Bone sarcoma patient-derived xenografts are faithful and stable preclinical models for molecular and therapeutic investigations

Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches

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

HER2 cancer vaccine optimization by combining Drosophila S2 insect cell manufacturing with a novel VLP-display technology

Breast cancer is a widespread oncology indication affecting more than 1.3 million people worldwide annually, 20%-30% of which are HER2 positive. HER2 is a tyrosine kinase receptor that is frequently overexpressed in several solid-tumor cancers (incl. breast, prostate, gastric, esophageal and osteosarcoma) where it denotes an aggressive phenotype, high metastatic rate, and poor prognosis. In a human context, passive HER2-targeted immunotherapy using monoclonal antibodies (mAb, e.g. Trastuzumab and Pertuzumab) has proven to be an effective treatment modality, which has dramatically improved clinical outcomes. Unfortunately, mAb therapy is very expensive and the repeated injections of high doses can be associated with severe side-effects that reduce efficacy. Vaccines are highly cost-effective, but overall progress in development of anti-cancer vaccines based on cancer-associated antigens (e.g. HER2) has been hampered by inherent immune-tolerogenic mechanisms rendering the immune system incapable of reacting against the body’s own cells/proteins (i.e. self-antigens). Consequently, many attempts to develop anti-cancer vaccines have failed in clinical trials due to insufficient immunogenicity. To circumvent this central issue, we have developed a proprietary virus-like particle (VLP)-based vaccine delivery platform. Notably, the VLP-platform is currently the only available technology to effectively facilitate multivalent “virus-like” display of large/complex vaccine antigens. This is key to overcome immune-tolerance and enable induction of therapeutically potent antibody responses directed against cancer-associated self-antigens. In this talk I will discuss the non-viral Drosophila S2 insect cell production system and how it was applied to the production of hHer2/neu antigen, including using advanced production methods such as perfusion for clinical material manufacture. Furthermore, I will present our data from a transgenic mouse model for spontaneous breast cancer development, where high-density display of the HER2 extracellular domain on the surface of virus-like particles (VLPs) enables induction of therapeutically potent anti-HER2 responses. Split-protein tag/catcher conjugation was used to facilitate directional covalent attachment of HER2 to the surface of icosahedral bacteriophage-derived VLPs, thereby harnessing the VLP platform to effectively overcome B-cell tolerance. Vaccine efficacy was demonstrated both in prevention and therapy of mammary carcinomas in HER2 transgenic mice. Thus, the HER2-VLP vaccine shows promise as a new strategy for treatment of HER2-positive cancer. The modular VLP system may also represent an effective tool for development of self-antigen based vaccines against other non-communicable diseases

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

Multiorgan Metastasis of Human HER-2+ Breast Cancer in Rag2−/−;Il2rg−/− Mice and Treatment with PI3K Inhibitor

In vivo studies of the metastatic process are severely hampered by the fact that most human tumor cell lines derived from highly metastatic tumors fail to consistently metastasize in immunodeficient mice like nude mice. We describe a model system based on a highly immunodeficient double knockout mouse, Rag2−/−;Il2rg−/−, which lacks T, B and NK cell activity. In this model human metastatic HER-2+ breast cancer cells displayed their full multiorgan metastatic potential, without the need for selections or additional manipulations of the system. Human HER-2+ breast cancer cell lines MDA-MB-453 and BT-474 injected into Rag2−/−;Il2rg−/− mice faithfully reproduced human cancer dissemination, with multiple metastatic sites that included lungs, bones, brain, liver, ovaries, and others. Multiorgan metastatic spread was obtained both from local tumors, growing orthotopically or subcutaneously, and from cells injected intravenously. The problem of brain recurrencies is acutely felt in HER-2+ breast cancer, because monoclonal antibodies against HER-2 penetrate poorly the blood-brain barrier. We studied whether a novel oral small molecule inhibitor of downstream PI3K, selected for its penetration of the blood-brain barrier, could affect multiorgan metastatic spread in Rag2−/−; Il2rg−/− mice. NVP-BKM120 effectively controlled metastatic growth in multiple organs, and resulted in a significant proportion of mice free from brain and bone metastases. Human HER-2+ human breast cancer cells in Rag2−/−;Il2rg−/− mice faithfully reproduced the multiorgan metastatic pattern observed in patients, thus allowing the investigation of metastatic mechanisms and the preclinical study of novel antimetastatic agents

Immune targeting of autocrine IGF2 hampers rhabdomyosarcoma growth and metastasis

Abstract Background Insulin-like Growth Factor Receptor-1 (IGF1R) system sustains the genesis of rhabdomyosarcoma through IGF2 autocrine overexpression. While several IGF1R-targeted strategies have been investigated to interphere with rhabdomyosarcoma growth, no attempt to neutralize IGF2 has been reported. We therefore studied the possibility to hamper rhabdomyosarcoma growth with passive and active immune approaches targeting IGF2. Methods A murine model developing IGF2-overexpressing pelvic rhabdomyosarcoma, along with IGF2-independent salivary carcinoma, was used to investigate the efficacy and specificity of passive anti-IGFs antibody treatment. Active vaccinations with electroporated DNA plasmids encoding murine or human IGF2 were performed to elicit autochthonous anti-IGF2 antibodies. Vaccinated mice received the intravenous injection of rhabdomyosarcoma cells to study the effects of anti-IGF2 antibodies against developing metastases. Results Passive administration of antibodies neutralizing IGFs delayed the onset of IGF2-overexpressing rhabdomyosarcoma but not of IGF2-independent salivary carcinoma. A DNA vaccine against murine IGF2 did not elicit antibodies, even when combined with Treg-depletion, while a DNA vaccine encoding the human IGF2 gene elicited antibodies crossreacting with murine IGF2. Mice with anti-IGF2 antibodies were partially protected against the metastatic growth of IGF2-addicted rhabdomyosarcoma cells. Conclusions Immune targeting of autocrine IGF2 inhibited rhabdomyosarcoma genesis and metastatic growth

Genetic prevention of lymphoma in p53 knockout mice allows the early development of p53-related sarcomas

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