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Supplementary Figure 1 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

Microarray cluster analysis

This study evaluated the ability of cytokine-engineered allogeneic (H-2q) HER-2/neu-positive cells to prevent tumor development in mammary cancer-prone virgin female BALB/c (H-2d) mice transgenic for the transforming rat HER-2/neu oncogene (BALB-neuT mice). Repeated vaccinations with cells engineered to release interleukin (IL)-2, IL-12, IL-15, or IFN-γ showed that IL-12-engineered cell vaccines had the most powerful immunopreventive activity, with >80% of 1-year-old BALB-neuT mice free of tumors. On the contrary all of the untreated mice and all of the mice vaccinated with IL-12-engineered cells lacking either HER-2/neu or allogeneic antigens developed mammary carcinomas within 22 or 33 weeks, respectively. Whole mount, histology, immunohistochemistry, and gene expression profile analysis showed that vaccination with IL-12-engineered cells maintained 26-week mammary glands free of neoplastic growth, with a gene expression profile that clustered with that of untreated preneoplastic glands. The IL-12-engineered cell vaccine elicited a high production of IFN-γ and IL-4 and a strong anti-HER-2/neu antibody response. Immune protection was lost or markedly impaired in BALB-neuT mice lacking IFN-γ or antibody production, respectively. The protection afforded by the IL-12-engineered cell vaccine was equal to that provided by the systemic administration of recombinant IL-12 in combination with HER-2/neu H-2q cell vaccine. However, IL-12-engineered cell vaccine induced much lower circulating IL-12 and IFN-γ, and therefore lower potential side effects and systemic toxicity.

Supplementary Figure 2 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

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Supplementary Figure Legend from In silico Modeling and In vivo Efficacy of Cancer-Preventive Vaccinations

Supplementary Methods from In silico Modeling and In vivo Efficacy of Cancer-Preventive Vaccinations

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Supplementary Figure 5 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

Supplementary Figure 6 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

Supplementary Methods from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

Supplementary Figure Legends 1-4, Tables Legends 1-8 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

Cancer vaccine feasibility would benefit from reducing the number and duration of vaccinations without diminishing efficacy. However, the duration of in vivo studies and the huge number of possible variations in vaccination protocols have discouraged their optimization. In this study, we employed an established mouse model of preventive vaccination using HER-2/neu transgenic mice (BALB-neuT) to validate in silico–designed protocols that reduce the number of vaccinations and optimize efficacy. With biological training, the in silico model captured the overall in vivo behavior and highlighted certain critical issues. First, although vaccinations could be reduced in number without sacrificing efficacy, the intensity of early vaccinations was a key determinant of long-term tumor prevention needed for predictive utility in the model. Second, after vaccinations ended, older mice exhibited more rapid tumor onset and sharper decline in antibody levels than young mice, emphasizing immune aging as a key variable in models of vaccine protocols for elderly individuals. Long-term studies confirmed predictions of in silico modeling in which an immune plateau phase, once reached, could be maintained with a reduced number of vaccinations. Furthermore, that rapid priming in young mice is required for long-term antitumor protection, and that the accuracy of mathematical modeling of early immune responses is critical. Finally, that the design and modeling of cancer vaccines and vaccination protocols must take into account the progressive aging of the immune system, by striving to boost immune responses in elderly hosts. Our results show that an integrated in vivo–in silico approach could improve both mathematical and biological models of cancer immunoprevention. Cancer Res; 70(20); 7755–63. ©2010 AACR.

Supplementary Figure 7 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

Supplementary Tables 1-4 from In silico Modeling and In vivo Efficacy of Cancer-Preventive Vaccinations

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Supplementary Figure 1 from In silico Modeling and In vivo Efficacy of Cancer-Preventive Vaccinations

Supplementary Figure 4 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

Supplementary Figure 3 from A Better Immune Reaction to Erbb-2 Tumors Is Elicited in Mice by DNA Vaccines Encoding Rat/Human Chimeric Proteins

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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.

(1) Background: Human epidermal growth factor receptor 2 (HER2)/neu-driven carcinogenesis is delayed by preventive vaccines able to elicit autochthonous antibodies against HER2/neu. Since cooperation between different receptor tyrosine kinases (RTKs) can occur in human as well as in experimental tumors, we investigated the set-up of DNA and cell vaccines to elicit an antibody response co-targeting two RTKs: HER2/neu and the Insulin-like Growth Factor Receptor-1 (IGF1R). (2) Methods: Plasmid vectors carrying the murine optimized IGF1R sequence or the human IGF1R isoform were used as electroporated DNA vaccines. IGF1R plasmids were transfected in allogeneic HER2/neu-positive IL12-producing murine cancer cells to obtain adjuvanted cell vaccines co-expressing HER2/neu and IGF1R. Vaccination was administered in the preneoplastic stage to mice prone to develop HER2/neu-driven, IGF1R-dependent rhabdomyosarcoma. (3) Results: Electroporated DNA vaccines for murine IGF1R did not elicit anti-mIGF1R antibodies, even when combined with Treg-depletion and/or IL12, while DNA vaccines carrying the human IGF1R elicited antibodies recognizing only the human IGF1R isoform. Cell vaccines co-expressing HER2/neu and murine or human IGF1R succeeded in eliciting antibodies recognizing the murine IGF1R isoform. Cell vaccines co-targeting HER2/neu and murine IGF1R induced the highest level of anti-IGF1R antibodies and nearly significantly delayed the onset of spontaneous rhabdomyosarcomas. (4) Conclusions: Multi-engineered adjuvanted cancer cell vaccines can break the tolerance towards a highly tolerized RTK, such as IGF1R. Cell vaccines co-targeting HER2/neu and IGF1R elicited low levels of specific antibodies that slightly delayed onset of HER2/neu-driven, IGF1R-dependent tumors.

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.

The TS/A cell line was established in 1983 from a spontaneous mammary tumor arisen in an inbred BALB/c female mouse. Its features (heterogeneity, low immunogenicity and metastatic ability) rendered the TS/A cell line suitable as a preclinical model for studies on tumor–host interactions and for gene therapy approaches. The integrated biological profile of TS/A resulting from the review of the literature could be a path towards the description of a precision experimental model of mammary cancer.

Mitochondrial DNA mutations are currently investigated as modifying factors impinging on tumor growth and aggressiveness, having been found in virtually all cancer types and most commonly affecting genes encoding mitochondrial complex I subunits. However, it is still unclear whether they exert a pro- or anti-tumorigenic effect. We here analyzed the impact of three homoplasmic mtDNA mutations (m.3460G>A/MT-ND1, m.3571insC/MT-ND1 and m.3243A>G/MT-TL1) on osteosarcoma progression, chosen since they induce different degrees of oxidative phosphorylation impairment. In fact, the m.3460G>A/MT-ND1 mutation caused only a reduction of complex I activity, whereas the m.3571insC/MT-ND1 and the m.3243A>G/MT-TL1 mutations induced a severe structural and functional complex I alteration. As a consequence, this severe complex I dysfunction determined an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation. Osteosarcoma cells carrying such marked complex I impairment displayed a reduced tumorigenic potential both in vitro and in vivo, when compared to cells with mild complex I dysfunction, suggesting that mtDNA mutations may display diverse impact on tumorigenic potential depending on the type and severity of the resulting oxidative phosphorylation dysfunction. The modulation of tumor growth was independent from reactive oxygen species production but correlated with hypoxia inducible factor 1α stabilization, indicating that structural and functional integrity of complex I and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.

The oncogenic versus suppressor roles of mitochondrial genes have long been debated. Peculiar features of mitochondrial genetics such as hetero/homoplasmy and mutation threshold are seldom taken into account in this debate. Mitochondrial DNA (mtDNA) mutations generally have been claimed to be protumorigenic, but they are also hallmarks of mostly benign oncocytic tumors wherein they help reduce adaptation to hypoxia by destabilizing hypoxia-inducible factor-1α (HIF1α). To determine the influence of a disassembling mtDNA mutation and its hetero/homoplasmy on tumorigenic and metastatic potential, we injected mice with tumor cells harboring different loads of the gene MTND1 m.3571insC. Cell cultures obtained from tumor xenografts were then analyzed to correlate energetic competence, apoptosis, α-ketoglutarate (α-KG)/succinate (SA) ratio, and HIF1α stabilization with the mutation load. A threshold level for the antitumorigenic effect of MTND1 m.3571insC mutation was defined, above which tumor growth and invasiveness were reduced significantly. Notably, HIF1α destabilization and downregulation of HIF1α-dependent genes occurred in cells and tumors lacking complex I (CI), where there was an associated imbalance of α-KG/SA despite the presence of an actual hypoxic environment. These results strongly implicate mtDNA mutations as a cause of oncocytic transformation. Thus, the antitumorigenic and antimetastatic effects of high loads of MTND1 m.3571insC, following CI disassembly, define a novel threshold-regulated class of cancer genes. We suggest these genes be termed oncojanus genes to recognize their ability to contribute either oncogenic or suppressive functions in mitochondrial settings during tumorigenesis. Cancer Res; 71(19); 6220–9. ©2011 AACR.

Oncolytic viruses infect, replicate in and kill cancer cells. HSV has emerged as a most promising candidate because it exerts a generally moderate pathogenicity in humans; it is amenable to attenuation and tropism retargeting; the ample genome provides space for heterologous genes; specific antiviral therapy is available in a worst case scenario. The first strategy to convert HSV into an oncolytic agent consisted in deletion of the γ(1) 34.5 gene which counteracts the protein kinase R (PKR) response, and of the UL39 gene which encodes the large ribonucleotide reductase subunit. Tumor specificity resided in low PKR activity, and high deoxyribonucleotides content of cancer cells. These highly attenuated viruses have been and presently are in clinical trials with encouraging results. The preferred route of administration has been intratumor or in tissues adjacent to resected tumors. Although the general population has a high seroprevalence of antibodies to HSV, studies in animals and humans demonstrate that prior immunity is not an obstacle to systemic routes of administration, and that oncolytic HSV (o-HSVs) do populate tumors. As the attenuated viruses undergo clinical experimentation, the research pipeline is developing novel, more potent and highly tumor-specific o-HSVs. These include viruses which overcome tumor heterogeneity in PKR level by insertion of anti-PKR genes, viruses which reinforce the host tumor clearance capacity by encoding immune cytokines (IL-12 or granulocyte-macrophage colony-stimulating factor), and non-attenuated viruses fully retargeted to tumor specific receptors. A strategy to generate o-HSVs fully retargeted to human epidermal growth factor receptor-2 (HER-2) or other cancer-specific surface receptors is detailed.

Ten years after the publication of the position paper “The hallmarks of cancer” (Hanahan and Weinberg Cell 100:57–70, 2000), it has become increasingly clear that mutated cells on their way to giving rise to a tumor have also to learn how to thrive in a chronically inflamed microenvironment, evade immune recognition, and suppress immune reactivity. Genetic and molecular definition of these three immune hallmarks of cancer offers the opportunity to learn how to deploy specific countermeasures to reverse the situation in favor of the immune system and, eventually, the patient. This new information could be channeled to address what seem to be the three major hallmarks for the immune control of cancer progression: effective procedures to activate immune reactivity; characterization of not-disposable oncoantigens; and counteraction of immune suppression.

The prevention of mammary carcinoma by immunological strategies targeting the HER-2/neu receptor has proved to be effective in preclinical models. Thus, a well-characterized HER-2/neu oncogene-driven mammary carcinogenesis model was analysed by various profiling strategies following "triplex" vaccination to identify new candidate targets for breast cancer immunoprevention. 2-DE-based proteomic profiling of preneoplastic and tumour lesions versus normal and aged mammary tissue demonstrated that tumour progression was associated with an up-regulation of molecular chaperones including glucose-regulated protein (GRP)78 and of proteins favouring cell motility, which was in line with the corresponding transcriptomic profiling data. Furthermore, PROTEOMEX analyses suggested that naturally induced autoantibody responses occur during early phases of mammary cancer progression. Most of the cancer progression-induced antibodies targeted proteins of normal and preneoplastic mammary glands. However, three proteins were only recognized by sera obtained from vaccinated mice, including 2 isoforms of annexin A6. The distinct expression patterns for annexin A6 and GRP78 during tumour progression were further verified by western blot and/or immunoprecipitation. In addition, an inhibitor-mediated blockade of GRP78 expression in a model cell line caused a reduced cell growth. Thus, the proteome-based approaches applied in the murine BALB-NeuT model might indeed provide candidates for immunoprevention strategies in breast cancer.

Human tumors are dynamic entities that undergo variation, selection and progression within the patient. How well patient-derived xenografts (PDX) recapitulate tumor dynamics? To investigate these aspects we established a collection of breast cancer PDX, representative of the main intrinsic subtypes. From 66 primary breast cancer specimens, we obtained 5 transplantable PDXs (8%). The highest rate of PDX stabilization was obtained for HER2-positive (40%) followed by triple negative (17%) and luminal B (14%) subtypes. In 3/66 cases a human lymphoma developed without any further evidence of breast cancer. Two HER-2-positive and one non-amplified, HER-2++, luminal B PDXs were serially transplanted in mice for 7-25 passages over a period of 2-4 years. Morphological and immunohistochemical (ER, PR, Ki67, p53, HER2, HER1, HER3, IGFR) features were highly stable over serial passages of PDXs, which retained the histology and the expression pattern of the tumor of origin. After the second passage, one HER-2++ amplified PDX, named BO-HAT4, was split in six different sublines, which were then studied separately to analyze random and selective events in long-term evolution. Two sublines progressively acquired a marked acceleration of tumor growth rate, whereas the remaining four did not. Metastatic spread was absent in early passages and appeared sporadically in late passages. In vitro cultures derived from early in vivo passages showed a rapid onset of cell senescence, whereas late in vivo passages gave rise to long-term in vitro cultures. However, we have not yet been able to obtain continuous cell lines from breast cancer PDX. To study the onset of resistance to HER-2 targeted therapies, sequential in vivo passages of BO-HAT4 were treated with trastuzumab, leading to a progressive loss of sensitivity. After one year of treatment BO-HAT4 was completely resistant to trastuzumab. We are currently investigating the molecular changes underlying trastuzumab resistance. Interestingly, both trastuzumab-sensitive and -resistant tumors were highly inhibited by neratinib. In conclusion, the low take rate as PDX prevents the generalized analysis of human breast cancer patients. Individual PDX allow the analysis of target therapy response and onset of resistance, however long-term study of transplantable breast cancer PDX show that tumor progression in these model systems is a late and random event. Supported by grants from the Italian Association for Cancer Research (AIRC) and the University of Bologna, Italy. Citation Format: Lorena Landuzzi, Marianna L. Ianzano, Claudio Ceccarelli, Enrico Di Oto, Giordano Nicoletti, Veronica Giusti, Roberta Laranga, Tania Balboni, Carla De Giovanni, Massimiliano Dall'Ora, Sofia Asioli, Arianna Palladini, Donatella Santini, Maria Pia Foschini, Mario Taffurelli, Pier-Luigi Lollini, Patrizia Nanni. Functional stability, progression and evolution of targeted drug sensitivity of HER-2-positive breast cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 216.

This study evaluated the effects of combining an OX40 agonistic antibody (aOX40) with a cell vaccine targeting HER2/neu, called “Triplex”. Such HER2/neu cell vaccine included two biological adjuvants (interleukin 12 (IL12) and allogeneic histocompatibility antigens) and was previously found able to prevent autochthonous HER2/neu-driven mammary carcinogenesis. Timing of aOX40 administration, concomitantly or after cell vaccination, gave opposite results. Unexpectedly, vaccine efficacy was hampered by concomitant OX40 triggering. Such decreased immunoprevention was likely due to a reduced induction of anti-HER2/neu antibodies and to a higher level of Treg activation. On the contrary, aOX40 administration after the completion of vaccination slightly but significantly increased immunopreventive vaccine efficacy, and led to increased production of GM-CSF and IL10. In conclusion, OX40 triggering can either impair or ameliorate immunoprevention of HER2/neu-driven mammary carcinogenesis depending on the schedule of aOX40 administration.

Apc(Min) mice are widely used for mechanism and efficacy studies associated with the development of chemopreventive agents. APC10.1 cells have been derived from Apc(Min) mouse adenomas and retain the heterozygous Apc genotype. We tested the hypothesis that this cell type may provide an in vitro model to predict chemopreventive activity of agents in the Apc(Min) mouse in vivo. The growth inhibitory properties of 14 putative colorectal cancer chemopreventive agents, tricin, apigenin, 3',4',5',5,7-pentamethoxyflavone, resveratrol, curcumin, 3,4-methylenedioxy-3',4',5'-trimethoxychalcone (DMU135), 3,4,5,4'-tetramethoxystilbene (DMU212), celecoxib, aspirin, piroxicam, all-trans-retinoic acid, difluoromethylornithine (DFMO), quercetin and cyanidin-3-glucoside, were studied in this cell line, and the IC(50) values were calculated. The IC(50) values were plotted against previously published data of reduction of adenoma numbers caused by these agents in Apc(Min) mice. The correlation co-efficient was 0.678 (p

Rhabdomyosarcoma is a group of soft-tissue sarcomas that share features of skeletal myogenesis, but show extensive heterogeneity in histology, age and site of onset, and prognosis. This review matches recent molecular data with biological features of rhabdomyosarcoma. Alterations in molecular pathways, animal models, cell of origin and potential new therapeutic targets are discussed.

Our studies have shown that constitutive interactions between hyaluronan and CD44 on tumor cells induces various anti-apoptotic cell survival pathways through the formation of a multimeric signaling complex that contains activated receptor tyrosine kinases. Inhibition of the hyaluronan-CD44 interactions on tumor cells by hyaluronan-CD44 interaction antagonists suppresses these activities by disassembling the complex. Although the anti-tumor activity of hyaluronan-oligosaccharides, a hyaluronan-CD44 interaction antagonist, is effective in sensitizing tumor cells to chemotherapeutic agents and reducing tumor growth in xenografts, hyaluronan-oligosaccharide alone was not effective in reducing tumor progression in Apc Min/+ mice. We now show in vitro and in vivo that targeted inhibition of the expression of CD44v6 depletes the ability of the colon tumor cells to signal through hyaluronan-CD44v6 interactions. First, we cloned oligonucleotides coding CD44v6 shRNA into a conditionally silenced pSico vector. Second, using pSico-CD44v6 shRNA and a colon-specific Fabpl promoter-driven Cre recombinase expression vector packaged into transferrin-coated nanoparticles, we successfully delivered the CD44v6 shRNA within pre-neoplastic and neoplastic colon malignant cells. Third, using the Apc Min/+ mice model, we demonstrated that inhibition of the CD44v6 expression reduces the signaling through a hyaluronan/CD44v6-pErbB2-Cox-2 interaction pathway and reduced adenoma number and growth. Together, these data provide insight into the novel therapeutic strategies of short hairpin RNA/nanoparticle technology and its potential for silencing genes associated with colon tumor cells.

The possible interference of tamoxifen with anti-tumor vaccines was studied in a translational view of combined preventive approaches. Tamoxifen treatment of HER-2/neu transgenic mice combined to anti-HER-2/neu cell vaccine did not hamper the efficacy of cancer immunoprevention, and caused a significantly increased production of interferon-gamma. These data suggest that tamoxifen could even have a positive impact on the efficacy of cancer immunoprevention.

Effective prevention of human cancer with vaccines against viruses, such as HBV and HPV, raises the question whether also non-virus related tumors could be prevented with immunological means. Studies in HER-2-transgenic mice showed that powerful anti-HER-2 vaccines, could almost completely prevent the onset of mammary carcinoma. Protective immune responses were orchestrated by T cells and their cytokines, and effected by antibodies against HER-2 gene product p185. Analogous findings were reported in a variety of other cancer immunoprevention systems, thus leading to the definition of oncoantigens, optimal target antigens that are causally involved in carcinogenesis and cancer progression. Prophylactic HER-2 vaccines were also effective in preventing metastasis outgrowth, indicating that concepts and approaches developed for cancer immunoprevention could prove fruitful in cancer immunotherapy as well. The availability of cancer-prone mice carrying a human HER-2 transgene is now fostering the design of novel vaccines against human p185. A further bridge toward human cancer was recently provided by novel immunodeficient models, like Rag2(-/-);Il2rg(-/-) mice, which are permissive for metastatic spread of human HER-2+ cancer cells and can be engrafted with a functional human immune system, allowing for the first time the study of vaccines against oncoantigens to elicit human immune responses against human cancer cells in vivo.

Several autocrine and paracrine growth factor circuits have been found in human rhabdomyosarcoma cells. In this study we show that endothelin-3 (ET-3), a vasoactive peptide, is produced by human rhabdomyosarcoma cell lines, whereas it is not expressed by human sarcoma cell lines of non-muscle origin. We did not find evidence of a significant autocrine loop; nevertheless ET-3 produced by rhabdomyosarcoma cells can act as a paracrine factor, since it promotes migration of endothelial cells. Moreover ET-3 is present in plasma of mice bearing xenografts of human rhabdomyosarcoma cells, and may be potential new marker of the human rhabdomyosarcoma to be studied further.