Quantification Of Proliferation Research Articles

Abstract 2976: The potential use of butyrate and propionate to inhibit oncogenic phenotypes in oral squamous cell carcinoma and triple negative breast cancer

Abstract Background Oral squamous cell carcinoma (OSCC) and triple-negative breast cancer (TNBC) are aggressive cancers with low survival rates, particularly in Hispanics. Since the underpinning mechanisms of oncogenesis for both OSCC and TNBC are multifactorial, the development of novel targeted therapies is limited. The tumor microbiome has been shown to modulate the microenvironment. The microbial product short-chain fatty acids (SCFA) may represent a putative target for treatment as they can potentially inhibit cell growth, proliferation, migration, and induce morphologic changes in cancer cells. We evaluated the impact of the SCFAs butyrate, acetate, and propionate on the oncogenic phenotypes of OECM-1 and MDA-231 cell line models for OSCC and TNBC respectively. Methods OECM-1 and MDA-231 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS. Cytotoxicity was determined using an alamarBlue assay by treating the cells with SCFA in a series of 7 concentrations starting at 125 mM (1:2 dilution factor) for 24, 48, and 72 hours (h). Proliferation quantification was performed using trypan blue dye after treatment with SCFA for 24h, 48h and 72h. Migration and invasion were measured after treatment for 24h with SCFAs. Comparative analysis of our results was performed using GraphPad Prism v10. Results Higher concentrations of all SCFA decreased cell viability in OSCC and TNBC at all time points. In OECM-1 cells, the average inhibitory concentration of 50% (IC50) was 3mM for butyrate, 382mM for acetate, and 64mM for propionate while in MDA-MB-231 cells, the average IC50 was 4.3mM for butyrate, 462mM for acetate, and 20mM for propionate. Both OECM-1 and MDA-MB-231 cells treated with butyrate and propionate at 5mM resulted in a slower migration after 24h of treatment. Additionally, there was a significant reduction of cell number and proliferation rate after 48h and 72h of butyrate treatment at the concentrations 1mM (p=0.0425) and 5mM (p=0.0007) in OSCC. Conclusions Our results show that butyrate and propionate can reduce oncogenic phenotypes in OSCC and TNBC and may represent a potential target for the development of alternative treatment strategies for both cancers. Future work includes measuring the impact of these SCFA in epithelial-mesenchymal transition, loss of adhesion, and cell death. Citation Format: Angel J. Amaral Maisonet. The potential use of butyrate and propionate to inhibit oncogenic phenotypes in oral squamous cell carcinoma and triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2976.

Stromal tissue segmentation in Ki67 histology images based on cytokeratin-19 stain translation.

The diagnosis and prognosis of breast cancer relies on histopathology image analysis. In this context, proliferation markers, especially Ki67, are increasingly important. The diagnosis using these markers is based on the quantification of proliferation, which implies the counting of Ki67 positive and negative tumoral cells in epithelial regions, thus excluding stromal cells. However, stromal cells are often very difficult to distinguish from negative tumoral cells in Ki67 images and often lead to errors when automatic analysis is used. We study the use of automatic semantic segmentation based on convolutional neural networks (CNNs) to separate stromal and epithelial areas on Ki67 stained images. CNNs need to be accurately trained with extensive databases with associated ground truth. As such databases are not publicly available, we propose a method to produce them with minimal manual labelling effort. Inspired by the procedure used by pathologists, we have produced the database relying on knowledge transfer from cytokeratin-19 images to Ki67 using an image-to-image (I2I) translation network. The automatically produced stroma masks are manually corrected and used to train a CNN that predicts very accurate stroma masks for unseen Ki67 images. An -score value of 0.87 is achieved. Examples of effect on the KI67 score show the importance of the stroma segmentation. An I2I translation method has proved very useful for building ground-truth labeling in a task where manual labeling is unfeasible. With reduced correction effort, a dataset can be built to train neural networks for the difficult problem of separating epithelial regions from stroma in stained images where separation is very hard without additional information.

1505-P: CD44 Knockdown in Human Adipocytes—Effect on Adipocyte Proliferation and Differentiation

Introduction: eGWAS implicates the immune-cell receptor CD44 as the top candidate gene in the molecular pathogenesis of T2D1. We have shown that in human adipose tissue, CD44 expression is associated with inflammatory gene expression, M1 macrophage phenotype, and systemic insulin resistance2. Here we sought to determine whether CD44 directly impairs adipocyte function via siRNA knockdown in human preadipocytes with quantification of proliferation, differentiation, and gene expression. Methods: Abdominal subcutaneous adipose tissue was harvested from five healthy nondiabetic obese individuals during bariatric surgery. Preadipocytes were isolated via collagenase digestion and seeded at 1×10^4 cells/well and treated with siRNA-CD44 or mock condition (control) using siRNA Maxi Transfection Reagents (Invitrogen). Cell proliferation was assayed by MTS (Promega) on Days 1, 2, 3 post transfection. Differentiation was induced at confluence (as described3), and cells were harvested at days 3, 6, 12 after confluence for gene expression and at day 12 for oil red O staining (total lipid uptake) and immunofluorescence microscopy (% differentiation = bodipy/dapi*100). Results: CD44 knockdown decreased preadipocyte proliferation and increased adipocyte differentiation and lipid uptake. Conclusion: CD44 may contribute to human insulin resistance by impairing adipocyte differentiation and fat storage. Disclosure H.Chen: None. N.R.Saini: None. E.M.Ayhan: None. T.Mclaughlin: Board Member; January, Inc., Research Support; Merck & Co., Inc., Stock/Shareholder; Eiger Bio. Funding National Institutes of Health (1R21AI159024-01A1)

Abstract P5-13-14: Antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3-directed antibody drug conjugate (ADC) across a diverse panel of breast cancer (BC) patient-derived xenografts (PDXs)

Abstract Background: HER3 is overexpressed in 30-50% of breast cancers and has been associated with poor prognosis. Patritumab deruxtecan (HER3-DXd; U3-1402) is a HER3-directed ADC with a potent topoisomerase I (TOP1) inhibitor payload. A phase 1/2 study of HER3-DXd (NCT02980341) demonstrated promising antitumor activity in hormone receptor positive (HR+) metastatic BC patients with clinical activity observed across baseline levels of HER3 protein or mRNA expression. A window of opportunity clinical trial is currently ongoing to evaluate the biological activity of HER3-DXd in patients with treatment naïve BC according to HER3 mRNA/protein expression levels (NCT04610528). Here, we aimed to describe the activity of HER3-DXd in PDX models to identify robust biomarkers of response. Methods: The antitumor activity of HER3-DXd was assessed in 21 BC PDX models (14 HR+ and 7 triple negative). HER3-DXd sensitivity was established as a complete response that lasted longer than 120 days, following 4 weekly doses of 10 mg/kg. HER3-DXd antitumor activity was compared to the antitumor activity of irinotecan (50 mg/kg dosed once weekly), which was evaluated according to modified RECIST criteria in a subset of 14 BC PDX models. PDX models of acquired resistance to HER3-DXd were generated by repeated treatment cycles. Pharmacodynamic (PD) experiments were conducted by collecting tumor samples from PDXs after a single dose of HER3-DXd or irinotecan. Baseline HER3 expression was assessed by immunohistochemistry (IHC) and Western blot. mRNA expression of 72 genes including ERBB3 and genes from the PAM50 signature were measured using the nCounter platform. Two-class unpaired significance analysis of microarrays (SAM), using a false-discovery rate<5%, identified differential gene expression across response groups. Genetic alterations harbored by PDX models were determined using the MSK-IMPACTTM targeted exome panel. Quantification of proliferation (% of Ki67-positive cells) and of DNA damage during the S-phase of the cell cycle (γH2AX nuclear foci in geminin-positive cells) was evaluated in untreated/treated PD samples by IHC or immunofluorescence (IF), respectively. Western blot was used to assess HER3-pathway downmodulation and induction of apoptosis. Results: Eight out of 21 (38%) PDXs were highly sensitive to HER3-DXd, and in 5/14 (36%) models HER3-DXd showed a superior antitumor activity when compared to irinotecan. We observed an enrichment of basal-like models amongst the non-relapsed PDXs, compared to the relapsed ones (6/8 (75%) vs. 3/13 (20%), p=0.0195). Baseline levels of HER3/ERBB3 were not associated with treatment response and a model of acquired-resistance did not exhibit a reduction in baseline HER3 expression. Interestingly, relapsed models showed increased expression of genes related with chemotherapy-resistance (MDM2, NAT1, MAPT, GRB7, BCL-2). Mechanistically, treatment with HER3-DXd did not reduce the level of Ki67-proliferating cells but resulted in a significantly higher induction of S-phase DNA damage measured as γH2AX nuclear foci in non-relapsed models, compared to relapsed ones. This was accompanied by a reduction of HER3 protein levels and downmodulation of pERK1/2 T202/Y204, along with activation of PARP cleavage. Conclusions: HER3-DXd exerts a potent antitumor response in BC PDXs, independently of baseline HER3/ERBB3 levels, in line with the clinical data of NCT02980341. Basal-like tumors were more sensitive to HER3-DXd than luminal B models. Mechanistically, our data suggests that treatment with HER3-DXd results in parallel HER3/ERK signaling downmodulation and induction of S-phase DNA damage, resulting in tumor cell death. Citation Format: Andreu Òdena, Laia Monserrat, Fara Brasó-Maristany, Marta Guzmán, Judit Grueso, Olga Rodríguez, Maurizio Scaltriti, Sarat Chandarlapaty, Yang Qiu, Kumiko Koyama, Mafalda Oliveira, Aleix Prat, Violeta Serra. Antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3-directed antibody drug conjugate (ADC) across a diverse panel of breast cancer (BC) patient-derived xenografts (PDXs) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-14.

The cardiotomy reservoir - a preliminary evaluation of a new cell source for cardiovascular tissue engineering.

Cell sources for cardiovascular tissue engineering (TE) are scant. However, the need for an ideal TE cardiovascular implant persists. We investigated the cardiotomy reservoir (CR) as a potential cell source that is more accessible and less ethically problematic. CR (n = 10) were removed from the bypass system after surgery. Isolation was performed using different isolation methods: blood samples were taken from the cardiopulmonary bypass and centrifuged at low density. The venous filter screen was cut out and placed into petri dishes for cultivation. The spongelike filter was removed, washed and treated in the same way as the blood samples. After cultivation, cell lines of fibroblasts (FB) and endothelial cells (EC) were obtained for analysis. The cells were seeded on polyurethane patches and analyzed via scanning electron microscopy (SEM), Life/Dead assay and immunohistochemistry. No correlation between age, time of surgery and quality of cells was observed. The successful extraction of FB and was proven by positive staining results for TE-7, CD31 and vWF. Cell morphology, cytoskeleton staining and quantification of proliferation using WST-1 assay resembled the cells of the control group in all ways. The topography of a confluent and vital cell layer after cell seeding was displayed by SEM analysis, Life/Dead Assay and immunohistochemistry. The establishment of an extracellular matrix (ECM) was proven by positive staining for collagen IV, laminin, fibronectin and elastin. Viable FB and EC cell lines were extracted from the CR after surgery. Easy access and high availability make this cell source destined for widespread application in cardiovascular tissue engineering.

Epithelial response to a high-protein diet in rat colon

BackgroundHigh-protein diets (HPD) alter the large intestine microbiota composition in association with a metabolic shift towards protein degradation. Some amino acid-derived metabolites produced by the colon bacteria are beneficial for the mucosa while others are deleterious at high concentrations. The aim of the present work was to define the colonic epithelial response to an HPD. Transcriptome profiling was performed on colonocytes of rats fed an HPD or an isocaloric normal-protein diet (NPD) for 2 weeks.ResultsThe HPD downregulated the expression of genes notably implicated in pathways related to cellular metabolism, NF-κB signaling, DNA repair, glutathione metabolism and cellular adhesion in colonocytes. In contrast, the HPD upregulated the expression of genes related to cell proliferation and chemical barrier function. These changes at the mRNA level in colonocytes were not associated with detrimental effects of the HPD on DNA integrity (comet assay), epithelium renewal (quantification of proliferation and apoptosis markers by immunohistochemistry and western blot) and colonic barrier integrity (Ussing chamber experiments).ConclusionThe modifications of the luminal environment after an HPD were associated with maintenance of the colonic homeostasis that might be the result of adaptive processes in the epithelium related to the observed transcriptional regulations.

744 Genome-Wide Association of Composition and Diversity of the Intestinal Bacterial Phyla in Healthy First Degree Relatives (FDR) of Crohn's Disease (CD) Subjects

Figure 1: Sepp1-/enteroids display an increase in stem cell characteristics and increased ROS production, proliferation, and decreased survival in response to hydrogen peroxide administration. (A) Relative Sepp1 mRNA expression. (B) Enteroid growth properties. (C) ROS quantification after 2 hours of treatment (left). Representative images (100x magnification, right). (D) Proliferation quantification as determined by EdU+ cells per crypt area. (100x magnification, right). (E) Survival curves for WT (red), Sepp1 (blue), and Sepp1 (black) enteroids after daily treatment with 400 μM hydrogen peroxide. *P<0.05, **P<0.01, ***P<0.001.

Oxidative stress and proinflammatory role of pancreatic stellate cells in response to tobacco and alcohol

s / Pancreatolog S54 Aims: To investigate the effects of LPS and hyperglycemia on PSC activation and effects of simvastatin on LPS induced PSC activation. Materials & methods: Primary cultures of rat PSCs were exposed to various concentrations of glucose and LPS with/without simvastatin. Quantification of proliferation was performed by Brd-U assay. PSC activation was assessed by expression of a-SMA and extracellular signal-regulated kinase (ERK) using Western blot. Results: LPS induced activation and proliferation of PSC. However, glucose did not affect PSC proliferation. The proliferative effect of LPS on PSC was not affected by glucose concentration in culture media. Simvastatin inhibited LPS induced activation and proliferation of PSC. The proliferative effect of LPS was mediated by activation of Erk 1/2 pathwaywhich was effectively inhibited by simvastatin. Conclusion: This study shows that LPS is one of the activating factors toward PSC. Endotoxemia which is frequently encountered in the clinical settings of acute and chronic pancreatitis may act as a triggering factor for initiating and persisting pancreatic fibrosis. Simvastain could be a therapeutic agent for preventing pancreatic fibrosis.

PP.08 Manchester Advanced Maternal Age Study (MAMAS) – Does an Ageing Maternal Environment and Altered Placental Function Explain Higher Risk of Poor Pregnancy Out Come in Advanced Maternal Age?

Background Women of advanced maternal age (AMA; ≥35 years) have increased risk of fetal growth restriction and stillbirth. The aetiology is unknown; however both conditions are linked with placental dysfunction,...

IL-27 and B7-H1 Mediate Liver Plasmacytoid DC Ability to Regulate Alloreactive T Cell Responses and Suppress Delayed-Type Hypersensitivity

Background: IL-27 (p28/Ebi3 heterodimer) and B7-H1 (PD-L1 or programmed death ligand-1) are regulatory molecules known to modulate inflammatory T cell-mediated immune responses. Plasmacytoid (p) dendritic cells (DC) are a unique DC subset that exhibits potent immunoregulatory function in experimental organ and hematopoietic cell transplant models. pDC isolated from the liver show enhanced ability to regulate immune responses compared to pDC from the secondary lymphoid tissue. Here, we investigated the contribution of IL-27 and B7-H1 to the ability of murine liver pDC to regulate alloreactive T cell responses in vitro and to suppress delayed-type hypersensitivity (DTH) responses to ovalbumin (OVA) in vivo. Methods: PDCA-1+ pDC were immunobead-purified from livers and spleens of Flt3L-mobilized, C57BL/6J (B6) wild type (WT), Ebi3 knockout (KO), or B7-H1 KO mice. pDC phenotype was evaluated by multi-color flow cytometry. IL-27p28 was quantified by ELISA and intracellular flow cytometric analysis. Ebi3 protein levels in pDC were evaluated by Western blot. T cell allostimulatory capacity in MLR was determined by quantification of proliferation by CFSE dilution analysis, and quantification of Foxp3+ T cells was performed by intracellular flow cytometry. For DTH experiments, recipient B6 mice were immunized with OVA-pulsed autologous bone marrow-derived conventional DC (BMDC) with or without PDCA-1+ pDC isolated from mice that were fed OVA. 7 d after immunization, the mice were challenged with heat-aggregated OVA in the hind footpad. Footpad swelling (DTH) responses were quantified at 24 h. T cells from antigen (Ag)-challenged mice were restimulated ex vivo with OVA, and IFN-γ was quantified in culture supernatants. Results: Liver pDC express high levels of IL-27p28 and Ebi3 (the components of IL-27) but low levels of IL-12p35 (that forms IL-35 when combined with Ebi3). They also exhibit high surface expression of B7-H1 compared to spleen pDC. Ebi3 KO and B7-H1 KO pDC show enhanced T cell allostimulatory capacity compared to WT pDC in MLR. Liver but not spleen pDC significantly suppress DTH responses to OVA when co-injected with BMDC pulsed with OVA at the site and time of immunization. We found a significant decrease in footpad swelling and IFN-γ production by restimulated T cells in mice that received liver pDC compared to mice receiving OVA-pulsed BMDC alone or co-injected with spleen pDC. This phenomenon is dependent on IL-27 and B7-H1 expression by liver pDC, as Ebi3−/− and B7-H1−/− liver pDC failed to suppress DTH responses to OVA. Conclusion: Liver pDC exhibit high expression of IL-27 and B7-H1 that mediate their regulatory ability in vitro and in vivo. Liver pDC and these two critical regulatory molecules may be important therapeutic targets to promote or enhance tolerance to alloAgs and prolong allograft survival.

Digital Holographic Microscopy for Assessment of Experimental Colitis and Monitoring of Intestinal Wound Healing in vitro

Purpose: Intestinal epithelial barrier function is crucial for pathogenesis of inflammatory bowel disease and for evaluation of potential anti-inflammatory drugs in vitro. Digital holographic microscopy (DHM) enables marker-free quantitative phase contrast imaging and provides assessment of cell thickness and tissue density by measuring optical path length delay (OPD). Aim of this study was to evaluate DHM for assessment of experimental colitis in vivo and wound healing in vitro. Methods: Experimental colitis was induced by dextran sodium sulfate in CL57/B6 WT mice. Cryostat colonic sections (5 μm) of healthy (n=7) and colitic mice (n=9) were examined via DHM. To quantify tissue density, segmental OPD analysis of colonic wall (75 measurements/animal) was performed according to anatomic structure (epithelium (e), submucosa (sm) and stroma (st)). DHM was used for time lapse monitoring of wound healing in scratch assay of human intestinal fibroblasts and intestinal epithelial cells (Caco-2 cells) by automated measurements over time (digital holograms were recorded every 3 min). Results: Average optical OPD varied significantly within the different intestinal wall-layers in all mice (e: 1.50 ± 0.30, sm: 1.22 ± 0.59, st: 1.80 ± 0.60 rad; p < 0.001). In contrast, OPD in colitic mice (n=9) was significantly decreased reflecting a significant loss to density in all parts of the colonic wall compared to healthy mice (e: 1.62 ± 0.27 vs. 1.41 ± 0.29; p < 0.001, sm: 1.77 ± 0.42 vs. 0.80 ± 0.29; p < 0.001, st: 1.84 ± 0,53 vs. 1.76 ± 0.66 rad; p < 0.05). Wound assays via DHM from living Caco-2-cells and human intestinal fibroblasts allow quantification of proliferation over time (Caco-2: T1(12h)=34.2%, T2(24h)=53.2% and fibroblasts: T1=(24h)=15.0%, T2=(48h)=30.4% growth in plane axis) with simultaneous morphology imaging (Figure 1).Figure: [1622] Figure 1 Wound assay: time-dependent quantitative DHM phase contrast images of living Caco-2-cells (a-c) and fibroblasts (d-f) coded to 256 gray levels. The wound borders are marked with arrows at different time intervals (T0,T1,T2).Conclusion: DHM allows differentiation between healthy and inflamed intestines and correctly detects density changes of colonic wall during experimental colitis, reflecting loss of epithelial barrier function. In addition, DHM may serve as a valuable tool for quantitative live monitoring of wound healing in vitro for evaluation of novel potential drugs in different cell types.

Anticancer Effects of Astaxanthin and α-tocopherol in Esophageal Cancer Cell Lines

Background/Aims: Astaxanthin (AX) has been attributed with potential for protecting the organism against different types of cancer due to its anti-oxidant activity. Also several in vivo and in vitro studies suggest certain naturally occurring vitamin E (i.e. α-tocopherol) as promising anticancer agents. We assessed the effect of AX and α-tocopherol (AT) respectively and their combination on human esophageal cancer cell lines to investigate the mechanism of anticancer effect and their therapeutic potential. Materials and Methods: Two human esophageal cancer cell lines (TE-1, TE-4) were exposed to AX (6 to 10μg/mL) and AT (20 to 100μM) for 24 hours. Quantification of proliferation was performed by MTT assay. Cell cycle machinery proteins such as p-AKT, p-p38, cyclin D1, p27 and caspase-3 were investigated by Western blot. Results: Significant inhibition of cell proliferation of AX and AT was observed in TE-4 cell line by a dose-dependent manner. Furthermore, AX and AT as single agents increased the protein expression of p27 and cleaved caspase-3 in TE-4 cell line. The combination of the two agents decreased the expression of cyclin D1, however they did not demonstrate pro-apoptotic effect. Conclusions: AX and AT as single agents are effective at inhibition of cell proliferation and induce apoptosis by the modulation of cell cycle machinery proteins in esophageal cancer cell lines. However, our data could not suggest that their combination has any cooperative apoptotic effect. (Korean J Helicobacter Up Gastrointest Res 2011;11:170-175)

An advanced approach for the characterization of dendritic cell-induced T cell proliferation in situ

It is commonly accepted that dendritic cells (DCs) play a pivotal role in the induction of adaptive T cell-mediated immune responses. The clonal expansion of antigen-specific T cells within secondary lymphoid organs reflects the efficiency of antigen processing and presentation by DCs. Consequently, the quantification of proliferating antigen-specific T cells represents an important read-out for analyzing DC–T cell interactions. Standard proliferation assays are usually performed with cell suspensions of antigen-stimulated lymphocytes labelled with carboxyfluorescein diacetate succinimidyl ester, [ 3H] thymidine, or 5-bromo-2-deoxyuridine. However, these assays have important limitations, including the complete loss of information about the localization of proliferating cells in situ. Additionally, the ex vivo stimulation with antigen does not necessarily reflect the in vivo situation of cell proliferation. Therefore, an advanced approach for a detailed characterization of proliferating T cells in situ would be helpful for the interpretation of ongoing adaptive immune responses. Here, we describe the development of a fluorescence-based multicolour assay allowing the characterization and quantification of proliferation in different T cell subtypes in cryosections of lymphoid organs. Our approach combines the major benefits of flow cytometry and immunofluorescent-based histology, such as (i) multicolour phenotyping of cell populations and (ii) description of tissue-specific sites of cell proliferation.

The toxic benthic dinoflagellate Ostreopsis ovata: Quantification of proliferation along the coastline of Genoa, Italy

Toxic harmful algal bloom (HAB) occurrence is becoming more frequent and problematic in highly urbanized coastal zones. In summer 2005 along the urbanized Genoa coastline (Ligurian Sea, North western Mediterranean Sea), local first aid stations treated about 200 people, who all showed similar symptoms following exposure to marine aerosols. The link with proliferation of Ostreopsis ovata was made, and it highlighted for the first time, the risks that benthic HABs may represent in highly urbanised temperate areas. Subsequently, a specific monitoring plan was designed and implemented in the same area in July 2006, before the first signs of Ostreopsis proliferation were detected. Here we report on this quantification of an Ostreopsis ovata bloom in the Ligurian Sea. Cells were quantified both in the water column and in the epiphytic community on macrophytes. Our results suggest a role of sea water temperature and weather conditions in favouring bloom development.

A2B5 lineages of human astrocytic tumors and their recurrence

Astrocytomas are very common intracranial glial cell neoplasms with an inherent tendency to progress. However, the heterogeneity of the morphological features and clinical behavior of the tumors makes accurate prognosis based on the histopathological grading system very difficult. Studies demonstrated that astrocytes have two distinctive cell lineages, and tumors arisen from these two astrocytic lineages have been speculated to have different biological and clinical manifestations. The present study aimed to delineate these two astrocytic lineages in human astrocytomas by using different immunohistochemical markers and to correlate the cell lineages of the tumors with their recurrence. Three markers were used, namely the A2B5 antigen, which is present in type 2 astrocytes but absent in type 1 astrocytes, glial fibrillary acidic protein (GFAP), a marker for astrocytes, and galactocerebroside (GC), a marker for oligodendrocytes. It was found that astrocytomas sharing the A2B5+ lineage (A2B5 positive and GFAP positive) have a significantly higher recurrence rate than the tumors of the A2B5- lineage (A2B5 negative and GFAP positive). Immunohistochemical staining and PCR-single-stranded conformational polymorphism analysis showed that p53 overexpression and p53 mutations were closely associated with the recurrent astrocytomas, and p53 abnormalities were more frequently detected in astrocytomas of the A2B5+ lineage. Quantification of proliferation by counting argyrophil nucleolar organizer regions (AgNORs) indicated a higher AgNOR count in the A2B5+ lineage than the A2B5- lineage. Our findings thus suggest that astrocytomas share similar antigenicity with astrocytes, and that the A2B5+ lineage exhibited a higher recurrence rate than the A2B5- lineage. The higher recurrence rate of the A2B5+ tumors may be in part related to the higher frequency of p53 abnormalities found in the tumors and the higher proliferative activity as reflected by the higher AgNOR count of the tumors.

Characterisation and quantification of angiogenesis in β-tricalcium phosphate implants by immunohistochemistry and transmission electron microscopy

The aim of this study is the histological characterisation of angiogenesis in a macroporous biomaterial with quantification techniques used in oncology. Porous tricalcium phosphate implants were seated in the tibias of 12 rabbits. This work allows (1) morphological study with photonic microscopy, transmission electron microscopic and immunohistochemistry labelling for (2) quantification of vascularisation using anti-CD31 monoclonal antibody (3) quantification of proliferation using anti-PCNA polyclonal antibody (4) study of two angiogenic growth factors: VEGF and FGF-2. Quantification of angiogenesis revealed an outbreak kinetic with early vascular growth in first several days and a second growth phase after 4 weeks. This study reveals in macropores many isolated cells without adjacent vascular lumen, with endothelial phenotype. Expression of angiogenic growth factors reveals that all endothelial cells were VEGF-negative throughout the test period. FGF-2 expression by endothelial cells began 2 weeks post-implantation. Osteoblasts strongly expressed two markers throughout the test period. Furthermore, the procedure described here can be used to compare angiogenesis in different biomaterials or in the same biomaterial with the influence of macroporosities.

Modelling of cell proliferation: nuclear versus membrane labelling

Cell proliferation is a fundamental process involved in growth, development and oncogenesis. Monitoring and quantification of proliferation are essential to analyse the behaviour of cells drug-treated or not. Flow cytometry assessment of cell proliferation requires mathematical models to extract information of interest from fluorescence distributions. Various methods are available for cell cycle analysis, including estimation of cell phase durations and doubling time. In this context, we compare widely used flow cytometric methods based on nuclear labelling (using BrdUrd incorporation in combination with DNA content) to membrane labelling (using intercalating dyes PKH).