Animal models of Soft Tissue Sarcoma for alternative anticancer therapy studies: characterization of the A-72 Canine Cell Line.

Canine Soft Tissue Sarcoma (STS) cell line A-72 has been largely employed for antiviral and antiproliferative studies. However, there are few information on their characteristics. Our aim was to evaluate A-72 expression level of genes and proteins involved in the innate immune response and cell cycle, their ability to respond to infective stressors and their possible use as a cellular model for anti-cancer studies in human and animal medicine. For this purpose, we evaluated the basal expression of immune-related, cell cycle and DNA repair genes on this cell line and tumoral tissues. A-72 ability to respond to a wild-type strain of Salmonella typhimurium was assessed. S. typhimurium showed ability to penetrate A-72 causing pro-inflammatory response accompanied by a decrease of cell viability. IL10 and IL18 genes were not expressed in A-72 while CXCL8, NOS2, CXCR4 and PTEN were highly expressed in all samples and TP53 was slightly expressed, as shown in human STS. Our results outline the ability of A-72 to respond to a bacterial agent by modifying the expression of important genes involved in innate immune response and provide a useful model for in vitro evaluation of new therapeutic approaches that could be translated into the human oncology.

Identification of infectious species after resection of soft-tissue sarcomas.

BACKGROUND: Pathogenic species in deep tissue infections after soft-tissue sarcoma (STS) resection is largely unstudied, particularly the role of anaerobic bacteria, risks factors for those pathogens, and the time course of infection presentation. METHODS: Retrospective analysis of 64 patients requiring operative debridement for deep tissue infection after STS resection was undertaken to identify infectious species and study risk factors for anaerobic infections. Kaplan-Meier methods examined the time course of infection presentation. RESULTS: STS subtypes were most commonly pleomorphic STS, myxofibrosarcoma, and undifferentiated STS. Staphylococcus aureus was the most common organism isolated (56%). Twenty (31%) infections were positive for ≥1 anaerobic organism. Twelve gram-positive and 10 gram-negative aerobic organisms were isolated. Most (90%) anaerobic-containing infections were polymicrobial, vs 52% of purely aerobic infections. No significant risk factors for anaerobic infections were identified. Median time from tumor resection until debridement was significantly greater for anaerobic infections (54.5 days) than for purely aerobic infections (29.5 days; P = 0.004), a difference so pronounced that using "presentation after 53 days" as a proxy for the presence of anaerobic pathogens had an accuracy of 81%. CONCLUSIONS: Because polymicrobial and anaerobic bacterial infections are common, we strongly support antibiotic use with anaerobic coverage at debridement, particularly for infections presenting later.

Tumor-targeting Salmonella typhimurium A1-R is a highly effective general therapeutic for undifferentiated soft tissue sarcoma patient-derived orthotopic xenograft nude-mouse models.

Undifferentiated soft tissue sarcoma (USTS) is a recalcitrant and heterogeneous subgroup of soft tissue sarcoma with high risk of metastasis and recurrence. Due to heterogeneity of USTS, there is no reliably effective first-line therapy. We have generated tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R), which previously showed strong efficacy on single patient-derived orthotopic xenograft (PDOX) models of Ewing's sarcoma and follicular dendritic cell sarcoma. In the present study, tumor resected from 4 patients with a biopsy-proven USTS (2 undifferentiated pleomorphic sarcoma [UPS], 1 undifferentiated sarcoma not otherwise specified [NOS] and 1 undifferentiated spindle cell sarcoma [USS]) were grown orthotopically in the biceps femoris muscle of mice to establish PDOX models. One USS model and one UPS model were doxorubicin (DOX) resistant. One UPS and the NOS model were partially sensitive to DOX. DOX is first-line therapy for these diseases. S. typhimurium A1-R arrested tumor growth all 4 models. In addition to arresting tumor growth in each case, S. typhimurium A1-R was significantly more efficacious than DOX in each case, thereby surpassing first-line therapy. These results suggest that S. typhimurium A1-R can be a general therapeutic for USTS and possibly sarcoma in general.

Inhibition of spontaneous and experimental lung metastasis of soft-tissue sarcoma by tumor-targeting Salmonella typhimurium A1-R.

Prognosis of patients with lung metastases of soft-tissue sarcoma is still poor. Therefore, novel systemic therapy is needed to improve the survival of soft-tissue sarcoma. In the present study, tumor-targeting therapy with a genetically-modified auxotrophic strain of Salmonella typhimurium, termed A1-R, was evaluated. Mouse models of primary soft tissue sarcoma and spontaneous lung metastasis were obtained by orthotopic intra-muscular injection of HT1080-RFP human fibrosarcoma cells. S. typhimurium A1-R was administered from day 14, once a week for two weeks. On day 28, lung samples were excised and observed with a fluorescence imaging system. The number of lung metastasis was 8.8 ± 3.4 in the untreated group and 0.8 ± 0.8 in the treated group (P = 0.024). A mouse model of experimental lung metastasis was obtained by tail vein injection of HT1080-RFP cells. The mice were treated with S. typhimurium A1-R (i.v.) on day 7, once a week for three weeks. S. typhimurium A1-R significantly reduced lung metastases and improved overall survival (P = 0.004). S. typhimurium A1-R bacterial therapy has future potential for treating advanced soft tissue sarcoma and improving prognosis of patients with lung metastasis.

Influence of MHC on the oncogenicity and immunogenicity of polyoma virus BK in rats.

The oncogenicity of the human papovavirus, type BK, was examined in the Lewis strain of inbred rats, possessing the recombinant alleles "a" and "u" on the MHC TR1 locus. It was found that only haplotypes containing "a" within the B/D locus but not within the A and the C locus of the RT1 region were associated with resistance to the oncogenic potential of BKV and with the capacity of animals to form BKV T antigen antibody. By contrast, the presence of "u" or "l" within the MHC B/D region was linked to the reverse phenotype characterized by sensitivity to the oncogenic effect of BKV and failure to yield T antibody response up to 6 months after inoculation with BK. These results present further evidence of the function as an immune control gene of the B/D region of the RT1 locus of rat MHC.

Characterization of BK virus variants rescued from human tumours and tumour cell lines.

Episomal BK virus (BKV) DNA was detected in primary human brain tumours, in Kaposi's sarcoma and in cell lines from brain tumours. Ewing sarcoma and osteogenic sarcoma. Infectious BKV was rescued from several tumours and tumour cell lines by transfection of total cellular DNA into human embryonic fibroblasts. Restriction endonuclease and nucleotide sequence analysis showed that all the rescued viruses are similar to BKV-IR, a BK variant previously isolated from a human tumour of pancreatic islets, indicating that a specific BKV strain may be associated with certain types of human tumours. All the variants contain a putative transposable elements in the regulatory region of the viral genome. This region has mutagenic properties and enhancing activity in transformation, suggesting a possible role of these variants in tumour induction or progression.

Experimental transmission of a dermal sarcoma in fingerling walleyes (Stizostedion vitreum vitreum).

Dermal sarcoma is a benign skin tumor of adult walleyes (Stizostedion vitreum vitreum) with a suspected viral etiology. A laboratory study was initiated to determine if the tumor could be experimentally transmitted by inoculating young walleyes with materials prepared from tumors from adult fish. Eighty walleye fingerlings were divided into four groups of 20 fish each. Two groups were inoculated intramuscularly at 4 months of age either with live tumor cells or with cell-free filtrates of sonicated tumor cells. The two other groups were used as controls and were inoculated either with cultured cells from normal walleye fry or with tissue culture media. Neoplasms, similar to the dermal sarcoma affecting adult walleyes, were observed after 4 months only in fingerlings inoculated with cell-free filtrates of sonicated tumor cells. Like the tumor affecting wild adult walleyes, the transmitted tumors were restricted to the dermis and originated from the superficial surface of scales. They never invaded locally and never metastasized. The transmitted tumors differed from tumors of adult walleyes in their severity and the absence of osteoid. The multicentric origin of transmitted walleye dermal sarcoma suggests that the virus spreads systemically and that tumor cells are polyclonal. This successful transmission of the lesion, along with the presence of C-type virus particles budding from tumor cells in two of seven tumor-bearing fingerlings, supports a retroviral etiology.

Retrovirus-like particles associated with myeloproliferative disease in the dog.

A case of granulocytic myeloproliferative disease with hypercalcaemia of malignancy in a 6-year-old Golden Retriever dog is described. Numerous retrovirus-like budding particles were observed at the cell surface of the neoplastic granulocytes, suggesting the presence of a new oncogenic virus. Several attempts by other workers to demonstrate the presence of an oncogenic retrovirus in canine lymphosarcoma have produced minimal results. This study suggests that non-lymphoid canine myeloproliferative disorders warrant further investigation.

Characterization of tumor cell lines from a spontaneous rat sarcoma expressing an endogenous retrovirus.

We have characterized various biologic, immunologic and growth properties of several cell lines established from a spontaneous rat sarcoma that was discovered more than 60 yr ago. The tumors consisted of mixed cell types with no detectable host cellular immune response. Cultures derived from single-cell clones of the parental cell line were non-invasive but highly tumorigenic even in adult rats. The cultured cells spontaneously released replication-competent endogenous rat type C virus which did not carry a transforming gene in its genome. Since normal cells from the same rat strain did not produce a retrovirus, it is possible that production of the endogenous retrovirus may have triggered specific cellular changes necessary for the oncogene expression and development of this tumor.

Feline leukaemia viruses: molecular biology and pathogenesis.

The feline leukaemia virus (FeLV) group represents one of the most important viral pathogens of the domestic cat. In addition, this virus - host system is one of the major experimental models for retroviral pathogenesis. Under natural conditions, the virus is horizontally transmitted through the cat population. The outcome of infection depends on a variety of factors including the virus does encountered and the age and immune status of the host. FeLVs can establish persistent infection, either overt or latent. Degenerative diseases of the haemopoietic system are the most common result of persistent infection and immunosuppression with secondary infection accounts for more deaths than does neoplastic disease. However, more is known about the molecular mechanisms of oncogenesis in this system and there are now numerous examples of field case tumours where FeLV has transduced an oncogene or acted as an insertional mutagen. The factors affecting the relative frequency of these mechanisms are considered as is the possibility that recombinant env gene recombinants play a role in FeLV pathogenesis.

Alveolar soft part sarcoma following radiotherapy for a spinal hemangioma. A case report.

A case of alveolar soft part sarcoma arising some 20 years later in a site previously irradiated as a treatment for a spinal cord hemangioma is described. This is the first known case of radiation-associated alveolar soft part sarcoma, and it fulfills the criteria for a tumor to be radiation-induced. The coincidental finding of "viral-like" particles within some of the tumor cells was noted.

Nucleotide sequence of the feline retroviral oncogene v-fms shows unexpected homology with oncogenes encoding tyrosine-specific protein kinases.

The nucleotide sequence encoding the transforming polyprotein of the McDonough strain of feline sarcoma virus was determined. This sequence includes 231 nucleotides specifying a leader peptide, 1,377 nucleotides encoding most of the feline leukemia virus-derived gag gene, and 2,969 nucleotides representing the viral transforming gene v-fms. A single open reading frame was predicted to encode a fusion polyprotein of 160,000 daltons (P160gag-fms). Fourteen potential sites for glycosylation were predicted within the v-fms-encoded portion of the protein, consistent with previous observations that the primary translation product is rapidly glycosylated. The presence of hydrophobic signal peptides within the amino-terminal leader sequence and in the middle of the v-fms-encoded moiety suggests that the transforming glycoprotein becomes oriented with its amino terminus within the lumen of the rough endoplasmic reticulum and its carboxyl terminus protruding across the membrane of the rough endoplasmic reticulum into the cytoplasm. The latter portion of the protein shows unexpected homology to tyrosine-specific protein kinases encoded by several of the known retroviral oncogenes.

Antiviral, anticellular and enzyme-inducing activities of interferons in RD-114 cells.

RD-114 is a human sarcoma-derived cell line which is chronically infected with the RD-114 retrovirus. In a previous study, we found that treatment of these cells with human interferon-alpha or human interferon-gamma causes a marked inhibition of RD-114 virus production, but that the replication of exogenous vesicular stomatitis or encephalomyocarditis virus is not impaired. In the present study, we report that neither type of interferon has strong inhibitory effects on DNA synthesis or on multiplication of the cells. We also failed to detect a double-stranded RNA-dependent protein kinase activity in extracts of both interferon-treated and untreated cells. However, a low level of 2',5'-oligoadenylate [2,5(A)] synthetase activity was detectable in extracts of interferon-treated cells. 2,5(A)-dependent endonuclease L activity was detectable in extracts of both untreated and interferon-treated cells. This was probably responsible for the inhibition of protein synthesis observed upon introduction of 2,5(A) to RD-114 cells. In many cells, interferon has been found to induce synthesis of several proteins demonstrable by autoradiographic analysis of slab gels on which extracts of interferon-treated and radiolabelled cells are separated. Using a similar method, no such induced protein synthesis was detectable in interferon-treated RD-114 cells. Our results indicate that RD-114 cells are resistant to most known actions of interferons except for the antiretroviral action to which they are as sensitive as any other cell line.

Search for proviral simian sarcoma associated virus sequences in bone marrow cells from children with neoplasms of mesenchymal origin.

DNAs obtained from bone marrow cells of 31 children with neoplasms of mesenchymal origin were tested for the presence of proviral simian sarcoma associated virus by southern blot-hybridization. The lower limit of detection in this method was one provirus per 20-30 cells. Applying stringent conditions of hybridization, no proviral DNA could be detected in any of the samples tested, most of which were from children with leukemia or with bone marrow invading lymphosarcomas. Relaxed conditions of hybridization revealed a diffuse pattern of hybridizing fragments which was similar in all DNAs tested, including normal human DNA.