Development of Preclinical Models of Mammary Carcinogenesis: Functional Role of Her2 and its Isoforms in Tumor Progression and in Drug Resistance

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

Evolution of HER2-positive mammary carcinoma: HER2 loss reveals claudin-low traits in cancer progression

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

Vaccines against human HER2 prevent mammary carcinoma in mice transgenic for human HER2

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

HER2 isoforms co-expression differently tunes mammary tumor phenotypes affecting onset, vasculature and therapeutic response

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

Early stability and late random tumor progression of a HER2-positive primary breast cancer patient-derived xenograft

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

Three-Dimensional Printed Custom-Made Prostheses after Partial Scapulectomy: A Case Report

This case study focuses on scapula reconstruction using three-dimensional printing in a patient with low-grade osteosarcoma. Malignant tumors originating from the scapula often lead to destructive surgery, with poor functional status and quality of life for the patients. Using custom prosthetic technology through three-dimensional printing could be a possible solution for reconstruction with greater long-term functional outcomes. This study aims to assess the functional outcomes of the reconstruction. A 39-year-old patient with low-grade central osteosarcoma involving the lateral two-thirds of the scapula underwent a custom prosthetic reconstruction. The patient subsequently followed a rehabilitation protocol for 12 months. The results indicate that even though there was a slight decrease in the range of movement, and an increase in the disabilities of the arm, shoulder, and hand (DASH) score, no relevant increase in activities of daily living (ADL) disability was present at follow-up. The patient returned to carry out his daily activities without pain and with a minimal functional reduction in movement. In conclusion, three-dimensional prosthetic reconstruction is a valid alternative for scapula reconstruction, allowing excellent functional and aesthetic results in oncological cases

Primary High-Grade Myxoid Liposarcoma of the Extremities: Prognostic Factors and Metastatic Pattern

(1) Background: This retrospective study aimed to analyze the history and treatment outcomes of localized, high-grade MLS of the extremities. (2) Methods: We retrospectively reviewed 82 patients with primary high-grade MLS of the extremities. OS, LRFS, MFS, PRS, and DFS were analyzed. (3) Results: Five-year OS and LRS were 96% (95% CI: 86–98) and 94% (95% CI: 85–98), respectively. Statistical analysis indicated no risk factors for OS and LFRS. MFS was 77% (65–85) at 5-year follow-up. Size (p = 0.0337) was the only risk factor statistically significant for MFS (HR = 0.248, 95% CI: 0.07–0.84). Median PRS after distant metastasis was 34 months (range: 1–127 months). Five-year PRS was 79% (48–93). Overall, the 5-year DFS was 76% (65–85). (4) Conclusions: Patients with MLS were found to have a good prognosis. In high-grade deep-seated tumors, common risk factors for MLS do not correlate with survival. Tumor size appears to be the only predictor of long-term DSF and MSF

Evaluation of metastatic burden and recovery of human metastatic cells from a mouse model

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

Retroviral and Lentiviral Vectors for the Induction of Immunological Tolerance

Retroviral and lentiviral vectors have proven to be particularly efficient systems to deliver genes of interest into target cells, either in vivo or in cell cultures. They have been used for some time for gene therapy and the development of gene vaccines. Recently retroviral and lentiviral vectors have been used to generate tolerogenic dendritic cells, key professional antigen presenting cells that regulate immune responses. Thus, three main approaches have been undertaken to induce immunological tolerance; delivery of potent immunosuppressive cytokines and other molecules, modification of intracellular signalling pathways in dendritic cells, and de-targeting transgene expression from dendritic cells using microRNA technology. In this review we briefly describe retroviral and lentiviral vector biology, and their application to induce immunological tolerance