Automated stitching of microscope images of fluorescence in cells with minimal overlap.

The morphology of tumor cells is highly related to their phenotype and activity. To verify the drug response of a brain tumor patient, fluorescence microscope images of drug-treated patient-derived cells in each well are analyzed. Due to the limitation of the field of view (FOV), a large number of small FOVs are acquired to compose one complete microscope well. Here, we propose an automated method for accurately stitching tile-scanned fluorescence microscope images, even with noise and a narrow overlapping region between adjacent fields. The proposed method is based on intensity-based normalized cross-correlation (NCC) and a triangular method-based threshold. The proposed method's quantitative accuracy and the sensitivity of the input was compared to other existing stitching tools, MIST and FijiIS, setting manually stitched images as the ground truth. The test images were 20 samples of 3 × 3 grid images in three versions of the fluorescence channel. The distance between the location of each field and number of cells was determined for different input field overlap ranges (1%, 3%, 5%, and 10%), while the actual value was about 1.15%. The proposed method had a distance error of 1.5 pixels at an input overlap of 1%, showing the lowest minimum error at all channels. Regarding the difference in cell numbers, although the number of overlapping cells was always small because of the narrow overlapping range, the proposed method was able to generate the resultant image with the smallest difference. In addition, to confirm the size limitation of the proposed algorithm, the accuracy of stitching images of grid structures 3 × 3, 5 × 5, 10 × 10-20 × 20 was tested, showing consistent results. In conclusion, quantitative evaluation of the performance of the method proved its improved accuracy compared to other current state-of-art techniques, and it showed robust performance even with noise and a narrow overlapping region between adjacent fields.

Characteristics of multicellular tumor spheroids formed by pancreatic cells expressing different adhesion molecules.

AIMS: Multicellular tumor spheroids (MCTS) produced by different methods vary in forms, sizes, and properties. The aim of this work was to characterize MCTS formed by six pancreatic cell lines on a non-adherent surface. MATERIALS AND METHODS: Human pancreatic cells were grown in 2D and 3D conditions and compared for the expression of E- and desmosomal cadherins (PCR, confocal microscopy), growth, cell cycling, apoptosis (flow cytometry), and a response to antitumor drugs doxorubicin and gemcitabine (MTT-assay). KEY FINDINGS: Three types of MCTS were identified: BxPC-3, T3M4 formed small number of large and dense spheroids representing type I MCTS; COLO-357 and AsPC-1 generated type II multiple and loose MCTS of different sizes while MiaPaCa-2 and PANC-1 represented type III cultures which grew almost as floating monolayer films. Formation of type I MCTS depended on the simultaneous expression of DSG3 and several DSC proteins; II MCTS expressed solely DSG2-DSC2 but not DSG3, while type III cells either did not express E-cadherin or a pair of DSG and DSC proteins. Cells in type I MCTS but not in types II and III ones quickly became quiescent which correlated with a decrease in the proliferation, increased apoptosis, and a higher resistance to antitumor drugs doxorubicin and gemcitabine. SIGNIFICANCE: Taken collectively, pancreatic cells significantly vary in the expression of desmosomal cadherins, resulting in the formation of MCTS with different characteristics. The sensitivity of MCTS to various drugs depends on the type of cells and the method of spheroid preparation used.

Extremely thin layer plastification for focused-ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells.

Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 µm2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.

Culturing substrates influence the morphological, mechanical and biochemical features of lung adenocarcinoma cells cultured in 2D or 3D.

Alternative models such as three-dimensional (3D) cell cultures represent a distinct milestone towards capturing the realities of cancer biology in vitro and reduce animal experimentation in the preclinical stage of drug discovery. Significant work remains to be done to understand how substrates used in in vitro alternatives influence cancer cells phenotype and drug efficacy responses, so that to accurately link such models to specific in vivo disease scenarios. Our study describes how the morphological, mechanical and biochemical properties of adenocarcinoma (A549) cells change in response to a 3D environment and varying substrates. Confocal Laser Scanning (LSCM), He-Ion (HIM) and Atomic Force (AFM) microscopies, supported by ELISA and Western blotting, were used. These techniques enabled us to evaluate the shape, cytoskeletal organization, roughness, stiffness and biochemical signatures of cells grown within soft 3D matrices (PuraMatrix™ and Matrigel™), and to compare them to those of cells cultured on two-dimensional glass substrates. Cell cultures are also characterized for their biological response to docetaxel, a taxane-type drug used in Non-Small-Cell Lung Cancer (NSCLC) treatment. Our results offer an advanced biophysical insight into the properties and potential application of 3D cultures of A549 cells as in vitro alternatives in lung cancer research.

Lattice light sheet imaging of membrane nanotubes between human breast cancer cells in culture and in brain metastases.

Membrane nanotubes are cytosolic protrusions with diameters <1 µm that extend between cells separated by tens of µm. They mediate several forms of intercellular communication and are upregulated in diverse diseases. Difficulties in visualizing and studying nanotubes within intact tissues have, however, prompted skepticism regarding their in vivo relevance, and most studies have been confined to cell culture systems. Here, we introduce lattice-light sheet imaging of MDA-MB-231 human breast cancer cells genetically engineered to brightly express membrane-targeted GFP as a promising approach to visualize membrane nanotubes in vitro and in situ. We demonstrate that cultured cells form multiple nanotubes that mediate intercellular communication of Ca2+ signals and actively traffic GFP-tagged membrane vesicles along their length. Furthermore, we directly visualize nanotubes in situ, interconnecting breast cancer cells in live acute brain slices from an experimental mouse model of breast cancer brain metastasis. This amenable experimental system should facilitate the transition of the study of intercellular communication by membrane nanotubes from cell culture to the whole animal.

Directing the Self-assembly of Tumour Spheroids by Bioprinting Cellular Heterogeneous Models within Alginate/Gelatin Hydrogels.

Human tumour progression is a dynamic process involving diverse biological and biochemical events such as genetic mutation and selection in addition to physical, chemical, and mechanical events occurring between cells and the tumour microenvironment. Using 3D bioprinting we have developed a method to embed MDA-MB-231 triple negative breast cancer cells, and IMR-90 fibroblast cells, within a cross-linked alginate/gelatin matrix at specific initial locations relative to each other. After 7 days of co-culture the MDA-MB-231 cells begin to form multicellular tumour spheroids (MCTS) that increase in size and frequency over time. After ~15 days the IMR-90 stromal fibroblast cells migrate through a non-cellularized region of the hydrogel matrix and infiltrate the MDA-MB-231 spheroids creating mixed MDA-MB-231/IMR-90 MCTS. This study provides a proof-of-concept that biomimetic in vitro tissue co-culture models bioprinted with both breast cancer cells and fibroblasts will result in MCTS that can be maintained for durations of several weeks.

[CRITERIA FOR SELECTION OF TUMOR CELLS AND PROSPECTS FOR SPECIFIC IMMUNOTHERAPY FOR BLADDER CANCER].

Specific antitumor immunotherapy with autologous dendritic cell vaccines is one of the new approaches of modern medicine. For activation of dendritic cells highly immunogenic antigens are used, however optimal antigens in case of bladder cancer (BC) are still not researched. Cancer-testis antigens (CTA) are the most promising target in the context of creation of antitumor vaccines, because they are distinguished by pronounced immunogenicity, they are detected in different types of tumors and have limited pattern of expression in healthy tissues of grown-up organism. Regarding the level of mutational load, bladder cancer (BC) holds the third position among all malignant growths, which creates particular opportunities for use of immunotherapy in case of this disease. At chromosomal level most times the following cytogenetic anomalies specific for BC are detected: hyperploidies at 3, 7 and 17 chromosomes and deletion of 9p 21 locus. Besides, in the literature there is information about possible monosomy at 2, 3, 6, 8, 13, 14, 17 and frequent loss of Y chromosome in case of BC. Development of personified dendritic cell antitumor vaccines (PDAV) against bladder cancer (BC) is a relevant problem, which covers many aspects, necessary for its standardization. In particular, in case of cultivation of tumor cells under in vitro conditions their transformation goes at higher pace in comparison with in vivo tumor development. Moreover, the article presents the results of the study of molecular-genetic features of BC of tumor cultures in case of long-term cultivation, the level of expression of CTA (MAGE, NY-ESO-1, GAGE, BAGE) by urothelial carcinoma cells (UCC). There has been described the karyotypes of cells of urothelial low differentiated carcinoma of high malignant potential at various passages with prolonged cultivation, as well as the correlation between cytogenetic profile and expression of tumor-specific cancer-testis antigens has been identified. There have been developed two verified cell line cultures of muscle invasive and muscle-non-invasive urothelial carcinoma, that are potentially useful for the producing of tumor-associated vaccines against BC.

Direct preparation protocol to obtain mitotic chromosomes from canine mammary tumors.

Currently, mammary neoplasms in female canines are a serious problem in veterinary clinics. In addition, the canine species is an excellent disease model for human oncology because of the biological and genetic similarities between the species. Cytogenetics has allowed further study of the characterization of neoplasms in canines. We hypothesized that the use of a direct preparation protocol for mitotic chromosome analysis would provide a simple and low cost protocol for use in all laboratories. The objective of this method is to display in a few hours of dividing cells just like the time of collection since cell division in tissue can be obtained. Ten female canines with the spontaneous occurrence of mammary neoplasia were used to test a pioneering direct preparation protocol to obtain mitotic chromosomes. The excised breast tumor tissue fragments were subjected to the protocol consisting of treatment with colchicine, treatment with hypotonic solution, and fixation. Mitotic chromosomes were absent in cell suspensions of only two samples among the 10 materials analyzed, based on the analysis of five blades for each preparation obtained. So, the cell suspension obtained allowed for the observation of eight tissue samples viable for cytogenetic analysis, five of which had excellent numbers of mitotic chromosomes. However, the technique was unsuccessful in producing high-quality cell suspensions because of inadequate condensation and scattering of chromosomes. While adjustments to methodological procedures are needed, this protocol represents a low cost and simplified method to study the cytogenetics of canine tumors.

Morphological differences between circulating tumor cells from prostate cancer patients and cultured prostate cancer cells.

Circulating tumor cell (CTC) enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile.

Mammary fibroblasts regulate morphogenesis of normal and tumorigenic breast epithelial cells by mechanical and paracrine signals.

Stromal factors play a critical role in the development of the mammary gland. Using a three dimensional-coculture model we demonstrate a significant role for stromal fibroblasts in the regulation of normal mammary epithelial morphogenesis and the control of tumor growth. Both soluble factors secreted by fibroblasts and fibroblast-derived modifications of the matrix compliance contribute to the regulation of epithelial cell morphogenesis. Readjustment of matrix tension by fibroblasts can even induce a phenotypic reversion of breast carcinoma cells. These data offer a basis to develop new strategies for the normalization of the tumor stroma as an innovative target in cancer therapy.

Specific high-affinity binding of thiazole orange to triplex and G-quadruplex DNA.

Interaction of Thiazole Orange (TO) with double-, triple-, and quadruple-stranded forms of DNA was studied. We have demonstrated by UV-vis absorption, circular dichroism (CD), and fluorescence spectroscopy that TO binds with much higher affinity to triplex and G-quadruplex DNA structures compared to double-stranded (ds) DNA. Complexes of the dye with DNA triplexes and G-quadruplexes are very stable and do not dissociate during chromatography and gel electrophoresis. TO binding to either triple- or quadruple-stranded DNA structures results in a >1000-fold increase in dye fluorescence. The fluorescence titration data showed that TO to triad and tetrad ratios, in tight complexes with the triplex and the G-quadruplex, are equal to 0.5 and 1, respectively. Preferential binding of TO to triplexes and G-quadruplexes enables selective detection of only these DNA forms in gels in the absence of free TO in electrophoresis running buffer. We have also demonstrated that incubation of U2OS cells with submicromolar concentrations of TO results in preferential staining of certain areas in the nucleus in contrast to DAPI which binds to dsDNA and efficiently stains regions that are unstained with TO. We suggest that TO staining may be useful for the detection of noncanonical structural motifs in genomic DNA.

Ultrastructural changes in tumor cells treated with liposomal forms of anticancer drugs.

AIM: To determine the main ultrastructural changes in MCF-7 sublines sensitive and resistant to cytotoxic action of anticancer drugs, resulting from the treatment with conventional and liposomal forms of cisplatin and doxorubicin. METHODS: Electron microscopy, light microscopy, MTT-test. RESULTS: It has been shown that the phenomenon of drug resistance is associated with complication of ultrastructural organization of cells and more high differentiation by the main cytomorphologic characteristics which promote their resistance to cytotoxic action of anticancer preparations. Cytoarchitectonics of all resistant cells possesses common patterns and doesn't depend on the particular drugs toward which the resistance has been developed. It has been shown that the cells of the parental form MCF-7 line are more sensitive to cytotoxic action of doxorubicin than to cisplatin. Liposomal forms of anticancer drugs used at the same concentrations that the conventional ones, especially that of doxorubicin, caused more expressed alterations in ultrastructural organization of cells of all studied sublines with dominance of apoptotic processes. CONCLUSION: Evaluating an effect of equal concentrations of cisplatin and doxorubicin in conventional and liposomal forms, one may conclude on higher cytotoxic action of doxorubicin vs. cisplatin that is expressed in a wider spectrum of ultrastructural changes of cell architectonics in different sublines of MCF-7 cells and higher rate of apoptosis.

Ro60 and La ribonucleoproteins become self-aggregated by cell stress.

Ro and La antigens are of clinical interest in subacute cutaneous lupus erythematosus because skin lesions appear after UV irradiation, which induces the translocation of intracellular Ro and La ribonucleoproteins and trigger autoantibody production. Present studies address the question whether cellular stressors modify molecular characteristics and distribution of Ro60 and La proteins. To accomplish our goal HEp-2 cells were stressed by heat and UV irradiation and Ro and La expression was studied by indirect immunofluorescence and Western blot and crossed-immunoprecipitation using monoclonal anti-Ro/La or anti-HSP70 linked to CNBr-Sepharose 4B. Results of present studies confirm that Ro60 and La were located in the nuclei of non stressed cells; however under stress, both ribonucleoproteins were redistributed within cytoplasm and nucleoplasm, interestingly the stress induces self aggregation of both ribonucleoproteins, as demonstrated the Western blot assays. Ro and La proteins interact with the cytoskeleton protein via HSP70. In conclusion, the cell stress redistributes Ro and La proteins whiting nucleo-cytoplasmic compartments. This redistribution is accompanied by self aggregation of Ro and La which became associated with HSP70. Finally, the cell stress is an important factor for antigenic redistribution.

Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgV(H) status and immunophenotyping.

In CLL data from chromosome banding analysis (CBA) have been scarce due to the low proliferative activity of CLL cells in vitro. We improved the cultivation technique using an immunostimulatory CpG-oligonucleotide DSP30 and IL-2. A total of 506 CLL samples were analysed with CBA and interphase FISH using probes for the detection of trisomy 12, IgH rearrangements and deletions of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319) and 17p13 (TP53). A total of 500 of 506 (98.8%) cases were successfully stimulated for metaphase generation and are subject to this study. Aberrations were detected in 415 of 500 (83.0%) cases by CBA and in 392 of 500 (78.4%) cases by FISH. CBA detected 832 abnormalities and FISH only 502. Therefore, CBA offers important information in addition to FISH. (1) CLL is characterized mainly by genomic imbalances and reciprocal translocations are rare. (2) A subgroup with complex aberrant karyotype (16.4%) is identified which is associated with an unmutated IgV(H) status and CD38 expression (P=0.034 and 0.02, respectively). (3) Additional abnormalities are detectable providing new biological insights into different CLL subclasses revealing a much more heterogeneous pattern of cytogenetic abnormalities as assumed so far based on FISH data only. Therefore, prospective clinical trials should evaluate the prognostic impact of newly available CBA data.

Variable pressure and environmental scanning electron microscopy: imaging of biological samples.

The use of elevated gas pressures in the sample chamber of a scanning electron microscope (i.e., variable pressure SEM, or VPSEM) together with specialized electron detectors create imaging conditions that allow biological samples to be examined without any preparation. Specific operating conditions of elevated pressures combined with sample cooling (usually restricted to the environmental SEM range) can allow hydrated samples to be maintained in a pristine state for long periods of time. Dynamic processes also can be easily observed. A wider range of detector options and imaging parameters introduce greater complexity to the VPSEM operation than is present in routine SEM. The current instrumentation with field emission electron sources has nanometer-scale beam resolution (approx 1 nm) and low-voltage beam capability (0.1 kV). However, under the more extreme variable pressure conditions, useful biological sample information can be achieved by skilled operators at image resolutions to 2 to 4 nm and with primary electron beam voltages down to 1.0 kV. Imaging relating to electron charge behavior in some biological samples, generally referred to as charge contrast imaging, provides information unique to this VPSEM and environmental SEM that closely relates to luminescence imaged by confocal microscopy.

Artificially introduced aneuploid chromosomes assume a conserved position in colon cancer cells.

BACKGROUND: Chromosomal aneuploidy is a defining feature of carcinomas. For instance, in colon cancer, an additional copy of Chromosome 7 is not only observed in early pre-malignant polyps, but is faithfully maintained throughout progression to metastasis. These copy number changes show a positive correlation with average transcript levels of resident genes. An independent line of research has also established that specific chromosomes occupy a well conserved 3D position within the interphase nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated whether cancer-specific aneuploid chromosomes assume a 3D-position similar to that of its endogenous homologues, which would suggest a possible correlation with transcriptional activity. Using 3D-FISH and confocal laser scanning microscopy, we show that Chromosomes 7, 18, or 19 introduced via microcell-mediated chromosome transfer into the parental diploid colon cancer cell line DLD-1 maintain their conserved position in the interphase nucleus. CONCLUSIONS: Our data is therefore consistent with the model that each chromosome has an associated zip code (possibly gene density) that determines its nuclear localization. Whether the nuclear localization determines or is determined by the transcriptional activity of resident genes has yet to be ascertained.

Expression of multidrug resistance (MDR) proteins and in vitro drug resistance in acute leukemias.

The expression of the multidrug resistance (MDR) proteins may influence the outcome of treatment in patients with acute leukemia. The aim of this study was to determine the IC50 of cytotoxic drugs (cytosine arabinoside, ara-C and daunorubicin, dnr) using the in vitro 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay method. A total of 82 newly diagnosed acute leukemia cases (43 adult myeloid leukaemia, AML cases and 39 acute lymphoblastic leukaemia, ALL cases) and 16 relapsed cases (8 AML cases and 8 ALL cases) were studied. The MTS assay was performed using two cytotoxic drugs, dnr and ara-C. Cells were incubated with different concentrations of drugs for 4 days and the IC50 was extrapolated from the viability curve. In newly diagnosed cases, we found that childhood ALL samples showed higher IC50 values of dnr (0.040 +/- 2.320) compared to adult AML samples (0.021 +/- 0.158). In contrast, newly diagnosed adult AML samples showed higher IC50 values of ara-C (0.157 +/- 0.529) compared to childhood ALL samples (0.100 +/- 2.350). In relapsed cases, two samples of childhood ALL showed IC50 values of dnr (0.910 +/- 1.760) and ara-C (1.310 +/- 2.390), which was higher compared to childhood AML samples (0.129 +/- 0.214 and 0.210 +/- 0.003, respectively). However, there was no correlation between IC50 values of these drugs tested with clinical outcome. In conclusion, we found that MTS assay is an easy, rapid and non laborious method to study in vitro drug resistance in acute leukaemia cases.

Independent motile microplast formation correlates with glioma cell invasiveness.

Diffuse brain invasion contributes to the poor prognosis for patients with gliomas. Analyzing glioma cell migration in vitro, we have demonstrated the spontaneous shedding of anucleate cell fragments that separate from glioma cell bodies and maintain viability from hours to days. Unlike previously described cell fragments that are released from cells as diffusible vectors, glioma cell fragments are independently motile. We used computerized time-lapse microscopy to characterize the formation of these independent motile microplasts (IMMPs) in human cell cultures derived from the most highly invasive glial tumor, glioblastoma. IMMPs were larger than previously described cell fragments, ranging in size from approximately 2% to nearly half of the area of their parent cells. Complex cell-like behaviors-including establishment of polarity, extension of lamellipodia and filopodia, and change in direction of movement-remained intact in IMMPs. The average direction and velocity of the IMMPs were indistinguishable from those of their parent cells. IMMPs formed at a significantly higher rate in glioma cell lines rendered more invasive by overexpression of invasion-related genes than in vector-transfected controls. The correlation with cell invasiveness indicates that IMMP formation may be related to the cell-invasive phenotype. Further investigation will determine whether IMMPs represent a novel addition to the growing list of viable cell fragments with biological relevance.

Regulation and characterization of the polarity of cells embedded in a reconstructed basement matrix using a three-dimensional micro-culture system.

Three cell lines, that is, the human breast cancer cell line (MCF-7) and the human mammary epithelial cell line (S-1) and its malignant form (T4-2) were embedded in a reconstituted basement membrane (Matrigel) that had 20-nL pyramid-shaped silicon microstructures. The proliferative behavior of the MCF-7 cells was dependent on the surrounding conditions (2-D, collagen gel, or Matrigel), whereas the respiratory activity of a single cell (F(c)) was almost identical under different culture conditions. The F(c) value changed with cellular polarity. The F(c) value for the S-1 cells was observed to decrease slightly, whereas that of the T4-2 cells increased 2 days after cultivation in the microstructures within the Matrigel. However, when the T4-2 cells were cultured in the presence of tyrphostin AG 1478 (T4-2 tyr) to inhibit epidermal growth factor (EGF) signaling, the F(c) value decreased slightly and remained almost constant for an additional 1 week; this was similar to the behavior of the S-1 cells. Further, fluorescence images showed that the T4-2 tyr cells formed polar structures that were similar to those formed by the S-1 cells whereas the T4-2 cells did not form such structures. These results indicate that cellular polarity can be assessed by measuring cellular respiratory activity.

Evaluation of particulate systems supporting tumor cell fractions in a preventive vaccination against intracranial rat glioma.

OBJECT: Irradiated autologous tumor cells are commonly used as a source of antigens in antiglioma vaccinations to activate the immune system. As cell number is often a limiting factor in these cells' preparation, the aim of the present study was to find a means that can lower the amount of cells required. Among strategies currently developed, adjuvant particulate systems offer a promising means to improve the antitumor immune response. In this study, the authors were interested in evaluating the role of particulate systems containing biodegradable microspheres that carry tumor cell fractions on their surfaces in the induction of a protective immunity in the 9L/Fischer 344 rat glioma model. The efficiency of these particulate systems was compared to that of irradiated 9L cells. METHODS: Particulate systems composed of poly(D,L-lactide-co-glycolide) (PLGA) microspheres that support 9L cell fractions on their surfaces (cell lysates or plasma membranes) or irradiated 9L cells alone were injected subcutaneously into the flanks of syngeneic Fischer 344 rats. Eighteen days later, the rats were intracranially injected with nonirradiated 9L cells. A study of survival in these animals and an analysis of the resulting immune response were then conducted. For the same amount of protein (50 microg) injected, irradiated 9L cells provided long-term survival in 30% of animals, whereas 9L plasma membranes adsorbed onto PLGA microspheres provided long-term survival in 10% of animals and cell lysates adsorbed onto microspheres provided long-term survival in 0%. Accordingly, particulate systems induced a lower T helper cell Type 1 (Th1) peripheral immune response than irradiated 9L cells. However, greater secretion of Th1 cytokines was observed when particulate systems were used than when cell fractions separated from microspheres were used, indicating the adjuvant property of these particulate systems. CONCLUSIONS: Particulate systems have adjuvant properties but are still less efficient than irradiated whole tumor cells for vaccinations. Encapsulation of an activating molecule in the microsphere will be the next developmental step in the search for efficient antiglioma vaccinations.