Index
1. Abstract ... 1
2. Aim of the thesis ... 2
3. Introduction ... 3
3.1 Breast Cancer and Risk factors ... 3
3.2 Variety of breast cancer subtypes ... 6
3.3 Breast Cancer treatments currently used in clinic ... 9
3.4 Oncogenes and tumor suppressors, “drivers” for Breast Cancer ... 10
3.5 Targeted therapy and oncogene dependence... 12
3.6 Targeted therapy in clinical use: the problem of resistance and relapses ... 13
3.7 Transgenic mouse models to study oncogene dependence ... 14
3.8 Oncogenes chosen for the mouse model: Her2 and Myc ... 16
3.9 In vitro complementary approach ... 17
3.10 Survival and polarity pathways: possible targets to reduce the frequency of relapses ... 19
3.11 Test in vivo for the treated cells: Rag1-/- mice and transplantation ... 25
4. Materials and methods ... 27
4.1 DNA extraction protocol from mouse tails ... 27
4.2 PCR Protocols ... 27
4.3 H&E protocol for paraffin sections ... 28
4.4 Mammary glands digestion ... 28
4.5 Reseeding of single epithelial cells from in 3D gels growing acini ... 30
4.6 Trizol RNA Extraction (from 3D Matrigel) ... 31
4.7 cDna Synthesis ... 31
4.8 Quantitative PCR ... 33
4.9 Immunohistochemistry ... 33
4.10 Carmine Alum whole mount mammary gland staining ... 34
4.11 Immunofluorescence of cysts embedded in a collagen:matrigel mix ... 35
4.12 Nuclear Fractionation Protocol from matrigel/collagen gels ... 35
4.13 Protein collection from 3D gels ... 36
4.14 Protein Assay ... 36
4.15 Western Blot... 37
5. Results ... 39
5.1 In vivo tumor progression ... 39
5.3 3D cultures of mammary gland epithelial cells... 44
5.4 Immunofluorescence staining ... 47
5.5 Real-Time PCR for in vitro cultures ... 49
5.6 Treatment to induce cell death in the surviving cells: Part 1 Candidates ... 49
5.7 Treatments to induce cell death in the surviving cells: Part 2 cell culture ... 55
5.8 Analysis of the effects of different inhibitors treatments ... 58
5.9 Immunofluorescence staining of the molecules inhibited ... 59
5.10 Control for the efficiency of the inhibitors: Western blot ... 61
5.11 Clearing the Fat Pad of Rag-/- mice ... 62
5.12 Injection of the treated cells in cleared Rag-/-mice ... 64
6. Discussion ... 69
6.1 Mouse model ... 69
6.2 In vitro system, oncogene dependence, survival of residual cells and relapse ... 69
6.3 Investigations on potential candidates for inhibition of cell survival during targeted treatment ... 71
