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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

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.

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

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.

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.

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.

// 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.

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.

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.

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 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.

Cancer immunoprevention is based on the fact that a functioning immune system controls tumor onset and development in humans and animals, thus leading to the idea that the enhancement of immune responses in healthy individuals could effectively reduce cancer risk later in life. Successful primary immunoprevention of tumors caused by hepatitis B and papilloma viruses is already implemented at the population level with specific vaccines. The immunoprevention of human tumors unrelated to infectious agents is an outstanding challenge. Proof-of-principle preclinical studies in genetically-modified or in carcinogen-exposed mice clearly demonstrated that vaccines and other immunological treatments induce host immune responses that effectively control tumor onset and progression, eventually resulting in cancer prevention. While a straightforward translation to healthy humans is currently unfeasible, a number of pioneering clinical trials showed that cancer immunoprevention can be effectively implemented in human cohorts affected by specific cancer risks, such as preneoplastic/early neoplastic lesions. Future developments will see the implementation of cancer immunoprevention in a wider range of conditions at risk of tumor development, such as the exposure to known carcinogens and genetic predispositions.

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.