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PubMed2003

Use of laser scanning cytometry to study tumor microenvironment.

The study of phenomena occurring in the tumor microenvironment is a challenging task because of technical difficulties, particularly when dealing with hypocellular specimens. Laser scanning cytometry (LSC) is a new laboratory technology that has been recently introduced to overcome the limitations of other traditional technologies. By combining the properties and the advantages of flow cytometry (FC) and immunohistochemistry (IHC), LSC allows the investigator to obtain objective information on DNA content, protein expression and cellular localization is combination with morphological features. It has been already shown that LSC results are reliable compared to more traditional technologies, and its implementation in the clinical routine is under way. Its use in oncology, which is rapidly expanding, spans from apoptosis analysis to DNA content quantitation and tumor cell phenotyping. Here we describe the technology underlying this novel fluorescence-based device, review its use in oncology by dissecting the phenomena occurring in the tumor microenvironment and propose its application for the immunological follow-up of malignant lesions undergoing immunotherapeutic manipulation.

Animals↗
PubMed2006

Retinal ganglion cell line apoptosis induced by hydrostatic pressure.

Cellular responses to changes in pressure are implicated in numerous disease processes. In glaucoma apoptosis of retinal ganglion cells (RGCs) is associated with elevated intra-ocular pressure, however, the exact cellular mechanisms remain unclear. We have previously shown that pressure can induce apoptosis in B35 and PC12 neuronal cell lines, using an in vitro model for pressure elevation. A novel RGC line allows us to study the effects of pressure on retinal neurons. 'RGC-5' cultures were subjected to elevated ambient hydrostatic pressure conditions in our model. Experimental pressure conditions were 100 mm Hg and 30 mm Hg, representing acute (high) and chronic (lower-pressure) glaucoma, and 15 mm Hg for normal intra-ocular pressure, set above atmospheric pressure for 2 h. Negative controls were treated identically except for the application of pressure, while positive controls were generated by treatment with a known apoptotic stimulus. Apoptosis was determined by a combination of cell morphology and specific TUNEL and Annexin V fluorescent markers. These were assessed simultaneously by laser scanning cytometry (LSC), which also enabled quantitative marker analysis. RGC-5 neurons showed a significantly increased proportion of apoptotic cells compared with controls; maximal at 100 mm Hg, moderate at 30 mm Hg and not statistically significant at 15 mm Hg. This graded response, proportionate to the level of pressure elevation, is representative of the severity of analogous clinical settings (acute, chronic glaucoma and normal). These results complement earlier findings of pressure-induced apoptosis in other neuronal cultures. They suggest the possibility of novel mechanisms of pressure-related mechanotransduction and cell death, relevant to the pathogenesis of diseases such as glaucoma.

Animals↗
PubMed1999

Functional assay of NF-kappaB translocation into nuclei by laser scanning cytometry: inhibitory effect by dexamethasone or theophylline.

Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of cytokine genes in epithelial cells. We assessed accumulation of fluorescence-stained NF-kappaB into propidium iodide-stained nuclei using laser scanning cytometry. "Activity-specific" antibodies to the Rel A (p65) and NF-kappaB1 (p50) subunits of NF-kappaB were detected in the nuclei of A549 cells (an immortalized human type II alveolar epithelial cell line). Exposure to TNF-alpha caused p65 and p50 to translocate into nuclei in a dose- and time-dependent manner. Preincubation with dexamethasone for 24 h or 30 min, or with theophylline for 30 min before TNF-alpha stimulation attenuated the nuclear translocation of Rel A and p50. These findings suggest that theophylline, as well as dexamethasone, may inhibit translocation of NF-kappaB.

Antineoplastic Agents, Hormonal↗
PubMed1994

[DNA ploidy and prognosis of cardia and stomach carcinomas. A comparative flow and image cytometric analysis pf paraffin-embedded tumor sections].

The prognostic relevance of DNA stem line ploidy was ascertained by comparative DNA analysis performed by flow cytometry (FCM) and by Feulgen image cytometry (ICM) of histologic slides on paraffin-embedded resection material from 221 carcinomas of the cardia and stomach. While flow cytometric detection of DNA stem line aneuploidy was rather insecure in smaller or diffuse carcinomas, the methodological restrictions of ICM were seen in a reduced measuring accuracy varying from one case to the other. This resulted in a lower security in distinguishing mere peridiploidy from real hyperdiploid DNA aneuploidy. Despite the difference of methodology, the results were principally concurring. The assessment of DNA stem line ploidy offered no appreciable information about the prognosis of Lauren's diffuse carcinomas. For intestinal carcinomas, however, there was a significant positive correlation between DNA stem line aneuploidy and the evidence of regional nodal metastases, which might explain to a considerable extent the significantly unfavorable clinical course of tumor cases with recorded DNA aneuploidy. The correlation between DNA stem line ploidy, nodal status, and clinical outcome was distinctly less strong in intestinal carcinomas of the cardia than in those of the stomach, because nodal metastatic spread of cardia carcinomas is strongly influenced by local conditions of lymph drainage and the special mechanical strain.

Aneuploidy↗
PubMed2000

Relationship between cytogenetic aberrations by CGH coupled with tissue microdissection and DNA ploidy by laser scanning cytometry in head and neck squamous cell carcinoma.

BACKGROUND: The relationship between DNA sequence copy number aberrations (DSCNAs) and DNA ploidy in head and neck squamous cell carcinomas (HNSCCs) is still controversial. Materials and Methods We analyzed DSCNAs by comparative genomic hybridization (CGH) combined with microdissection and DNA ploidy by laser scanning cytometry (LSC) in 18 surgically removed HNSCCs and compared the data. RESULTS: Copy number increases were most frequently observed on chromosomes 3q (16 cases), 8q (13 cases), and 12p (11 cases). Copy number decreases were observed on chromosome 3p (14 cases). LSC revealed DNA aneuploidy in 10 of the 18 cases. All DNA aneuploid tumors exhibited gain or amplification of DNA copy number at 12p11-12.1, whereas gain of DNA copy number was found in only 1 of 8 diploid tumors. DSCNAs were more frequent in DNA aneuploid tumors than in diploid tumors (P < 0.005). CONCLUSIONS: The present observations indicate a close relationship between DSCNAs and DNA ploidy in HNSCCs.

Aged↗
PubMedDate not supplied

Laser scanning cytometry (LCS) allows detailed analysis of the cell cycle in PI stained human fibroblasts (TIG-7).

We have demonstrated a method for the in situ determination of the cell cycle phases of TIG-7 fibroblasts using a laser scanning cytometer (LSC) which has not only a function equivalent to flow cytometry (FCM) but also has a capability unique in itself. LSC allows a more detailed analysis of the cell cycle in cells stained with propidium iodide (PI) than FCM. With LSC it is possible to discriminate between mitotic cells and G2 cells, between post-mitotic cells and G1 cells, and between quiescent cells and cycling cells in a PI fluorescence peak (chromatin condensation) vs. fluorescence value (DNA content) cytogram for cells stained with PI. These were amply confirmed by experiments using colcemid and adriamycin. We were able to identify at least six cell subpopulations for PI stained cells using LSC; namely G1, S, G2, M, postmitotic and quiescent cell populations. LSC analysis facilitates the monitoring of effects of drugs on the cell cycle.

Cell Cycle↗
PubMed2002

Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry.

A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both R-phycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.

Animals↗
PubMed1993

Detection of aneuploidy and polyploidy in non-Hodgkin's lymphoma by computer video image analysis.

Thirty-six cases of histologically intermediate and low grade non-Hodgkin's lymphoma, classified according to the International Working Formulation, were examined using image analysis cytometry. Aneuploidy was detected in 21 cases (58%). No significant difference in either the incidence or extent of aneuploidy was seen for cases that entered remission when compared to cases that did not. Bone marrow examinations, by either aspiration or trephine biopsy, were conducted on 34 cases, with 11 showing lymphocytic infiltration. Aneuploidy was detected in 6 of the 11 cases having bone marrow involvement, while 5 cases with bone marrow infiltration were euploid. Polyploid cell distributions were detected in 3 cases, and the remaining 12 cases were diploid. The biologic significance of polyploidy among non-Hodgkin's lymphomas is not well understood, and its detection is limited to static techniques for DNA quantitation. With further investigation, awareness of its presence may reveal some insight into the clonal evolution of this neoplasm.

Adolescent↗
PubMed2003

DNA ploidy and markovian analysis of neoplastic progression in experimental pancreatic cancer.

Computer-assisted analysis of DNA ploidy and nuclear morphology were used to elucidate changes in the cell nucleus that occur during the development of experimental pancreatic cancer. Ductal pancreatic adenocarcinoma was induced in 49 Syrian hamsters by SC injection of N-nitrosobis (2-oxopropyl) amine; twenty hamsters served as controls. Groups of animals were sacrificed every 4 weeks for 20 weeks and adjacent sections of pancreatic tissue were H&E and Feulgen-stained for light microscopy and computer assisted cytometry. Pancreatic ductal cells were classified as normal, atypical, or malignant; tissue inflammation (pancreatitis) was also noted when present. DNA ploidy and nuclear morphology evaluation (Markovian analysis) identified an atypical cell stage clearly distinguishable from either normal or malignant cells; pancreatitis preceded this atypia. The DNA ploidy histogram of these atypical cells revealed a major diploid peak and a minor aneuploid peak. The receiver operator characteristic curve areas for a logistic regression model of normal vs atypical cells was 0.94 and for atypical vs malignant was 0.98, numbers indicative of near-perfect discrimination among these three cell types. The ability to identify an atypical cell population should be useful in establishing the role of these cells in the progression of human pancreatic adenocarcinoma.

Adenocarcinoma↗
PubMed1998

Cytometric study of intracellular P-gp expression and reversal of drug resistance.

Expression of the multidrug resistance (MDR) phenotype is responsible for chemotherapy failure in numerous cancers. This phenotype is generally due to the expression of the mdr1 gene-encoded P-gp. Modulation of P-gp activity by chemotherapy has limited possibilities because of toxicity and poor specificity. In contrast, specific transcription blockage of the mdr1 gene can be obtained by oligonucleotides forming a triple helix structure at the DNA level. We used here immunofluorescence and both flow cytometry and image analysis to evaluate surface and total P-gp content in K562 MDR cells. The mdr1 mRNA content was measured by RT-PCR. We confirm the capacity of a 27-mer oligodeoxynucleotide, targeted to an mdr1 DNA fragment, to cause a 10-fold decrease in mdr1 mRNA level. However, this specific genetic inhibition was functionally limited because cellular growth was not modified in a cytotoxic environment. We found that total P-gp content was reduced in resistant cells treated with the mdr1-targeted oligonucleotide, while it remained in high levels on the cell surface, suggesting the existence of a large cytoplasmic pool of P-gp (approximately 50% of the total cellular P-gp). Moreover, when cycloheximide was used for 72 h to suppress protein synthesis, surface P-gp expression showed no decrease, whereas total P-gp was considerably lowered. A rapid 35% decrease in surface P-gp level was reached when resistant cells were treated for 24 h with brefeldin A, an inhibitor of intracellular protein trafficking. Simultaneously, the total P-gp level remained stable, thus indicating a probable accumulation of cytoplasmic P-gp, in agreement with the interruption of protein migration. We propose that the cytoplasmic P-gp pool could be a storage pool consumed for maintaining a steady-state level of surface P-gp. Cytometry could be a useful tool to study such a mechanism of P-gp trafficking and cellular distribution, which could explain the difficulties encountered in achieving stable and rapid effects of MDR reversal with oligonucleotides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Cell array coupled with laser scanning cytometry allows easy analysis of changes in cyclin expression during the cell cycle. An application of cell array system.

To assess the potential of cell array technology, a cell array slide with 50 spots was designed specifically for automated analysis of changes in expression of cyclins A and B1 during the cell cycle at the cellular level. Cells harvested every 1 hour from 0 through 23 hours after synchronization by mitotic selection were spotted in duplicate on a glass slide. Each slide contained 48 spots representing 24 different cell cycle phases, and the remaining two spots were peripheral blood lymphocytes, which were included as negative control. This cell array provided temporal and spatial information related to changes in cyclin expression during the cell cycle in a single experiment. The present study indicates that expression analysis by cell array is novel approach for cell cycle studies. Furthermore, sophisticated multiplexed cell array technology has great potential for analyses of expression of specific genes during diverse cellular events at the cellular level.

Cell Cycle↗
PubMed2006

Increasing the luminescence of lanthanide complexes.

This review compares the chemical and physical properties of lanthanide ion complexes and of other narrow-emitting species that can be used as labels for cytometry. A series of luminescent lanthanide ion macrocyclic complexes, Quantum Dyes, which do not release or exchange their central lanthanide ion, do accept energy transfer from ligands, and are capable of covalent binding to macromolecules, including proteins and nucleic acids, is described and their properties are discussed. Two methods are described for increasing the luminescence intensity of lanthanide ion complexes, which intrinsically is not as high as that of standard fluorophores or quantum dots. One method consists of adding a complex of a second lanthanide ion in a micellar solution (columinescence); the other method produces dry preparations by evaporation of a homogeneous solution containing an added complex of a second lanthanide ion or an excess of an unbound antenna ligand. Both methods involve the Resonance Energy Transfer Enhanced Luminescence, RETEL, effect as the mechanism for the luminescence enhancement.

Animals↗
PubMedDate not supplied

Ultrastructure of the Ewing's sarcoma family of tumors.

Specimens of 47 tumors diagnosed by routine light microscopy as Ewing's sarcoma of bone, and 5 similar soft tissue tumors (extraskeletal Ewing's sarcomas), were examined by transmission electron microscopy. Immunohistochemical stains were performed on all the tumors, and pre-therapy and post-therapy specimens from 5 of the patients were compared. Cell and nuclear areas were assessed in 41 cases by cytomorphometry by using low-magnification electron micrographs. DNA ploidy was determined by static cytometry on 51 of the tumors. None of the methods revealed differences between the bone and soft tissue tumors. The ultrastructural spectrum extended imperceptibly from the typical forms to markedly irregular variants, and was much broader than could be anticipated from the light microscopy. Neural features were observed but they were not common. Comparison of the Ewing's sarcomas with a group of other small round cell tumors (rhabdomyosarcoma, neuroblastoma, small cell carcinoma) using the same techniques showed that they have similar cell and nuclear areas despite the obvious differences in their immunophenotypes and ultrastructure. The collective findings are in keeping with the currently favored view that Ewing's sarcoma and peripheral primitive neuro-ectodermal tumor are the extremes in a morphologic continuum within which neural differentiation ranges from absent to prominent.

Biomarkers, Tumor↗
PubMed1994

DNA ploidy in prostate cancer: potential measurement as a surrogate endpoint biomarker.

Measurements of DNA ploidy have considerable promise for serving as surrogate endpoint biomarkers (SEBs) for prostate carcinoma. Studies of benign prostatic hypertrophy (BPH) tissue by flow cytometry (FCM), image analysis, and fluorescent in situ hybridization (FISH) for ploidy invariably show normal DNA content and chromosome numbers. In contrast, most aggressive prostate cancers are aneuploid, particularly when new, sensitive methodologies are applied. Certain tumors which have appeared to be DNA diploid by FCM can be shown to be aneuploid with only one or a few abnormal chromosomes by FISH studies. Certain cases of prostatic intraepithelial neoplasia (PIN) are also known to be aneuploid by FISH ploidy using alpha satellite probes for centromere counting. The latest data suggest abnormalities of chromosomes 7 and 8 are most frequently detected. Therefore, a surrogate endpoint assay which detects aneusomies of chromosomes 7 or 8 in histologically normal or dysplastic prostate tissue might serve as a very significant intermediate endpoint biomarker to identify neoplastic prostate cells on their way to becoming clinically aggressive prostate carcinomas.

Biomarkers, Tumor↗
PubMed1991

[Clinical studies on pathobiological characteristics of early hepatocellular carcinoma].

On the basis of the study of animal hepatocellular carcinoma (HCC) model, the DNA content was measured with flow cytometry and image analyzer in 62 cases of human HCC, in which 30 HCCs were smaller than 3 cm in diameter, and the pathobiological behaviors of 79 cases of small HCC were observed. The results showed that most of less than 3 cm HCCs had DNA diploid (2C) level and most of greater than 3 cm HCCs had DNA aneuploid (AN) level (P less than 0.05). Along with the increase of tumor size, the incidences of varied invasions were raised, but the post operative survival rate was relatively lowered. We conclude that in human HCC, the DNA content changes from 2C to AN when HCC grows to about 3 cm in size with marked change of relative biological characteristics. This period is of clinical importance in increasing the postoperative survival rate of HCC patients. The mechanism and clinical significance of early appearance of malignant. behaviors in some small HCCs are also discussed.

Adult↗
PubMed2001

Malignant teratoma of the thyroid with primitive neuroepithelial and mesenchymal sarcomatous components.

A 15-year-old black girl had a near total resection of a malignant thyroid teratoma with bilateral nodal involvement and mediastinal extension. A predominant neuroepithelial pattern had ependymal rosettes and mitoses, stained for neuron-specific enolase, neuron-specific B tubulin, and synaptophysin. A malignant spindle cell component stained for smooth-muscle actin, muscle actin, and to a lesser extent S-100. Loose myxoid tissue resembled primitive cartilage. Epithelial membrane antigen and cytokeratin identified epithelial foci. Chromogranin A, MIC2, glial fibrillary acidic protein, and thyroid stimulating hormone receptor stains were negative. There was focal anaplasia. DNA ploidy by laser scanning cytometry was 1.2. The tumor from the left and right thyroid lobes exhibited trisomy 8, the right also had hyperdiploid cell lines. She was treated with aggressive combination chemotherapy and radiation. Presently there is no residual disease 16 months after diagnosis. Malignant thyroid teratoma is an aggressive tumor, with 15 of 27 reported patients dying 2 weeks to 3 years after diagnosis. Survivors have been treated with total or subtotal resection, combination chemotherapy with agents effective in the treatment of germ cell tumors as well as sarcomas, and radiation for either recurrent or residual disease. The heterologous elements, lacking MIC2 staining and t(11;22), support the diagnosis of malignant teratoma rather than a neuroepithelial tumor. Trisomy 8 is the first cytogenetic abnormality described in malignant thyroid teratoma. Therapy should be tailored to the management of all transformed histologies.

Adolescent↗
PubMed1998

Activation of nuclear factor kappa B (NF-kappaB) assayed by laser scanning cytometry (LSC).

Nuclear factor kappa B (NF-kappaB)/rel is the family of ubiquitous transcriptional activators involved in regulation of diverse immune and inflammatory responses. It also plays a role in control of cell growth and apoptosis. In its inactive form NF-kappaB remains in the cytoplasm sequestered through interaction with IkappaB protein. Rapid translocation of NF-kappaB from cytoplasm to nucleus that occurs in response to extracellular signals is considered to be a hallmark feature of its activation. The translocation of NF-kappaB in HL-60, U-937 and Jurkat leukemic cells as well as in human fibroblasts induced by tumor necrosis factor alpha (TNF-alpha) or phorbol myristate acetate (PMA) was presently measured by laser scanning cytometry (LSC). NF-kappaB was detected immunocytochemically with FITC-tagged antibody and its presence in the nucleus vis-a-vis cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus, which was counterstained with propidium iodide (PI), and over the cytoplasm, respectively. Activation of NF-kappaB led to a rapid increase in NF-kappaB-associated fluorescence measured over the nucleus (FN) concomitant with a decrease in fluorescence over the cytoplasm (F(C)), which was reflected by an increase in F(N)/F(C) ratio. This rapid assay of NF-kappaB activation can be combined with morphological identification of the activated cells or with their immunophenotype. Bivariate analysis of NF-kappaB expression versus cellular DNA content makes it possible to correlate its activation with the cell cycle position. The described method has a potential to be used as a functional assay to monitor intracellular translocation of other transcriptional activators such as p53 tumor suppressor protein or signal transduction molecules.

Cell Cycle↗
PubMed2001

Preferential loss of 5q14-21 in intestinal-type gastric cancer with DNA aneuploidy.

BACKGROUND: Little is known about the genetic changes associated with DNA ploidy in gastric cancer (GC). The aim of this study was to identify recurrent or specific chromosomal regions of DNA sequence copy number aberrations (DSCNAs) that might harbor genes associated with DNA aneuploidy in GC. METHODS: We analyzed DSCNAs with comparative genomic hybridization and DNA ploidy by laser scanning cytometry in 16 primary intestinal-type GCs. RESULTS: All GCs examined showed at least one DSCNA (loss or gain); eight were DNA diploid (DD) tumors and eight were DNA aneuploid (DA) tumors. The frequent (>30%) DSCNAs were loss of 5q14-21 and gains of 7p11-14, 8q, 20q, and Xq25-26. Recurrent amplifications (>10%) were detected at chromosomal regions 6p, 7p, and 13q. The overall number of DSCNAs was significantly greater in DA than in DD tumors (P = 0.006). Furthermore, the number of aberrations was clearly greater with 5q loss than without 5q loss (P = 0.002). Losses of 5q14-21, 9p21-pter, 16q, and 18q21-qter were preferentially detected in DA tumors. CONCLUSION: The present observations indicate that there is a close relationship between DSCNA and DNA ploidy in intestinal-type GC and that gene(s) at 5q14-21, 9p21-pter, 16q, and/or 18q21-qter may play important roles in acquisition of DNA aneuploidy.

Adult↗