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IL-1 family members have multiple pleiotropic functions affecting various tissues and cells, including the regulation of the immune response, hematopoietic homeostasis, bone remodeling, neuronal physiology, and synaptic plasticity. Many of these activities are involved in various pathological processes and immunological disorders, including tumor initiation and progression. Indeed, IL-1 family members have been described to contribute to shaping the tumor microenvironment (TME), determining immune evasion and drug resistance, and to sustain tumor aggressiveness and metastasis. This review addresses the role of IL-1 family members in bone sarcomas, particularly the highly metastatic osteosarcoma (OS) and Ewing sarcoma (EWS), and discusses the IL-1-family-related mechanisms that play a role in bone metastasis development. We also consider the therapeutic implications of targeting IL-1 family members, which have been proposed as (i) relevant targets for anti-tumor and anti-metastatic drugs; (ii) immune checkpoints for immune suppression; and (iii) potential antigens for immunotherapy.

Abstract Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness. Accordingly, in experimental in vitro models, low lamin A expression correlated with enhanced cell migration and invasiveness and, in vivo, with an increased metastatic load. At the molecular level, this condition was linked to altered expression and anchorage of nuclear envelope proteins and increased nuclear retention of YAP/TAZ, a mechanosignaling effector. Conversely, overexpression of lamin A rescued LINC complex organization, thus reducing YAP/TAZ nuclear recruitment and preventing cell invasiveness. These effects were also obtained through modulation of lamin A maturation by a statin-based pharmacological treatment that further elicited a more differentiated phenotype in EWS cells. These results demonstrate that drugs inducing nuclear envelope remodeling could be exploited to improve therapeutic strategies for EWS.

Cancer vaccines are increasingly being studied as a possible strategy to prevent and treat cancers. While several prophylactic vaccines for virus-caused cancers are approved and efficiently used worldwide, the development of therapeutic cancer vaccines needs to be further implemented. Virus-like particles (VLPs) are self-assembled protein structures that mimic native viruses or bacteriophages but lack the replicative material. VLP platforms are designed to display single or multiple antigens with a high-density pattern, which can trigger both cellular and humoral responses. The aim of this review is to provide a comprehensive overview of preventive VLP-based vaccines currently approved worldwide against HBV and HPV infections or under evaluation to prevent virus-caused cancers. Furthermore, preclinical and early clinical data on prophylactic and therapeutic VLP-based cancer vaccines were summarized with a focus on HER-2-positive breast cancer.

Synovial sarcoma (SyS) is a rare aggressive soft tissue sarcoma carrying the chromosomal translocation t(X;18), encoding the fusion transcript SS18::SSX. The fusion oncoprotein interacts with both BAF enhancer complexes and polycomb repressor complexes, resulting in genome-wide epigenetic perturbations and a unique altered genetic signature. Over 80% of the patients are initially diagnosed with localized disease and have a 5-year survival rate of 70–80%, but metastatic relapse occurs in 50% of the cases. Advanced, unresectable, or metastatic disease has a 5-year survival rate below 10%, representing a critical issue. This review summarizes the molecular mechanisms behind SyS and illustrates current treatments in front line, second line, and beyond settings. We analyze the use of immune check point inhibitors (ICI) in SyS that do not behave as an ICI-sensitive tumor, claiming the need for predictive genetic signatures and tumor immune microenvironment biomarkers. We highlight the clinical translation of innovative technologies, such as proteolysis targeting chimera (PROTAC) protein degraders or adoptive transfer of engineered immune cells. Adoptive cell transfer of engineered T-cell receptor cells targeting selected cancer/testis antigens has shown promising results against metastatic SyS in early clinical trials and further improvements are awaited from refinements involving immune cell engineering and tumor immune microenvironment enhancement.

Abstract 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. MamBo89HER2 stable cell line displayed high and stable HER2 expression, which was maintained upon in vivo passages, whereas MamBo43HER2 labile cell line gave rise to HER2-negative tumors from which MamBo38HER2 loss cell line was derived. Both low-density seeding and in vitro trastuzumab treatment of MamBo43HER2 labile cells induced the loss of HER2 expression. MamBo38HER2 loss 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 MamBo38HER2 loss cell tumorigenic ability. Sunitinib inhibited MamBo38HER2 loss 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.

The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Overexpression of either PAX-FOXO1s results in related cell transformation in animal models. Yet, in patients the two structural genetic aberrations they derived from are associated with distinct pathological manifestations. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 binding results in greater recruitment of the H3K27ac activation mark than PAX3-FOXO1 binding and is accompanied by greater transcriptional activation of neighbouring genes. These effects are associated with a PAX-FOXO1-specific alteration in the expression of genes regulating cell shape and the cell cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into S phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into M phase, and causes increased DNA damage. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the genomic occupancy and transcriptional activity of PAX3-FOXO1 and PAX7-FOXO1.

Abstract 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.

Synovial sarcomas (SyS) are rare malignant tumors predominantly affecting children, adolescents, and young adults. The genetic hallmark of SyS is the t(X;18) translocation encoding the SS18-SSX fusion gene. The fusion protein interacts with both the BAF enhancer and polycomb repressor complexes, and either activates or represses target gene transcription, resulting in genome-wide epigenetic perturbations and altered gene expression. Several experimental in in vivo models, including conditional transgenic mouse models expressing the SS18-SSX fusion protein and spontaneously developing SyS, are available. In addition, patient-derived xenografts have been estab-lished in immunodeficient mice, faithfully reproducing the complex clinical heterogeneity. This review focuses on the main molecular features of SyS and the related preclinical in vivo and in vitro models. We will analyze the different conditional SyS mouse models that, after combination with some of the few other recurrent alterations, such as gains in BCL2, Wnt-β-catenin signaling, FGFR family, or loss of PTEN and SMARCB1, have provided additional insight into the mechanisms of synovial sarcomagenesis. The recent advancements in the understanding of SyS biology and improvements in preclinical modeling pave the way to the development of new epigenetic drugs and immunotherapeutic approaches conducive to new treatment options.

Abstract Background The treatment of metastatic osteosarcoma (OS) remains a challenge for oncologists, and novel therapeutic strategies are urgently needed. An understanding of the pathways that regulate OS dissemination is required for the design of novel treatment approaches. We recently identified Rho-associated coiled-coil containing protein kinase 2 (ROCK2) as a crucial driver of OS cell migration. In this study, we explored the impact of ROCK2 disruption on the metastatic capabilities of OS cells and analyzed its functional relationship with Yes-associated protein-1 (YAP), the main transcriptional mediator of mechanotransduction signaling. Methods The effects of ROCK2 depletion on metastasis were studied in NOD Scid gamma (NSG) mice injected with U-2OS cells in which ROCK2 expression had been stably silenced. Functional studies were performed in vitro in human U-2OS cells and in three novel cell lines derived from patient-derived xenografts (PDXs) by using standard methods to evaluate malignancy parameters and signaling transduction. The nuclear immunostaining of YAP and the evaluation of its downstream targets Cysteine Rich Angiogenic Inducer 6, Connective Tissue Growth Factor and Cyclin D1 by quantitative PCR were performed to analyze YAP activity. The effect of the expression and activity of ROCK2 and YAP on tumor progression was analyzed in 175 OS primary tumors. Results The silencing of ROCK2 markedly reduced tumor growth and completely abolished the metastatic ability of U-2OS cells. The depletion of ROCK2, either by pharmacological inhibition or silencing, induced a dose- and time-dependent reduction in the nuclear expression and transcriptional activity of YAP. The nuclear expression of YAP was observed in 80/175 (46%) tumor samples and was significantly correlated with worse patient prognosis and a higher likelihood of metastasis and death. The use of verteporfin, a molecule that specifically inhibits the TEAD–YAP association, remarkably impaired the growth and migration of OS cells in vitro. Moreover to inhibiting YAP activity, our findings indicate that verteporfin also affects the ROCK2 protein and its functions. Conclusions We describe the functional connection between ROCK2 and YAP in the regulation of OS cell migration and metastasis formation. These data provide support for the use of verteporfin as a possible therapeutic option to prevent OS cell dissemination.

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.

Abstract 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.

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

Abstract 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.

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.

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.

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.

Treatment with inhibition of programmed cell death 1 (PD-1) or its ligand (PD-L1) improves survival in advanced non-small-cell lung cancer (NSCLC). Nevertheless, only a subset of patients benefit from treatment and biomarkers of response to immunotherapy are lacking. Expression of PD-L1 on tumor cells is the primary clinically-available predictive factor of response to immune checkpoint inhibitors, and its relevance in cancer immunotherapy has fostered several studies to better characterize the mechanisms that regulate PD-L1 expression. However, the factors associated with PD-L1 expression are still not well understood. Genomic alterations that activate KRAS, EGFR, and ALK, as well as the loss of PTEN, have been associated with increased PD-L1 expression. In addition, PD-L1 expression is reported to be increased by amplification of CD274, and decreased by STK11 deficiency. Furthermore, PD-L1 expression can be modulated by either tumor extrinsic or intrinsic factors. Among extrinsic factors, the most prominent one is interferon-γ release by immune cells, while there are several tumor intrinsic factors such as activation of the mechanistic target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK) and Myc pathways that can increase PD-L1 expression. A deeper understanding of PD-L1 expression regulation is crucial for improving strategies that exploit inhibition of this immune checkpoint in the clinic, especially in NSCLC where it is central in the therapeutic algorithm. We reviewed current preclinical and clinical data about PD-L1 expression regulation in NSCLC.

Gastric cancer (GC) is one of the deadliest malignancies worldwide. Complex disease heterogeneity, late diagnosis, and suboptimal therapies result in the poor prognosis of patients. Besides genetic alterations and environmental factors, it has been demonstrated that alterations of the epigenetic machinery guide cancer onset and progression, representing a hallmark of gastric malignancies. Moreover, epigenetic mechanisms undergo an intricate crosstalk, and distinct epigenomic profiles can be shaped under different microenvironmental contexts. In this scenario, targeting epigenetic mechanisms could be an interesting therapeutic strategy to overcome gastric cancer heterogeneity, and the efforts conducted to date are delivering promising results. In this review, we summarize the key epigenetic events involved in gastric cancer development. We conclude with a discussion of new promising epigenetic strategies for gastric cancer treatment.

Oncolytic herpes simplex viruses (oHSVs) showed efficacy in clinical trials and practice. Most of them gain cancer-specificity from deletions/mutations in genes that counteract the host response, and grow selectively in cancer cells defective in anti-viral response. Because of the deletions/mutations, they are frequently attenuated or over-attenuated. We developed next-generation oHSVs, which carry no deletion/mutation, gain cancer-specificity from specific retargeting to tumor cell receptors-e.g. HER2 (human epidermal growth factor receptor 2)-hence are fully-virulent in the targeted cancer cells. The type of immunotherapy they elicit was not predictable, since non-attenuated HSVs induce and then dampen the innate response, whereas deleted/attenuated viruses fail to contrast it, and since the retargeted oHSVs replicate efficiently in tumor cells, but spare other cells in the tumor. We report on the first efficacy study of HER2-retargeted, fully-virulent oHSVs in immunocompetent mice. Their safety profile was very high. Both the unarmed R-LM113 and the IL-12-armed R-115 inhibited the growth of the primary HER2-Lewis lung carcinoma-1 (HER2-LLC1) tumor, R-115 being constantly more efficacious. All the mice that did not die because of the primary treated tumors, were protected from the growth of contralateral untreated tumors. The long-term survivors were protected from a second contralateral tumor, providing additional evidence for an abscopal immunotherapeutic effect. Analysis of the local response highlighted that particularly R-115 unleashed the immunosuppressive tumor microenvironment, i.e. induced immunomodulatory cytokines, including IFNγ, T-bet which promoted Th1 polarization. Some of the tumor infiltrating cells, e.g. CD4+, CD335+ cells were increased in the tumors of all responders mice, irrespective of which virus was employed, whereas CD8+, Foxp3+, CD141+ were increased and CD11b+ cells were decreased preferentially in R-115-treated mice. The durable response included a breakage of tolerance towards both HER2 and the wt tumor cells, and underscored a systemic immunotherapeutic vaccine response.

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.

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.

Years of unsuccessful attempts at fighting established tumors with vaccines have taught us all that they are only able to truly impact patient survival when used in a preventive setting, as would normally be the case for traditional vaccines against infectious diseases. While true primary cancer prevention is still but a long-term goal, secondary and tertiary prevention are already in the clinic and providing encouraging results. A combination of immunopreventive cancer strategies and recently approved checkpoint inhibitors is a further promise of forthcoming successful cancer disease control, but prevention will require a considerable reduction of currently reported toxicities. These considerations summed with the increased understanding of tumor antigens allow space for an optimistic view of the future.

(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.

Oncolytic viruses aim to specifically kill tumor cells. A major challenge is the effective targeting of disseminated tumors in vivo. We retargeted herpes simplex virus (HSV) tropism to HER-2 oncoprotein p185, overexpressed in ovary and breast cancers. The HER-2-retargeted R-LM249 exclusively infects and kills tumor cells expressing high levels of human HER-2. Here, we assessed the efficacy of systemically i.p. delivered R-LM249 against disseminated tumors in mouse models that recapitulate tumor spread to the peritoneum in women. The human ovarian carcinoma SK-OV-3 cells implanted intraperitoneally (i.p.) in immunodeficient Rag2⁻/⁻;Il2rg⁻/⁻ mice gave rise to a progressive peritoneal carcinomatosis which mimics the fatal condition in advanced human patients. I.p. administration of R-LM249 strongly inhibited carcinomatosis, resulting in 60% of mice free from peritoneal diffusion, and 95% reduction in the total weight of neoplastic nodules. Intraperitoneal metastases are a common outcome in breast cancer: i.p. administration of R-LM249 strongly inhibited the growth of ovarian metastases of HER-2+ MDA-MB-453 breast cells. Brain metastases were also reduced. Cumulatively, upon i.p. administration the HER-2-redirected oncolytic HSV effectively reduced the growth of ovarian and breast carcinoma disseminated to the peritoneal cavity.

Metformin, a well-known insulin-sensitizer commonly used for type 2 diabetes therapy, has recently emerged as potentially very attractive drug also in oncology. It is cheap, it is relatively safe and many reports have indicated effects in cancer prevention and therapy. These desirable features are particularly interesting for pediatric sarcomas, a group of rare tumors that have been shown to be dependent on IGF and insulin system for pathogenesis and progression. Metformin exerts anti-mitogenic activity in several cancer histotypes through several molecular mechanisms. In this paper, we analyzed its effects against osteosarcoma, Ewing sarcoma and rhabdomyosarcoma, the three most common pediatric sarcomas. Despite in vitro metformin gave remarkable antiproliferative and chemosensitizing effects both in sensitive and chemoresistant cells, its efficacy was not confirmed against Ewing sarcoma xenografts neither as single agent nor in combination with vincristine. This discrepancy between in vitro and in vivo effects may be due to hypoxia, a common feature of solid tumors. We provide evidences that in hypoxia conditions metformin was not able to activate AMPK and inhibit mTOR signaling, which likely prevents the inhibitory effects of metformin on tumor growth. Thus, although metformin may be considered a useful complement of conventional chemotherapy in normoxia, its therapeutic value in highly hypoxic tumors may be more limited. The impact of hypoxia should be considered when novel therapies are planned for pediatric sarcomas.

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