Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells.

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

Epithelial/Mesenchymal Characteristics and PD-L1 Co-Expression in CTCs of Metastatic Breast Cancer Patients Treated with Eribulin: Correlation with Clinical Outcome.

We aimed to evaluate the co-expression of PD-L1 and epithelial-mesenchymal markers in CTCs from metastatic breast cancer (MBC) patients and to determine if there is any relationship with patients' outcome after eribulin treatment. Using cytospin preparations of peripheral blood mononuclear cells (PBMCs) from MBC patients treated with eribulin and a combination of immunocytochemistry and immunofluorescence, we quantified PD-L1, keratins and vimentin in single and cluster CTCs on days 1 and 8 of the first-treatment cycle. CTCs (n = 173) were found in 31 out of 38 patients. At baseline, the presence of cluster CTCs (p = 0.048), cluster mesenchymal CTCs (mCTCs) (p = 0.0003) or cluster PD-L1+mCTCs (p = 0.006) was associated with shorter overall survival (OS). In multivariate cox regression analysis, the detection of cluster mCTCs was the only parameter associated with increased risk of death (p = 0.024). On day 8 post-eribulin administration, PD-L1+mCTCs and especially single PD-L1+mCTCs decreased in 75% and 89% of patients, respectively. The detection of single PD-L1+mCTCs after eribulin treatment was correlated with shorter PFS (p = 0.047) and OS (p = 0.020). In conclusion, our study identified for the first time that cluster and single PD-L1+mCTCs subpopulations are of clinical significance in patients with MBC and highlighted the importance of CTC phenotyping during treatment with eribulin.

Hyperthermia During Intraperitoneal Chemotherapy With Paclitaxel or Docetaxel for Ovarian Cancer: Is There Any Benefit?

BACKGROUND/AIM: Intraperitoneal chemotherapy with taxanes provides high locoregional drug concentrations. Regarding their synergy with hyperthermia, results have been inconclusive. In this in vitro study, the thermal enhancement of the effect of paclitaxel and docetaxel on ovarian cancer cells under conditions mimicking those during hyperthermic intraperitoneal chemotherapy (HIPEC) is evaluated. MATERIALS AND METHODS: Cisplatin-resistant SKOV-3 and OVCAR-3 ovarian cancer cells were exposed for 2 h to 0.1, 1 and 3 µΜ of paclitaxel and docetaxel at 37°C (normothermia) and 41.5°C (hyperthermia). Cell proliferation and cell-cycle distribution were evaluated after 24 h, 3 days and 7 days. RESULTS: A concentration-dependent cytotoxic effect on cell proliferation was observed. Concurrent hyperthermia caused an increased arrest of cells in the G2/M phase. At 7 days, thermal enhancement of drug effect was shown only for treatment of OVCAR-3 cells with 1 µM paclitaxel. CONCLUSION: The concentration-dependent cytotoxic effect of paclitaxel and docetaxel supports their intraperitoneal use. Due to the lack of or only minimal thermal enhancement, normothermic may be as effective as hyperthermic intraoperative intraperitoneal chemotherapy with taxanes, avoiding, however, potential oncological and treatment-related adverse effects of concurrent hyperthermia.

Nuclear localization of PD-L1: artifact or reality?

BACKGROUND: The levels of expression and membrane localization of programmed cell death ligand 1 (PD-L1), an immune checkpoint type I transmembrane glycoprotein, are related to the clinical response of anti-PD-L1/PD-1 therapy. Although the biologically relevant localization of PD-L1 is on the plasma membrane of cancer cells, it has also been reported to be in the cytoplasm and sometimes in the nucleus. Furthermore, it has been claimed that chemotherapeutics can modify PD-L1 expression and/or its nuclear localization. RESULTS: Data from our group suggest that the nuclear localization of PD-L1, and other plasma membrane proteins as well, could be an artifact resulting from inadequate experimental conditions during immunocytochemical studies. Mild detergent and rigorous fixation conditions should be used in order to preserve the membrane localization and to prevent an erroneous translocation of PD-L1 and other non-interconnected membrane proteins, such as CD24, into other cellular compartments including the nucleus, of untreated and chemotherapeutically treated breast cancer cells. CONCLUSION: We propose that well-specified and rigorously followed protocols should be applied to immunocytochemical diagnostic techniques, especially to those related to individualized diagnosis and treatment.

BCMA (TNFRSF17) Induces APRIL and BAFF Mediated Breast Cancer Cell Stemness.

Recent advances in cancer immunology revealed immune-related properties of cancer cells as novel promising therapeutic targets. The two TNF superfamily members, APRIL (TNFSF13), and BAFF (TNFSF13B), which are type II membrane proteins, released in active forms by proteolytic cleavage and are primarily involved in B-lymphocyte maturation, have also been associated with tumor growth and aggressiveness in several solid tumors, including breast cancer. In the present work we studied the effect of APRIL and BAFF on epithelial to mesenchymal transition, migration, and stemness of breast cancer cells. Our findings show that both molecules increase epithelial to mesenchymal transition and migratory capacity of breast cancer cells, as well as cancer stem cell numbers, by increasing the expression of pluripotency genes such as ALDH1A1, KLF4, and NANOG. These effects are mediated by their common receptor BCMA (TNFRSF17) and the JNK signaling pathway. Interestingly, transcriptional data analysis from breast cancer cells and patients revealed that androgens can increase APRIL transcription and subsequently, in an autocrine/paracrine manner, enhance its pluripotency effect. In conclusion, our data suggest a possible role of APRIL and BAFF in breast cancer disease progression and provide evidence for a new possible mechanism of therapy resistance, that could be particularly relevant in aromatase inhibitors-treated patients, were local androgen is increased.

Stem cell technology in breast cancer: current status and potential applications.

Breast cancer, the leading cause of cancer among females, is supported by the presence of a rare subset of undifferentiated cells within the tumor, identified as breast cancer stem cells (BCSCs). BCSCs underlie the mechanisms of tumor initiation and sustenance and are implicated in the dissemination of the primary tumor to metastatic sites, as they have been found circulating in the blood of breast cancer patients. The discovery of BCSCs has generated a great amount of interest among the scientific community toward their isolation, molecular characterization, and therapeutic targeting. In this review, after summarizing the literature on molecular characterization of BCSCs and methodologies used for their isolation, we will focus on recent data supporting their molecular and functional heterogeneity. Additionally, following a synopsis of the latest approaches for BCSC targeting, we will specifically emphasize on the therapeutic use of naïve or engineered normal stem cells in the treatment of breast cancer and present contradictory findings challenging their safety.

Variable expression levels of keratin and vimentin reveal differential EMT status of circulating tumor cells and correlation with clinical characteristics and outcome of patients with metastatic breast cancer.

BACKGROUND: CTCs expressing variable levels of epithelial and mesenchymal markers in breast cancer have previously been reported. However, no information exists for keratin expression levels of CTCs in association with disease status, whereas assays for the characterization of transitional EMT phenotypes of CTCs in breast cancer are rather lacking. We investigated the correlation between keratin expression of CTCs and patients' outcome and characterized the EMT status of CTCs via the establishment of a numerical "ratio" value of keratin and vimentin expression levels on a single cell basis. METHODS: Keratin expression was evaluated in 1262 CTCs from 61 CTC-positive patients with metastatic breast cancer, using analysis of images obtained through the CellSearch System. For the determination of vimentin/keratin (vim/K) ratios, expression levels of keratin and vimentin were measured in cytospin preparations of luminal (MCF-7 and T47D) and basal (MDA.MB231 and Hs578T) breast cancer cell lines and 110 CTCs from 5 CTC-positive patients using triple immunofluorescence laser scanning microscopy and image analysis. RESULTS: MCF-7 and T47D displayed lower vim/K ratios compared to MDA.MB231 and Hs578T cells, while MCF-7 cells that had experimentally undergone EMT were characterized by varying intermediate vim/K ratios. CTCs were consisted of an heterogeneous population presenting variable vim/K values with 46% of them being in the range of luminal breast cancer cell lines. Keratin expression levels of CTCs detected by the CellSearch System correlated with triple negative (p = 0.039) and ER-negative (p = 0.025) breast cancer, and overall survival (p = 0.038). CONCLUSIONS: Keratin expression levels of CTCs correlate with tumor characteristics and clinical outcome. Moreover, CTCs display significant heterogeneity in terms of the degree of EMT phenotype that probably reflects differential invasive potential. The assessment of the vim/K ratios as a surrogate marker for the EMT status of CTCs merits further investigation as a prognostic tool in breast cancer.

Cancer stem cells in solid and liquid tissues of breast cancer patients: characterization and therapeutic perspectives.

Breast cancer stem cells (BCSCs) represent a heterogeneous subpopulation of rare cells within breast cancer tumors, displaying an enhanced tumor initiating capability and underlying disease progression and therapy resistance. Unraveling their phenotypic, biological and functional profile is a major challenge in the context of diminishing patient mortality. In this review, following a brief description on how cancer stem cells (CSCs) and their microenvironment contribute to tumor preservation and heterogeneity, we summarize the current literature regarding the molecular signature of BCSCs either localized in the primary tumor or circulating in the blood of breast cancer patients. We present recent data on specific stem and epithelial-to-mesenchymal transition (EMT) markers designating the BCSC subpopulation and underline their pathogenic significance. The molecular characterization of BCSCs has promoted the design of novel therapeutic approaches targeting the BCSC subpopulation which are currently being experimentally and clinically evaluated. We highlight recent advances on the development of novel BCSC-targeting therapeutic strategies including the inhibition of cell signaling pathways, differentiation therapy, metabolic interference and nucleotide-, bio- and nano-technology based approaches. Eliminating the chemo- and radio-resistance properties of breast cancer tumor cells via BCSC-directed therapies, combined to conventional therapeutic approaches, will augment the effectiveness of breast cancer treatment and improve the clinical outcome of breast cancer patients.

Differential nuclear shape dynamics of invasive andnon-invasive breast cancer cells are associated with actin cytoskeleton organization and stability.

Cancer cells often exhibit characteristic aberrations in their nuclear architecture, which are indicative of their malignant potential. In this study, we have examined the nuclear and cytoskeletal composition, attachment configuration dynamics, and osmotic or drug treatment response of invasive (Hs578T and MDA-MB-231) and non-invasive (MCF-10A and MCF-7) breast cancer cell lines. Unlike MCF-10A and MCF-7, Hs578T and MDA-MB-231 cells showed extensive nuclear elasticity and deformability and displayed distinct kinetic profiles during substrate attachment. The nuclear shape of MCF-10A and MCF-7 cells remained almost unaffected upon detachment, hyperosmotic shock, or cytoskeleton depolymerization, while Hs578T and MDA-MB-231 revealed dramatic nuclear contour malformations following actin reorganization.

Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer.

The CD44(+)/CD24(-/low) and ALDH1(+) cell phenotypes are associated with stemness and enhanced tumorigenic potential in breast cancer. We assessed the expression of CD44, CD24 and ALDH1 on tumor cells circulating in the peripheral blood (CTCs) of patients with metastatic breast cancer using triple-marker immunofluorescence microscopy. Among a total of 1439 CTCs identified in 20 (66.7%) out of 30 patients, 35.2% had the stem-like/tumorigenic phenotype CD44(+)/CD24(-/low), whereas 17.7% of the CTCs analyzed in seven patients, were ALDH1(high)/CD24(-/low). In conclusion, we report the existence of a subpopulation of CTCs with putative stem cell progenitor phenotypes in patients with metastatic breast cancer.

Nucleocytoplasmic shuttling of soluble tubulin in mammalian cells.

We have investigated the subcellular distribution and dynamics of soluble tubulin in unperturbed and transfected HeLa cells. Under normal culture conditions, endogenous alpha/beta tubulin is confined to the cytoplasm. However, when the soluble pool of subunits is elevated by combined cold-nocodazole treatment and when constitutive nuclear export is inhibited by leptomycin B, tubulin accumulates in the cell nucleus. Transfection assays and FRAP experiments reveal that GFP-tagged beta-tubulin shuttles between the cytoplasm and the cell nucleus. Nuclear import seems to occur by passive diffusion, whereas exit from the nucleus appears to rely on nuclear export signals (NESs). Several such motifs can be identified by sequence criteria along the beta-tubulin molecule and mutations in one of these (NES-1) cause a significant accumulation in the nuclear compartment. Under these conditions, the cells are arrested in the G0-G1 phase and eventually die, suggesting that soluble tubulin interferes with important nuclear functions. Consistent with this interpretation, soluble tubulin exhibits stoichiometric binding to recombinant, normally modified and hyper-phosphorylated/acetylated histone H3. Tubulin-bound H3 no longer interacts with heterochromatin protein 1 and lamin B receptor, which are known to form a ternary complex under in vitro conditions. Based on these observations, we suggest that nuclear accumulation of soluble tubulin is part of an intrinsic defense mechanism, which tends to limit cell proliferation under pathological conditions. This readily explains why nuclear tubulin has been detected so far only in cancer or in transformed cells, and why accumulation of this protein in the nucleus increases after treatment with chemotherapeutic agents.

Short-term exposure of cancer cells to micromolar doses of paclitaxel, with or without hyperthermia, induces long-term inhibition of cell proliferation and cell death in vitro.

BACKGROUND: During intraoperative hyperthermic intraperitoneal chemotherapy for primary or secondary peritoneal malignancies, tumor cells are exposed to high drug concentrations for a relatively short period of time. We investigated in vitro the effect of paclitaxel and hyperthermia on cell proliferation, cell cycle kinetics and cell death under conditions resembling those during intraoperative hyperthermic intraperitoneal chemotherapy. METHODS: Human breast MCF-7, ovarian SKOV-3 and hepatocarcinoma HEpG2 cells were exposed to 10 and 20 microM paclitaxel at 37, 41.5 or 43 degrees C for 2 h. Cell proliferation, cell cycle kinetics, necrosis and apoptosis were evaluated. RESULTS: Hyperthermia exerted a cytostatic effect to all cell lines and at 43 degrees C a cytotoxic effect on MCF-7 cells. MCF-7 and SKOV-3 cells treated under normothermic conditions with paclitaxel were arrested at G2/M or M phase for at least 3 days. Most of MCF-7 cells and approximately half of SKOV-3 cells were in interphase and became multinucleated without properly completing cytokinesis. Hyperthermia at 41.5 degrees C altered cell cycle distribution and affected the paclitaxel-related effect on cell cycle kinetics of MCF-7 and SKOV-3 cells. Analysis of the mode of cell death showed that cell necrosis prevailed over apoptosis. Hyperthermia at 43 degrees C increased paclitaxel-mediated cytotoxicity in MCF-7 cells and to a lesser extent in SKOV-3 and HEpG2 cells. CONCLUSIONS: Short-time treatment of carcinoma cells with high (micromolar) concentrations of paclitaxel in normothermic and hyperthermic conditions is highly efficient for cell growth arrest and could be of clinical relevance in locoregional chemotherapy.

Optimized detection of circulating anti-nuclear envelope autoantibodies by immunofluorescence.

BACKGROUND: Antinuclear antibodies are useful diagnostic tools in several autoimmune diseases. However, the routine detection of nuclear envelope autoantibodies using immunofluorescence (IF) is not always easy to perform in patients' sera because of the presence of autoantibodies to other nuclear and cytoplasmic components which could mask the characteristic rim-like pattern of nuclear envelope autoantibodies. This is particularly common in sera from patients with primary biliary cirrhosis (PBC), which generaly have high titres of anti-mitochondrial antibodies. Therefore, we have assayed a number of commercial slides and alternative fixation conditions to optimize the detection of anti-nuclear envelope antibodies (ANEA) in PBC sera. METHODS: We have explored the presence of ANEA in 33 sera from patients with established PBC using three different Hep2 commercial slides and home-made slides with HeLa and Hep2 cells fixed with methanol, ethanol, 1% or 4% formaldehyde. RESULTS: We observed that the IF pattern was related to the cell type used (Hep2 or HeLa), the manufacturer and the cell fixation scheme. When both cell lines were fixed with 1% formaldehyde, the intensity of the cytoplasmic staining was considerably decreased regardless to the serum sample, whereas the prevalence of cytoplasmic autoantibodies was significantly lowered, as compared to any of the Hep2 commercial slide and fixation used. In addition, the prevalence of ANEA was importantly increased in formaldehyde-fixed cells. CONCLUSION: Immunofluorescence using appropriately fixed cells represent an easy, no time-consuming and low cost technique for the routine screening of sera for ANEA. Detection of ANEA is shown to be more efficient using formaldehyde-fixed cells instead of commercially available Hep2 cells.