Immunohistochemistry was performed using a DXT automat (Ventana Medical Systems, Roche Diagnostics, Grenzacherstrasse, Switzerland) with the streptavidin-biotin-peroxidase complex method with 3,3 0 -diaminobenzidine (DAB) as a substrate and hematoxylin counterstaining. The primary antibody was a rabbit monoclonal antibody (Ki67 [SP6], RM-9106-S1, Thermo Fisher Scientific, Waltham, MA, USA) at 1/200. For the assessment of Ki67, the slides were digitized and areas devoid of necrosis were selected. Counting was performed manually on at least 500 tumoral epithelial cells per tumor. The number of tumoral epithelial cells with positive nuclear labeling and the total number of tumoral epithelial cells were calculated via ZEN software (Carl Zeiss Microscopy, Oberkochen, Germany). For each tumor, the Ki67 index was calculated by dividing the number of labeled tumoral cells by the total number of tumoral epithelial cells and expressed as a percentage.

Fresh biopsy samples of canine mammary tumors were collected immediately after surgical excision and incubated in 48-well plates in Iscove's modified Dulbecco's medium, 20% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 40 mg/mL gentamycin. Biopsies were incubated at 37 C with a 5% CO 2 level. Biopsy samples were cut into fragments approximately 8 mm in diameter and 4 mm in thickness. To assess susceptibility of canine mammary tumor explants to VACV, three biopsies were infected with 10 6 PFUs of VVTG17990 for 4 days. Evaluation of fluorescence was performed every day using a fluorescence microscope (Zeiss LSM 700 confocal microscope, Oberkochen, Germany). An uninfected biopsy was used as control. After 4 days, biopsies were fixed in 10% neutral buffered formalin for 24 h, and immunohistochemical analyses were performed using a rabbit anti-VACV polyclonal antibody (dilution 1/500) (B65101R, Meridian Life Science, Memphis, TN, USA). Immunohistochemistry analyses were performed using a DXT automat (Ventana Medical Systems, Roche Diagnostics, Grenzacherstrasse, Switzerland) with the streptavidin-biotin-peroxidase complex method with DAB as a substrate and hematoxylin counterstaining formalin-fixed tissue sections.

Paraffin-embedded HeLa cell lines (3 Â 10 5 cells) infected for 24 h with VVTG17990 at a MOI of 10 À1 were used as positive control. Evaluation of oncolytic potency of TG6002 was performed by infection of canine mammary tumor biopsies at the dose of 10 6 or 10 7 PFUs for 6 days. On the second day of infection, 5-FC was added in the culture medium at the concentration of 10 À2 M. Six days after infection, biopsies were fixed in 10% neutral buffered formalin for 24 h. Histological analyses were performed on hematoxylin-eosinsaffron-stained tissue sections. Oncolytic potency of TG6002 was determined by the evaluation of tubular necrosis. Slides were evaluated blindly by a board-certified veterinary pathologist. Tubular necrosis was classified into four groups: 0% to 25%, 25% to 50%, 50% to 75% and 75% to 100%.

To assess viability of biopsies in our culture conditions, histological analysis of the control, cultured 6 days in the same condition without TG6002, was performed. To confirm susceptibility of canine mammary tumors to 5-FU, tubular necrosis was evaluated by histological analysis on a sample cultured with 5-FU (10 À2 M) for 4 days and fixed in 10% neutral buffered formalin for 24 h. Evaluation of cytosine deaminase activity was also quantified by measuring the amount of 5-FU released in the culture media. After 48 h of infection by TG6002 at 10 6 and 10 7 PFUs, 1 mM 5-FC was added to the culture medium containing the biopsies. Every day for 5 days, 5-FC and 5-FU concentrations in the media were measured by high-performance liquid chromatography. Fifty microliters of media were quenched with 50 mL acetonitrile. The samples were vortexed and centrifuged. The organic supernatant was evaporated to dryness and reconstituted in 50 mL water and analyzed by high-performance liquid chromatography using a mobile phase of 50 mM phosphoric acid adjusted to a pH of 2.1. Results are expressed as the percentage of 5-FU relative to the total amount of 5-FC plus 5-FU after various incubation times with 5-FC.

Statistical Analyses

Statistical analyses of surviving cells were performed using a Student's t test. A p value <0.05 was considered to be statistically significant. Statistical analyses of tumor volume were performed using the nonparametric Mann-Whitney U test (Statistica v.7.1 software, Stat-Soft). A p value <0.05 was considered to be statistically significant.

Figure 1 .

 1 Figure 1. Characterization of In Vitro Infection, Replication, and Oncolytic Activity of the Double-Deleted VACV on Canine Cancer Cells Using 2D Monolayer Cultures (A) Amplification factor of VVTG17990 in two canine tumor cell lines (A72 and REM134) and two human tumor cell lines (HeLa and MDA-MB-231) infected at MOIs of 10 À5 and 10 À4 and collected 4 days post-infection. The results are presented as a mean of triplicate experiments ± SD. (B) Susceptibility of REM134 canine tumor cell line to VACV infection. REM134 tumor cells were infected at the indicated MOI with VVTG17990, and the percentage of GFPpositive cells was determined by cytometry at 24 h postinfection. The results were obtained from three separate experiments ± SD. (C) Viral replication kinetics of TG6002 on canine tumor cells. Replication capacity was analyzed through viral growth curve by recovering infected REM134 monolayers (300 PFUs per well) at different time points. The titers of progeny virus were determined by serial dilution titration on Vero cells. The results are presented as a mean of triplicate experiments ± SD. (D) Oncolytic effect of the double-deleted VACV on REM134 canine tumor cells. Cells were infected at MOIs of 10 À5 , 10 À4 , 10 À3 , or 10 À2 with TG6002, and cell viability was measured 5 days later by trypan blue exclusion. The results are presented as a mean of triplicate experiments ± SD. (E) Combination of oncolytic and prodrug cytotoxicity. REM134 canine tumor cells were infected at a MOI of 10 À4 with TG6002. After 48 h, 5-FC was added in a range of concentrations, and cell survival was determined 3 days later by trypan blue exclusion. Results were standardized against values for wells lacking virus and drug, which represented 100% viability. Values are represented as means ± SD of three individual determinations.

Figure 2 .

 2 Figure 2. Characterization of Replication and Oncolytic Activity of TG6002 on a 3D In Vitro REM134 Model (A) Viral replication kinetics of TG6002 on REM134 spheroids. Replication capacity was analyzed through viral growth curve by recovering infected REM134 spheroids (100 PFUs per well) at different time points. The titers of progeny virus were determined by serial dilution titration on Vero cells. The results are presented as a mean of triplicate experiments ± SD. (B) Combination of oncolytic and prodrug cytotoxicity on REM134 spheroids. Spheroids were infected with the indicated doses of TG6002. After 48 h, 5-FC was added at 1 mM, and cell survival was determined 8 days later using the CellTiter-Blue Cell Viability Assay. Results were standardized against values for wells lacking virus and drug, which represented 100% viability. The asterisks indicate a significant difference (p < 0.05) between groups (Student's t test). Values are represented as means ± SD of three individual determinations.

Figure 3 .

 3 Figure 3. In Vivo Anti-tumor Activity of VACV on a REM134 Canine Tumor Xenograft Model (A) In vivo detection of VACV and apoptosis in the REM134 xenograft tumors. Untreated mice (1, 2, and 3) and mice treated intratumorally (10 6 PFUs; 4, 5, and 6) or intravenously (10 7 PFUs; 7, 8, and 9) with VVTG17990 were sacrificed at day 14 post-infection, and macroscopic tumors were removed, formalin fixed, and analyzed by immunohistochemistry. Cellular DNA was stained in blue with DAPI (1-9), GFP was stained in green (1, 4, and 7), CD31 was stained in red (1, 4, and 7), VACV was stained in purple (2, 5, and 8), and activated caspase-3 was stained in orange (3, 6, and 9). Scale bars, 100 mm. (B) Mean tumor volume after intratumoral or systemic administration of TG6002 with 5-FC. Mice bearing REM134 subcutaneous xenografts were treated with three intratumoral (10 6 PFUs) or intravenous (10 7 PFUs) injections of TG6002 (indicated by vertical arrows). Three days after the last virus injection, the animals were treated twice daily with per os administration of water or 5-FC (200 mg/kg/day) for 3 weeks. The data represent mean ± SD of 11 animals.

Figure 4 .

 4 Figure 4. In Vitro Evaluation of VACV Transduction in Canine Mammary Tumor Explants (A) Transduction of VACV in canine mammary tumor explants by fluorescence microscope detecting GFP. Three biopsies were infected with 10 6 PFUs of VVTG17990 per well, and transduction was monitored 2, 3, and 4 days after infection. From left to right, each column corresponds to days 0, 2, 3, and 4. From top to bottom, each line fits to a canine mammary tumor explant (first tumor, 1-4; second tumor, 5-8; and third tumor, 9-12). GFP expression was observed and increased during 4 days after infection with VVTG17990, confirming the susceptibility of three canine grade I tubular complex mammary adenocarcinomas to VACV (magnification, 200Â). (B) Microphotographs: immunohistochemistry for VACV on uninfected canine mammary tumor explant (1) and two canine mammary tumor explants infected with 10 6 PFUs of VVTG17990 (2 and 3); viral antigen is detected in the cytoplasm of tumoral cells (brown staining). Scale bars, 20 mm.

Figure 5 .

 5 Figure 5. In Vitro Evaluation of the Oncolytic Potency of TG6002 with 5-FC in Canine Mammary Tumor Explants (A) Histological microphotographs of uninfected canine mammary tumor explants and infected canine mammary tumor explants with TG6002 and 5-FC. (1) Microphotograph: biopsy of canine mammary tumor collected after surgical excision diagnosis of tubular complex mammary adenocarcinoma, grade I, without necrosis. (2) Microphotograph: explant of canine mammary tumor cultured in culture medium for 6 days after surgical collection. (3 and 4) Microphotographs: explants of canine mammary tumor cultured with TG6002 and 5-FC. Six days after infection by TG6002-10 6 PFUs (3) or 10 7 PFUs (4)-with 5-FC, nuclear pyknosis (arrowheads), cell-to-cell detachment (arrows), and cell debris (asterisks) in the lumen of tubules were noticed. Tumor necrosis ranged from 75% to 100%, regardless of TG6002 dosage. Hematoxylin-eosin-saffron (HES) staining was used. Scale bars, 20 mm. (B) 5-FU generated by canine mammary tumor explant infected by TG6002 upon conversion of 5-FC. A canine mammary tumor biopsy was infected with 10 6 or 10 7 PFUs of TG6002 and then incubated with 1 mM 5-FC from day 2 to day 6 postinfection. The relative concentration of 5-FC and 5-FU in the culture supernatant was measured by high-performance liquid chromatography. The results are expressed as the percentage of 5-FU in the media relative to the total amount of 5-FC plus 5-FU.

Molecular Therapy: Oncolytics Vol. 19 December 2020

Molecular Therapy: Oncolytics Vol. 19 December 2020

ACKNOWLEDGMENTS J.B. is recipient of an Industrial Training Convention for Research (CIFRE) doctoral fellowship.

AUTHOR CONTRIBUTIONS

J.B., J.F., C.M., E.Q., B.K., and P.E. designed the study and analyzed the data. J.B., J.F., E.L., J.H., V.N., C.P., S.C., P.C., and H.H. performed the experiments. J.B., J.F., C.M., B.K., and P.E. wrote the manuscript.

CONFLICTS OF INTEREST

J.B., J.F., J.H., V.N., C.P., S.C., P.C., E.Q., and P.E. were employees of Transgene when the work was performed. Transgene is a publicly traded French biopharmaceutical company, with Institut Merieux as the major shareholder. The other authors declare no other competing interests.