Erbb2 transgenic mice: a tool to evaluate the potential efficacy of vaccination in the prevention and cure of carcinomas
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1 1 st International MUGEN Conference on Animal Models for Human Immunological Disease Athens, 11 September 2006 Erbb2 transgenic mice: a tool to evaluate the potential efficacy of vaccination in the prevention and cure of carcinomas elena.quaglino@unito.it
2 Mouse models: To what extent do they predict clinical events? Preservation of the relationship between the tumor and the surrounding tissues Genetic predestination to develop a specific cancer Slowness of tumor progression Natural occurence of metastasis Long-lasting interaction between the tumor and the host immune system
3 Mouse models: They are not devoid of pitfalls Gene alteration foreign to the human setting Pathogenic alteration has no exact equivalence in human tumors Short lifespan of mouse Long telomeres Oncogene expression pattern due to the trasgene promoter Kind and intensity of the immune tolerance to the oncogene protein product
4 MMTV rat neu BALB/c mice (BALB-neuT mice) a- mammary carcinogenesis b- trasgenic for the mutated rat Erbb2 oncogene under the MMTV promoter c- rat Erbb2 expression is evident in the rudimentary mammary glands 3 weeks Di Carlo et al., Lab Invest., 1999
5 MMTV rat neu BALB/c mice (BALB-neuT mice) d- 100% penetrance e- carcinogenesis is completed over the course of 5 months f- 7 months of age: a palpable ca in each mammary gland 33 weeks tumor free mice (%) saline i.v. untreated weeks Di Carlo et al., Lab Invest., 1999
6 MMTV rat neu BALB/c mice (BALB-neuT mice) a- trasgenic for the mutataed rat Erbb2 oncogene under the MMTV promoter b- rat Erbb2 expression is evident in the rudimentary mammary glands c- mammary carcinogenesis d- 100% penetrance e- carcinogenesis is completed over the course of 5 months f- 7 months of age: a palpable ca in each mammary gland g- multistep progression
7 BALB/c BALB-neuT
8 In BALB-neuT mice the mammary lesions are multifocal and scattered all over the gland BALB-neuT tumors As in the human monoclonal disease, these multiple cell clones share the same first mutational lesion Their independent progression replicates the clonal diversification that takes place in human cancer over the decades of its progression, and thus reproduce another key issue of human tumors Pannellini, Univ Chieti
9 BALB-neuT mice week 6 Hyperplastic lesions week 15 week 19 Neoplastic lesions Metastatic lesions Whole mount Erbb2 PCNA
10 Erbb2 expression impacts the immune system of BALB-neuT mice: a- They express rat Erbb2 in the thymus and overexpress it in the mammary glands during tumor progression BALB-neuT BALB/c BALB-neuT BALB/c TUBO cells TSA cel ls rat Erbb2 G3PDH 2 days-old 4 weeks-old
11 ErbB2 expression impacts the immune system of BALB-neuT mice: a- They express rat Erbb2 in the thymus and overexpress it in the mammary glands during tumor progression b- They lack high affinity CTL recognizing the dominant H-2 d class I Erbb2 peptide (TYVPANASL; Nagata et al., J. Immunol. 1997)
12 DNA-vax vs an inexorable genetic predestination to mammary carcinogenesis Amp-R pcmv EC-TM extracellular and transmembrane domains of the rat mutated Erbb2 pcdna3-neo BGH polya ColE1 f1ori SV40 ori 6 wks 10 wks 16 wks
13 The timeframe of prevention tumor free mice (%) wks 16 & 18 One year of age wks 10 & 12 wks 6 & weeks 6 wks 10 wks 16 wks
14 EC-TM electroporation and long lasting memory tumor free mice (%) wks Quaglino et al., Cancer Res., 2004
15 EC-TM electroporation and long lasting memory tumor free mice (%) One year of age wks Quaglino et al., Cancer Res., 2004
16 Mechanisms leading to the blockade of Erbb2 carcinogenesis in BALB-neuT mice a- No protection in IFN-γ KO BALB-neuT mice b- No protection in B cells KO BALB-neuT mice c- Lower protection in FcRγI/III KO BALB-neuT mice d- No protection in CD4+ cell-depleted BALB-neuT mice e- Additive protection afforded by the combined passive transfer of immune sera and CD4+ cells
17 Anti-rat Erbb2 antibodies are the most prominent features associated with carcinoma protection 200 sbp (x10-3 ) Ab4 pcdna3 EC-TM 1 EC-TM 2 rat Her2/neu 0 IgM IgA IgG1 IgG2a IgG2b IgG3 4 C 37 C 37 C Quaglino et al., J. Clin. Invest., 2004
18 Anti-rat Erbb2 antibodies inhibit tumor cell proliferation Empty vector EC-TM Erbb2 PCNA Quaglino et al., J. Clin. Invest., 2004
19 ErbB2 expression impacts the immune system of BALB-neuT mice: a- They express rat Erbb2 in the thymus and overexpress it in the mammary glands during tumor progression b- They lack high affinity CTL recognizing the dominant H-2 d class I Erbb2 peptide c- Myeloid immature suppressor and Treg cells expand during carcinogenesis
20 Erbb2 carcinogenesis in BALB-neuT transgenic mice expands immature myeloid cells Melani et al., Blood, 2003
21 Foxp3+ Treg cells accumulate at the tumor site and in the tumor draining LNs during Erbb2 carcinogenesis Atypical hyperplasia Advanced tumor Tumor draining lymph node Ambrosino et al., Cancer Res., 2006
22 Treatment with PC61 mab & EC-TM weeks of age (50 µg) EC-TM PC61 (0.5 mg) % of CD25 + cells among CD4 + spleen cells mice age 13 wks 17 wks 19 wks 21 wks untreated ctrl PC61 treated
23 Treg depletion enhance the protection in EC-TM vaccinated BALB-neuT mice tumor free mice (%) EC-TM PC wks EC-TM & PC-61
24 EC-TM DNA vaccination in BALB-neuT mice: CONCLUSIONS EC-TM vaccination by intramuscular electroporation is effective in halting tumor progression in BALB-neuT mice Tumor inhibition is mainly due to humoral immune response, while CTL reactive clones are wiped out by tolerance Repeated vaccination boosts are required to induce a long lasting anti-tumor immune memory Treg cell depletion shortly after vaccination is an effective way to induce a long lasting protection Treg cells have a major role in maintaining tolerance towards rat Erbb2 in BALB-neuT mice
25 The BALB-neuT/p53172R-H model a- BALB-neuT mice transgenic for mutated p53 (Arg-His substitution at aa 172) driven by the WAP promoter b- Males and females develop fast-growing and highly metastasizing parotid gland carcinomas c- Progression of mammary carcinomas in females is similar to that of parental BALB-neuT mice Pannellini et al., J. Immunol., 2006
26 Acinar and ductal typical hyperplasia Typical and atypical ductal hyperplasia In situ carcinoma Erbb2 negative poorly differentiated carcinoma rat Erbb2 expression Weeks of age Pannellini et al., J. Immunol., 2006
27 BALB-neuT/p53172 R-H parotid gland carcinomas 7 week 8 week 10 week a b c Erbb2 d e f PCNA Pannellini et al., J. Immunol., 2006
28 Immunopreventive and curative treatment of BALB-neuT/p53172 R-H salivary cancer EC-TM (50 µg) ril12 (100 ng) Preventive vaccination: 5 weeks,, diffuse atypical hyperplasia weeks of age
29 Immunopreventive and curative treatment of BALB-neuT/p53172 R-H salivary cancer EC-TM (50 µg) ril12 (100 ng) Curative vaccination: 7 weeks,, in situ carcinoma weeks of age
30 Timely vaccination prevents parotid carcinomas Treatment started at week Mouse treatment Tumor free/ total mice Tumor multiplicity Preventive vaccination none ril-12 EC-TM EC-TM & ril-12 ril-12 0/19 (0%) 0/10 (0%) 0/10 (0%) 8/11 (73%) 0/8 (0%) 2.00 (38/19) 2.00 (20/10) 1.20 (12/10) 0.27 (3/11) 2.00 (16/8) 7 EC-TM 0/10 (0%) 1.40 (14/10) 7 EC-TM & ril-12 7/24 (29%) 1.00 (24/24) Pannellini et al., J. Immunol., 2006
31 Timely vaccination cures parotid carcinomas Treatment started at week Mouse treatment Tumor free/ total mice Tumor multiplicity - none 0/19 (0%) 2.00 (38/19) 5 ril-12 0/10 (0%) 2.00 (20/10) 5 EC-TM 0/10 (0%) 1.20 (12/10) Curative vaccination EC-TM & ril-12 ril-12 EC-TM EC-TM & ril-12 8/11 (73%) 0/8 (0%) 0/10 (0%) 7/24 (29%) 0.27 (3/11) 2.00 (16/8) 1.40 (14/10) 1.00 (24/24) Pannellini et al., J. Immunol., 2006
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