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(NSCLCs). Several tyrosine kinase inhibitors (TKIs) have shown high efficacy in patients whose tumors harbour a ROS1 fusion. However, the limited availability of preclinical models of ROS1-positive NSCLC hinders the discovery of new drugs and the understanding of the mechanisms underlying drug resistance and strategies to overcome it. Methods: The ADK-VR2 cell line was derived from the pleural effusion of a treatment-naïve NSCLC patient bearing SDC4-ROS1 gene fusion. The sensitivity of ADK-VR2 and its crizotinib-resistant clone ADK-VR2 AG143 (selected in 3D culture in the presence of crizotinib) to different TKIs was tested in vitro, in both 2D and 3D conditions. Tumorigenic and metastatic ability was assessed in highly immunodeficient mice. In addition, crizotinib efficacy on ADK-VR2 was evaluated in vivo. Results: 2D-growth of ADK-VR2 cells was partially inhibited by crizotinib. On the contrary, the treatment with other TKIs, such as lorlatinib, entrectinib and DS-6051b, did not result in cell growth inhibition. TKIs showed dramatically different efficacy on ADK-VR2 cells, depending on the cell culture conditions. In 3D culture, ADK-VR2 growth was indeed almost totally inhibited by lorlatinib and DS-6051b. The clone ADK VR2 AG143 showed higher resistance to crizotinib treatment in vitro, compared to its parental cell line, in both 2D and 3D cultures. Similarly to ADK-VR2, ADK-VR2 AG143 growth was strongly inhibited by lorlatinib in 3D conditions. Nevertheless, ADK-VR2 AG143 sphere formation was less affected by TKIs treatment, compared to the parental cell line. In vivo experiments highlighted the high tumorigenic and metastatic ability of ADK-VR2 cell line, which, once injected in immunodeficient mice, gave rise to both spontaneous and experimental lung metastases while the crizotinib-resistant clone ADK-VR2 AG143 showed a slower growth in vivo. In addition, ADK-VR2 tumor growth was significantly reduced but not eradicated by crizotinib treatment. Conclusions: The ADK-VR2 cell line is a promising NSCLC preclinical model for the study

Additional materials and methods

Silencing of DNMTs inhibits cell proliferation, induces osteoblastic differentiation and enhances DXR sensitivity

Inhibitory activity of MC3343 against human DNMT1 and human DNMT3a

Weight variations of Rag2 -/-;IL2rg -/- mice in single and combined treatments

The identification of new therapeutic strategies against osteosarcoma, the most common primary bone tumor, continues to be a primary goal to improve the outcomes of patients refractory to conventional chemotherapy. Osteosarcoma originates from the transformation of mesenchymal stem cells (MSC) and/or osteoblast progenitors, and the loss of differentiation is a common biological osteosarcoma feature, which has strong significance in predicting tumor aggressiveness. Thus, restoring differentiation through epigenetic reprogramming is potentially exploitable for therapeutic benefits. Here, we demonstrated that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 affected tumor proliferation by blocking osteosarcoma cells in G1 or G2–M phases and induced osteoblastic differentiation through the specific reexpression of genes regulating this physiologic process. Although MC3343 has a similar antiproliferative effect as 5azadC, the conventional FDA-approved nucleoside inhibitor of DNA methylation, its effects on cell differentiation are distinct. Induction of the mature osteoblast phenotype coupled with a sustained cytostatic response was also confirmed in vivo when MC3343 was used against a patient-derived xenograft (PDX). In addition, MC3343 displayed synergistic effects with doxorubicin and cisplatin (CDDP), two major chemotherapeutic agents used to treat osteosarcoma. Specifically, MC3343 increased stable doxorubicin bonds to DNA, and combined treatment resulted in sustained DNA damage and increased cell death. Overall, this nonnucleoside DNMTi is an effective novel agent and is thus a potential therapeutic option for patients with osteosarcoma who respond poorly to preadjuvant chemotherapy. Mol Cancer Ther; 17(9); 1881–92. ©2018 AACR.

Osteogenesis RT2 Profiler TM PCR Array in Saos-2 cell line after 7 and 14 days of induced osteogenesis and DNMT inhibitors treatment

Supplementary Data from NVP-BEZ235 as a New Therapeutic Option for Sarcomas

Purpose: To evaluate the in vitro and in vivo effects of NVP-BEZ235, a dual pan-phosphoinositide 3-kinase–mammalian target of rapamycin inhibitor in the three most common musculoskeletal tumors (osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma).Experimental Design: Antiproliferative activity as well as the effects on migration and metastasis were evaluated in a panel of osteosarcoma, Ewing's sarcoma, as well as rhabdomyosarcoma cell lines. Moreover, simultaneous and sequential treatments were done in association with two of the most important conventional drugs in the treatment of sarcoma, doxorubicin and vincristine.Results: NVPBEZ235 effectively blocked the pathway in in vitro and in vivo settings. Under the experimental conditions tested, the compound induced disease stasis, by arresting cells in G1 phase of cell cycle, without remarkable effects on apoptosis. As a consequence, to obtain the maximum exploitation of its therapeutic potential, NVP-BEZ235 has been evaluated in combination with conventional cytotoxic agents, thus showing promising efficacy with either doxorubicin and vincristine. Inhibition of the phosphoinositide 3-kinase/mammalian target of rapamycin pathway increased activation of extracellular signal-regulated kinase 1/2, likely due to the presence of autocrine circuits shifting growth factor signaling toward the mitogen-activated protein kinase pathway. This supports the combined use of NVP-BEZ235 with other small signaling inhibitors. Here, we showed synergistic effects when the compound was associated with a anti–insulin-like growth factor-I receptor tyrosine kinase inhibitor. NVP-BEZ235 also inhibited cell migration and metastasis. Combination with vincristine further potentiated the antimetastatic effects.Conclusions: NVP-BEZ235 displays the features to be considered for sarcoma therapy to potentiate the activity of other anticancer agents. The drug is currently undergoing phase I/II clinical trials in advanced cancer patients. Clin Cancer Res; 16(2); 530–40

Supplementary Data from Integrated Molecular Characterization of Patient-Derived Models Reveals Therapeutic Strategies for Treating CIC-DUX4 Sarcoma

Capicua-double homeobox 4 (CIC-DUX4)–rearranged sarcomas (CDS) are extremely rare, highly aggressive primary sarcomas that represent a major therapeutic challenge. Patients are treated according to Ewing sarcoma protocols, but CDS-specific therapies are strongly needed. In this study, RNA sequencing was performed on patient samples to identify a selective signature that differentiates CDS from Ewing sarcoma and other fusion-driven sarcomas. This signature was used to validate the representativeness of newly generated CDS experimental models—patient-derived xenografts (PDX) and PDX-derived cell lines—and to identify specific therapeutic vulnerabilities. Annotation analysis of differentially expressed genes and molecular gene validation highlighted an HMGA2/IGF2BP/IGF2/IGF1R/AKT/mTOR axis that characterizes CDS and renders the tumors particularly sensitive to combined treatments with trabectedin and PI3K/mTOR inhibitors. Trabectedin inhibited IGF2BP/IGF2/IGF1R activity, but dual inhibition of the PI3K and mTOR pathways was required to completely dampen downstream signaling mediators. Proof-of-principle efficacy for the combination of the dual AKT/mTOR inhibitor NVP-BEZ235 (dactolisib) with trabectedin was obtained in vitro and in vivo using CDS PDX-derived cell lines, demonstrating a strong inhibition of local tumor growth and multiorgan metastasis. Overall, the development of representative experimental models (PDXs and PDX-derived cell lines) has helped to identify the unique sensitivity of the CDS to AKT/mTOR inhibitors and trabectedin, revealing a mechanism-based therapeutic strategy to fight this lethal cancer.Significance:This study identifies altered HMGA2/IGF2BP/IGF2 signaling in CIC-DUX4 sarcomas and provides proof of principle for combination therapy with trabectedin and AKT/mTOR dual inhibitors to specifically combat the disease.

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

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 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

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

Vaccines are a promising therapeutic alternative to monoclonal antibodies against HER-2

BackgroundMetastatic prostate cancer (PC) is a leading cause of cancer death in men worldwide. Targeting of the culprits of disease progression is an unmet need. Interleukin (IL)-30 promotes PC onset and development, but whether it can be a suitable therapeutic target remains to be investigated. Here, we shed light on the relationship between IL30 and canonical PC driver genes and explored the anti-tumor potential of CRISPR/Cas9-mediated deletion of IL30.MethodsPC cell production of, and response to, IL30 was tested by flow cytometry, immunoelectron microscopy, invasion and migration assays and PCR arrays. Syngeneic and xenograft models were used to investigate the effects of IL30, and its deletion by CRISPR/Cas9 genome editing, on tumor growth. Bioinformatics of transcriptional data and immunopathology of PC samples were used to assess the translational value of the experimental findings.ResultsHuman membrane-bound IL30 promoted PC cell proliferation, invasion and migration in association with STAT1/STAT3 phosphorylation, similarly to its murine, but secreted, counterpart. Both human and murine IL30 regulated PC driver and immunity genes and shared the upregulation of oncogenes, BCL2 and NFKB1, immunoregulatory mediators, IL1A, TNF, TLR4, PTGS2, PD-L1, STAT3, and chemokine receptors, CCR2, CCR4, CXCR5. In human PC cells, IL30 improved the release of IGF1 and CXCL5, which mediated, via autocrine loops, its potent proliferative effect. Deletion of IL30 dramatically downregulated BCL2, NFKB1, STAT3, IGF1 and CXCL5, whereas tumor suppressors, primarily SOCS3, were upregulated. Syngeneic and xenograft PC models demonstrated IL30’s ability to boost cancer proliferation, vascularization and myeloid-derived cell infiltration, which were hindered, along with tumor growth and metastasis, by IL30 deletion, with improved host survival. RNA-Seq data from the PanCancer collection and immunohistochemistry of high-grade locally advanced PCs demonstrated an inverse association (chi-squared test,p = 0.0242) between IL30 and SOCS3 expression and a longer progression-free survival of patients with IL30NegSOCS3PosPC, when compared to patients with IL30PosSOCS3NegPC.ConclusionsMembrane-anchored IL30 expressed by human PC cells shares a tumor progression programs with its murine homolog and, via juxtacrine signals, steers a complex network of PC driver and immunity genes promoting prostate oncogenesis. The efficacy of CRISPR/Cas9-mediated targeting of IL30 in curbing PC progression paves the way for its clinical use.

ES2B-C001 is a virus-like particle (VLP) vaccine against human HER-2, developed for the therapy of breast cancer. We show here that ES2B-C001 effectively inhibits mammary carcinoma growth and metastasis in a transgenic mouse model expressing activated human HER-2. ES2B-C001 vaccine is a tag/catcher conjugation system: Acinectobacter phage 205 (AP205) capsid VLP, each with 180 tag peptides, are conjugated with catcher-HER-2 extracellular domain. The vaccine was administered alone, thanks to the intrinsic adjuvanticity of the VLP, or with Montanide ISA 51. QD is a HER-2-positive cell line established from a mammary carcinoma of a Delta16 (FVB background) transgenic mouse, bearing the human HER-2 splice variant Delta16. FVB female mice were challenged in the mammary fat pad with 1 million QD cells; vaccinations started 2 weeks after cell challenge and were repeated every 2 weeks. Untreated mice developed progressive tumors within one month, whereas 70% of mice vaccinated without adjuvant and all mice vaccinated with adjuvant were still tumor-free one year after cell challenge. Mice challenged intravenously (i.v.) developed more than 300 lung metastases, whereas all vaccinated mice remained metastasis-free. Delta16 transgenic mice are immunologically tolerant to human HER-2 and develop aggressive mammary carcinomas with a median latency of 5 months. Vaccination of young, tumor-free Delta16 mice completely prevented tumor onset for more than one year. Delta16 mice challenged i.v. with QD cells mice developed a mean of 68 lung nodules, whereas 73% of mice therapeutically vaccinated without adjuvant, and all mice vaccinated with E2SB-C001+ISA 51, were free from metastases. ES2B-C001 induced copious anti-HER-2 antibodies of all IgG subclasses, ranging 1-10 mg/mL in FVB mice and 0.1-1 mg/mL in Delta16 mice; antibody titers remained very high for 6-10 months after the last vaccination. Antibodies inhibited the 3D growth of human HER-2+++ and HER-2++ breast cancer cells, of trastuzumab resistant cells and of gastric carcinoma cells. Vaccination increased interferon-gamma secreting cells in the spleen, as evaluated by ELISPot (21±3 spots/2x105 cells). The results warrant further development of ES2B-C001 for the therapy of HER-2 positive human breast cancer and of other tumors expressing HER-2. Citation Format: Francesca Ruzzi, Arianna Palladini, Stine Clemmensen, Annette Strobaek, Nicolaas Buijs, Tanja Domeyer, Jerzy Dorosz, Vladislav Soroka, Dagmara Grzadziela, Christina Jo Rasmussen, Ida Busch Nielsen, Max Soegaard, Maria Sofia Semprini, Laura Scalambra, Stefania Angelicola, Lorena Landuzzi, Pier-Luigi Lollini, Mette Thorn. Preclinical activity of ES2B-C001, a human candidate HER-2 virus-like particle (VLP) vaccine, against mammary carcinoma onset and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 687.

ROS1 fusions are driver molecular alterations in 1-2% of non-small cell lung cancers (NSCLCs). Several tyrosine kinase inhibitors (TKIs) have shown high efficacy in patients whose tumors harbour a ROS1 fusion. However, the limited availability of preclinical models of ROS1-positive NSCLC hinders the discovery of new drugs and the understanding of the mechanisms underlying drug resistance and strategies to overcome it.The ADK-VR2 cell line was derived from the pleural effusion of a treatment-naïve NSCLC patient bearing2D-growth of ADK-VR2 cells was partially inhibited by crizotinib. On the contrary, the treatment with other TKIs, such as lorlatinib, entrectinib and DS-6051b, did not result in cell growth inhibition. TKIs showed dramatically different efficacy on ADK-VR2 cells, depending on the cell culture conditions. In 3D culture, ADK-VR2 growth was indeed almost totally inhibited by lorlatinib and DS-6051b. The clone ADK-VR2 AG143 showed higher resistance to crizotinib treatmentThe ADK-VR2 cell line is a promising NSCLC preclinical model for the study of novel targeted therapies against ROS1 fusions and the mechanisms of resistance to TKI therapies.

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 are a promising therapeutic alternative to monoclonal antibodies against HER-2+ breast cancer. We present the preclinical activity of an ES2B-C001, a VLP-based vaccine being developed for human breast cancer therapy. FVB mice challenged with HER-2+ mammary carcinoma cells QD developed progressive tumors, whereas all mice vaccinated with ES2B-C001+Montanide ISA 51, and 70% of mice vaccinated without adjuvant, remained tumor-free. ES2B-C001 completely inhibited lung metastases in mice challenged intravenously. HER-2 transgenic Delta16 mice developed mammary carcinomas by 4–8 months of age; two administrations of ES2B-C001+Montanide prevented tumor onset for >1 year. Young Delta16 mice challenged intravenously with QD cells developed a mean of 68 lung nodules in 13 weeks, whereas all mice vaccinated with ES2B-C001+Montanide, and 73% of mice vaccinated without adjuvant, remained metastasis-free. ES2B-C001 in adjuvant elicited strong anti-HER-2 antibody responses comprising all Ig isotypes; titers ranging from 1–10 mg/mL persisted for many months. Antibodies inhibited the 3D growth of human HER-2+ trastuzumab-sensitive and -resistant breast cancer cells. Vaccination did not induce cytokine storms; however, it increased the ELISpot frequency of IFN-γ secreting HER-2-specific splenocytes. ES2B-C001 is a promising candidate vaccine for the therapy of tumors expressing HER-2. Preclinical results warrant further development towards human clinical studies.

Ewing sarcoma, a highly aggressive pediatric tumor, is driven by EWS–FLI1, an oncogenic transcription factor that remodels the tumor genetic landscape. Epigenetic mechanisms play a pivotal role in Ewing sarcoma pathogenesis, and the therapeutic value of compounds targeting epigenetic pathways is being identified in preclinical models. Here, we showed that modulation of CD99, a cell surface molecule highly expressed in Ewing sarcoma cells, may alter transcriptional dysregulation in Ewing sarcoma through control of the zyxin–GLI1 axis. Zyxin is transcriptionally repressed, but GLI1 expression is maintained by EWS–FLI1. We demonstrated that targeting CD99 with antibodies, including the human diabody C7, or genetically inhibiting CD99 is sufficient to increase zyxin expression and induce its dynamic nuclear accumulation. Nuclear zyxin functionally affects GLI1, inhibiting targets such as NKX2–2, cyclin D1, and PTCH1 and upregulating GAS1, a tumor suppressor protein negatively regulated by SHH/GLI1 signaling. We used a battery of functional assays to demonstrate (i) the relationship between CD99/zyxin and tumor cell growth/migration and (ii) how CD99 deprivation from the Ewing sarcoma cell surface is sufficient to specifically affect the expression of some crucial EWS–FLI1 targets, both in vitro and in vivo, even in the presence of EWS–FLI1. This article reveals that the CD99/zyxin/GLI1 axis is promising therapeutic target for reducing Ewing sarcoma malignancy.

Osteosarcoma (OS) is a rare malignant primary tumor of mesenchymal origin affecting bone. It is characterized by a complex genotype, mainly due to the high frequency of chromothripsis, which leads to multiple somatic copy number alterations and structural rearrangements. Any effort to design genome-driven therapies must therefore consider such high inter- and intra-tumor heterogeneity. Therefore, many laboratories and international networks are developing and sharing OS patient-derived xenografts (OS PDX) to broaden the availability of models that reproduce OS complex clinical heterogeneity. OS PDXs, and new cell lines derived from PDXs, faithfully preserve tumor heterogeneity, genetic, and epigenetic features and are thus valuable tools for predicting drug responses. Here, we review recent achievements concerning OS PDXs, summarizing the methods used to obtain ectopic and orthotopic xenografts and to fully characterize these models. The availability of OS PDXs across the many international PDX platforms and their possible use in PDX clinical trials are also described. We recommend the coupling of next-generation sequencing (NGS) data analysis with functional studies in OS PDXs, as well as the setup of OS PDX clinical trials and co-clinical trials, to enhance the predictive power of experimental evidence and to accelerate the clinical translation of effective genome-guided therapies for this aggressive disease.

Ewing sarcoma (EWS) is a rare malignant pediatric tumor and patient derived xenografts (PDXs) could represent a possibility to increase the number of available models to study this disease. Compared to cell derived xenografts (CDX), PDXs are reported to better recapitulate tumor microenvironment, heterogeneity, genetic and epigenetic features and are considered reliable models for their better predictive value when comparing preclinical efficacy and treatment response in patients. In this chapter, we extensively describe a method for generating Ewing sarcoma PDX models, for their validation and molecular characterization.

Rhabdomyosarcomas (RMS) are tumors of the skeletal muscle lineage. Two main features allow for distinction between subtypes: morphology and presence/absence of a translocation between the PAX3 (or PAX7) and FOXO1 genes. The two main subtypes are fusion-positive alveolar RMS (ARMS) and fusion-negative embryonal RMS (ERMS). This review will focus on the role of receptor tyrosine kinases of the human epidermal growth factor receptor (EGFR) family that is comprised EGFR itself, HER2, HER3 and HER4 in RMS onset and the potential therapeutic targeting of receptor tyrosine kinases. EGFR is highly expressed by ERMS tumors and cell lines, in some cases contributing to tumor growth. If not mutated, HER2 is not directly involved in control of RMS cell growth but can be expressed at significant levels. A minority of ERMS carries a HER2 mutation with driving activity on tumor growth. HER3 is frequently overexpressed by RMS and can play a role in the residual myogenic differentiation ability and in resistance to signaling-directed therapy. HER family members could be exploited for therapeutic approaches in two ways: blocking the HER member (playing a driving role for tumor growth with antibodies or inhibitors) and targeting expressed HER members to vehiculate toxins or immune effectors.

Simple Summary Hyperprogressive disease (HPD) is a pattern of paradoxical tumor progression that has been reported in patients treated with immune checkpoint inhibitors (ICIs). Although a large number of studies have investigated HPD and several associated factors have been reported, the mechanisms that drive the acceleration of tumor growth remain unknown. In this review, we discuss the possible role of IFN-γ and CD38 in the development of HPD, and we report the main findings in the scientific literature on the signaling pathways in which these factors take part, and their involvement in response and resistance to ICI therapy. Abstract Immune checkpoint inhibitors (ICIs) improve the survival of patients with multiple types of cancer. However, low response rates and atypical responses limit their success in clinical applications. The paradoxical acceleration of tumor growth after treatment, defined as hyperprogressive disease (HPD), is the most difficult problem facing clinicians and patients alike. The mechanisms that underlie hyperprogression (HP) are still unclear and controversial, although different factors are associated with the phenomenon. In this review, we propose two factors that have not yet been demonstrated to be directly associated with HP, but upon which it is important to focus attention. IFN-γ is a key cytokine in antitumor response and its levels increase during ICI therapy, whereas CD38 is an alternative immune checkpoint that is involved in immunosuppressive responses. As both factors are associated with resistance to ICI therapy, we have discussed their possible involvement in HPD with the conclusion that IFN-γ may contribute to HP onset through the activation of the inflammasome pathway, immunosuppressive enzyme IDO1 and activation-induced cell death (AICD) in effector T cells, while the role of CD38 in HP may be associated with the activation of adenosine receptors, hypoxia pathways and AICD-dependent T-cell depletion.

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.

We could recently demonstrate an important role of receptor interacting protein-2 (RIP2), an activator of nuclear factor kappa B (NF-κB) and a target of activated receptors of the tumor necrosis factor receptor (TNFR) type, in myogenic differentiation and regeneration. Here, we analyze a potential role of TNFR associated factor 6 (TRAF6), which also associates with the cytoplasmic domain of TNFR type, but also IL-1-R and TLR type receptors, and activates NF-κB, in these processes. Specifically, we show that during myogenic differentiation in vitro, traf6 gene expression is downregulated in normal myoblasts, but not in rhabdomyosarcoma cells, suggesting a role of the TRAF6 protein in this process. Inhibition of traf6 expression using specific siRNAs led to an inhibition of both myoblast proliferation and differentiation, whereas inhibition of the TRAF6 effector NF-κB alone in our system only blocked proliferation. Finally, we demonstrate that the traf6 gene is downregulated in skeletal muscle tissue of the dystrophic mdx mouse. Taken together, these data argue for a role of TRAF6 in the regulation of skeletal muscle differentiation and regeneration. © 2010 International Society of Differentiation.

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.

Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in about 20% of invasive breast cancers. Anti-HER-2 monoclonal antibody therapy is effective, but its utility is limited by high costs, side effects and development of resistance, thus underlining the need of new therapeutic approaches. A novel anti-HER-2 vaccine made of virus-like particles (VLPs) displaying the extracellular domain (ECD) of the human oncogene/antigen HER-2 induced protective immune responses against transgenic mouse mammary carcinomas expressing human HER-2. We have developed a versatile antigen display platform that, unlike existing technologies, effectively facilitates directional covalent attachment of large antigens at high density on the surface of VLPs (J. Nanobiotechnology 14: 30, 2016). The system uses a tag/catcher conjugation system that was developed by splitting the CnaB2 domain from the fibronectin-binding protein FbaB of Streptococcus pyogenes into a highly reactive peptide (SpyTag) and a protein (SpyCatcher) binding partner. Interaction between SpyTag and SpyCatcher results in the spontaneous formation of an isopeptide bond, occurring at high efficiency in a wide variety of protein contexts and buffer conditions. Here, we genetically fused with SpyCatcher the full extracellular domain (subdomains I-IV) of human HER-2, and bound the fusion antigen (SpyCatcher-HER2) to the surface of VLPs (derived from the AP205 phage), each presenting 360 SpyTag peptides. The vaccine, referred to as HER2-VLP, effectively overcame immune tolerance and induced Th1 cytokines and high-titer, high affinity, therapeutically potent anti-HER-2 antibodies which inhibited tumor growth in wild-type FVB mice implanted with transgenic mammary carcinomas expressing human HER-2. Furthermore, vaccination with HER2-VLP prevented spontaneous development of human HER2-positive mammary carcinomas in tolerant transgenic mice. Vaccination with a control preparation of untagged VLP and HER-2 ECD did not induce protective immune responses. Polyclonal IgG antibodies elicited by HER2-VLP vaccination had an affinity for human HER-2 comparable to trastuzumab and inhibited the 3D growth in vitro of both trastuzumab-sensitive and trastuzumab-resistant BT-474 human breast cancer cells. In conclusion, the HER2-VLP vaccine has the potential to become a tool in the fight against HER-2-positive human cancer. The results also provide strong proof-of-concept for the use of the versatile VLP platform to develop a variety of vaccines against other tumor antigens. Supported by grants from the Italian Association for Cancer Research (AIRC), the University of Bologna, the Danish Research Council, the Eurostars program and the European Research Council (ERC). Citation Format: Arianna Palladini, Susan Thrane, Christoph M. Janitzek, Jessica Pihl, Stine B. Clemmensen, Wilhelm A. de Jongh, Thomas M. Clausen, Giordano Nicoletti, Lorena Landuzzi, Manuel L. Penichet, Tania Balboni, Marianna L. Ianzano, Veronica Giusti, Thor H. Theander, Morten A. Nielsen, Ali Salanti, Pier-Luigi Lollini, Patrizia Nanni, Adam F. Sander. A novel virus-like particle vaccine presenting HER-2 extracellular domain elicits strong immune responses against mammary carcinoma [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 716.

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.

Starting from an experimental model that accounts for the 2 most important adverse processes to successful therapy of Ewing's sarcoma (EWS), chemoresistance and the presence of metastasis at the time of diagnosis, we defined a molecular signature of potential prognostic value. Functional annotation of differentially regulated genes revealed 3 major networks related to cell cycle, cell-to-cell interactions and cellular development. The prognostic impact of 8 genes, representative of these 3 networks, was validated in 56 EWS patients. High mRNA expression levels of HINT1, IFITM2, LGALS3BP, STOML2 and c-MYC were associated with reduced risk to death and lower risk to develop metastasis. At multivariate analysis, LGALS3BP, a matricellular protein with a role in tumor progression and metastasis, was the most important predictor of event-free survival and overall survival. The association between LGALS3BP and prognosis was confirmed at protein level, when expression of the molecule was determined in tumor tissues but not in serum, indicating a role for the protein at local tumor microenvironment. Engineered enhancement of LGALS3BP expression in EWS cells resulted in inhibition of anchorage independent cell growth and reduction of cell migration and metastasis. Silencing of LGALS3BP expression reverted cell behavior with respect to in vitro parameters, thus providing further functional validation of genetic data obtained in clinical samples. Thus, we propose LGALS3BP as a novel reliable indicator of prognosis, and we offer genetic signatures to the scientific communities for cross-validation and meta-analysis, which are indispensable tools for a rare tumor such as EWS.

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.

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

The highly aggressive cancer syndrome of female mice carrying a p53 knockout allele and a rat HER-2/neu (Neu) transgene (BALB-p53Neu) can be prevented by a cell vaccine presenting three components: Neu, interleukin (IL)-12 production, and allogeneic major histocompatibility complex (MHC) alleles (Triplex cell vaccine). Here we tested a second-generation Triplex DNA-based vaccine (Tri-DNA), consisting of the combination of three gene components (a transmembrane-extracellular domain fragment of the Neu gene, IL-12 genes, and the H-2D(q) allogeneic MHC gene), carried by separate plasmids. The Tri-DNA vaccine was at least as effective as the Triplex cell vaccine for cancer immunoprevention, giving a similar delay in the onset of mammary cancer and complete protection from salivary cancer. Both vaccines induced anti-Neu antibodies of the murine IgG2a isotype at similar levels. The Tri-DNA vaccine gave more restricted immunostimulation, consisting of a fully helper T cell type 1 (Th1)-polarized response, with effective production of interferon (IFN)-gamma in response to the vaccine but no spontaneous production, and no induction of anti-Neu IgG3 antibodies. On the other hand, the Triplex cell vaccine induced both Th1 and Th2 cytokines, a strong increase in spontaneous IFN-gamma production, and high levels of IgG3 antibodies recognizing Neu-positive syngeneic cells. In conclusion, the Tri-DNA vaccine is as effective as Triplex cell vaccine, exploiting a more restricted immune stimulation.

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.

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.

Purpose: Autocrine/paracrine stimulation of c-kit has been recently observed in Ewing’s sarcoma (ES) cell lines. In this study, we tested the prognostic and therapeutic role of the receptor in this tumor. Methods: One hundred one ES tumor biopsies were evaluated for the expression of c-kit by the avidin-biotin-peroxidase procedure. Effectiveness of STI-571 (Gleevec; Novartis, Basel, Switzerland), a selective inhibitor of specific tyrosine kinases, was analyzed with respect to in vitro growth and migration inhibition, as single agent or in combination with doxorubicin. Results: Approximately 30% of patients expressed c-kit in their primary tumors. No significant association between the expression of the receptor and the clinical outcome was observed. In vitro growth of ES cell lines showing high levels of c-kit demonstrated limited inhibition by exposure to STI-571 (10 μmol/L is required to obtain 40% to 50% of growth inhibition). A decrease of stem-cell factor–mediated ES cell migration was also found. The drug acted additively with doxorubicin in inhibiting ES cell growth. Conclusion: The negative prognostic findings and the limited in vitro therapeutic activity of STI-571 indicate that the putative aberrant signaling provided by c-kit overexpression may be dispensable for ES development and unlikely to constitute a critical therapeutic target. Accordingly, the dose of STI-571 required to give a significant ES growth inhibition is much higher than for those tumors in which mutations of c-kit constitute a relevant pathogenetic event. Nevertheless, in the subset of ES patients showing a high level of c-kit expression, the activity of the drug may be exploited in combination with standard therapy.

FVB-NeuN (N#202) female mice transgenic for the HER-2/neu protooncogene driven by the murine mammary tumor virus (MMTV) promoter develop mammary carcinomas with a progression from focal atypical hyperplasia to in situ carcinoma and to invasive carcinoma that closely resembles that of human neoplasia. Here we report that the combination of tamoxifen plus interleukin 12 (IL-12) results in a very effective prevention of mammary carcinogenesis, significantly higher than those obtained with either tamoxifen or IL-12 alone. At 1 year of age, 20% of control mice resulted tumor-free, whereas 80% of mice receiving the combined treatment were tumor-free. At 2 years of age, less than 5% of control mice were tumor-free, as opposed to 70% of mice treated with tamoxifen plus IL-12. The combined treatment inhibited mammary carcinogenesis mainly through a reduction in the number of mammary cells at risk of progression, a reduction in estrogen receptors (ERs) expression and a reduction in the angiogenic support to mammary development, likely due to cross-talk between tamoxifen and interferon-gamma (IFN-gamma) (the main downstream mediator elicited in vivo by IL-12). The addition of IL-12 to the tamoxifen treatment more than doubled mouse lifetime and did not exacerbate known side effects of tamoxifen.

Overexpression of human epidermal growth factor receptor-2 (HER2) occurs in 20–30% of invasive breast cancers. Monoclonal antibody therapy is effective in treating HER2-driven mammary carcinomas, but its utility is limited by high costs, side effects and development of resistance. Active vaccination may represent a safer, more effective and cheaper alternative, although the induction of strong and durable autoantibody responses is hampered by immune-tolerogenic mechanisms. Using a novel virus-like particle (VLP) based vaccine platform we show that directional, high-density display of human HER2 on the surface of VLPs, allows induction of therapeutically potent anti-HER2 autoantibody responses. Prophylactic vaccination reduced spontaneous development of mammary carcinomas by 50%-100% in human HER2 transgenic mice and inhibited the growth of HER2-positive tumors implanted in wild-type mice. The HER2-VLP vaccine shows promise as a new cost-effective modality for prevention and treatment of HER2-positive cancer. The VLP platform may represent an effective tool for development of vaccines against other non-communicable diseases.

IGF-IR plays an essential role in the establishment and maintenance of the transformed phenotype of ES cells and interference with the IGF-IR pathways causes reversal of the malignant potential of this neoplasm. In this report, we stably transfected a dominant negative IGF-IR expression plasmid in an ES cell line to determine the effectiveness of this strategy against the in vitro and in vivo growth of ES cells. DXR sensitivity of TC-71 cells expressing dominant negative mutants of IGF-IR was also examined. The mutated IGF-IR that we used carries a mutation in the ATP-binding domain of the intracellular β subunit, while the extracellular, ligand-binding α subunit remains unchanged. Cells carrying the dominant mutant IGF-IR had a marked decrease in proliferation, a significant increase in anoikis-induced apoptosis and a severely reduced ability to form colonies in soft agar. In vivo, when cells carrying dominant negative IGF-IR were injected into nude mice, the tumor formation and metastatic abilities of ES cells were reduced and survival increased. Furthermore, transfected clones showed significantly higher sensitivity to DXR, a major drug in the treatment of ES. These results indicate that the IGF/IGF-IR stimulation of ES cells may be inhibited by expression of mutated IGF-IR on their surfaces and that this strategy may be considered a possible alternative to impair this important target of ES cells, whose therapeutic potential was further confirmed. © 2002 Wiley-Liss, Inc.

TS/A spontaneous mouse mammary adenocarcinoma cells were engineered to release interferon-gamma (IFN-gamma), a Th1 cytokine (TS/A-IFNgamma) and interleukin-13 (IL-13), a Th2 cytokine (TS/A-IL13). Mice bearing lung micrometastases induced by parental TS/A cells received repeated subcutaneous vaccinations with TS/A-IFN-gamma admixed with TS/A-IL13 engineered cells. This combined treatment cured up to 75% of mice, whereas vaccinations with either TS/A-IFNgamma or TS/A-IL13 alone cured only 20-40% of mice. Combined TS/A-IL13 and TS/A-IFNgamma therapeutic vaccinations elicited a reactive infiltrate of CD4+ and CD8+ lymphocytes in lung metastases and an increased production of IFN-gamma in the spleen and lung, suggesting a shift of the immune response toward the Th1 type. The type of infiltrating cells along with the lack of efficacy in T cell-deficient mice point to a major role of T cells. In conclusion, no antagonism but a synergistic and effective definitive cure stems from the combined vaccination with tumor cells engineered to release a Th1 and a Th2 cytokine.

The expression and biological function of Nerve Growth Factor (NGF) receptors was studied in a panel of rhabdomyosarcoma cell lines derived from embryonal and alveolar histotype. All the cell lines expressed both the high affinity receptor TrkA and the low affinity receptor p75NTR. Treatment with exogenous NGF did not considerably alter rhabdomyosarcoma cell growth or differentiation, but significantly inhibited spontaneous apoptosis as well as apoptosis, and induced by serum starvation or apoptosis induced by treatment with cycloheximide (CHX). Rhabdomyosarcoma cell lines expressed NGF and other neurotrophins and trace amounts of NGF protein were found in the supernatants of rhabdomyosarcoma cell cultures. Blocking the putative autocrine loop with an anti-NGF antibody resulted in an increase in apoptosis compared with control cultures. These data suggest that the simultaneous presence of both high and low affinity NGF receptors engaged by endogenous or exogenous NGF might contribute to the escape from apoptosis exhibited by the rhabdomyosarcoma cells.

Cancer immunoprevention posits that the enhancement of immune defenses in healthy individuals could control tumor onset. Immunoprevention of viral tumors is already implemented at the population level for human hepatocellular and cervical carcinomas. Altogether, viral vaccines could prevent more than 10% of all human tumors. The big question is whether immunoprevention can be applied to nonviral tumors, including breast cancer. Promising results were obtained in preclinical models, in particular in HER-2/neu transgenic mice, which are prone to mammary carcinoma development, using vaccines against HER-2/neu oncoprotein p185. The life expectancy of vaccinated mice was more than doubled. Protective immune mechanisms elicited by effective vaccines were mainly based on helper T cell cytokines, in particular γ-interferon, and anti-p185 antibodies. The term “oncoantigens” was coined to define those antigenic molecules that, like HER-2, are indispensable for tumor growth, thus representing the best class of targets for cancer immunoprevention. The study of immunopreventive vaccines against subsequent phases of neoplastic progression showed a dramatic loss of efficacy against established mammary carcinomas, whereas the prevention of micrometastasis growth was successful. Preclinical results provide useful indications for the translation of cancer immunoprevention to humans, and useful hints for cancer immunotherapy.

Transgenic FVB-NeuN mice (N202) bearing the rat neu protooncogene driven by the mouse mammary tumor virus promoter/enhancer develop focal mammary carcinomas overexpressing the neu-encoded p185neu protein. In vitro expression of p185neu among mammary carcinoma cultures was heterogeneous, and we could establish some cell lines and clones displaying a complete loss of p185neu expression, along with others with very high p185neu protein level. Upon in vivo injection, p185neu-positive cells gave rise to fast-growing tumors with a short latency, while p185neu-negative cells required a very long latency time, and the resulting tumors were invariably p185neu-positive. The lower growth ability of p185neu-negative cells in vivo was also confirmed in athymic nude mice. In vitro, analysis of anchorage-independent growth in soft agar revealed colony formation from p185neu-positive but not p185neu-negative cells. The direct involvement of p185neu in clonogenicity was demonstrated by the inhibition of p185neu-positive colony growth in soft agar in the presence of an anti-p185neu monoclonal antibody. By contrast, a higher level of anchorage-dependent clonogenic growth and proliferation was observed in p185neu-negative cells as compared to p185neu-positive cells, thus explaining the relative ease with which p185neu-negative cell lines and clones were established in vitro. Together, the results indicate that p185neu expression can lead to tumor formation and metastasis through the modification of intrinsic properties of cells related to anchorage-independent growth ability rather than to proliferation or host-dependent mechanisms. Int. J. Cancer 87:186–194, 2000. © 2000 Wiley-Liss, Inc.

Five human rhabdomyosarcoma cell lines were used to investigate the surface expression of HER/erbB receptors, particularly of HER-2, HER-3 and HER-4, by flow cytometry. HER-2 was expressed in 3/5 rhabdomyosarcoma cell lines. HER-3 was expressed in all rhabdomyosarcoma cell lines. None of the rhabdomyosarcoma cell lines investigated showed HER-4 surface expression. To study the biological activity of HER/erbB receptors in rhabdomyosarcomas, cells were cultured in the presence of glial growth factor 2 (GGF-2), a specific ligand of HER-3 that is able to stimulate myogenesis in normal myoblasts. In 3 of 3 human rhabdomyosarcoma cell lines positive for both HER-2 and HER-3 receptors, the treatment with GGF-2 induced a significant increase in myogenic differentiation.