Systemic delivery of HER2-retargeted oncolytic-HSV by mesenchymal stromal cells protects from lung and brain metastases.

Fully retargeted oncolytic herpes simplex viruses (o-HSVs) gain cancer-specificity from redirection of tropism to cancer-specific receptors, and are non-attenuated. To overcome the hurdles of systemic delivery, and enable oncolytic viruses (o-viruses) to reach metastatic sites, carrier cells are being exploited. Mesenchymal stromal cells (MSCs) were never tested as carriers of retargeted o-viruses, given their scarse-null expression of the cancer-specific receptors. We report that MSCs from different sources can be forcedly infected with a HER2-retargeted oncolytic HSV. Progeny virus spread from MSCs to cancer cells in vitro and in vivo. We evaluated the organ distribution and therapeutic efficacy in two murine models of metastatic cancers, following a single i.v. injection of infected MSCs. As expected, the highest concentration of carrier-cells and of viral genomes was in the lungs. Viral genomes persisted throughout the body for at least two days. The growth of ovarian cancer lung metastases in nude mice was strongly inhibited, and the majority of treated mice appeared metastasis-free. The treatment significantly inhibited also breast cancer metastases to the brain in NSG mice, and reduced by more than one-half the metastatic burden in the brain.

Interleukin-15 is required for immunosurveillance and immunoprevention of HER2/neu-driven mammary carcinogenesis.

INTRODUCTION: We previously demonstrated that HER2/neu-driven mammary carcinogenesis can be prevented by an interleukin-12 (IL-12)-adjuvanted allogeneic HER2/neu-expressing cell vaccine. Since IL-12 can induce the release of interleukin-15 (IL-15), in the present study we investigated the role played by IL-15 in HER2/neu driven mammary carcinogenesis and in its immunoprevention. METHODS: HER2/neu transgenic mice with homozygous knockout of IL-15 (here referred to as IL15KO/NeuT mice) were compared to IL-15 wild-type HER2/neu transgenic mice (NeuT) regarding mammary carcinogenesis, profile of peripheral blood lymphocytes and splenocytes and humoral and cellular responses induced by the vaccine. RESULTS: IL15KO/NeuT mice showed a significantly earlier mammary cancer onset than NeuT mice, with median latency times of 16 and 20 weeks respectively, suggesting a role for IL-15 in cancer immunosurveillance. Natural killer (NK) and CD8+ lymphocytes were significantly lower in IL15KO/NeuT mice compared to mice with wild-type IL-15. The IL-12-adjuvanted allogeneic HER2/neu-expressing cell vaccine was still able to delay mammary cancer onset but efficacy in IL-15-lacking mice vanished earlier: all vaccinated IL15KO/NeuT mice developed tumors within 80 weeks of age (median latency of 53 weeks), whereas more than 70 % of vaccinated NeuT mice remained tumor-free up to 80 weeks of age. Vaccinated IL15KO/NeuT mice showed less necrotic tumors with fewer CD3+ lymphocyes and lacked perforin-positive infiltrating cells compared to NeuT mice. Concerning the anti-vaccine antibody response, antibody titer was unaffected by the lack of IL-15, but less antibodies of IgM and IgG1 isotypes were found in IL15KO/NeuT mice. A lower induction by vaccine of systemic interferon-gamma (IFN-γ) and interleukin-5 (IL-5) was also observed in IL15KO/NeuT mice when compared to NeuT mice. Finally, we found a lower level of CD8+ memory cells in the peripheral blood of vaccinated IL15KO/NeuT mice compared to NeuT mice. CONCLUSIONS: We demonstrated that IL-15 has a role in mammary cancer immunosurveillance and that IL-15-regulated NK and CD8+ memory cells play a role in long-lasting immunoprevention, further supporting the potential use of IL-15 as adjuvant in immunological strategies against tumors.

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

Homozygous knockout of p53 in mice leads to early mortality from lymphoma, with almost complete penetrance, thus hampering studies of other tumor histotypes related to p53 alterations. To avoid lymphoma development, we crossed p53 knockout mice (BALB-p53 mice) with alymphocytic BALB/c Rag2-/-;Il2rg-/- (RGKO) mice. We compared the tumor spectrum of homozygous (BALB-p53-/-) and heterozygous (BALB-p53+/-) mice with alymphocytic mice (RGKO-p53-/- and RGKO-p53+/-). Lymphoma incidence in BALB-p53-/- mice exceeded 80%, whereas in RGKO-p53-/- it was strongly reduced. The prevalent tumor of RGKO-p53-/- mice was hemangiosarcoma (incidence over 65% in both sexes, mean latency 18 weeks), other tumors included soft tissue sarcomas (incidence ~10%), lung and mammary carcinomas. Tumor spectrum changes occurred also in p53 heterozygotes, in which lymphomas are relatively rare (~20%). RGKO-p53+/- had an increased incidence of hemangiosarcomas, reaching ~30%, and females had an increased incidence of osteosarcomas, reaching ~20%. Osteosarcomas shared with the corresponding human tumors the involvement of limbs and a high metastatic ability, mainly to the lungs. Specific alterations in the expression of p53-related genes (p16Ink4a, p19Arf, p15Ink4b, p21Cip1) were observed. Genetic prevention of lymphoma in p53 knockout mice led to new models of sarcoma development, available for studies on hemangiosarcoma and osteosarcoma onset and metastatization.

Vaccines against human HER2 prevent mammary carcinoma in mice transgenic for human HER2.

INTRODUCTION: The availability of mice transgenic for the human HER2 gene (huHER2) and prone to the development of HER2-driven mammary carcinogenesis (referred to as FVB-huHER2 mice) prompted us to study active immunopreventive strategies targeting the human HER2 molecule in a tolerant host. METHODS: FVB-huHER2 mice were vaccinated with either IL-12-adjuvanted human HER2-positive cancer cells or DNA vaccine carrying chimeric human-rat HER2 sequences. Onset and number of mammary tumors were recorded to evaluate vaccine potency. Mice sera were collected and passively transferred to xenograft-bearing mice to assess their antitumor efficacy. RESULTS: Both cell and DNA vaccines significantly delayed tumor onset, leading to about 65% tumor-free mice at 70 weeks, whereas mock-vaccinated FVB-huHER2 controls developed mammary tumors at a median age of 45 weeks. In the DNA vaccinated group, 65% of mice were still tumor-free at about 90 weeks of age. The number of mammary tumors per mouse was also significantly reduced in vaccinated mice. Vaccines broke the immunological tolerance to the huHER2 transgene, inducing both humoral and cytokine responses. The DNA vaccine mainly induced a high and sustained level of anti-huHER2 antibodies, the cell vaccine also elicited interferon (IFN)-γ production. Sera of DNA-vaccinated mice transferred to xenograft-carrying mice significantly inhibited the growth of human HER2-positive cancer cells. CONCLUSIONS: Anti-huHER2 antibodies elicited in the tolerant host exert antitumor activity.

Tumor suppressor genes promote rhabdomyosarcoma progression in p53 heterozygous, HER-2/neu transgenic mice.

Human sarcomas arise suddenly, thus preempting the study of preneoplastic and early neoplastic lesions. To explore the natural history of these tumors we studied male mice carrying a heterozygous deletion of p53 and an activated HER-2/neu transgene (BALB-p53Neu mice), that develop urethral rhabdomyosarcomas with nearly full penetrance and early onset (4 months of age). Among genes prominently upregulated in preneoplastic tissue, and more highly expressed in tumors, we found the insulin-like growth factor 2 (Igf2) and tumor suppressors, p19Arf and p21Cip1. In urethral tissues of male mice p53 was less expressed than in female mice, whereas HER-2/neu was more expressed, a combination not found in other skeletal muscles of the same mice that could contribute to the anatomic and sexual specificity of BALB-p53Neu rhabdomyosarcoma. Upregulation of p19Arf and p21Cip1 was additively determined by HER-2/neu activation and by p53 inactivation. Silencing of p19Arf or p21Cip1 in rhabdomyosarcoma cell lines can inhibit cell growth and motility, thus suggesting that these genes can contribute to growth autonomy and malignancy of tumor cells. In vivo injection of gene-silenced cells highlighted selective variations in organ-specific metastatic ability, indicating that overexpression of p19Arf and p21Cip1 controlled both tumor cell-intrinsic properties and microenvironmental interactions. The onset of pelvic rhabdomyosarcoma in BALB-p53Neu male mice is triggered by the coincidental overexpression of HER-2/neu and hypoexpression of the residual p53 allele, that foster p53 loss, Igf2 autocriny and overexpression of p19Arf and p21Cip1, a phenotype that could provide novel potential targets for cancer prevention and therapy.

Modulation of cytochrome P450 and induction of DNA damage in Cyprinus carpio exposed in situ to surface water treated with chlorine or alternative disinfectants in different seasons.

Epidemiological studies have shown an association between consumption of disinfected drinking water and adverse health outcomes. The chemicals used to disinfect water react with occurring organic matter and anthropogenic contaminants in the source water, resulting in the formation of disinfection by-products (DBPs). The observations that some DBPs are carcinogenic in animal models have raised public concern over the possible adverse health effects for humans. Here, the modulation of liver cytochrome P450-linked monooxygenases (MFO) and the genotoxic effects in erythrocytes of Cyprinus carpio fish exposed in situ to surface drinking water in the presence of disinfectants, such as sodium hypochlorite (NaClO), chlorine dioxide (ClO(2)) and peracetic acid (PAA), were investigated in winter and summer. A complex induction/suppression pattern of CYP-associated MFOs in winter was observed for all disinfectants. For example, a 3.4- to 15-fold increase was recorded of the CYP2B1/2-linked dealkylation of penthoxyresorufin with NaClO (10 days) and PAA (20 days). In contrast, ClO(2) generated the most notable inactivation, the CYP2E1-supported hydroxylation of p-nitrophenol being decreased up to 71% after 10 days' treatment. In summer, the degree of modulation was modest, with the exception of CYP3A1/2 and CYP1A1 supported MFOs (62% loss after 20 days PAA). The micronucleus (MN) induction in fish circulating erythrocytes was also analysed as an endpoint of genotoxic potential in the same fish population. Significant increases of MN induction were detected at the latest sampling time on fish exposed to surface water treated with chlorinate-disinfectants, both in winter (NaClO) and summer (NaClO and ClO(2)), while no effect was observed in fish exposed to PAA-treated water. These results show that water disinfection may be responsible for harmful outcomes in terms of MFO perturbation and DNA damage; if extrapolated to humans, they ultimately offer a possible rationale for the increased urinary cancer risk recorded in regular drinking water consumers.