Radio/ Oncol 1996; 30: 116-9.
Effect of the type of application of Newcastle disease virus on the Ehrlich ascites tumor
Tomaž Milanez, Robert Košak, Anton Cerar
Experimental Department, Institute of Pathology, Medical School of Ljubljana, Slovenia
Newcastle disease virus (NDV) Iws been shown to have an inhibitory effect on the tumours. Most authors use perituinoral application of virus. The purpose of our study was to compare the effects of the ip in contrast to sc application of the vims on the ip and sc transplanted Ehrlich ascites tumor (EAT) in CBA/H mouse. We measured the length of survival, the tumor cure rates, the metastatic rale, and the frequency of ascites and sc tumors in the site of ip EAT injection.
Prolongation of survival after the therapy with NDV in ip transplanted EAT was found. The average lime of survival in control group wcs 70.5 days, ancl 107 and 79.9 days with ip and sc NDV virus therapy respectively. The differences were significant only between control group and the group treated with ip application of NDV. Tumor cure rates were: ipNDV group 30%, scNDV group 20 % and control group 5 %. NDV therapy in sc transplanled EAT prolonged the time of survival; in control group it was 63.3 days, and 75.2 and 65.9 days with ip and sc NDV therapy respectively.
NDV therapy inhibited meiastalic rale ofip transplanted EAT. !nhibition was more effective with ip application of NDV. Virus lherapy also lowered thefrequency of appearance tfasciies and sc tumour in the site ofip EAT injection. In sc transplanted EAT ip application of NDV inhibited the meiastalic mle while in sc applied NDV some stimulalion of metastasation was found.
Ip application of NDV was found to be superior in contrasl to sc applicalion in all ils therapeutic: effects against EAT. Our results show thal lhe liimor inhibition of NDV, in the syslem we used, lws the characteristics ofthe biological response modifiers.
Key words: Carcinoma, Ehrlich tumor; Newcastle di.sea.se virus; survival rate; mice
Introduction
incidental infection,-1 or in therapeutic trial.-4- 5 Reichard et al.'' have found the selective effect on tumor in contrast to normal cells oflive NDV in vino.
Newcastle disease virus (NDV) is a paramyxovirus pathogenic to birds and only slightly to men.1- 2 As an inhibitor of tumor growth it has commanded continuous attention ever since the '50s until recently. The tumour inhibition was found in in vitro systems, on experimental animals, ancl in men after
In in vivo experiments the authors use various modes of virus application but no study compares the effect of different modes. In our experiment we tried to find out if there is a difference in the effect between sc and ip virus application on sc and ip transplanted EAT of mouse. In the case that the differences in the effect are found it would to some degree explain the mechanism of NDV's tumor inhibition.
Correspondence to: Assist. Prof. Anton Cerar, M.D„ Ph. D., Institut of Pathology, Korytkova 2, 1000 Ljubljana, Slovenia.
Abbreviations: BRM-biological response modilier(s), EAT Ehrlich ascites tumor. ipEAT-iiUraperitonealy transplanted Ehrlich ascites tumor. scEAT-subcutaneously transplanted Ehrlich ascites tumor, NDV-Newcaslle disease virus, ipNDV-intraperitonealy applied Newcastle disease virus, scNDV-subcutaneously applied Newcastle disease virus.
We used 120 inbred mice, 8 to I O weeks old, males of CBA/1-1 strain, which were obtained from the Institute Ruder Boskovic, Zagreb. The animals were
Experimental animals
Materials and methods
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Effect oftiie type of application (¡(Newcastle disease virus oil the Ehrlich a.weites tunwr
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provided pelleted Knapka food and tap water ad libintm. The light regime was natural.
Experimental tumor
We used EAT, composed of predominantly hyper-diploicl cells, in ascitic form. EAT was transplanted ip to a donor animal 14 clays before. Tumor cells were counted in a hemocytometer with Trypan blue exclusion test.
The same number of tumor cells was implanted in both experimental ancl control groups. For ip transplantation we used 7.9 x 10J cell s in 0,5 ml of sterile 0.9% NaCl while for se transplantation we used 18.9 x l 0-1 cells in 0.3 ml of sterile 0.9% NaCl per animal. Tumor cells were inoculated into the right inguinal region.
Virus
Wilcl type NDV strain was used. Virus was obtained ancl titrated by the Viral laboratory, Faculty of Veterinary Medicine, Ljubljana. It was cultured in chorioallantoic fluid of 10-clay old embryonated SPF chicken eggs, EID,o was 1075 It was stored at -70"C until application. Before application it was diluted in Hanks' solution in the ratio l:15. For ip and for se application 0.2 ml of viral solution was used. Se application in scEAT groups of animals was in the peritumoral region.
Experimental course
We used 120 mice which were divided in 6 groups, each consisting of 20 animals. EAT was transplanted ip to group 1, 2 and 3 and se to group 4, 5, and 6. The first ancl the fourth group were control, ancl the other 4 were experimental groups, which received NDV either ip or se (Table l ).
The therapy of the groups with ipEAT started on the 7'" day after transplantation, ancl of scEAT groups when the tumor reached the average diameter of 8 mm. The therapy was applied twice a week, during
the total length of the experiment. Mice died spontaneously until 149'" clay when we finished the experiment. The animals which survived were sacrificed by the method of cervical dislocation.
Morphological. Techniques
All the animals were autopsiecl to check the presence, site, and location of the tumor growth and presence of ascitic fluid. The organs, except the brain, were removed ancl fixed in l 0% buffered formalin for macroscopic evaluation of tumor growth in fixed tissues and for the histologic examination. The presence of tumor tissue was confirmed histologically in all animals in at least one specimen. In the cases where animals were sacrificed and the tumor was not found macroscopically, we examined histologically all the organs.
Slatistical Melhods
The result were statistically evaluated with computer statistical package SOLO (BMDP), ancl Logrank test.
Results
The effect of the site of NDV application on survival
In ipEAT groups evident differences in survival of animals were found (Fig.l). In control group the average survival was 70.15 clays, in ipNDV group 106,15 days and in scNDV group 79.9 clays. The difference between control group ancl ipNDV group is statistically significant (p=0.008), but there is no such difference between control group ancl scNDV group (p=0.36).
The influence of application site is also reflected in the number of animals which survived the whole length of experiment (149 days) ancl no tumor was found at morphological analysis: there were 30% such animals in ipNDV group, 20 % in scNDV group and 5 % in the control group.
Table l. Groups of CBA/I-I mice and experimental design of NDV treatment.
* 7,9 x l0' EAT cells for ip and 18.9 x 10' EAT cells for sc transplantation were used per animal. The original virus liter was EID I ()''; 0,2 ml of NDV diluted 1: 15 with Hanks' solution was applied per animal twice weekly.
Group No.,	Mode of EAT	Solution	Mode of solution
(20 mice in each)	transplanlalion	applied	application
I (control)	iP	0,9% NaCl	ip
2	ip	NDV	ip
3	ip se	NDV	se
4 (control)		0,9% NaCl	sc
.5	se	NDV	.sc
6	se	NDV	ip
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group 1 group —6— group 3
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Days after ipEAT transplantation
Figure l. .Survival of ipEAT groups (group 1-control, group 2-ipNDV, group 3-scNDV). The animals that survived 147'" day were sacrificed and showed histologically no tumor growth. 7,9 x 10' EAT cells were transplanted per animal. The original virus liter was E1D 1075; 0,2 ml of NDV diluted 1:15 with Hanks' solution was applied per animal twice weekly.
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Days after scEAT transplantation
Figure 2. Survival of scEAT groups (group 4-control, group 5-ipNDV, group 6-scNDV). The animals died spontaneously. 18,9 x lO4 EAT cells were transplanted per animal. The original virus liter was EID 1075; 0,2 ml of NDV diluted 1:15 with Hanks' solution was applied per animal twice weekly.
Among scEAT groups the differences in survival were smaller than in ipEAT groups (Fig. 2). The average time of survival in control group was 63.3 days, and in ipNDV group 75.2 days and in scNDV group 65.9 days. The differences are not signifi-
cant. All the animals died spontaneously with tumour by l03"' day of the experiment.
The effect of the site of NDV application on the number of metastases
In ipEAT the greatest number of tumors was found in mesentery, pancreas and respiratory diaphragm, and smaller number in organs of small pelvic cavity, kidneys, suprarenal glands and liver. Metastases outside abdominal cavity were found in the lungs, and the lymph nodes (inguinal, axillary). The total number of tumors and metastases found by groups was: control 44, ipNDV 25 and scNDV 34. Ascitic fluid was found in 60 % of animals in the control group and in 25 % and 45 % of animals in ipNDV and scNDV group respectively. The sc tumor in the place of ip EAT injection was found in 70 % of animals in control group and in 45 % and 60 % of animals in ipNDV and scNDV group respectively.
In scEAT groups most of the metastases were in the abdominal organs, while in lungs and lymph nodes they were rare. The total number of metastases per groups are: control 222, ipNDV 16 and scNDV 32.
Discussion
'lie in vivo tumor therapeutic effect of live NDV has been found to depend on many factors of which the virus dose, the virus strain, the regime of application, and the tumor mass seem most known. The authors have also found that the tumor inhibitory effect was best if the NDV was injected into the tumor.-'
The mechanism of tumor inhibition by NDV has been studied quite extensively. One of the first ideas was that virus incorporates into the membranes of tumor cells in the process of budding and in this way changes antigenicity of tumor cells.7 On the other hand, there is no objective evidence, except in tumor-adapted NDV strain," that NDV multiplies in tumor cells. The most argued findings are NDV's effects on the immune system generally through interferon induction," TNF induction and the sensibilization of tumor cells to TNF.10 Some authors have found a selective cytotoxic effect of NDV on tumor cells in vitro."
Usually a few millions of cells are used in the experiments with ipEAT. We used only a few thousands of cells to prolong survival, to allow appearance of more metastases and to obtain a more sensi-
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Effect oflhe lype (»(application ofNewcaslle di.sea.se virus on tile Ehrlich ascites tumor
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ble model lor therapy testing. The regime of NDV application was thai proposed for the biological response modifiers (BRM),11 because viruses are also treated as BRMs.12
Our finding is that ip NDV application has stronger tumor inhibitory effects than sc application. Because virus is a diffusible panicle, which is absorbed afler application by mesothelial pores and endothelial capillary cells into blood system, and the blood supply area of peritoneum is much larger than subcutaneous area, we can expect that in ip application there is much higher concentration of virus in the blood. This is probably why the influence on the involved mechanisms of tumor inhibition is stronger.
The length of survival of experimental groups in ipEAT was longer than in scEAT. This is most probably a consequence of bigger tumor masses in the begining of the virus therapy in scEAT groups. Other authors have found the same effect of tumor mass, using NDV' or TNF therapy.J It is also the guideline for the use of BRMs that they should not be used lor advanced neoplastic diseases.IJ
According to our results, NDV inhibits tumor metastatic rate which is also reflected in the reduced incidence of ascites appearance. Both could be the consequence of the reduced tumor mass on the peritoneal surfaces, as a consequence of NDV influence. It was found in vitro that NDV activates periloneal macrophages which showed a strong cytostatic effect against tumor cells.7- 15 It was also found that the reduction of ascites appearance is an effect of increased immunologic potency.1'' In the scEAT group we found increased metastatic rate after scNDV. This is probably the result of repeated periiumoral injections where we pricked the tumour cells and introduced them into the vessels.
Acknowledgement
Our thanks are due to Prof. Z. Zeleznik lor virus supply and titration, Mrs M. Prebil and Mrs. T Klemenc for preparation of histologic slides. The work was supported by The Ministry of .Science and Technology of The Republic of .Slovenia.
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