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Metastatic dissemination is the major cause of death in cancer. Xenotransplantation of tumor tissue into immunodeficient mice is a widespread preclinical technique to study tumor development. However, preclinical studies on the spreading of metastases were so far hampered by the poor dissemination of malignant human tumors in conventional immunodeficient hosts, like the nude mouse. The development of highly immunodeficient knockout mice spurred a new wave of metastatic model systems. It was recently shown that human HER-2-positive breast cancer cells, which do not metastasize in nude mice, when implanted in knockout mice with severe immunodeficiency, give rise to multiorgan metastatic patterns resembling those observed in human patients. The growth of metastatic nodules in a variety of locations, including brain, lungs, liver, kidneys, adrenals, ovaries, and bone marrow, opens up the problem of quantifying metastatic burden and recovering human metastatic cells from mouse organs for cellular and molecular studies in vitro .Here we used the Mouse Cell Depletion Kit (Miltenyi Biotec) to enrich and quantify metastatic human cells, derived from human breast carcinoma, in a model of brain metastases1 in NOD scid gamma (NOD.Cg- Prkdc scid Il2rg tm1Wjl/SzJ, NSG) mice.

Overexpression of huHER2 occurs in nearly 15–20% of breast cancers, and it is generally associated with poor patient survival. Existing therapies such as trastuzumab and lapatinib are currently used in the treatment of HER2-positive cancers, although issues with high recurrence and acquired resistance still remain. Elucidation of the molecular mechanisms underlying resistance is leading to the identification of therapies and strategies to manage resistance to HER2-targeted therapies. In addition to intrinsic and acquired resistance associated to HER2 oncogene, the induction of bypass pathways that reactivate growth factor-dependent signalling upon oncogene inhibition is likely pervasive across cancers and should be anticipated. Together, these findings underscore that many resistance mechanisms fall into predictable and therapeutically tractable themes, and can be effectively targeted with rationally designed combined therapies. It is, therefore, necessary to come back to dissect HER2 pathway and unravel key features contributing to its transforming capacity. The present thesis, is focused on the role played by HER2-loss variants and Delta16 isoform in mediating HER2 oncogenic activity and in conditioning the response to HER2 therapies in breast cancer. These HER2 phenotypes can drive differential drug responses of the tumor and of distant metastases. Thus, recent investigations on drug resistance and on tumor biology converged to the development of preclinical cancer models representative of cancer heterogeneity and able to mimic all possible scenarios observed in human tumors. In this project, thanks to the availability of several preclinical models representative of HER2 postive breast cancer, it was studied the contribute of HER2 and of its variants to cancer development and drug resistance. In addition, with the purpose of obtaining preclinical models that could best recapitulate human tumor heterogeneity a panel of breast cancer PDX was developed.

We established patient-derived xenografts (PDX) from human primary breast cancers and studied whether stability or progressive events occurred during long-term in vivo passages (up to 4 years) in severely immunodeficient mice. While most PDX showed stable biomarker expression and growth phenotype, a HER2-positive PDX (PDX-BRB4) originated a subline (out of 6 studied in parallel) that progressively acquired a significantly increased tumor growth rate, resistance to cell senescence of in vitro cultures, increased stem cell marker expression and high lung metastatic ability, along with a strong decrease of BCL2 expression. RNAseq analysis of the progressed subline showed that BCL2 was connected to three main hub genes also down-regulated (CDKN2A, STAT5A and WT1). Gene expression of progressed subline suggested a partial epithelial-to-mesenchymal transition. PDX-BRB4 with its progressed subline is a preclinical model mirroring the clinical paradox of high level-BCL2 as a good prognostic factor in breast cancer. Sequential in vivo passages of PDX-BRB4 chronically treated with trastuzumab developed progressive loss of sensitivity to trastuzumab while HER2 expression and sensitivity to the pan-HER tyrosine kinase inhibitor neratinib were maintained. Long-term PDX studies, even though demanding, can originate new preclinical models, suitable to investigate the mechanisms of breast cancer progression and new therapeutic approaches.

HER2-positive breast cancers may lose HER2 expression in recurrences and metastases. In this work, we studied cell lines derived from two transgenic mammary tumors driven by human HER2 that showed different dynamics of HER2 status. MamBo89HER2stable cell line displayed high and stable HER2 expression, which was maintained upon in vivo passages, whereas MamBo43HER2labile cell line gave rise to HER2-negative tumors from which MamBo38HER2loss cell line was derived. Both low-density seeding and in vitro trastuzumab treatment of MamBo43HER2labile cells induced the loss of HER2 expression. MamBo38HER2loss cells showed a spindle-like morphology, high stemness and acquired in vivo malignancy. A comprehensive molecular profile confirmed the loss of addiction to HER2 signaling and acquisition of an EMT signature, together with increased angiogenesis and migration ability. We identified PDGFR-B among the newly expressed determinants of MamBo38HER2loss cell tumorigenic ability. Sunitinib inhibited MamBo38HER2loss tumor growth in vivo and reduced stemness and IL6 production in vitro. In conclusion, HER2-positive mammary tumors can evolve into tumors that display distinctive traits of claudin-low tumors. Our dynamic model of HER2 status can lead to the identification of new druggable targets, such as PDGFR-B, in order to counteract the resistance to HER2-targeted therapy that is caused by HER2 loss.

// Arianna Palladini 1, * , Giordano Nicoletti 2, * , Alessia Lamolinara 3, * , Massimiliano Dall’Ora 1, * , Tania Balboni 1 , Marianna L. Ianzano 1 , Roberta Laranga 1 , Lorena Landuzzi 2 , Veronica Giusti 1 , Claudio Ceccarelli 1, 4 , Donatella Santini 4 , Mario Taffurelli 5 , Enrico Di Oto 6 , Sofia Asioli 6 , Augusto Amici 7 , Serenella M. Pupa 8 , Carla De Giovanni 1 , Elda Tagliabue 8 , Manuela Iezzi 3 , Patrizia Nanni 1 and Pier-Luigi Lollini 1 1 Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy 2 Rizzoli Orthopedic Institute, Laboratory of Experimental Oncology, Bologna, Italy 3 Aging Research Centre, “Gabriele d’Annunzio” University, Chieti, Italy 4 Pathology Unit, Policlinico S.Orsola-Malpighi University Hospital, Bologna, Italy 5 Department of Medical and Surgical Sciences of Bologna, Bologna, Italy 6 Anatomic Pathology, Department of Biomedical and Neuromotor Sciences, Bellaria Hospital, University of Bologna, Bologna, Italy 7 University of Camerino, Camerino, Italy 8 Istituto Nazionale Tumori, Milano, Italy * These authors have contributed equally to this work Correspondence to: Patrizia Nanni, email: patrizia.nanni@unibo.it Keywords: HER2, Delta16, trastuzumab, breast cancer, model of host-tumor interactions Received: December 16, 2016 Accepted: March 22, 2017 Published: April 13, 2017 ABSTRACT Full-length HER2 oncoprotein and splice variant Delta16 are co-expressed in human breast cancer. We studied their interaction in hybrid transgenic mice bearing human full-length HER2 and Delta16 (F1 HER2/Delta16) in comparison to parental HER2 and Delta16 transgenic mice. Mammary carcinomas onset was faster in F1 HER2/Delta16 and Delta16 than in HER2 mice, however tumor growth was slower, and metastatic spread was comparable in all transgenic mice. Full-length HER2 tumors contained few large vessels or vascular lacunae , whereas Delta16 tumors presented a more regular vascularization with numerous endothelium-lined small vessels. Delta16-expressing tumors showed a higher accumulation of i.v. injected doxorubicin than tumors expressing full-length HER2. F1 HER2/Delta16 tumors with high full-length HER2 expression made few large vessels, whereas tumors with low full-length HER2 and high Delta16 contained numerous small vessels and expressed higher levels of VEGF and VEGFR2. Trastuzumab strongly inhibited tumor onset in F1 HER2/Delta16 and Delta16 mice, but not in full-length HER2 mice. Addiction of F1 tumors to Delta16 was also shown by long-term stability of Delta16 levels during serial transplants, in contrast full-length HER2 levels underwent wide fluctuations. In conclusion, full-length HER2 leads to a faster tumor growth and to an irregular vascularization, whereas Delta16 leads to a faster tumor onset, with more regular vessels, which in turn could better transport cytotoxic drugs within the tumor, and to a higher sensitivity to targeted therapeutic agents. F1 HER2/Delta16 mice are a new immunocompetent mouse model, complementary to patient-derived xenografts, for studies of mammary carcinoma onset, prevention and therapy.

INTRODUCTION: The availability of mice transgenic for the human HER2 gene (huHER2) and prone to the development of HER2-driven mammary carcinogenesis (referred to as FVB-huHER2 mice) prompted us to study active immunopreventive strategies targeting the human HER2 molecule in a tolerant host. METHODS: FVB-huHER2 were vaccinated with either IL-12-adjuvanted human HER2-positive cancer cells or DNA vaccine carrying chimeric human-rat HER2 sequences. Onset and number of mammary tumors were recorded to evaluate vaccine potency. Mice sera were collected and passively transferred to xenograft-bearing mice to assess their antitumor efficacy. RESULTS: Both cell and DNA vaccines significantly delayed tumor onset, leading to about 65% tumor-free mice at 70 weeks, whereas mock-vaccinated FVB-huHER2 controls developed mammary tumors at a median age of 45 weeks. In the DNA vaccinated group, 65% of mice were still tumor-free at about 90 weeks of age. The number of mammary tumors per mouse was also significantly reduced in vaccinated mice. Vaccines broke the immunological tolerance to the huHER2 transgene, inducing both humoral and cytokine responses. The DNA vaccine mainly induced a high and sustained level of anti-huHER2 antibodies, the cell vaccine also elicited interferon (IFN)-gamma production. Sera of DNA-vaccinated mice transferred to xenograft-carrying mice significantly inhibited the growth of human HER2-positive cancer cells. CONCLUSIONS: Anti-huHER2 antibodies elicited in the tolerant host exert antitumoral activity.

Homozygous knockout of p53 in mice leads to early mortality from lymphoma, with almost complete penetrance, thus hampering studies of other tumor histotypes related to p53 alterations. To avoid lymphoma development, we crossed p53 knockout mice (BALB-p53 mice) with alymphocytic BALB/c Rag2-/-;Il2rg-/- (RGKO) mice. We compared the tumor spectrum of homozygous (BALB-p53-/-) and heterozygous (BALB-p53+/-) mice with alymphocytic mice (RGKO-p53-/- and RGKO-p53+/-). Lymphoma incidence in BALB-p53-/- mice exceeded 80%, whereas in RGKO-p53-/- it was strongly reduced. The prevalent tumor of RGKO-p53-/- mice was hemangiosarcoma (incidence over 65% in both sexes, mean latency 18 weeks), other tumors included soft tissue sarcomas (incidence ~10%), lung and mammary carcinomas. Tumor spectrum changes occurred also in p53 heterozygotes, in which lymphomas are relatively rare (~20%). RGKO-p53+/- had an increased incidence of hemangiosarcomas, reaching ~30%, and females had an increased incidence of osteosarcomas, reaching ~20%. Osteosarcomas shared with the corresponding human tumors the involvement of limbs and a high metastatic ability, mainly to the lungs. Specific alterations in the expression of p53-related genes (p16Ink4a, p19Arf, p15Ink4b, p21Cip1) were observed. Genetic prevention of lymphoma in p53 knockout mice led to new models of sarcoma development, available for studies on hemangiosarcoma and osteosarcoma onset and metastatization.