Loss of atrx cooperates with p53-deficiency to promote the development of sarcomas and other malignancies.

The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously reported p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR/Cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. These cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably mimics a role for ATRX-loss in the early pathogenesis of these human cancer types. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development.

Impact of infrared radiation on UVB-induced skin tumourigenesis in wild type C57BL/6 mice.

Although infrared radiation (IR) represents more than 50% of the solar radiation reaching the Earth's surface, this waveband has been hardly investigated in terms of tumourigenesis. The objective of the present study was to investigate the influence of IR on ultraviolet B (UVB)-induced carcinogenesis in male and female wild type mice. For this purpose, male and female C57BL/6N mice were subjected to a long-term irradiation protocol. Mice were irradiated once neonatally and from the age of eight weeks for 36 weeks with a cumulative dose of 576 kJ m-2 UVB and/or 78 895 kJ m-2 IR. In order to resemble natural sun irradiation, exposure to physiological doses of UVB and IR was performed simultaneously. Mice were screened for arising lesions twice a week. Lesions were excised and histologically diagnosed. Kaplan-Meier analyses were carried out and lesion counts and cumulated hazard rates for the development of lesions in the UVB and IR + UVB-exposed groups in male and female mice were compared. We found that IR-exposure did not change the number of epithelial malignant tumours in UVB-exposed wild type mice. In combination with IR there was a tendency of more tumours with increased malignancy: 23 vs. seven spindle cell shaped sarcomas and seven vs. two MelanA+/S100+ tumours in groups of 35 C57BL/6 mice. IR did not influence UVB-induced carcinogenesis differently in male and female mice. However, comparing UVB and sham irradiated animals irrespective of IR exposure, UVB-induced non-epithelial tumours arose significantly earlier in male mice than in female mice.

Immunohistochemical Characterization of Sarcomas in Trp53+/- Haploinsufficient Mice.

The use of immunohistochemical (IHC) staining in determining and/or confirming the cellular origin of poorly differentiated sarcomas was evaluated in this study. Sarcomatous neoplasms were evaluated in a research study conducted in 2 strains of p53+/- haploinsufficient mice. The most common neoplasms were undifferentiated sarcomas, followed by osteosarcomas and rhabdomyosarcomas (RMSs). The RMSs were poorly differentiated and appeared similar to the pleomorphic, or adult type, RMS of humans. All sarcomas stained positive by IHC for the mesenchymal cell intermediate filament vimentin. The RMSs were identified by positive IHC staining for myogenin, a transcription factor specific to skeletal muscle. Osteosarcomas were easily identifiable on hematoxylin and eosin-stained slides; no generally accepted IHC stain specific for bone is presently available. Some of the undifferentiated sarcomas contained numerous macrophages that stained positive for F4/80, a macrophage marker; the positive-staining cells were considered to be infiltrating macrophages. One-third of the neoplasms observed in this study were associated with subcutaneous implanted electronic microchips used for animal identification. Based upon histopathologic evaluation and IHC staining, it was not possible to distinguish neoplasms associated with subcutaneous microchips from neoplasms not associated with microchips.

The soluble glycoprotein NMB (GPNMB) produced by macrophages induces cancer stemness and metastasis via CD44 and IL-33.

In cancer, myeloid cells have tumor-supporting roles. We reported that the protein GPNMB (glycoprotein nonmetastatic B) was profoundly upregulated in macrophages interacting with tumor cells. Here, using mouse tumor models, we show that macrophage-derived soluble GPNMB increases tumor growth and metastasis in Gpnmb-mutant mice (DBA/2J). GPNMB triggers in the cancer cells the formation of self-renewing spheroids, which are characterized by the expression of cancer stem cell markers, prolonged cell survival and increased tumor-forming ability. Through the CD44 receptor, GPNMB mechanistically activates tumor cells to express the cytokine IL-33 and its receptor IL-1R1L. We also determined that recombinant IL-33 binding to IL-1R1L is sufficient to induce tumor spheroid formation with features of cancer stem cells. Overall, our results reveal a new paracrine axis, GPNMB and IL-33, which is activated during the cross talk of macrophages with tumor cells and eventually promotes cancer cell survival, the expansion of cancer stem cells and the acquisition of a metastatic phenotype.

Tumor etiology and chromosome pattern.

Fibrosarcomas induced in Chinese hamsters and rats by Rous sarcomla virus and 7,12-dimethylbenz(a)anthracene are associated with nonrandom chromosome variation. Although histologically indistinguishable, the tumors induced by the virus or chemical in each host species are characterized by completely different karyotypic patterns.

"Sontaneous" neoplastic transformation in vitro: a form of foreign body (smooth surface) tumorigenesis.

Explants of subcutaneous connective tissue from adult BALB/c mice into plastic petri dishes were serially subcultured and tested for tumorigenicity in two ways: by the subcutaneous implantation of cells attached to plastic plates (1 by 5 by 10 millimeters), and by the subcutaneous injection of cells suspended in saline. Cells grown in vitro for 18 or more days before being implanted attached to a plastic plate (2.4 x 10(4) to 3.4 x 10(5) cells per plate) formed tumors after 24 to 79 weeks. The latent period before tumor appearance correlated inversely with the time spent by the cells in tissue culture. Cells inoculated in saline suspension (10 to 100 times the above number per plate) did not form tumors until after 84 days in vitro; plates alone did not induce tumor formation within more than 1 1/2 years of implantation. The tumors arising from the plate-attached cells were transplantable without plates and histologically appeared to be undifferentiated sarcomas. It is well established that smooth-surfaced foreign bodies, regardless of their chemical composition, will produce sarcomas when transplanted subcutaneously in rodents. We interpret our data, particularly the decrease in tumor latent period with time spent in tissue culture, as indicating that a smooth surface was acting as a carcinogen first in vitro (the surface of the tissue culture dish) and then in vivo (the surface of the plastic plate).

Preparation and characterization of technetium and rhenium tricarbonyl complexes bearing the 4-nitrobenzyl moiety as potential bioreductive diagnostic radiopharmaceuticals. In vitro and in vivo studies.

The synthesis of a ligand containing a nitrobenzyl group as bioreductive pharmacophore and the preparation of the corresponding technetium and rhenium complexes are presented. (99m)Tc labelling was performed in high yield (>90%) by ligand substitution using fac-[(99m)Tc(CO)(3)(H(2)O)(3)](+) as precursor. The structure of the technetium complex was established by chromatographic comparison with the analogous rhenium compound which was fully characterized by elemental analysis, spectroscopic methods and X-ray crystallography. Reduction potential of the rhenium complex was in the characteristic range for bioreductive compounds. Biodistribution in normal mice was characterized by fast blood and soft tissue depuration and combined excretion via the hepatobiliary and urinary systems. Tumour uptake was low, probably due to low lipophilicity but tumour/muscle ratios were favourable as a consequence of high excretion.

Injury promotes sarcoma development in a genetically and temporally restricted manner.

Cancer results from the accumulation of genetic mutations in a susceptible cell of origin. We and others have also shown that injury promotes sarcoma development, but how injury cooperates with genetic mutations at the earliest stages of tumor formation is not known. Here, we utilized dual recombinase technology to dissect the complex interplay of the timing of KrasG12D activation, p53 deletion, and muscle injury in sarcomagenesis using a primary mouse model of soft tissue sarcoma. When mutations in oncogenic Kras and p53 are separated by 3 weeks, few sarcomas develop without injury. However, the transformation potential of these tumor-initiating cells can be unmasked by muscle injury. In the absence of Kras mutations, injury of the muscle with global deletion of p53 results in sarcomas with amplification of chromosomal regions encompassing the Met or Yap1 gene. These findings demonstrate a complex interplay between the timing of genetic mutations and perturbations in the tumor microenvironment, which provides insight into the earliest stages of sarcoma development.

Tumor induced by Moloney sarcoma virus causes periosteal osteogenesis engaging osteopontin, fibronectin, stromelysin-1 and tenascin.

Excessive bone formation occurring in such conditions as paravertebral ligamentous ossification, hallux osteophytes or some neoplastic tumors, presents a significant problem, both epidemiological and clinical. Since pathogenesis of this disorder is still unclear, we studied its mechanism in experimental model utilizing inducible orthotopic osteogenesis. Periosteal bone apposition stimulated by Moloney sarcoma is characterized by unusually high volume of new bone tissue appearing subperiosteally in the bone adjacent to the tumor. Genes engaged in this growth have not been characterized so far. Here we show the results of mRNA Representation Difference Analysis in Moloney sarcoma, which reveal high expression of four genes coding extracellular matrix proteins: osteopontin, fibronectin, stromelysin-1 and tenascin. These findings suggest that the uncommon dynamics of the Moloney sarcoma-induced osteogenesis depends on high expression of these extracellular matrix proteins.

Relation of particle dimension to carcinogenicity in amphibole asbestoses and other fibrous minerals.

In 72 experiments, durable minerals in the form of particles on respirable size and of wide chemical and structural varieties, were implanted in the pleurae of outbred female Osborne- Mendel rats for periods of more than 1 year. The incidence of induced malignant mesenchymal neoplasms correlated well with the dimensional distribution of the particles. The probability of pleural sarcoma correlated best with a number of fibers that measured 0.25 micro or less in diameter and more than 8 micrometer in length, but relatively high correlations were also noted with fibers in other size categories having diameters up to 1.5 micrometer and lengths greater than 4 micrometer. Morphologic observations indicated that short fibers and large-diameter fibers were inactivated by phagocytosis and that negligible phagocytosis of long, thin fibers occurred. The wide variety of compounds used in these experiments suggested that the carcinogenicity of fibers depended on dimension and durability rather than on physicochemical properties.

Effects of gonadectomy on foreign-body tumorigenesis in CBA/H mice.

Sarcomas were induced by sc implantation of unplasticized polyvinylchloride-vinylacetate films in gonadectomized and normal male and female CBA/H mice. Gonadectomy did not demonstrably influence tumor incidence and tumor latencies in males but significantly prolonged tumor latencies in females. The results suggest that estrogen influences the pace of foreign-body tumorigenesis in CBA/H mice.

Intratibial Moloney sarcoma virus-induced osteosarcoma in the rat: tumor incidence and pathologic evaluation.

Osteosarcomas were induced in approximately 80% of young New Zealand Black rats by the intratibial inoculation of Moloney murine sarcoma virus from day 1 to day 5 after birth. The neoplasms were composed of a spectrum of well-differentiated to poorly differentiated osteoblasts, osteocytes, and osteoclasts. Budding of C-type viral particles was associated with tumor induction. Compared to rats inoculated on day 1 after birth, rats inoculated at 4 days of age developed consistently more osteoproliferative bone tumors that often were associated with hypercalcemia, increased serum alkaline phosphatase, and elevated urinary hydroxyproline.

Chemical carcinogenesis in nude mice: comparison between nude mice from homozygous matings and heterozygous matings and effect of age and carcinogen dose.

The incidence and latency periods for local tumor development after sc injection of 3-methylcholanthrene (MCA) into 30-day-old nude mice (nu/nu partially inbred on the CBA/H background) derived from homozygous matings (nu/nu times nu/nu) or heterozygous matings (nu/+ times nu/+) were comparable and did not differ with the immunologically normal controls, even when the carcinogen dosages ranged from 0.01 to 0.10 mg. Similarly, no differences in tumor incidence or latency periods between nude mice from homozygous or heterozygous matings as well as their immunologically normal controls were observed when weight-adjusted doses of MCA equivalent to 0.02 to 0.10 mg in the 30-day-old mice were administered at 120, 210, or 360 days of age. Tumor incidence was lower in nude mice and normal mice when MCA was administered at 210 and 360 days of age, especially in mice given the lower dose of MCA. The lower dosages of MCA (0.01-0.05 mg) had no detectable immunodepressive effects in normal mice. Thus the "normal" tumor incidence in nude mice after MCA administration could not be attributed to: 1) the effect of humoral thymus gland function (in the nude mice derived from heterozygous matings), 2) the immunodepressive effects of the carcinogen (the lower MCA dosages are not immunodepressive), or 3) the age of the mice at administration. These results argue against the thymus dependency of immunologic surveillance.

Immunization of Rous sarcoma virus-inoculated marmosets with BCG and transformed allogeneic cells.

The effects of specific immunotherapy with allogeneic cells transformed by Schmidt-Ruppin Rous sarcoma virus (SR-RSV), of treatment with BCG, and of surgery on the growth of SR-RSV-induced sarcomas in white-lipped marmosets were studied. Tumor incidence, tumor progression, and survival did not differ between control and treated animals. Animals immunized with BCG developed lymphocyte reactivity to tuberculin, which remained until the animals died. BCG was isolated from the spleen of one tumor-bearing animal.

Effects of subcutaneous administration of 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene on spontaneous reticulum cell neoplasms in SJL/J mice.

Inbred 4-week-old and 16-week-old SJL/J mice were treated sc with either 150 or 450 microgram 3-methylcholanthrene (MCA). No difference was noted in their subcutaneous tumor response related to age. Although the incidences of reticulum cell neoplasms (RCN's) were the same in the controls for the 2 age groups, more 16-week-old animals developed RCN's after carcinogen treatment than did 4-week-old SJL/J mice. More 16-week-old mice developed both subcutaneous tumors at the site of MCA injection and systemic RCN's than did mice treated at 4 weeks of age. This incidence was related to the coinciding of the chronologic age of the mice for RCN development with the latency for chemical carcinogen-induced subcutaneous tumor development. Mice treated with 7,12-dimethylbenz[a]anthracene at 16 weeks of age responded in the same way as did those treated with MCA.

Foreign-body tumors of mice: strain and sex differences in latency and incidence.

Sarcomas were induced by sc implantation of unplasticized polyvinylchloride acetate films in female and male mice of strains AKR/J, BALB/cJ, BALB/cWat, CBA/H and CBA/H-T6, C3H/HeJ, C57BL/10ScSn, C57BL/6J-bgj, C57BL/cdJ, DBA/-1J l/LnJ, LP/J, SJL/J, X/Gf, 129/J, and hybrids (CBA/H-T6 X AKR/J)F1, (C57BL/10ScSn x CBA/H or CBA/H-T6)F1, (C57BL/6J-bgj x C57BL/6J)F1. The strains and sexes showed marked differences in incidence and mean latency of resulting tumors. Crucial information was provided for the selection of appropriate mouse strains for the study of interrelationships between genotypes, defined somatic properties, and the multifactorial process of foreign-body tumorigenesis.

In situ Fc receptor-bearing cells in two murine tumors. I. Isolation and identification.

The in situ inflammatory response was studied in 2 different tumors, the T1699 mammary adenocarcinoma in inbred DBA/2 mice and a sarcoma induced in inbred C57BL/6 mice by murine sarcoma virus. The presence of Fc receptors (FcR's) on infiltrating host cells was determined by rosette formation with antibody-coated sheep red blood cells (EA). Differences in FcR-binding activity among various cells were detected with the use of EA prepared with a range of antibody concentrations. Cells bearing FcR represented one-third or more of the inflammatory cell population in both tumors. The FcR-positive cells were heterogeneous in regard to both cell type and FcR activity. Monocytes and macrophages had greater FcR activity than did lymphocytes. Different populations of FcR-positive cells could be isolated by their EA-binding characteristics.

Delayed foreign-body tumorigenesis in mice infected with lactate dehydrogenase-elevating virus: Brief communication.

Female and male CBA/H mice were infected with lactate dehydrogenase virus (LDV). Two weeks later, these mice and noninfected controls received double sc implants of unplasticized vinyl chloride-vinyl acetate copolymer films (0.2 X 15 X 22 mm). Foreign-body (FB) tumorigenesis was delayed in LDV-infected females and males by 2 months. This result could not be explained by an effect of LDV on cellular immunity, inasmuch as cellular immunity does not influence the course of FB tumorigenesis.

Evaluation of Moloney murine sarcoma and leukemia virus complex as a model for airborne oncogenic virus biohazards: survival of airborne virus and exposure of mice.

Aerosols of the Moloney murine sarcoma virus (MuSV-M) and leukemia virus (MuLV-M) complex (MuSV-M/MuLV-M) were generated from refluxing atomizers and then aged in rotating drums at 21 degrees C holding temperature with relative humidities ranging from 25 to 76%. The MuSV-M and MuLV-M aerosolized from the same tumor extract preparation survived almost equally at the four humidity levels. Both viruses remained viable in the airborne state for at least 2 hours after aerosolization. When mice were exposed to airborne MuSV-M/MuLV-M, no macroscopic lesions were observed in lungs or other tissues examined during the 2-month postexposure period. On the basis of this study, MuSV-M was determined unsuitable as a "model system" in which a simple aerosol dose response could be used for biohazard evaluation of oncogenic virus aerosols.

Influence of regenerative capacity and innervation on oncogenesis in the adult frog (Rana pipiens).

Twenty-two sarcomas were induced in 19 adult frogs (Rana pipiens) treated with 3-methylcholanthrene pellets. Thirteen of these tumors arose first in a denervated forelimb, and only 2 arose first in normal or nerve-supplemented control forelimbs (P = 0.004). The remaining tumors developed either as a second tumor in a tumor-bearing frog or in hindlimbs. The critical role of innervation in regenerative capacity suggests that the predilection to tumor formation in the denervated limbs may have resulted from their lessened regenerative capacity.