Preniche as missing link of the metastatic niche concept explaining organ-preferential metastasis of malignant tumors and the type of metastatic disease

Here we attempt to supplement the metastatic niche concept with a stage of “preniche” that determines the site of development of a premetastatic niche and of a subsequent metastasis. The “preniche” includes all cellular and molecular events in the site of a prospective metastasis preceding the entrance of myeloid progenitor cells. The “preniche” integrates an activation of vascular endothelium of the microcirculatory vessels of target organs in the site of a future metastasis under conditions of chronic persistent productive inflammation that can be induced by cytokines from the primary tumor and independently of it. The endothelium activation is responsible for adhesion and clustering of the recruited myeloid progenitor cells and also for the retention of cells of malignant tumors. The preniche easily arises in organs enriched with organspecific macrophages (lungs, liver, brain, etc.) where the endothelium is predisposed for intensive recruiting of myeloid progenitor cells of macrophages, especially under conditions of inflammation. Introduction of the “preniche” concept allows us to avoid difficulties associated with the development of the metastatic niche concept, especially concerning the problem of organ-preferential localization of metastases, and to make some predictions for experimental verification and potential approaches for preventing metastasizing in some oncologic patients.     

FT-ICR-MS characterization of intermediates in the biosynthesis of the α-methylbutyrate side chain of lovastatin by the 277 kDa polyketide synthase LovF

There are very few fungal polyketide synthases that have been characterized by mass spectrometry. In this paper we describe the in vitro reconstitution and FT-ICR-MS verification of the full activity of an intact 277 kDa fungal polyketide synthase LovF of the lovastatin biosynthetic pathway. We report here both the verification of the reconstitution of fully functional holo-LovF by using (13)C-labeled malonyl-CoA to form α-methylbutyrate functionality and also detection of five predicted intermediates covalently bound to the 4'-phosphopantetheine at the acyl carrier protein (ACP) active site utilizing the phosphopantetheine ejection assay and high-resolution mass spectrometry. Under in vitro conditions, the diketide acetoacetyl intermediate did not accumulate on the ACP active site of holo-LovF following incubation with malonyl-CoA substrate. We found that incubation of holo-LovF with acetoacetyl-CoA served as an effective means of loading the diketide intermediate onto the ACP active site of LovF. Our results demonstrate that subsequent α-methylation of the acetoacetyl intermediate stabilizes the intermediate onto the ACP active site and facilitates the formation and mass spectrometric detection of additional intermediates en route to the formation of α-methylbutyrate.     

MIP-3alpha transfection into a rodent tumor cell line increases intratumoral dendritic cell infiltration but enhances (facilitates) tumor growth and decreases immunogenicity

Dendritic cells are powerful APCs for activation of specific antitumor T lymphocytes. To present tumor Ags efficiently, they have first to migrate to the tumor site, engulf Ag, and then process them. To attract immature DCs to the tumor site, we transfected tumor cells with MIP-3alpha which is strongly chemotactic for DCs. Surprisingly, MIP-3alpha-transfected tumor cells grew faster than the mock-transfected tumor cells. Histological analysis and tumor dissociation confirmed that the MIP-3alpha-transfected tumors contain three to four times more DCs than mock-transfected tumors. FACS analysis of the intratumor DCs showed that they were predominantly immature. Functional analysis showed that the alloreactivity mediated by these infiltrating MIP-3alpha-transfected tumor DCs is strongly reduced. In conclusion, MIP-3alpha is an efficient chemokine for attracting DCs in vivo, but the high density of DCs in the tumor site injection is not a sufficient condition to induce an immune response. Furthermore, this attraction of immature DCs may always have an adverse effect by inducing a tolerance to the tumor cells.     

采用花器喷雾法并基于Cre/lox系统定点整合拟南芥Rps2基因的技术体系研究

To improve site?specific integration technology system, site?specific integration of Rps2 target gene in Arabidopsis thaliana (Linn.) Heynh. was carried out based on Cre/lox system by floral spraying method. The results show that 1495 site?specific integration candidate plants are obtained by this method with a site?specific integration efficiency of about 0076%. After PCR and histochemical staining experiment verification, the positive plants of precise integration account for 8604%, in which, 6334% positive plants are single copy transformed plants. The results of quantitative real?time PCR (qRT?PCR) and hypersensitive reaction (HR) show that the site?specific integrated Rps2 gene can be transcribed and expressed normally. It is suggested that this system can greatly improve the stability and efficiency of site?specific integration genetic transformation system in plants.     

Rapid verification of lambda-cDNA clones using mixed-base oligonucleotide as screening probe and sequencing primer

A rapid procedure has been developed for the isolation and verification of cDNA clones isolated from a cDNA library based on lambda vectors. Using information of the partial amino acid sequence of a protein, synthetic mixed-base oligonucleotides are first employed as a screening probe using the plaque hybridization procedure. The cDNA inserts of the clones obtained are then directly amplified by polymerase chain reaction (PCR) using primers flanking the cloning site of the vector. Besides being used for cloning into a plasmid vector, the amplified DNA's are also subjected to nucleotide sequence analysis using the same mixed-base oligonucleotides as sequencing primers. This approach allows sequencing through the region of the known amino acid sequence for direct verification of the authenticity of the clones obtained. This procedure has successfully been used for cloning and partial characterization of the gene coding for a platelet aggregation inhibitor.     

High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.     

A Mathematical Model for the Regulation of Tumor Dormancy Based on Enzyme Kinetics

In this paper we present a two-compartment model for tumor dormancy based on an idea of Zetter [1998, Ann. Rev. Med. 49, 407–422] to wit: The vascularization of a secondary (daughter) tumor can be suppressed by an inhibitor originating from a larger primary (mother) tumor. We apply this idea at the avascular level to develop a model for the remote suppression of secondary avascular tumors via the secretion of primary avascular tumor inhibitors. The model gives good agreement with the observations of [De Giorgi et al., 2003, Derm. Surgery 29, 664–667]. These authors reported on the emergence of a polypoid melanoma at a site remote from a primary polypoid melanoma after excision of the latter. The authors observed no recurrence of the melanoma at the primary site, but did observe secondary tumors at secondary sites 5–7 cm from the primary site within a period of 1 month after the excision of the primary site. We attempt to provide a reasonable biochemical/cell biological model for this phenomenon. We show that when the tumors are sufficiently remote, the primary tumor will not influence the secondary tumor while, if they are too close together, the primary tumor can effectively prevent the growth of the secondary tumor, even after it is removed. It should be possible to use the model as the basis for a testable hypothesis.     

Lung cancer xenografting alters microRNA profile but not immunophenotype

Lung tumor xenografts grown in immunocompromised mice provide a renewable source of tumor tissue for research and a means to study individualized response to chemotherapy. Critical to this utility is verification that the xenograft cells retain core phenotypic characteristics of the original tumor. We compared eight non-small cell lung carcinomas with their corresponding xenografts grown in mice with severe combined immunodeficiency by way of histology, immunohistochemistry, and microRNA expression profiling. Six of the eight xenografts closely resembled their original tumor by light microscopy. The xenografts also largely retained key immunophenotypic features. With expression profiling of human microRNAs, however, xenografts clustered separately from the original tumors. While this may be partly due to contamination by non-neoplastic human and mouse stroma, the results suggest that miRNA expression may be altered in xenografts and that this possibility should be further evaluated.     

mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa

Broad host-range mini-Tn7 vectors facilitate integration of single-copy genes into bacterial chromosomes at a neutral, naturally evolved site. Here we present a protocol for employing the mini-Tn7 system in bacteria with single attTn7 sites, using the example Pseudomonas aeruginosa. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. aeruginosa by either transformation or conjugation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. From start to verification of the insertion events, the procedure takes as little as 4 d and is very efficient, yielding several thousand transformants per microgram of input DNA or conjugation mixture. In contrast to existing chromosome integration systems, which are mostly based on species-specific phage or more-or-less randomly integrating transposons, the mini-Tn7 system is characterized by its ready adaptability to various bacterial hosts, its site specificity and its efficiency. Vectors have been developed for gene complementation, construction of gene fusions, regulated gene expression and reporter gene tagging.     

Is the Testis a Chemo-Privileged Site? Is There a Blood-Testis Barrier?

The incidence of testicular cancer, primarily seminoma, has been increasing in many countries, including the United States. The testis is often the site of residual cancer after adequate treatment with systemic chemotherapy. The blood-testis barrier is commonly cited as the explanation for residual tumor within the gonad after chemotherapy and as the indication for delayed orchiectomy. Conversely, complete eradication of viable tumor from the primary site is common and argues against the testis as a "tumor sanctuary." Residual tumor is also demonstrated within metastatic foci, and the disparity between the histopathologic response of the primary tumor and metastatic sites may be best explained by tumor heterogeneity and multiple tumor clones. Regardless of the scientific and academic arguments, delayed radical orchiectomy remains an important part of treatment for patients undergoing primary chemotherapy.     

Comprehensive computational design of mCreI homing endonuclease cleavage specificity for genome engineering

Homing endonucleases (HEs) cleave long (～ 20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements. In order to determine whether comprehensive computational design could be used as a general strategy to engineer new HE target site specificities, we used RosettaDesign (RD) to generate 3200 different variants of the mCreI LAGLIDADG HE towards 16 different base pair positions in the 22 bp mCreI target site. Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved. These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.     

A Comparison of Scanning and Two-Wavelength Microspectrophotometry

Because of the absence of suitable standards, microspectrophotometry suffers from a lack of objective verification. An approach to this problem is suggested which is based on the comparison of results obtained when different techniques or instruments are applied to the same objects. The application of this approach to scanning versus two-wavelength photometry has been justified by the widely different bases of these two methods. A group of ascites tumor cells stained with gallocyanin-chrome alum was measured successively with both methods and a high degree of proportionality between the two sets of results was found. This suggests that the two methods measure the same quality of the cells within a standard deviation of 3.39 per cent. This degree of correlation is a verification of the accuracy of both of the methods and shows that either one is suitable for resolving differences in stain content between cell nuclei of the order of 10 per cent.     

A Mathematical Model to Elucidate Brain Tumor Abrogation by Immunotherapy with T11 Target Structure

T11 Target structure (T11TS), a membrane glycoprotein isolated from sheep erythrocytes, reverses the immune suppressed state of brain tumor induced animals by boosting the functional status of the immune cells. This study aims at aiding in the design of more efficacious brain tumor therapies with T11 target structure. We propose a mathematical model for brain tumor (glioma) and the immune system interactions, which aims in designing efficacious brain tumor therapy. The model encompasses considerations of the interactive dynamics of glioma cells, macrophages, cytotoxic T-lymphocytes (CD8+ T-cells), TGF-β, IFN-γ and the T11TS. The system undergoes sensitivity analysis, that determines which state variables are sensitive to the given parameters and the parameters are estimated from the published data. Computer simulations were used for model verification and validation, which highlight the importance of T11 target structure in brain tumor therapy.     

Interleukin-2 inhibits proliferation of HPV-associated tumor cells and halts tumor growth in vivo

Previous studies have shown inhibition of cervical cancer cell growth by treatment with high concentrations of IL-2. In the present study, we evaluated the in vitro and in vivo effects of recombinant human IL-2 on HPV-associated tumor cells (3T3-16). Treatment of 3T3-16 cells with rhIL-2 for 72 h inhibited cell growth in a dose-dependent manner and this effect was evidenced at nanomolar concentrations. These tumor cells expressed mRNA for beta and gamma subunits of the IL-2 receptor, which are required for signal transduction. In experiments to explore the effect of IL-2 on the growth of the HPV-associated tumor, mice received rhIL-2 through different routes: (i) intraperitoneal; (ii) subcutaneous, at the tumor inoculation site; or (iii) subcutaneous, distant from the tumor inoculation site. An effective antitumor response was observed only in those animals that received IL-2 at the tumor site (P<0.01). These results indicate the potential adequacy of therapeutic strategies based on local administration of rhIL-2 for cervical carcinoma, not only based on the ability of this cytokine to stimulate cellular-mediated immunity but also because of its direct effects on tumor cells.     

Site-dependent angiogenic cytokine production in human tumor xenografts

Tumor microenvironment plays a critical role in tumor growth, angiogenesis, and metastasis. Differences in site of tumor implantation result in differences in tumor growth, metastasis, as well as response to chemotherapy. We hypothesized that tumor-induced angiogenic growth factor production into the plasma will also be influenced by site of tumor implantation. We evaluated the site-dependent production of angiogenic growth factors in the plasma of tumor bearing animals at two different sites of implantation. Plasma levels of tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were evaluated in nude mice bearing A2780, SKOV-3, or OVCAR-3 human ovarian tumors, as well as Panc-1, AsPC-1, or BxPC-3 human pancreatic tumors grown as subcutaneous (SC) xenografts or in the intraperitoneal (IP) cavity. Plasma VEGF and bFGF levels produced by two ovarian tumor lines and two pancreatic tumor lines were substantially higher when the tumors were implanted in the IP cavity than in the SC space. These studies indicated that the site of tumor implantation was an important determinant in the production of plasma VEGF and bFGF levels. As more and more anti-angiogenic agents are developed, the need for appropriate animal models becomes apparent. These results suggest the demand for an appropriate model for the in vivo evaluation of anti-angiogenesis.     

模式识别受体介导的肿瘤免疫耐受参与肿瘤转移

肿瘤细胞与宿主相互作用经历免疫警戒、免疫平衡和免疫逃逸过程,称为肿瘤免疫编辑。具有免疫逃逸能力是肿瘤细胞的标志性改变,也是肿瘤生长失控、转移和治疗失效的重要原因。病原微生物所含病原相关模式分子和肿瘤组织释放的损伤相关模式分子与肿瘤细胞及周围组织细胞和免疫细胞表达的模式识别受体相互作用,引起抑制性免疫微环境是导致肿瘤免疫耐受的关键。以肿瘤免疫耐受为药靶,使用小分子化合物或具有免疫刺激活性的生物制剂如单克隆抗体,可逆转肿瘤抑制性免疫微环境,打破肿瘤免疫耐受,抑制肿瘤细胞的生长和侵袭能力,从而降低肿瘤细胞转移和肿瘤致死率。     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.     

Production of affinity purified antipeptide antibodies to survivin for structural and functional studies of the protein

Tumor-associated protein survivin is a bifunctional protein that can participate either in the regulation of cell division or in the inhibition of apoptosis depending on its location in the cell and its structural state. The aim of the present work was to obtain monospecific antibodies to survivin targeting various sites in the protein and suitable for the study of its structural and functional features. The ability of the antibodies to detect survivin in tumor cells and mammary tumor tissues has been assessed. Antibodies targeting the (1–22) and (95–105) fragments of the protein have been shown to have the highest specific activity. Antibodies targeting the (1–22) site preferentially bound to survivin-containing complex (presumably a dimer of survivin) in immunoblotting, whereas antibodies targeting the (95–105) site detected only the monomeric form of the protein. Analysis of malignant mammary tumor samples showed that monomeric survivin was present only in tumor tissue samples, whereas survivin-containing complex was expressed both in tumor tissues and in the tissues adjacent to the tumor. Antibodies targeting the (1–22) site were shown to preferentially detect survivin localized in the cell nuclei (and involved in the regulation of mitosis) in immunocytochemistry, while those targeting the (95–105) site stained the nucleoplasm and cytoplasm at all stages of the cell cycle. Therefore, the antibodies obtained can serve as a useful tool for structural and functional research on survivin.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on itsin vitro andin vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.