Breeding of the transgenic C57Bl6 Wnt-1 mice with the transgenic mouse strain B6.129P2-Axltm1Dgen/J in order to study the role of Axl in mammary tumor development
1. Study purpose
AXL is a tyrosine kinase transmembrane receptor belonging to the TAM family, which regulates a whole range of cellular responses. In adult tissues, AXL expression is restricted to smooth muscles cells, but elevated levels are found in various disease states such as cancer. In cancer patients, AXl expression is associated with widespread metastasis and poor overall survival. We have previously bred B6.129P2-Axltm1Dgen/J strain to elucidate the role AXL in absence of expression (FOTS 11928). In this project, we want to crossbreed the B6.129P2-Axltm1Dgen/J mouse strain with C57Bl6 Wnt-1 strain where the latter strain develop mammary tumours. Crossing these strains will allow us to assess the tumour incidence and relapse in the absence or presence of AXL expression. Preliminary data suggests to corroborate our hypothesis (FOTS 11927, FOTS 6638).
2. Expected harm to animals
Animals with malignant phenotypes are expected to suffer moderate distress and/or pain. However, measures are taken as to lessen this burden as mush as possible. The animals will be monitored daily for signs of pain and/or distress. They will be sacrificed before they reach human endpoint specified in the score sheet.
3. Expected benefits to scientific community and society
We hypothesize that AXL expression in cancer is associated with metastasis and overall poor prognosis. This project will provide insights into tumour signaling in the presence or absence of AXL expression. These insights can contribute to a larger understanding of cancer malignancies and potentially targets for therapy in the future.
4. Amount of animals and species/strain
We plan to use 500 of each of the B6.129P2-Axltm1Dgen/J and C57Bl6 Wnt-1 strains.
5. Complying with replacement, reduction, and refinement
The purpose of these studies are to understand the role AXL has in cancer malignancies, and to unveil the AXL signaling in the microenvironment. In vitro experiments are done to reduce the number of animals, but to mimic the whole body complexity it is necessary to perform in vivo studies. Furthermore, animals will be used both for in vivo and ex vivo studies, and the breeding will be planned according to the experiments needed in order to reduce amount of animals. As this projects build on previous projects (FOTS 6638, and FOTS 11927), we take advantage of already having established a functional breeding strategy. Homozygous wild type animals are excluded from the colony in order to exclude unwanted phenotype and to decrease the number of not-targeted alleles amongst newborn pups. In order to select animals with targeted genotypes, small biopsies are taken from the ear. This approach allow reduction of animals used in experiments. As the colony is established, backcrossing will be performed using C57bl6/J from The Jackson Laboratory with particular focus on limiting inbred mice in order to maintain healthy animals in the colony. The normal state in these animals does not cause pain and no painful procedures will be performed during breeding. We do however expect some of the wnt-1 positive animals to develop tumours from the age of 10 weeks.
AXL is a tyrosine kinase transmembrane receptor belonging to the TAM family, which regulates a whole range of cellular responses. In adult tissues, AXL expression is restricted to smooth muscles cells, but elevated levels are found in various disease states such as cancer. In cancer patients, AXl expression is associated with widespread metastasis and poor overall survival. We have previously bred B6.129P2-Axltm1Dgen/J strain to elucidate the role AXL in absence of expression (FOTS 11928). In this project, we want to crossbreed the B6.129P2-Axltm1Dgen/J mouse strain with C57Bl6 Wnt-1 strain where the latter strain develop mammary tumours. Crossing these strains will allow us to assess the tumour incidence and relapse in the absence or presence of AXL expression. Preliminary data suggests to corroborate our hypothesis (FOTS 11927, FOTS 6638).
2. Expected harm to animals
Animals with malignant phenotypes are expected to suffer moderate distress and/or pain. However, measures are taken as to lessen this burden as mush as possible. The animals will be monitored daily for signs of pain and/or distress. They will be sacrificed before they reach human endpoint specified in the score sheet.
3. Expected benefits to scientific community and society
We hypothesize that AXL expression in cancer is associated with metastasis and overall poor prognosis. This project will provide insights into tumour signaling in the presence or absence of AXL expression. These insights can contribute to a larger understanding of cancer malignancies and potentially targets for therapy in the future.
4. Amount of animals and species/strain
We plan to use 500 of each of the B6.129P2-Axltm1Dgen/J and C57Bl6 Wnt-1 strains.
5. Complying with replacement, reduction, and refinement
The purpose of these studies are to understand the role AXL has in cancer malignancies, and to unveil the AXL signaling in the microenvironment. In vitro experiments are done to reduce the number of animals, but to mimic the whole body complexity it is necessary to perform in vivo studies. Furthermore, animals will be used both for in vivo and ex vivo studies, and the breeding will be planned according to the experiments needed in order to reduce amount of animals. As this projects build on previous projects (FOTS 6638, and FOTS 11927), we take advantage of already having established a functional breeding strategy. Homozygous wild type animals are excluded from the colony in order to exclude unwanted phenotype and to decrease the number of not-targeted alleles amongst newborn pups. In order to select animals with targeted genotypes, small biopsies are taken from the ear. This approach allow reduction of animals used in experiments. As the colony is established, backcrossing will be performed using C57bl6/J from The Jackson Laboratory with particular focus on limiting inbred mice in order to maintain healthy animals in the colony. The normal state in these animals does not cause pain and no painful procedures will be performed during breeding. We do however expect some of the wnt-1 positive animals to develop tumours from the age of 10 weeks.
Etterevaluering
We consider the administration of substance via oral gavage daily for as long as 2 months to be a procedure with a high risk of physical harm to the animals.
The application describes clear humane endpoints, and we therefore classify the project as moderate, cf. the Regulation annex B. However, we have decided that we will retrospectively assess the experiments.
The application describes clear humane endpoints, and we therefore classify the project as moderate, cf. the Regulation annex B. However, we have decided that we will retrospectively assess the experiments.
Begrunnelse for etterevalueringen
The objective of the experiment was to breed the wnt1 strain with B6.129P2-Axltm1Dgen/J mouse strain in order to check the effect of tumor development in axl knockout mice. The mice heterozygous mice developped tumors as the control mice. The wnt1 mice, homozigous for the truncated axl form showed no axl expression and showed a lower tumor incidence. This finding was included in a publication (Engelsen AST, Wnuk-Lipinska K, Bougnaud S, Pelissier Vatter FA, Tiron C, Villadsen R, Miyano M, Lotsberg ML, Madeleine N, Panahandeh P, Dhakal S, Tan TZ, Peters SD, Grøndal S, Aziz SM, Nord S, Herfindal L, Stampfer MR, Sørlie T, Brekken RA, Straume O, Halberg N, Gausdal G, Thiery JP, Akslen LA, Petersen OW, LaBarge MA, Lorens JB. AXL Is a Driver of Stemness in Normal Mammary Gland and Breast Cancer. iScience. 2020 Oct 7;23(11):101649.) The cells and tumors were collected and may be used for more in vitro characterization of the cells.
654mice were used, none of them had hiegher severity that what was stated in the form. In fact fewer mice than expected had tumors and therefore most of the mice did not develop tumors and therefore display less severity.
We collected the results as doing the experiment. When we reached the wanted results for publication, with significant differences, the experiment was stopped.
The distress of animals was reduced to the minimum but the in vivo experiment was not possible to avoid in order to get the data on tumor incidence we expected. When the data were obtained, the experiment was stopped
654mice were used, none of them had hiegher severity that what was stated in the form. In fact fewer mice than expected had tumors and therefore most of the mice did not develop tumors and therefore display less severity.
We collected the results as doing the experiment. When we reached the wanted results for publication, with significant differences, the experiment was stopped.
The distress of animals was reduced to the minimum but the in vivo experiment was not possible to avoid in order to get the data on tumor incidence we expected. When the data were obtained, the experiment was stopped