Androgen receptor expression and DNA content of paraffin-embedded archival human prostate tumors

BACKGROUND: Androgen receptors (AR) are expressed in human prostate cells and immunohistochemistry has been used for qualitative analysis of AR expression in prostate tumor cells. Quantitative and multiparametric analysis of receptor expression could be of diagnostic and prognostic value in the management of patients on antiandrogen therapy. Multiparametric flow cytometric methods have been developed for analysis of hormone receptor expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded human solid tumors. The present study was undertaken for analysis of AR expression and DNA content in archival human prostate tumors. METHODS: AR expression and DNA content were measured in nuclei isolated by enzyme digestion from thick sections cut from 51 paraffin-embedded human prostate tumors. AR expression in different subpopulations was studied by gated analysis. The relationship among AR activity, DNA content, and histopathological grade was analyzed. RESULTS: Distinct aneuploid populations were observed in 23% of tumors examined. AR activity was observed in all the specimens and the percentage of AR- positive nuclei in the 48 samples analyzed was <10% (n = 4), 11-50% (n = 39), and >51% (n = 5). Tumor subpopulations with aneuploid DNA content had higher AR expression (percent AR-positive cells and mean log fluorescence) than the diploid subpopulations. No strong correlation was seen between AR expression and histopathological grade of the tumors. CONCLUSIONS: Flow cytometric analysis of archival prostate tumor can be used for rapid determination of aneuploid DNA content and AR expression in subpopulations of nuclei isolated from formalin-fixed/paraffin-embedded prostate tumor blocks.     

Distinction between HLA class I-positive and -negative cervical tumor subpopulations by multiparameter DNA flow cytometry

BACKGROUND: The study of the molecular-genetic basis of heterogeneity of HLA class I expression in solid tumors is hampered by the lack of reliable rapid cell-by-cell isolation techniques. Hence, we studied the applicability of a flow cytometric approach (Corver et al.: Cytometry 2000;39;96-107). METHODS: Cells were isolated from five fresh cervical tumors and simultaneously stained for CD45 or vimentin (fluorescein isothiocyanate fluorescence), Keratin (R-phycoerythrin fluorescence), HLA class I (APC fluorescence), and DNA (propidium iodide fluorescence). A dual-laser flow cytometer was used for fluorescence analysis. Tissue sections from the corresponding tumors were stained for HLA class I antigens, keratin, vimentin, or CD45. RESULTS: Flow cytometry enabled the simultaneous measurement of normal stromal cells (vimentin positive), inflammatory cells (CD45 positive), epithelial cells (keratin positive), and DNA content readily. Normal stromal/inflammatory cells served as intrinsic HLA class I-positive as well as DNA-diploid references. Good DNA histogram quality was obtained (average coefficient of variation < 4%). Intratumor keratin positive subpopulations differing in HLA class I expression as well as DNA content could be clearly identified. Losses of allele-specific HLA class I expression found by immunohistochemistry were also detected by flow cytometry. CONCLUSIONS: We conclude that multiparameter DNA flow cytometry is a powerful tool to study loss of HLA class I expression in human cervical tumors. The method enables flow-sorting of discrete tumor and normal cell subpopulations for further molecular genetic analysis.     

Cytoskeletal proteins as tissue-specific markers in cytopathology

Antibodies to intermediate filament proteins react in a tissue-specific manner and can be used to characterize tumor cells present in thin-needle aspirates from solid tumors, from palpable lymph nodes and cells present in samples from peritoneal and pleural effusions. From our studies so far the following conclusions can be drawn: Polyclonal antisera to cytokeratins can identify carcinoma metastases in thin-needle aspirates from palpable lymph nodes and distinguish them from malignant lymphomas and nonmalignant lesions such as chronic lymphadenitis, which show only vimentin-positive cells. Monoclonal antibodies to specific cytokeratin polypeptides are able to distinguish between different types of epithelial tumor metastases, i.e. metastases from adenocarcinomas and metastases from squamous cell carcinomas. Cells present in peritoneal and pleural effusions can be partly characterized using intermediate filament antisera. We have found that metastatic adenocarcinoma cells from breast, ovary, endometrium, cervix, colon and stomach, as well as squamous cell carcinomas and malignant mesothelioma stain specifically with antibodies to cytokeratin while mesenchymally derived tumors such as malignant lymphomas, malignant melanomas, and fibrosarcomas, are positive for vimentin only. Metastatic tumor cells of epithelial origin present in aspirates from human serous body cavity fluids may coexpress vimentin next to their original cytokeratin intermediate filaments. Benign mesothelial cells present in body cavity fluids frequently coexpress cytokeratins and vimentin. Tumor cells present in thin-needle aspirates from solid tumors such as pleomorphic adenomas of the parotid gland can be identified as such because of their typical patterns of intermediate filament (co-)expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric DNA analysis using cytokeratin labeling for identification of tumor cells in carcinomas of the breast and the female genital tract.

Flow cytometric assessment of DNA-ploidy and S-phase fraction in malignant tumors is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g., tumor, stromal and inflammatory cells. To evaluate the effect on quality of DNA cell cycle analysis and determination of DNA ploidy, cytokeratin labeling of epithelial cells was used for tumor cell enrichment in breast, ovarian, cervical and endometrial cancer prior to DNA analysis. In a prospective study, tumor cell subpopulations of 620 malignant tumors were labeled by a FITC-conjugated cytokeratin antibody (CK 5, 6, CK18 and CK 5, 6, 8 and CK 17, respectively) prior to flow cytometric cell cycle analysis. Compared to total cell analysis, detection rate of DNA-aneuploid tumors following cytokeratin labeling was increased from 62% to 76.5% in breast cancer, from 68% to 77% in ovarian cancer, from 60% to 80% in cervical cancer and from 30% to 53% in endometrial cancer. Predominantly in DNA-diploid tumors, a significantly improved detection of S-phase fraction of the tumor cells was shown due to the elimination of contaminating nonproliferating "normal cells". S-phase fraction following tumor cell enrichment was increased by 10% (mean) following cytokeratin staining in ovarian and endometrial cancer, by 30% in breast cancer and even by 70% in cervical cancer compared to total cell analysis. Thus, diagnostic accuracy of DNA-analysis was enhanced by cytokeratin labeling of tumor cells for all tumor entities investigated.     

Reliable differential diagnosis between osteosarcoma and regenerative bone cells in rats through simultaneous analysis of nuclear DNA content and size

OBJECTIVE: To evaluate the difference between benign proliferative lesions of regenerative bone calluses and malignant tumors in rats by simultaneous analysis of nuclear DNA content and size. STUDY DESIGN: Nuclei were stained with DAPI and measured by cytofluorometry. Five regenerative bones and five osteosarcoma samples were used. Two-dimensional dispersion diagrams between relative nuclear DNA content and nuclear size were drawn up and their regression lines were compared. RESULTS: It was possible to distinguish between osteosarcomas and regenerative bones by simultaneous analysis of nuclear size and DNA content during the cell cycle. The results yielded coefficients of correlation and regression. All five regression lines of osteosarcomas were situated dramatically higher on dispersiongrams than those of regenerative bones. CONCLUSION: Differential diagnosis between regenerative lesions and malignant tumors may now be possible using simultaneous measurement of nuclear DNA content and size.     

Correlation of micro vessel density and c-Myc expression in breast tumor of mice following mesenchymal stem cell therapy

Stem cell therapy for degenerative diseases has been established; however there are controversies over the treatment of solid tumors with stem cell transplantation. In the present study, the anti-tumor action of mesenchymal stem cells (MSCs) has been examined in a mouse model of breast cancer with emphasize on tumor growth, angiogenesis and c-Myc expression in breast tumors. For this purpose, MSCs were isolated from bone marrow of Balb/c mice and characterized. A Balb/c mouse model of breast cancer was developed and subjected to cell therapy intra venous (I.V) or intra tumor (I.T) with MSCs. Tumor growth was measured using a digital caliber for until the end of experiment (30 days). Then the mice were sacrificed and their tumors were removed and processed for histopathological examination, immunohistochemical assay of CD31 and measuring of c-Myc expression using quantitative PCR. Detection of the labeled-MSCs in tumors following injection of the cells (I.V or I.T) clearly showed the homing of MSCs into tumors. Tumor growth in case of MSC-treated mice by I.V and I.T routes was inhibited by approximately 28% and 34% respectively compared to controls. The suppression of angiogenesis was reflected in Micro Vessel Density (MVD) following I.V or I.T delivery of the MSCs. c-Myc gene expression in tumor tissues of mice treated I.V or IT with MSCs was down-regulated to 28.0% and 16.0% respectively compare to control groups. In conclusion, growth inhibition of breast tumors in mice due to MSC therapy is associated with modulation of c-Myc activation and angiogenesis markers.     

Size-dependent B lymphocyte subpopulations: relationship of cell volume to surface phenotype, cell cycle, proliferative response, and requirements for antibody production to TNP-Ficoll and TNP-BA

Murine splenic B lymphocytes were separated into size-dependent subpopulations by using counterflow centrifugation. Spleen cells were rigorously depleted of T lymphocytes to yield a population of cells that were greater than 90% surface immunoglobulin (Ig)-positive and that had a mean cell volume of 136.6 +/- 3.3 microns. From this population, five fractions of cells were obtained with mean cell volumes that ranged from 115.8 +/- 3.7 microns in fraction 1 to 168.0 +/- 6 microns in fraction 5. The cells in these five subpopulations were characterized by analysis on a fluorescence-activated cell sorter after staining with acridine orange to evaluate RNA and DNA content, and with fluorescein-conjugated anti-mu, anti-delta, and anti-Ia antibodies to evaluate their surface membrane phenotypes. DNA analysis revealed that virtually all of the cells in fractions 1 to 4 had 2 N DNA. Between 7 and 21% of fraction 5 cells were either in S-phase or contained 4 N DNA. In contrast, RNA content increased through the fractions, suggesting a transition from G0 to G1 in the subpopulations with increasing B cell size. As another measure of cell activation seen with increasing cell size, we observed a progressive increase in the expression of surface Ia and a decrease in the expression of surface IgD. In the absence of in vitro stimulation, the larger cells showed significantly higher levels of thymidine incorporation. When polyclonal B cell activators such as LPS or anti-Ig antibody were added, peak proliferative responses were similar in all of the fractions, but the time necessary to achieve a maximal response was shorter for the larger-sized cell subpopulations than it was for the smaller-sized cell subpopulations. Unprimed, size-dependent B lymphocyte subpopulations exhibited spontaneous or "background" antibody formation that occurred primarily in the subpopulations containing the largest cells. T cell factors present in EL4 supernatant enhanced the efficiency of in vitro differentiation of these same subpopulations. When cultured in the absence of T cell help, the thymus-independent type 1 (TI-1) antigen TNP-Brucella abortus (TNP-BA) or the thymus-independent type 2 (TI-2) antigen TNP-Ficoll induced the largest anti-TNP plaque-forming cell (PFC) responses in the fractions containing intermediate-sized cells, suggesting that in vitro, antigen-specific responses came primarily from B cells that have been influenced in vivo to leave their small resting state. The subpopulations containing the smallest size B cells required the presence of both a TI antigen and EL4 supernatant for efficient differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative in vitro microdosimetric study of murine- and human-derived cancer cells exposed to alpha particles

Diffusing alpha-emitter radiation therapy (DaRT) is a proposed new form of brachytherapy using α particles to treat solid tumors. The method relies on implantable 22?Ra-loaded sources that continually release short-lived α-particle-emitting atoms that spread inside the tumor over a few millimeters. This treatment was demonstrated to have a significant effect on tumor growth in murine and human-derived models, but the degree of tumor response varied across cell lines. Tumor response was found to correlate with the degree of radionuclide spread inside the tumor. In this work we examined the radiosensitivity of individual cells to determine its relationship to tumor response. Cells were irradiated in vitro by α particles using a 22?Th irradiator, with the mean lethal dose, D?, estimated from survival curves generated by standard methods. The results were further analyzed by microdosimetric tools to calculate z?, the specific energy resulting in a survival probability of 1/e for a single cell, which is considered to better represent the intrinsic radiosensitivity of individual cells. The results of the study demonstrate that, as a rule, tumors that respond more favorably to the DaRT treatment are also characterized by higher intrinsic cellular radiosensitivities, with D? ranging from 0.7 Gy to 1.5 Gy for the extreme cases and z? following the same trend.     

Cell surface molecules and tumor metastasis: Regulation of metastatic phenotypic diversity

Metastatic tumor cells are characterized by quantitative alterations in cell surface and other properties that confer to these cells their abilities to invade, disseminate, implant, survive and grow at secondary sites. Metastasis is also determined by a variety of host factors that prevent, allow or even stimulate metastatic processes. The emergence of diversified cell subpopulations in malignant tumors insures that some cells will ultimately become highly metastatic, resulting in tumor progression towards characteristics which are the most favorable for survival and growth. Unknown mechanisms appear to stimulate and then to control phenotypic diversification of tumor cell subpopulations. These mechanisms may be altered by genetic (mutational) and/or epigenetic (non-mutational) modifications that individually influence cells within a malignant neoplasm.     

Blocking tumor cell eicosanoid synthesis by GP x 4 impedes tumor growth and malignancy

Using tumor cell-restricted overexpression of glutathione peroxidase 4 (GP x 4), we investigated the contribution of tumor cell eicosanoids to solid tumor growth and malignant progression in two tumor models differing in tumorigenic potential. By lowering cellular lipid hydroperoxide levels, GP x 4 inhibits cyclooxygenase (COX) and lipoxygenase (LOX) activities. GP x 4 overexpression drastically impeded solid tumor growth of weakly tumorigenic L929 fibrosarcoma cells, whereas B16BL6 melanoma solid tumor growth was unaffected. Yet, GP x 4 overexpression did markedly increase the sensitivity of B16BL6 tumors to angio-destructive TNF-alpha therapy and abolished the metastatic lung colonizing capacity of B16BL6 cells. Furthermore, the GP x 4-mediated suppression of tumor cell prostaglandin E(2) (PGE(2)) production impeded the induction of COX-2 expression by the tumor stress conditions hypoxia and inflammation. Thus, our results reflect a PGE(2)-driven positive feedback loop for COX-2 expression in tumor cells. This was further supported by the restoration of COX-2 induction capacity of GP x 4-overexpressing L929 tumor cells when cultured in the presence of exogenous PGE(2). Thus, although COX-2 expression and eicosanoid production may be enabled by PGE(2) from the tumor microenvironment, our results demonstrate the predominant tumor cell origin of protumoral eicosanoids, promoting solid tumor growth of weakly tumorigenic tumors and malignant progression of strongly tumorigenic tumors.     

A new approach to phenotyping disseminated tumor cells: methodological advances and clinical implications

At the time of primary therapy (surgery, systemic chemotherapy and/or radiation), disseminated tumor cells in the bone marrow can be found in almost one-third of patients with cancer of the breast, ovary, esophagus, stomach, colon, and other solid tumors. Whereas the prognostic impact of the mere presence of these cells is still a matter of debate, it has been shown that expression of tumor-associated antigens in disseminated tumor cells is linked to more aggressive disease. Therefore, further characterization of disseminated tumor cells at the protein and gene level has become increasingly important. To date, the most common detection method for disseminated tumor cells in the bone marrow is an immunocytochemical approach using cytokeratin-directed antibodies for detection of epithelial cells and the APAAP system for their visualization. We have established a new double immunofluorescence technique enabling simultaneous detection, phenotyping, and antigen quantification of disseminated tumor cells. Mononuclear cells from bone marrow are enriched by Ficoll gradient centrifugation and cytospins are prepared. Double immunofluorescence is performed using antibodies against cytokeratins 8/18/19 (mAb A45B/B3) and the uPA receptor CD87 (pAb HU277). CD87 expression is recorded by confocal laser scanning microscopy (CLSM) using fluorescence labeled latex beads as the reference; staining intensities of all the scans are then summed and quantified (extended focus). This protocol, originally designed for disseminated tumor cells in bone marrow, can also be applied to disseminated tumor cells in blood, to leukapheresis cells or to cells present in malignant ascites or other malignant effusions. The tumor cells detected may be used for gene and mRNA analyses. Furthermore, disseminated tumor cells also represent interesting targets for clinical studies on patient prognosis or prediction of therapy response as well as for specific tumor-biological therapies.     

肿瘤类器官在药物筛选和个性化用药中的研究进展*

恶性肿瘤是影响人类生命健康的重大疾病之一,药物治疗是常见的治疗手段。近年来,“精准治疗”已经成为肿瘤治疗的趋势。要实现对恶性肿瘤有效、精准的药物治疗,药物筛选模型至关重要。肿瘤类器官是近年来新兴的一种三维细胞模型,具有经长期传代还保留亲本肿瘤的特征和异质性、培养成功率高、周期短和能够高通量筛选药物等优点,已被用于药物筛选、预测患者对治疗的反应以及为个性化用药提供指导等。重点介绍了肿瘤类器官在药物筛选及个性化用药中的研究进展和面临的挑战。     

DNA Demethylation and Carcinogenesis

DNA methylation plays an important role in the establishment and maintenance of the program of gene expression. Tumor cells are characterized by a paradoxical alteration of DNA methylation pattern: global DNA demethylation and local hypermethylation of certain genes. Hypermethylation and inactivation of tumor suppressor genes are well documented in tumors. The role of global genome demethylation in carcinogenesis is less studied. New data provide evidence for independence of DNA hypo- and hypermethylation processes in tumor cells. These processes alter expression of genes that have different functions in malignant transformation. Recent studies have demonstrated that global decrease in the level of DNA methylation is related to hypomethylation of repeated sequences, increase in genetic instability, hypomethylation and activation of certain genes that favor tumor growth, and increase in their metastatic and invasive potential. The recent data on the role of DNA demethylation in carcinogenesis are discussed in this review. The understanding of relationships between hypo- and hypermethylation in tumor cells is extremely important due to reversibility of DNA methylation and attempts to utilize for anti-tumor therapy the drugs that modify DNA methylation pattern.__________Translated from Biokhimiya, Vol. 70, No. 7, 2005, pp. 900–911.Original Russian Text Copyright © 2005 by Kisseljova, Kisseljov.This article was not published in the journal special issue devoted to the 70th anniversary of B. F. Vanyushin (Biochemistry (Moscow) (2005) 70, No. 5) because of the limiting volume of the journal.     

DNA ploidy in colorectal cancer, heterogeneity within and between tumors and relation to survival

Flow cytometry was used to study the incidence of aneuploidy and to determine the significance of multiple sampling from colorectal tumors. DNA ploidy pattern has been proposed as a supplementary prognostic marker, but discrepancies in findings are major. DNA clonal heterogeneity, defined as two or more DNA aneuploid stemlines in the same tumor, is well established. However, most studies have been based on only one biopsy from each tumor. In our study multiple biopsies were taken from 163 patients (88 males and 75 females) electively operated for colorectal cancer. Tumor cells were harvested by fine needle aspiration from fresh frozen biopsies sampled at different sites of each tumor. DNA aneuploidy was detected in tumors from 145 patients (89%), and 18 patients (11%) had a solitary DNA diploid cell population. In a 79 month follow-up period 105 patients had died. Statistical analysis showed that distinction between diploidy and aneuploidy did not predict survival. However, grouping subpopulations into DNA diploid plus near diploid (DNA index (DI) 0. 97-1.15), DNA aneuploid with all aneuploid subpopulations in the interval 1.15-2.06, and DNA aneuploid with subpopulations with DI < 0.97 and/or DI > 2.06, showed a significant difference in survival in a Cox multivariate analysis including Dukes' stage P = 0.049 comparing the second group to the first and P = 0.01 comparing the third group to the first. In 21 (13%) patients only one subpopulation was found, 57 (35%) had two, 44 (27%) had three, and 41 (25%) had four or more different subpopulations. The association of DNA ploidy to survival is shown to be dependent on the number of biopsies analysed.     

The potential of DNA vaccination against tumor-associated antigens for antitumor therapy

Conventional treatment approaches for malignant tumors are highly invasive and sometimes have only a palliative effect. Therefore, there is an increasing demand to develop novel, more efficient treatment options. Increased efforts have been made to apply immunomodulatory strategies in antitumor treatment. In recent years, immunizations with naked plasmid DNA encoding tumor-associated antigens have revealed a number of advantages. By DNA vaccination, antigen-specific cellular as well as humoral immune responses can be generated. The induction of specific immune responses directed against antigens expressed in tumor cells and displayed e.g., by MHC class I complexes can inhibit tumor growth and lead to tumor rejection. The improvement of vaccine efficacy has become a critical goal in the development of DNA vaccination as antitumor therapy. The use of different DNA delivery techniques and coadministration of adjuvants including cytokine genes may influence the pattern of specific immune responses induced. This brief review describes recent developments to optimize DNA vaccination against tumor-associated antigens. The prerequisite for a successful antitumor vaccination is breaking tolerance to tumor-associated antigens, which represent "self-antigens." Currently, immunization with xenogeneic DNA to induce immune responses against self-molecules is under intensive investigation. Tumor cells can develop immune escape mechanisms by generation of antigen loss variants, therefore, it may be necessary that DNA vaccines contain more than one tumor antigen. Polyimmunization with a mixture of tumor-associated antigen genes may have a synergistic effect in tumor treatment. The identification of tumor antigens that may serve as targets for DNA immunization has proceeded rapidly. Preclinical studies in animal models are promising that DNA immunization is a potent strategy for mediating antitumor effects in vivo. Thus, DNA vaccines may offer a novel treatment for tumor patients. DNA vaccines may also be useful in the prevention of tumors with genetic predisposition. By DNA vaccination preventing infections, the development of viral-induced tumors may be avoided.     

Directing CAR T cells towards the tumor vasculature for the treatment of solid tumors

For successful application of chimeric antigen receptor (CAR) T cell therapy in solid tumors, major hurdles have to be overcome. CAR T cells have to cross the vascular barrier, which is hampered by the anergic state of the tumor vasculature, characterized by suppressed levels of leukocyte adhesion molecules on the endothelium. Additional immunosuppressive mechanisms in the solid tumor microenvironment can affect infiltration, activity and persistence of CAR T cells. Redirecting CAR T cells towards the tumor vasculature poses a possible solution, as molecular targets of tumor endothelial cells can be directly engaged from within the blood.In this review, we discuss recent advances in CAR T cell therapy against solid tumors, with a focus on targeting the tumor vasculature. Furthermore, we discuss opportunities to overcome challenges and barriers through engineering of CAR T cells to enhance trafficking, safety and efficacy.     

肿瘤浸润淋巴细胞在实体肿瘤中作用的研究进展

肿瘤是21世纪威胁人类健康的主要疾患之一。临床上,实体瘤治疗仍以手术切除、放化疗和靶向治疗为主,但这些方法往往不能根除肿瘤病灶,易导致肿瘤复发和进展。肿瘤免疫治疗是利用人体的免疫系统,通过增强或恢复抗肿瘤免疫力实现控制和杀伤肿瘤的一种新的治疗模式。肿瘤免疫治疗能够在众多患者中产生持久反应,过继性免疫治疗和免疫检查点阻断剂治疗均可产生显著的抗原特异性免疫反应。肿瘤浸润淋巴细胞(TILs)是一种存在于肿瘤组织内部具有高度异质性的淋巴细胞,在宿主抗原特异性肿瘤免疫应答中发挥关键作用。最新研究表明,在肿瘤发生和治疗过程中,TILs的亚群组成和数量与患者预后密切相关;抗肿瘤的TILs介导的过继性免疫治疗方法已在多种实体瘤中取得了良好的疗效。文中就实体肿瘤中TILs的研究进展作一综述。     

NOS-2 signaling and cancer therapy

The role of NO and cGMP signaling in tumor biology has been extensively studied during the past three decades. However, whether the pathway is beneficial or detrimental in cancer is still open to question. We suggest several reasons for this ambiguity: first, although NO participates in normal signaling (e.g., vasodilation and neurotransmission), NO is also a cytotoxic or apoptotic molecule when produced at high concentrations by inducible nitric-oxide synthase (iNOS or NOS-2). In addition, the cGMP-dependent (NO/sGC/cGMP pathway) and cGMP-independent (NO oxidative pathway) components may vary among different tissues and cell types. Furthermore, solid tumors contain two compartments: the parenchyma (neoplastic cells) and the stroma (nonmalignant supporting tissues including connective tissue, blood vessels, and inflammatory cells) with different NO biology. Thus, the NO/sGC/cGMP signaling molecules in tumors as well as the surrounding tissue must be further characterized before targeting this signaling pathway for tumor therapy. In this review, we focus on the NOS-2 expression in tumor and surrounding cells and summarized research outcome in terms of cancer therapy. We propose that a normal function of the sGC-cGMP signaling axis may be important for the prevention and/or treatment of malignant tumors. Inhibiting NOS-2 overexpression and the tumor inflammatory microenvironment, combined with normalization of the sGC/cGMP signaling may be a favorable alternative to chemotherapy and radiotherapy for malignant tumors.     

Niche-Dependent Gene Expression Profile of Intratumoral Heterogeneous Ovarian Cancer Stem Cell Populations

Intratumoral heterogeneity challenges existing paradigms for anti-cancer therapy. We have previously demonstrated that the human embryonic stem cells (hESC)-derived cellular microenvironment in immunocompromised mice, enables functional distinction of heterogeneous tumor cells, including cells which do not grow into a tumor in a conventional direct tumor xenograft platform. We have identified and characterized six cancer cell subpopulations each clonally expanded from a single cell, derived from human ovarian clear cell carcinoma of a single tumor, to demonstrate striking intratumoral phenotypic heterogeneity that is dynamically dependent on the tumor growth microenvironment. These cancer cell subpopulations, characterized as cancer stem cell subpopulations, faithfully recapitulate the full spectrum of histological phenotypic heterogeneity known for human ovarian clear cell carcinoma. Each of the six subpopulations displays a different level of morphologic and tumorigenic differentiation wherein growth in the hESC-derived microenvironment favors growth of CD44+/aldehyde dehydrogenase positive pockets of self-renewing cells that sustain tumor growth through a process of tumorigenic differentiation into CD44-/aldehyde dehydrogenase negative derivatives. Strikingly, these derivative cells display microenvironment-dependent plasticity with the capacity to restore self-renewal markers and CD44 expression. In the current study, we delineate the distinct gene expression and epigenetic profiles of two such subpopulations, representing extremes of phenotypic heterogeneity in terms of niche-dependent self-renewal and tumorigenic differentiation. By combining Gene Set Enrichment, Gene Ontology and Pathway-focused array analyses with methylation status, we propose a suite of robust differences in tumor self-renewal and differentiation pathways that underlie the striking intratumoral phenotypic heterogeneity which characterize this and other solid tumor malignancies.