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Cancer metastasis accounts for nearly 90% of all cancer deaths. Metastatic cancer progression requires both cancer cell migration to the site of the metastasis and subsequent proliferation after colonization. However, it has long been recognized that cancer cell migration and proliferation can be uncoupled; but the mechanism underlying this paradox is not well understood. Here we report that TNFAIP8 (tumor necrosis factor-α-induced protein 8), a "professional" transfer protein of phosphoinositide second messengers, promotes cancer cell migration or metastasis but inhibits its proliferation or cancer growth. TNFAIP8-deficient mice developed larger tumors, but TNFAIP8-deficient tumor cells completely lost their ability to migrate toward chemoattractants and were defective in colonizing lung tissues as compared to wild-type counterparts. Mechanistically, TNFAIP8 served as a cellular "pilot" of tumor cell migration by locally amplifying PI3K-AKT and Rac signals on the cell membrane facing chemoattractant; at the same time, TNFAIP8 also acted as a global inhibitor of tumor cell growth and proliferation by regulating Hippo signaling pathway. These findings help explain the migration-proliferation paradox of cancer cells that characterizes many cancers., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Vaccines are important for providing protection from infectious diseases. Vaccination initiates a process that stimulates development of a robust and long-lived immune response to the disease agents in the vaccine. Side effects are sometimes associated with vaccination. These vary from development of acute hypersensitivity responses to vaccine components to local tissue reactions that are annoying but not significantly detrimental to the patient. The pathogenesis of these responses and the consequent clinical outcomes are discussed. Overstimulation of the immune response and the potential relationship to autoimmunity is evaluated in relation to genetic predisposition., (Copyright © 2017 Elsevier Inc. All rights reserved.)

For decades, proteases have been associated with cancer progression due to the ability of some members of this large group of enzymes to degrade tumor cell surroundings, thereby facilitating cancer invasion and dissemination. However, the generation of mouse models deficient in proteases has revealed the existence of a great variety of functions among proteolytic enzymes in cancer biology, including important tumor-suppressive roles. Therefore, in this chapter, we describe methods to chemically induce different types of cancer (lung adenocarcinoma, hepatocellular carcinoma, oral and esophageal carcinoma, colorectal carcinoma, skin cancer, and fibrosarcoma) in genetically modified mouse models to efficiently evaluate the specific pro- or antitumoral function of proteases in cancer.

Diagnosis of cancer and photothermal therapy using optoelectronic properties of noble metal nanoparticles (NPs) has established a new therapeutic approach for treating cancer. Here we address the intrinsic properties of noble metal NPs (gold and silver) as well as the mechanism of their potential antitumor activity. For this, the study addresses the functional characterization of tumor associated macrophages (TAMs) isolated from murine fibrosarcoma induced by a chemical carcinogen, 3-methylcholanthrene (MCA). We have previously shown antitumor activity of both gold nanoparticles (AuNPs) and silver nanoparticle (AgNPs) in vivo in a murine fibrosarcoma model. In the present study, it has been seen that AuNPs and AgNPs modulate the reactive oxygen species (ROS) and reactive nitrogen species (RNS) production, suppressing the antioxidant system of cells (TAMs). Moreover, the antioxidant-mimetic action of these NPs maintain the ROS and RNS levels in TAMs which act as second messengers to activate the proinflammatory signaling cascades. Thus, while there is a downregulation of tumor necrosis factor-α (TNF-α) and Interleukin-10 (IL-10) in the TAMs, the proinflammatory cytokine Interleukin-12 (IL-12) is upregulated resulting in a polarization of TAMs from M2 (anti-inflammatory) to M1 (pro-inflammatory) nature., (Copyright © 2016. Published by Elsevier B.V.)

USP15 is a deubiquitinase that negatively regulates activation of naive CD4(+) T cells and generation of IFN-γ-producing T helper 1 (Th1) cells. USP15 deficiency in mice promotes antitumor T cell responses in a transplantable cancer model; however, it has remained unclear how deregulated T cell activation impacts primary tumor development during the prolonged interplay between tumors and the immune system. Here, we find that the USP15-deficient mice are hypersensitive to methylcholantrene (MCA)-induced fibrosarcomas. Excessive IFN-γ production in USP15-deficient mice promotes expression of the immunosuppressive molecule PD-L1 and the chemokine CXCL12, causing accumulation of T-bet(+) regulatory T cells and CD11b(+)Gr-1(+) myeloid-derived suppressor cells at tumor site. Mixed bone marrow adoptive transfer studies further reveals a T cell-intrinsic role for USP15 in regulating IFN-γ production and tumor development. These findings suggest that T cell intrinsic USP15 deficiency causes excessive production of IFN-γ, which promotes an immunosuppressive tumor microenvironment during MCA-induced primary tumorigenesis., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Epidemiologic studies depict a negative correlation between caffeine consumption and incidence of tumors in humans. The main pharmacological effects of caffeine are mediated by antagonism of the adenosine receptor, A2AR. Here, we examine whether the targeting of A2AR by caffeine plays a role in anti-tumor immunity. In particular, the effects of caffeine are studied in wild-type and A2AR knockout (A2AR(-/-)) mice. Tumor induction was achieved using the carcinogen 3-methylcholanthrene (3-MCA). Alternatively, tumor cells, comprised of 3-MCA-induced transformed cells or B16 melanoma cells, were inoculated into animal footpads. Cytokine release was determined in a mixed lymphocyte tumor reaction (MLTR). According to our findings, caffeine-consuming mice (0.1% in water) developed tumors at a lower rate compared to water-consuming mice (14% vs. 53%, respectively, p=0.0286, n=15/group). Within the caffeine-consuming mice, tumor-free mice displayed signs of autoimmune alopecia and pronounced leukocyte recruitment intocarcinogen injection sites. Similarly, A2AR(-/-) mice exhibited reduced rates of 3-MCA-induced tumors. In tumor inoculation studies, caffeine treatment resulted in inhibition of tumor growth and elevation in proinflammatory cytokine release over water-consuming mice, as depicted by MLTR. Addition of the adenosine receptor agonist, NECA, to MLTR resulted in a sharp decrease in IFNγ levels; this was reversed by the highly selective A2AR antagonist, ZM241385. Thus, immune response modulation through either caffeine or genetic deletion of A2AR leads to a Th1 immune profile and suppression of carcinogen-induced tumorigenesis. Taken together, our data suggest that the use of pharmacologic A2AR antagonists may hold therapeutic potential in diminishing the rate of cancer development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The present study aimed to assess the antitumor effect of glycosphingolipid-incorporated liposomes (glycosphingosomes) in combination with liposomal doxorubicin (Lip-Dox) in a mouse model of fibrosarcoma. Glycosphingosomes were prepared by incorporating glycosphingolipids isolated from Sphingomonas paucimobilis into the liposomes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, cholesterol, and cardiolipin. Tumors were induced by administering dimethyl-α-benzanthracene, and tumor-bearing mice were treated with various formulations of Dox, including free Dox, Lip-Dox, or glycosphingosomes + Lip-Dox. Mice were observed for 90 days to monitor their survival and tumor size. Free Dox, but not Lip-Dox or a combination of glycosphingosomes and Lip-Dox, caused the substantial depletion of leukocytes and significantly increased the levels of lactate dehydrogenase and creatinine kinase in mice. Tumor-bearing mice treated with a combination of glycosphingosomes and Lip-Dox showed restricted tumor growth and increased survival when compared to those treated with free Dox or Lip-Dox. The results of the present study suggest that a combination of glycosphingosomes and Lip-Dox may prove to be very effective in the treatment of tumors.

Foxp3(+) regulatory T cells (Tregs) are often highly enriched within the tumor-infiltrating T cell pool. Using a well-characterised model of carcinogen-induced fibrosarcomas we show that the enriched tumor-infiltrating Treg population comprises largely of CXCR3(+) T-bet(+) 'TH1-like' Tregs which are thymus-derived Helios(+) cells. Whilst IL-2 maintains homeostatic ratios of Tregs in lymphoid organs, we found that the perturbation in Treg frequencies in tumors is IL-2 independent. Moreover, we show that the TH1 phenotype of tumor-infiltrating Tregs is dispensable for their ability to influence tumor progression. We did however find that unlike Tconvs, the majority of intra-tumoral Tregs express the activation markers CD69, CD25, ICOS, CD103 and CTLA4 and are significantly more proliferative than Tconvs. Moreover, we have found that CD69(+) Tregs are more suppressive than their CD69- counterparts. Collectively, these data indicate superior activation of Tregs in the tumor microenvironment, promoting their suppressive ability and selective proliferation at this site.

Antitumor immunity is augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naive and effector T cells proliferate extensively and show enhanced antitumor effects in lymphopenic recipients. Although the impact of lymphodepletion on transferred donor T cells has been well evaluated, its influence on recipient T cells is largely unknown. The current study demonstrates that both regulatory T cells (Tregs) and effector CD8(+) T cells from lymphopenic recipients play critical roles in the development of antitumor immunity after lymphodepletion. Cyclophosphamide (CPA) treatment depleted lymphocytes more efficiently than other cytotoxic agents; however, the percentage of CD4(+)CD25(+) Foxp3(+) Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemoresistant Tregs following CPA treatment and transfer of naive CD4(+) T cells augmented the antitumor immunity and significantly suppressed tumor progression. Further analyses revealed that recipient CD8(+) T cells were responsible for this augmentation. Using Rag2(-/-) mice or depletion of recipient CD8(+) T cells after CPA treatment abrogated the augmentation of antitumor effects in CPA-treated reconstituted mice. The transfer of donor CD4(+) T cells enhanced the proliferation of CD8(+) T cells and the priming of tumor-specific CD8(+) T cells originating from the lymphopenic recipients. These results highlight the importance of the recipient cells surviving cytotoxic regimens in cancer immunotherapies., (Copyright © 2015 by The American Association of Immunologists, Inc.)

A TAT47-57 peptide was modified on the N-terminus by elongation with a 2,3-diaminopropionic acid residue and then by coupling of two histidine residues on its N-atoms. This branched peptide could bind to Ni under physiological conditions as a 1 : 1 complex. We demonstrated that the complex was quantitatively taken up by human fibrosarcoma cells, in contrast to Ni(2+) ions. Ni localization (especially at the nuclei) was confirmed by imaging using both scanning X-ray fluorescence microscopy and Newport Green fluorescence. A competitive assay with Newport Green showed that the latter displaced the peptide ligand from the Ni-complex. Ni(2+) delivered as a complex with the designed peptide induced substantially more DNA damage than when introduced as a free ion. The availability of such a construct opens up the way to investigate the importance of the nucleus as a target for the cytotoxicity, genotoxicity or carcinogenicity of Ni(2+).

The frequency of CD4(+)  Foxp3(+) regulatory T (Treg) cells is often significantly increased in the blood of tumour-bearing mice and people with cancer. Moreover, Treg cell frequencies are often higher in tumours compared with blood and lymphoid organs. We wished to determine whether certain chemokines expressed within the tumour mass selectively recruit Treg cells, thereby contributing to their enrichment within the tumour-infiltrating lymphocyte pool. To achieve this goal, the chemokine profile of carcinogen-induced fibrosarcomas was determined, and the chemokine receptor expression profiles of both CD4(+)  Foxp3(-) and CD4(+)  Foxp3(+) T cells were compared. These analyses revealed that the tumours are characterized by expression of inflammatory chemokines (CCL2, CCL5, CCL7, CCL8, CCL12, CXCL9, CXCL10 and CX3CL1), reflected by an enrichment of activated Foxp3(-) and Foxp3(+) T cells expressing T helper type 1-associated chemokine receptors. Notably, we found that CXCR3(+) T cells were significantly enriched in the tumours although curiously we found no evidence that CXCR3 was required for their recruitment. Instead, CXCR3 marks a population of activated Foxp3(-) and Foxp3(+) T cells, which use multiple and overlapping ligand receptor pairs to guide their migration to tumours. Collectively, these data indicate that enrichment of Foxp3(+) cells in tumours characterized by expression of inflammatory chemokines, does not occur via a distinct chemokine axis, thus selective chemokine blockade is unlikely to represent a meaningful therapeutic strategy for preventing Treg cell accumulation in tumours., (© 2014 The Authors. Immunology Published by John Wiley & Sons Ltd.)

Matrix metalloproteinase (MMP)-9 degrades type IV collagen in the basement membrane and plays crucial roles in several pathological implications, including tumorigenesis and inflammation. In this study, we analyzed the effect of flavonols on MMP-9 expression in phorbol-12-myristate-13-acetate (PMA)-induced human fibrosarcoma HT-1080 cells. Galangin and kaempferol efficiently decreased MMP-9 secretion, whereas fisetin only weakly decreased its secretion. Galangin and kaempferol did not affect cell viability at concentrations up to 30 μM. Luciferase reporter assays showed that galangin and kaempferol decrease transcription of MMP-9 mRNA. Moreover, galangin and kaempferol strongly reduce IκBα phosphorylation and significantly decrease JNK phosphorylation. These results indicate that galangin and kaempferol suppress PMA-induced MMP-9 expression by blocking activation of NF-κB and AP-1. Therefore, these flavonols could be used as chemopreventive agents to lower the risk of diseases involving MMP-9.

Phorbol ester (PMA or TPA), a tumor promoter, can cause either proliferation or cell cycle arrest, depending on cellular context. For example, in SKBr3 breast cancer cells, PMA hyper-activates the MEK/MAPK pathway, thus inducing p21 and cell cycle arrest. Here we showed that PMA-induced arrest was followed by conversion to cellular senescence (geroconversion). Geroconversion was associated with active mTOR and S6 kinase (S6K). Rapamycin suppressed geroconversion, maintaining quiescence instead. In this model, PMA induced arrest (step one of a senescence program), whereas constitutively active mTOR drove geroconversion (step two). Without affecting Akt phosphorylation, PMA increased phosphorylation of S6K (T389) and S6 (S240/244), and that was completely prevented by rapamycin. Yet, T421/S424 and S235/236 (p-S6K and p-S6, respectively) phosphorylation became rapamycin-insensitive in the presence of PMA. Either MEK or mTOR was sufficient to phosphorylate these PMA-induced rapamycin-resistant sites because co-treatment with U0126 and rapamycin was required to abrogate them. We next tested whether activation of rapamycin-insensitive pathways would shift quiescence towards senescence. In HT-p21 cells, cell cycle arrest was caused by IPTG-inducible p21 and was spontaneously followed by mTOR-dependent geroconversion. Rapamycin suppressed geroconversion, whereas PMA partially counteracted the effect of rapamycin, revealing the involvement of rapamycin-insensitive gerogenic pathways. In normal RPE cells arrested by serum withdrawal, the mTOR/pS6 pathway was inhibited and cells remained quiescent. PMA transiently activated mTOR, enabling partial geroconversion. We conclude that PMA can initiate a senescent program by either inducing arrest or fostering geroconversion or both. Rapamycin can decrease gero-conversion by PMA, without preventing PMA-induced arrest. The tumor promoter PMA is a gero-promoter, which may be useful to study aging in mammals.

Tumor recognition by immune effector cells is mediated by antigen receptors and a variety of adhesion and costimulatory molecules. The evidence accumulated since the identification of CD155 and CD112 as ligands for DNAM-1 in humans and mice has suggested that the interactions between DNAM-1 and its ligands play an important role in T cell- and natural killer (NK) cell-mediated recognition and lysis of tumor cells. We have previously demonstrated that methylcholanthrane (MCA) accelerates tumor development in DNAM-1-deficient mice, and the Cd155 level on MCA-induced tumors is significantly higher in DNAM-1-deficient mice than in wild-type (WT) mice. By contrast, Cd112 expression on the tumors is similar in WT and DNAM-1-deficient mice, suggesting that CD155 plays a major role as a DNAM-1 ligand in activation of T cells and NK cells for tumor immune surveillance. To address this hypothesis, we examined MCA-induced tumor development in CD155-deficient mice. Unexpectedly, we observed no significant difference in tumor development between WT and CD155-deficient mice. Instead, we found that Cd112 expression was significantly higher in the MCA-induced tumors of CD155-deficient mice than in those of WT mice. We also observed higher expression of DNAM-1 and lower expression of an inhibitory receptor, TIGIT, on CD8+ T cells in CD155-deficient mice. These results suggest that modulation of the expression of receptors and CD112 compensates for CD155 deficiency in immune surveillance against MCA-induced tumors.

Immune suppression is well documented during tumor progression, which includes loss of effect of T cells and expansion of T regulatory (Treg) cells. IL-7 plays a key role in the proliferation, survival and homeostasis of T cells and displays a potent antitumor activity in vivo. In the present study, we investigated the antitumor effect of IL-7 in Meth A model. IL-7 inhibited tumor growth and prolonged the survival of tumor-bearing mice with corresponding increases in the frequency of CD4 and CD8 T cells, Th1 (CD4(+)IFN-γ(+)), Tc1 (CD8(+)IFN-γ(+)) and T cells cytolytic activity against Meth A cells. Neutralization of CD4 or CD8 T cells reversed the antitumor benefit of IL-7. Furthermore, IL-7 decreased regulatory T Foxp3 as well as cells suppressive activity with a reciprocal increase in SMAD7. In addition, we observed an increase of the serum concentrations of IL-6 and IFN-γ, and a significant decrease of TGF-β and IL-10 after IL-7 treatment. Taken together, these results indicate that IL-7 augments T cell-mediated antitumor immunity and improves the effect of antitumor in Meth A model., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Toll-like receptors (TLRs) enable metazoans to mount effective innate immune responses to microbial and viral pathogens, as well as to endogenous host-derived ligands. It is understood that genetic background of the host can influence TLR responsiveness, altering susceptibility to pathogen infection, autoimmunity and cancer. Macrophage stimulatory protein (MSP), which activates the receptor tyrosine kinase recepteur d'origine nantais (RON), promotes key macrophage functions such as motility and phagocytic activity. MSP also acts via RON to modulate signaling by TLR4, which recognizes a range of pathogen or endogenous host-derived molecules. Here, we show that RON exerts divergent control over TLR4 activity in macrophages from different mouse genetic backgrounds. RON potently modulated the TLR4 response in macrophages from M2-prone FVB mice, as compared with M1-skewed C57Bl6 mice. Moreover, global expression analysis revealed that RON suppresses the TLR4-dependent type-I interferon gene signature only in FVB macrophages. This leads to attenuated production of the potent inflammatory mediator, tumor necrosis factor-α. Eliminating RON kinase activity markedly decreased carcinogen-mediated tumorigenesis in M2/Th2-biased FVB mice. We propose that host genetic background influences RON function, thereby contributing to the variability in TLR4 responsiveness in rodents and, potentially, in humans. These findings provide novel insight into the complex interplay between genetic context and immune function.

Cancer/testis antigens (CTAs) are known to be expressed in various cancer types but are minimally or not expressed in normal tissues except for germline tissues. CTAs are attractive targets for cancer immunotherapy and diagnosis because of their restricted expression. The mechanisms of CTAs expression are unclear because the inducers of CTAs expression remain to be elucidated. We hypothesized that carcinogens may induce the cellular expression of CTAs. To prove this, we attempted to inoculate Helicobacter pylori, a known carcinogen, in Meth-A cells, normal gastric cells, and normal splenocytes and induce the expression of a CTA. Melanoma antigen-encoding gene (Mage)-A3, one of the CTAs, was not expressed in both normal cells but in Meth-A cells inoculated with H. pylori. Furthermore, we performed limiting dilution using Meth-A cells inoculated with H. pylori and established derivative clone from Meth-A designated as Meth-A/pylori/3C3 which permanently express Mage-A3 after excluding H. pylori. We herein report the first successful induction of a CTA in a cell line via exposure to a carcinogenic agent. Furthermore, the establishment of Meth-A/pylori/3C3, which is Meth-A expressing Mage-A3, is considered to contribute to the resolution of the mechanism of CTAs expression.

The NLRP3 inflammasome acts as a danger signal sensor that triggers and coordinates the inflammatory response upon infectious insults or tissue injury and damage. However, the role of the NLRP3 inflammasome in natural killer (NK) cell-mediated control of tumor immunity is poorly understood. Here, we show in a model of chemical-induced carcinogenesis and a series of experimental and spontaneous metastases models that mice lacking NLRP3 display significantly reduced tumor burden than control wild-type (WT) mice. The suppression of spontaneous and experimental tumor metastases and methylcholanthrene (MCA)-induced sarcomas in mice deficient for NLRP3 was NK cell and IFN-γ-dependent. Focusing on the amenable B16F10 experimental lung metastases model, we determined that expression of NLRP3 in bone marrow-derived cells was necessary for optimal tumor metastasis. Tumor-driven expansion of CD11b(+)Gr-1(intermediate) (Gr-1(int)) myeloid cells within the lung tumor microenvironment of NLRP3(-/-) mice was coincident with increased lung infiltrating activated NK cells and an enhanced antimetastatic response. The CD11b(+)Gr-1(int) myeloid cells displayed a unique cell surface phenotype and were characterized by their elevated production of CCL5 and CXCL9 chemokines. Adoptive transfer of this population into WT mice enhanced NK cell numbers in, and suppression of, B16F10 lung metastases. Together, these data suggested that NLRP3 is an important suppressor of NK cell-mediated control of carcinogenesis and metastases and identify CD11b(+)Gr-1(int) myeloid cells that promote NK cell antimetastatic function., (©2012 AACR.)

Congenital fibrosarcoma (CFS) is a rare fibrous tissue malignancy that usually presents in the first few years of life. It is unique among human sarcomas in that it has an excellent prognosis. We describe a temporal clustering of a number of cases of CFS and investigate the possible associated prenatal risk factors. The Pediatric Environmental History, a questionnaire developed in our clinic that is instrumental in determining environmental risk factors for tumor-related disease, was essential in documenting the presence or absence of risk factors considered as human carcinogens. We found a history of exposure to petroleum products in four cases of CFS that occurred at a greater than expected rate in a short time frame-an apparent cancer cluster. We call attention to the possibility that exposure to petroleum products raises the risk of developing CFS. While future studies should focus on systematic investigation of CFS and its underlying mechanisms, this report suggests the need for proactive measures to avoid exposure to solvents and petroleum products during pregnancy.

Cancer immunoediting, the process by which the immune system controls tumor growth and shapes tumor immunogenicity, consists of 3 stages: elimination, equilibrium, and escape. The molecular mechanisms that underlie the equilibrium phase, during which the immune system maintains tumor dormancy, remain incompletely defined. Here, we investigated the length of the equilibrium phase during immune control of methylcholanthrene (MCA)-induced or p53 mutant cancers and showed the critical and opposing roles of interleukin (IL)-23 and IL-12 in maintaining cancer cells in a state of immune-mediated dormancy. Inhibition of IL-23p19 was shown to reduce the malignant potential of lesions established by MCA inoculation, whereas inhibition of IL-12/23p40 enhanced tumor outgrowth. Furthermore, agonistic anti-CD40 antibody treatment mimicked the effects of anti-IL-23p19 monoclonal antibody treatment. Other cytokines such as IL-4, IL-17, TNF, and IFNαβ, which are known to play important roles either in MCA tumorigenesis or in the elimination phase of cancer immunoediting, did not play critical roles in maintaining the equilibrium phase. Taken together, our findings show opposing roles for IL-23 and IL-12 in determining the outgrowth versus dormancy of occult neoplasia and suggest a potential long-term danger in using IL-12/23p40 antibodies for treating human autoimmune inflammatory disorders., (©2012 AACR.)

Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis.

CD73 is a cell surface 5'-nucleotidase that converts AMP to adenosine, an immune suppressive molecule. CD73 may promote immune escape in cancer by contributing to the degradation of extracellular ATP released by dying cancer cells in hypoxic tumors or following chemotherapy. However, whether CD73 exerts a critical oncogenic function during tumorigenesis is unknown. In this study, we used genetically deficient mice to investigate its contribution to autochthonous tumor formation. CD73 deficiency suppressed the development of 3-methylcholanthrene (MCA)-induced fibrosarcomas through a mechanism relying upon IFN-γ, natural killer (NK) cells, and CD8(+) T cells. Similarly, CD73 deficiency also suppressed prostate tumorigenesis in TRAMP transgenic mice. Importantly, treatment with an anti-CD73 monoclonal antibody effectively suppressed growth of established MCA-induced tumors or TRAMP-C1 prostate tumors and inhibited the development of TRAMP-C1 lung metastases. The therapeutic activity of anti-CD73 monoclonal antibody against primary tumors was dependent on CD8(+) T cells, whereas its antimetastatic activity was dependent on host CD73 expression independent of T cells or NK cells. Taken together, our findings indicate that CD73 is a critical factor in tumorigenesis and that anti-CD73 antibodies may offer a novel generalized strategy to blunt immune escape and treat cancer., (©2012 AACR)

Context: Elevated expression of the folate receptor (FR) occurs in many human malignancies. Thus, folate targeting is widely utilized in drug delivery purposes specially using nano-radioactive agents., Aims: In this work, we report production and biological evaluation of gallium-67 labeled superparamagnetic iron oxide nanoparticles, embedded by folic acid ( 67 Ga-SPION-folate) complex especially in tumor-bearing mice for tumor imaging studies., Settings and Design: The structure of SPION-folate was confirmed by X-ray diffraction (XRD), transmission electron microscopy (TEM) and foureir transform infrared spectroscopy (FT-IR) analyses. The radiolabeled SPION-folate formation was confirmed by instant thin layer chromatography (ITLC). Tumor induction was performed by the use of poly-aromatic hydrocarbon injection in rodents as reported previously., Materials and Methods: [ 67 Ga]-SPION-folate was shown to possess a particle size of ≈ 5-10 nm using instrumental methods followed by ITLC test. Biocompatibility of the compound was investigated using an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay followed by stability tests and tumor accumulation studies in fibrosarcoma-bearing mice after subcutaneous (s.c.) application., Statistical Analysis Used: All values were expressed as mean ± standard deviation (mean ± SD) and the data were compared using Student t-test. Statistical significance was defined as P<0.05., Results: [ 67 Ga]-SPION-folate was prepared by a modified co-precipitation method possessing a particle size of ≈ 5-10 nm using instrumental methods (>95% radiochemical purity). Biodistribution studies demonstrated tumor:blood, tumor:bone and tumor:muscle ratios of 4.23, 4.98 and 11.54 respectively after 24 h., Conclusions: Due to the nano-scale size and high-penetrative property of the developed folate-containing nano-complex, this system can be an interesting drug delivery modality with therapeutic applications and folate receptor-targeting behavior, while possessing paramagnetic properties for thermotherapy.

Interleukin-10 (IL-10) is a broadly acting immune inhibitory cytokine that is generally thought to support tumor growth. Here we challenge this view with evidence that genetic ablation of IL-10 in the mouse significantly heightens sensitivity to chemical carcinogenesis, growth of transplanted tumors, and formation of metastases. Tumor growth in IL-10-deficient (IL-10(-/-)) mice was associated with an increased level of myeloid-derived suppressor cells (MDSC) and CD4(+)Foxp3(+) regulatory T (Treg) cells in both the tumor microenvironment and the tumor-draining lymph nodes. IL-10(-/-) MDSCs express high levels of MHC and IL-1, and they efficiently induced formation of Treg cells. IL-1 signaling blockade reduced tumor growth mediated by IL-10 deficiency, associated with a partial rescue of tumor infiltration and function of effector T cells and a decrease in tumor angiogenesis and tumor infiltration by Treg cells. Taken together, our findings establish that endogenous IL-10 inhibits inflammatory cytokine production and hampers the development of Treg cells and MDSCs, two key components of the immunosuppressive tumor microenvironment, thereby inhibiting tumor development, growth, and metastasis.

This work reports the chemopreventive property of hydroalcoholic extract of the Trichosanthes dioica root (TDA) against 3-methylcholanthrene (3-MC)-induced carcinogenesis in Swiss albino mice. TDA was administered orally at 2 and 4 mg/kg for 45 days after 24 hours of a single subcutaneous administration of 3-MC (200 µg) in mice. The mice were observed for 15 weeks to record tumor incidence (fibrosarcoma) and survival. After 15 weeks the mice were killed for the evaluation of hematological profiles and hepatic biochemical parameters viz lipid peroxidation, reduced glutathione, glutathione-S-transferase, superoxide dismutase, and catalase. TDA treatment markedly reduced tumor incidence and prolonged the life span of sarcoma-bearing mice compared with 3-MC control mice. Hematological profiles of TDA-treated mice were restored significantly to normal levels. TDA treatment significantly modulated the liver biochemical parameters compared with 3-MC control. Therefore, TDA possesses remarkable cancer chemopreventive efficacy plausibly mediated by multiple mechanisms in Swiss albino mice.

Reducing cancer incidence and mortality by use of cancer-chemopreventive agents is an important goal. We have established an in vitro bioassay that is able to screen large numbers of candidate chemicals that are positive for prevention of inflammation-related carcinogenesis. To accomplish this we have added candidate chemicals or vehicles and freshly isolated, fluorescent dye-labeled inflammatory cells that were overlaid on TNF-alpha-stimulated mouse endothelial cells in a 96-well plate. Inhibition of inflammatory cell attachment to the endothelial cells by the chemicals was quantified by the intensity of fluorescence from the adherent inflammatory cells after removing unattached cells. Using this assay, we selected two chemicals, auraptene and turmerones, for further study. As an in vivo test, diets containing these test chemicals were administered to mice with a piece of foreign body, gelatin sponge, that had been implanted to cause inflammation, and we found that the number of inflammatory cells that infiltrated into the subcutaneously implanted gelatin sponge was reduced compared to that found in the mice fed with a control diet. Moreover, diets containing either of the two chemicals prevented inflammation-based carcinogenesis in a mouse model. We found that the compounds reduced not only the number of infiltrating cells but also the expression of inducible nitric oxide synthase (iNOS) or formation of 8-hydroxy-2'-deoxyguanine (8-OHdG) in the infiltrated cells. Moreover, both compounds but not controls sustained the reducing activity in the inflammatory lesion, and this finding was confirmed by using non-invasive in vivo electron spin resonance. The newly established in vitro screening assay will be useful for finding biologically effective chemopreventive agents against inflammation-related carcinogenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The dual peroxisome-proliferator-activated receptor (PPAR) α/γ agonist tesaglitazar has been shown to produce fibrosarcomas in rats. Here, the authors studied morphology, proliferation, differentiation, and inflammation markers in adipose tissue from rats exposed to 1, 3, or 10 µmol/kg tesaglitazar for 2 or 12 weeks, including recovery groups (12 weeks treatment followed by 12 weeks recovery), and 3 or 10 µmol/kg tesaglitazar for 24 weeks. Subcutaneous white and brown fat revealed reversible dose-related histopathological alterations and after 12 and 24 weeks developed areas of thickened skin (fatty lumps). There was a dose-dependent increase in proliferation of interstitial cells in white and brown fat as shown by increased mitotic index in all dose groups after 2 weeks. This was limited to the high dose after 12 and 24 weeks in white fat. Gene expression analyses showed that while tesaglitazar induced differentiation of adipose tissue characterized with a switch in cyclin D1 and D3 mRNA by 12 weeks, longer exposure at high doses reversed this differentiation concurrent with a reappearance of early adipocyte and inflammatory markers. These data suggest that sustained increased turnover of mesenchymal cells in adipose tissues, concomitant with onset of inflammation and fibrosis, drives development of fibrosarcomas in rats treated with tesaglitazar.

Background: Recent studies have confirmed that the expression of Ezrin, hepatocyte growth factor (HGF) and its receptor (C-met) is related to the genesis, progress, invasion and metastasis of some malignant tumors. Researches have also found that the biological function of Ezrin is closely related to HGF/C-met in malignant tumors. However, there is no report on the expression levels of Ezrin, HGF and C-met in rat pancreatic cancer induced by dimethylbenzanthracene (DMBA). This study aimed to detect the expression of Ezrin, HGF and C-met in rat pancreatic cancer and non-cancerous pancreatic tissues, and assess its effect in cancer induction by DMBA., Methods: Ninety Sprague-Dawley rats were divided into 3 groups randomly: 40 in a pancreatic cancer model group (group A), 40 in a trichostatin A (TSA) intervention group (group B), and 10 in a control group (group C). DMBA was directly implanted into the parenchyma of rat pancreas in group A+group B. The rats of group B were treated with 1 ml of TSA saline solution (1 μg/ml) via intraperitoneal injection weekly. The carcinogenesis of rats executed within 3-5 months in groups A and B was observed by macrograph and microscopy. Meanwhile, the rats in group C were executed within 5 months. The EnVisionTM immunohistochemistry for detecting the expression levels of Ezrin, HGF and C-met was used in paraffin-embedded sections of the pancreatic specimens., Results: The incidence of pancreatic cancer in group A was 48.6% and in group B 33.3%. The maximal diameter of tumor mass was significantly larger in group A than that in group B (P<0.05). No pathological changes were observed in the pancreas of group C and other main organs of groups A and B. The positive rates of Ezrin, HGF and C-met were significantly higher in ductal adenocarcinoma than in non-cancerous pancreatic tissues of groups A and B (P<0.01). The positive rates of Ezrin, HGF and C-met were significantly higher in ductal adenocarcinoma of group A than those in non-cancerous pancreatic tissues of group A (P<0.05), but there was no significant difference in group B (P>0.05). The positive rates of Ezrin, HGF and C-met in non-cancerous pancreatic tissues proved mild to severe atypical hyperplasia of the ductal epithelia. The pancreas of group C and 2 cases of fibrosarcoma showed the negative expression of Ezrin, HGF and C-met. There was a trend of consistency in the expression of Ezrin, HGF and C-met in ductal adenocarcinoma (P<0.05 or P<0.01)., Conclusions: DMBA directly implanted into the parenchyma of the pancreas can produce a model of pancreatic cancer with a high incidence in a short time. TSA might inhibit the carcinogenesis and growth of pancreatic cancer, and its effects may be related to the inhibition of the expression of Ezrin, HGF and C-met during the process. Ezrin, HGF and C-met may have positive effects on the carcinogenesis of rat pancreas.

Sialylation of tumor cells is involved in various aspects of their malignancy (proliferation, motility, invasion, and metastasis); however, its effect on the process of immunoediting that affects tumor cell immunogenicity has not been studied. We have shown that in mice with impaired immunoediting, such as in IL-1α(-/-) and IFNγ(-/-) mice, 3-methylcholanthrene-induced fibrosarcoma cells are immunogenic and concomitantly bear low levels of surface sialylation, whereas tumor cells derived from wild type mice are nonimmunogenic and bear higher levels of surface sialylation. To study immune mechanisms whose interaction with tumor cells involves surface sialic acid residues, we used highly sialylated 3-methylcholanthrene-induced nonimmunogenic fibrosarcoma cell lines from wild type mice, which were treated with sialidase to mimic immunogenic tumor cell variants. In vivo and in vitro experiments revealed that desialylation of tumor cells reduced their growth and induced cytotoxicity by NK cells. Moreover, sialidase-treated tumor cells better activated NK cells for IFN-γ secretion. The NKG2D-activating receptor on NK cells was shown to be involved in interactions with desialylated ligands on tumor cells, the nature of which is still not known. Thus, the degree of sialylation on tumor cells, which is selected during the process of immunoediting, has possibly evolved as an important mechanism of tumor cells with low intrinsic immunogenicity or select for tumor cells that can evade the immune system or subvert its function. When immunoediting is impaired, such as in IFN-γ(-/-) and IL-1α(-/-) mice, the overt tumor consists of desialylayed tumor cells that interact better with immunosurveillance cells.

NK cells interact with a wide variety of hazardous cells including pathogen-infected and tumor cells. NKp46 is a specific NK killer receptor that recognizes various influenza hemagglutinins and unknown tumor ligands. It was recently shown that NKp46 plays a significant role in the in vivo eradication of tumor cells; however, the role played by NKp46 in vivo with regard to tumor development is still unclear. In this study, we used the 3-methylcholanthrene (MCA)-induced fibrosarcoma model in NKp46-deficient mice to test the NKp46 recognition of carcinogen-induced tumors. We show that although the rate of MCA-induced tumor formation was similar in the presence and in the absence of NKp46, the expression of its unknown ligands was NKp46 dependent. The unknown NKp46 ligands were nearly absent in tumors that originated in wild-type mice, whereas they were detected in tumors that originated in the NKp46-deficient mice. We demonstrate that the interactions between NKp46 and its MCA tumor-derived ligands lead to the secretion of IFN-gamma but not to the elimination of the MCA-derived tumor cells. In addition, we show that the in vivo growth of MCA-derived tumor cells expressing high levels of the NKp46 ligands is NKp46 and IFN-gamma dependent. Thus, we present in this study a novel NKp46-mediated mechanism of tumor editing.

During the course of inflammation and its resolution, macrophages are exposed to various cytotoxic materials, including reactive oxygen species. Thus, macrophages require a protective machinery against oxidative stress to survive at the inflammatory site. Here, we showed that xCT, a component of transport system x(c)(-), was significantly up-regulated in activated infiltrating cells, including macrophages and neutrophils at the inflammatory site. System x(c)(-) mediates the uptake of extracellular L-cystine and is consequently responsible for maintenance of intracellular glutathione levels. We established a loss-of-function mouse mutant line of xCT by N-ethyl-N-nitrosourea mutagenesis. Macrophages from xCT(mu/mu) mice showed cell death in association with the excessive release of high mobility group box chromosomal protein 1 upon stimulation with LPS, suggesting that xCT deficiency causes unremitting inflammation because of the impaired survival of activated macrophages at the inflammatory site. Subcutaneous injection of 3-methylcholanthrene (3-MCA) induced the generation of fibrosarcoma in association with inflammation. When 3-MCA was injected s.c. into mice, xCT mRNA was up-regulated in situ. In xCT(mu/mu) mice, inflammatory cytokines (such as IL-1beta and TNFalpha) were overexpressed, and the generation of 3-MCA-induced fibrosarcoma was accelerated. These results clearly indicate that the defect of the protective system against oxidative stress impaired survival of activated macrophages and subsequently enhanced tumorigenecity.

We describe a fibrosarcoma in a 12-year-old Quarterhorse x Arabian gelding as a sequela to equine influenza vaccination. Shortly after the second vaccination, swelling at the site was noticed by the owner and it continued to increase in size over the following 6 months. Biopsy of the mass indicated a fibrosarcoma had developed at the vaccination site. It was approximately 20 cm in diameter and elevated well above the level of the skin. There was no clinical evidence of metastases to the lungs or local lymph nodes. Surgical resection of the mass was performed and the wound healed by first and second intention. Histopathological examination and immunohistochemical staining confirmed a myofibroblastic fibrosarcoma with multifocal osseous metaplasia. To the authors' knowledge, this is the first equine case of a vaccine-associated fibrosarcoma.

The balance between inflammation and immunity is cardinal for the outcome of the malignant process. Local attenuated inflammatory responses mediated by innate cells may provide accessory signals for the development of acquired immunity against malignant cells. In contrast, excessive inflammatory responses accompany tumorigenesis and tumor invasiveness, by the induction of immunosuppression. In the present study, we have assessed the role of tumor cell-derived IL-1 in determining the invasive versus immunostimulatory potential of tumor cells. For this purpose, we have used 3-MCA-induced fibrosarcoma cell lines from IL-1 knockout (KO) versus control mice. Cell lines with no IL-1 failed to establish tumors in intact mice, while lines obtained from control mice were invasive and induced a potent angiogenic response. In contrast, cell lines from IL-1KO mice were more immunogenic. SDF-1 and IL-6, each induced by IL-1, were the two major cytokines whose levels differed in cell lines with or without IL-1. We could not detect differences in cell surface markers related to immunogenicity, such as MHC Class I, co-stimulatory, or adhesion molecules between both types of cells. However, more T-cells were observed at the inoculation site of tumor cells devoid of IL-1 and more pronounced parameters related to anti-tumor immunity were observed in the spleen (IL-12 and IFNgamma) of these mice, compared to mice bearing tumors derived from control mice, where host-derived IL-1 is present. In addition, injection of tumor cells devoid of IL-1, which failed to grow in mice, induced an anti-tumor cell immune memory, while in mice injected with tumor cells from control mice; no immune memory could be detected. From the results, it seems that IL-1 is a crucial factor in determining the balance between immunity and inflammation in tumor-bearing mice. This suggests that manipulation of IL-1 could be useful in anti-tumor therapy, by reducing invasiveness and promoting immunity against the malignant cells.

To study the role of mutant p53 in the induction and cure of tumors, we generated transgenic mice carrying mutant p53 (mp53) containing a 9 bp deletion in exon 6 in addition to wild-type p53, expressing both p53 and mp53. The mp53 cDNA was cloned from a radiation-induced mouse tumor and ligated to the chicken beta-actin promoter/CMV-IE enhancer in the expression vector. The presence of mp53 suppressed p21 expression in primary fibroblasts after ionizing irradiation, indicating the dominant-negative activity of mp53 in the mice. These mice developed fibrosarcomas after the subcutaneous injection of 3-methylcholanthrene with an incidence 1.7-fold higher than that of wild-type mice (42% excess). The tumors were then treated via a potent atelocollagen delivery system with small interfering RNA (siRNA), that targeted the promoter/enhancer of the expression vector, resulting in the suppression of tumor growth in 30% of 44 autochthonous tumors, including four cures, and their transplants, the total fraction corresponding to the tumor excess. This suppressive effect involved the induction of apoptosis. These results indicate that mp53 activity causes tumors that can be suppressed by subsequent silencing of mp53 in the presence of wild-type p53 alleles.

We evaluated the patterns of sialylation on fibrosarcoma cell lines arising following 3-methylcholanthrene treatments of wild-type and IL-1alpha-deficient mice; the former induced progressive tumors, whereas the latter cell lines induced regressing tumors or failed to develop into tumors in mice due to immune rejection. In regressing tumors, terminating alpha2-6-Neu5Ac residues were present at lower levels than in progressively growing tumors. In both tumor cells, the amount of alpha2-6-Neu5Ac residues was higher by an order of magnitude relative to the amount expressed in primary fibroblasts harvested from IL-1alpha-deficient and wild-type mice. We focused on membrane proteins, which may interact with the immune system. Interestingly, HSP65, grp75, and gp96 were found on the surfaces of malignant cells and were shown to possess sialylated N-glycans. The amount of trisialylated glycans on gp96 and HSP65 and monosialylated glycans on grp75 of regressing cells was significantly lower than in progressively growing cells, suggesting a dependency of these specific glycoforms on anti-tumor immunity.

Purpose: Radiation-sensitive microcapsules composed of alginate and hyaluronic acid are being developed. We report the development of improved microcapsules that were prepared using calcium- and yttrium-induced polymerization. We previously reported on the combined antitumor effect of carboplatin-containing microcapsules and radiotherapy., Methods and Materials: We mixed a 0.1% (wt/vol) solution of hyaluronic acid with a 0.2% alginate solution. Carboplatin (l mg) and indocyanine green (12.5 microg) were added to this mixture, and the resultant material was used for capsule preparation. The capsules were prepared by spraying the material into a mixture containing a 4.34% CaCl(2) solution supplemented with 0-0.01% yttrium. These capsules were irradiated with single doses of 0.5, 1.0, 1.5, or 2 Gy (60)Co gamma-rays. Immediately after irradiation, the frequency of microcapsule decomposition was determined using a microparticle-induced X-ray emission camera. The amount of core content released was estimated by particle-induced X-ray emission and colorimetric analysis with 0.25% indocyanine green. The antitumor effect of the combined therapy was determined by monitoring its effects on the diameter of an inoculated Meth A fibrosarcoma., Results: Microcapsules that had been polymerized using a 4.34% CaCl(2) solution supplemented with 5.0 x 10(-3)% (10(-3)% meant or 10%(-3)) yttrium exhibited the maximal decomposition, and the optimal release of core content occurred after 2-Gy irradiation. The microcapsules exhibited a synergistic antitumor effect combined with 2-Gy irradiation and were associated with reduced adverse effects., Conclusion: The results of our study have shown that our liquid core microcapsules can be used in radiotherapy for targeted delivery of chemotherapeutic agents.

We recently reported that tumor-derived interleukin (IL)-1beta strongly promotes tumor growth by inducing myeloid-derived suppressor cell (MDSC) and regulatory T-cell (T(reg)) expansion. To see whether redirection of an immune response can be achieved through immune response-supporting IL-1alpha application, IL-1RI competent (IL-1RI(comp)) and IL-1RI-deficient (IL-1RI(-/-)) mice received IL-1alpha cDNA-transformed attenuated Salmonella typhimurium (SL-IL-1alpha) and/or lysates from methycholanthrene-induced IL-1(comp) or IL-1(-/-) fibrosarcoma cells. Vaccination with SL-IL-1alpha and/or tumor lysate exerted only a minor effect on the survival of IL-1alpha/beta(-/-) and none on IL-1alpha(comp) tumor-bearing mice despite induction of a potent antitumor response, that was overridden by intratumoral and systemic expansion of MDSC. Application of all-trans-retinoic acid together with anti-CD25 efficiently coped with MDSC and T(reg) expansion. Vaccination concomitantly with application of all-trans-retinoic acid and anti-CD25 treatment significantly increased the survival time and rate of IL-1alpha/beta(comp), but even of IL-1alpha(-/-)beta(comp) IL-1RI(comp) tumor-bearing mice. Instead, in IL-1RI(-/-) mice, though MDSC expansion was weaker, SL-IL-1alpha application hardly displayed any therapeutic efficacy, which implies signal transduction through IL-1alpha binding to the IL-1RI as an essential component for immune response induction. Taken together, IL-1alpha can efficiently support tumor vaccination, as far as expansion of MDSC and T(reg) is controlled. However, care should be taken to interfere with MDSC expansion/activation not through a blockade of the IL-1RI, which is the preferential target of IL-1alpha.

The chemical carcinogen MCA induces fibrosarcoma and tissue damage at the injection site. Despite the importance of ROS in the development of cancer, little is known about the pattern of expression of ROS in MCA-induced fibrosarcomas. To gain some insight into the biological significance of iNOS and Cu/Zn-SOD, comparative immunohistochemical analyses were performed to characterize their expression in MCA-induced fibrosarcomas. Cyclin A is overexpressed in various tumors, but its expression in MCA-induced fibrosarcoma in mice and its correlation to mitosis and apoptosis are unclear. The presence of apoptotic cell death was evaluated using the TUNEL method and findings were compared with cyclin A expression and mitotic count of fibrosarcomas. Subcutaneous application of MCA caused fibrosarcoma development in 14 of 20 mice (70%) in 26 weeks. Limited cytoplasmic Cu/Zn-SOD and iNOS immunostainings were detected in 13 of 14 and 9 of 14 tumors with median immunoreactive scores of 2 and 1, respectively. Prominent nuclear cyclin A immunostaining and TUNEL-positive reactions were seen in all the fibrosarcoma cases. Cyclin A immunoreaction significantly correlated with the TUNEL index (P<0.01) and MC (P<0.001). The present findings show a low level of iNOS expression in neoplastic cells indicating limited synthesizing capacity of tumor cells. Limited Cu/Zn-SOD reaction could be associated with an imbalance in between pro-oxidant/antioxidant levels. Furthermore, it was shown that cyclin A is overexpressed in MCA-induced fibrosarcomas and possibly plays a significant role in the pathogenesis of fibrosarcomas. Cyclin A could be useful for detecting the S phase of the cell cycle and could also indicate that cyclin A may induce S phase arrest associated with apoptosis in the MCA-induced fibrosarcomas.

Recent studies in human fibroblasts have provided a new general paradigm of tumor suppression according to which oncogenic signaling produces DNA damage and this, in turn, results in ATM/p53-dependent cellular senescence. Here, we have tested this model in a variety of murine experimental systems. Overexpression of oncogenic Ras in murine fibroblasts efficiently induced senescence but this occurred in the absence of detectable DNA damage signaling, thus suggesting a fundamental difference between human and murine cells. Moreover, lung adenomas initiated by endogenous levels of oncogenic K-Ras presented abundant senescent cells, but undetectable DNA damage signaling. Accordingly, K-Ras-driven adenomas were also senescent in Atm-null mice, and the tumorigenic progression of these lesions was only modestly accelerated by Atm-deficiency. Finally, we have examined chemically-induced fibrosarcomas, which possess a persistently activated DNA damage response and are highly sensitive to the activity of p53. We found that the absence of Atm favored genomic instability in the resulting tumors, but did not affect the persistent DNA damage response and did not impair p53-dependent tumor suppression. All together, we conclude that oncogene-induced senescence in mice may occur in the absence of a detectable DNA damage response. Regarding murine Atm, our data suggest that it plays a minor role in oncogene-induced senescence or in p53-dependent tumor suppression, being its tumor suppressive activity probably limited to the maintenance of genomic stability.

There is evidence that cell-associated IL-1 alpha supports immune response induction. Here we explored the impact of malignant cell-derived IL-1 on immunogenicity, immune response induction and tumor-induced immunosuppression using 3-methylcholanthrene-induced fibrosarcoma lines derived from wild-type (wt), IL-1 alpha-, IL-1 beta- or IL-1a beta-knockout (IL-1 alpha(-/-), IL-1 beta(-/-), IL-1 alphabeta(-/-)) C57BL6 mice. The wt, IL-1 alpha(-/-), IL-1 beta(-/-) and IL-1 alphabeta(-/-) fibrosarcoma lines express MHC class I molecules at a high level. The lines do not differ in their susceptibility toward NK cells, macrophages, and allogeneic CTL, or in their capacity as stimulators of an allogeneic response. However, IL-1 beta(-/-) tumors rarely grow in the syngeneic host, which is the consequence of a strong T helper and CTL response induction by IL-1 alpha-competent, IL-1 beta(-/-) tumors. On the other hand, IL-1 beta-competent, IL-1 alpha(-/-) tumors strongly assist CD11b(+)Gr-1(+) myeloid-derived suppressor cell and regulatory T cell expansion, which both suppress with high efficacy activated T helper cell proliferation and CTL lysis. In IL-1 alphabeta(-/-) tumors, the absence of IL-1 alpha becomes decisive, i.e. despite reduced suppressor cell recruitment, tumor growth was unimpaired due to inefficient immune response induction. Thus, sarcoma cell-derived IL-1 alpha and IL-1 beta do not act in concert. Induction of a strong immune response by IL-1 alpha demands therapeutic exploitation, which may become more efficient if systemic induction of immunosuppression by IL-1 beta can also be circumvented., ((c) 2008 Wiley-Liss, Inc.)

Pseudolaric acid B (PAB) exerted cytostatic activity on murine fibrosarcoma L929 cells. The cytostatic mechanism of PAB on L929 cells was investigated in this paper. At 36 h, after 80 microM PAB treatment, the inhibitory ratio was 65.37 +/- 4.12%, and the MDC staining ratio was strongest in L929 cells. 3-Methyladenine (3-MA), an inhibitor of autophagy, inhibited the generation of autolysosomes induced by PAB. The expression of autophagy-associated Beclin 1 protein was up-regulated, and microtubule-associated protein light chain 3 I (LC3 I) was cleaved into LC3 II after 80 microM PAB treatment from 12 h. Therefore, it was concluded that PAB exerted a cytostatic effect on L929 cells through autophagy. However, 80 microM PAB treatment did not induce apoptotic body formation, but 3 mM 3-MA promoted apoptosis, so autophagy might inhibit apoptosis. PAB had no effect on mitochondrial membrane potential (MMP), but up-regulated the expressions of Bax and Bcl-2. Immunoprecipitation analysis showed that PAB inhibited Bcl-2 binding with Beclin 1. Additionally, PAB inhibited the localization of Bax in mitochondria, but Bcl-2 still was in the mitochondria to sustain MMP. Meanwhile PAB promoted the phosphorylation of cytoplasmic Bcl-2. Therefore the phosphorylation of Bcl-2 in the cytoplasm and the mitochondrial location of Bcl-2 might be the reasons why PAB inhibited the binding of Bcl-2 and Beclin 1.

Ligands for the NKG2D stimulatory receptor are frequently upregulated on tumor lines, rendering them sensitive to natural killer (NK) cells, but the role of NKG2D in tumor surveillance has not been addressed in spontaneous cancer models. Here, we provided the first characterization of NKG2D-deficient mice, including evidence that NKG2D was not necessary for NK cell development but was critical for immunosurveillance of epithelial and lymphoid malignancies in two transgenic models of de novo tumorigenesis. In both models, we detected NKG2D ligands on the tumor cell surface ex vivo, providing needed evidence for ligand expression by primary tumors. In a prostate cancer model, aggressive tumors arising in NKG2D-deficient mice expressed higher amounts of NKG2D ligands than did similar tumors in wild-type mice, suggesting an NKG2D-dependent immunoediting of tumors in this model. These findings provide important genetic evidence for surveillance of primary tumors by an NK receptor.

Two cases of feline vaccine-associated fibrosarcoma (FVAF) are reported. The excised tumours were both characterised as well circumscribed, subcutaneous, firm and white with central necrosis. Histopathologically, they consisted of well-differentiated and variably sized and shaped anaplastic cells, characterised by marked nuclear and cellular pleomorphism including giant cells. The mitotic activity was low. Aluminium was demonstrated in the central necrosis and giant cells. Neoplastic cells were positive for vimentin and negative for desmin and cytokeratin. The presence of feline sarcoma virus and feline immunodeficiency virus could not be detected by PCR in either case.

Although side effects of cancer chemotherapy are well known, "opposite effects" of chemotherapy that enhance the malignancy of the treated cancer are not well understood. In this report, we describe the induction of intravascular proliferation, extravasation, and colony formation by cancer cells, critical steps of metastasis, by pretreatment of host mice with the commonly used chemotherapy drug cyclophosphamide. In contrast, in the unpretreated mice, most cancer cells remained quiescent in vessels without extravasation. HT1080 human fibrosarcoma cells, labeled in the nucleus with green fluorescent protein and red fluorescent protein in the cytoplasm for imaging, were injected into the epigastric cranialis vein of nude mice. Twenty-four hours before cancer cell injection, cyclophosphamide was given i.p. Double-labeled cancer cells were imaged at the cellular level in live mice with the Olympus OV100 Small Animal Imaging System with variable magnification. Cyclophosphamide seems to interfere with a host process that inhibits intravascular proliferation, extravasation, and extravascular colony formation. Cyclophosphamide does not directly affect the cancer cells because cyclophosphamide has been cleared by the time the cancer cells were injected. This report shows an important unexpected "opposite effect" of chemotherapy that enhances critical steps in malignancy rather than inhibiting them, suggesting that certain current approaches to cancer chemotherapy should be modified.

Up to date, most attempts to use immunotherapy to cause the regression of animal and human established tumors have not been successful. Former experiments have postulated that this failure could be attributed, at least in part, to a lack of immunogenicity of spontaneous tumors. In this paper, we have investigated whether this lack of immunogenicity can be attributed to the absence of tumor antigens or to the existence of tolerogenic mechanisms preventing such antigens from initiating an antitumor immune response. We have used two murine tumors a non-immunogenic spontaneous lymphoma (LB) and a strongly immunogenic methylcholanthrene-induced fibrosarcoma (MC-C) together with a vaccination strategy based on the inoculation of dendritic cells (DC) loaded with a tumor lysate. When DC were pulsed with LB lysate (DC+LB), no maturation of DC was achieved in vitro and no protection against LB implants after DC+LB inoculation was observed in vivo. On the other hand, when DC were pulsed with MC-C lysate (DC+MC-C), maturation of DC was observed along with a strong protection against MC-C implants after DC+MC-C inoculaton. Finally, when DC were pulsed with both LB and MC-C lysates (DC+LB+MC-C), maturation of DC and protection against LB implants were achieved. Since no immune cross reaction between MC-C and LB was ever observed, the most likely interpretation is that LB bears specific tumor antigens but lacks other signals to achieve DC maturation. These signals would be provided by MC-C which would enable DC to mature and to initiate an effective anti-LB immune response.

Peroxisome proliferator-activated receptors (PPAR) are involved in the pathogenesis of insulin resistance, diabetes, and related complications. Consequently, the identification of PPAR subtypes and the potential for their activation provides promising therapeutic targets for the management of type 2 diabetes mellitus. Available data from rodent carcinogenicity studies, however, demonstrate that PPAR agonists can be tumorigenic in one or more species of rodents at multiple sites. In 2005, the Health and Environmental Sciences Institute (HESI) PPAR Agonist Project Committee was established by a group of pharmaceutical companies to advance research on and to understand the modes of action and human relevance of this emerging rodent tumor data for PPAR agonists. Since the most commonly observed tumor types reported in rodents are hemangiosarcomas, fibrosarcomas and liposarcomas, the PPAR Agonist Project Committee approved a Pathology Working Group (PWG) to develop consensus of morphologic criteria for tumor diagnoses and consistency of diagnoses across multiple studies for hemangiosarcomas in mice and hamsters and liposarcomas/fibrosarcomas in rats. Therefore, the focus of the PWG review was to establish consistent tumor diagnostic criteria, to assess evidence of potentially preneoplastic changes and to identify distinguishing morphologic differences which may exist between spontaneous changes present in control animals with similar changes from treated animals. Specific diagnostic criteria and nomenclature are recommended for the classification of proliferative vascular lesions which may be present in mice or hamsters and for proliferative mesenchymal changes in rats in studies that are conducted with PPAR agonists.

A crossbred cat developed a subcutaneous fibrosarcoma on the left side of the thorax at the site of previous administration of a feline parvo-, herpes- and calicivirus vaccine. A few months later the cat developed a second mass on the right side of the thorax after a booster vaccine had been administered at this site. This unique case of bilateral fibrosarcomas in a cat shortly after vaccination with parvo-, herpes- and caliciviruses suggests an individual disposition for the development of vaccine-associated sarcomas and a possible triggering of this type of pathological response which could have precipitated the development of the second tumour. To the authors' knowledge, this is the first case of vaccine-induced fibrosarcomas occurring bilaterally after injection of a feline parvo-, herpes- and calicivirus containing vaccine at different sides of the thorax.