Ascites-derived pancreatic ductal adenocarcinoma primary cell cultures as a platform for personalised medicine.

BACKGROUND: Challenges in developing drugs for pancreatic ductal adenocarcinoma (PDAC) include obtaining metastatic cancer tissue for research and validating biomarkers predicative for personalised therapeutic decisions. We have recently developed a novel therapeutic model for PDAC to address these challenges based on the isolation of viable PDAC cells derived from ascites fluid. METHODS: Ascites fluid was obtained from PDAC patients undergoing palliative paracentesis. Ascites-derived PDAC primary cells were isolated, cultured and characterised in ovo and in vitro. RESULTS: We successfully established ascites-derived primary cell cultures within 2-7 days from 92% (93 out of 101) of the ascites fluid samples obtained (from 36 different patients). Homogeneous epithelial PDAC-enriched cell cultures were identified and characterised. We observed a wide range in doubling times and migration properties among the different patient-derived cell cultures. The diverse nature of each individual patient's cell cultures was further demonstrated by differences in therapeutic susceptibility and resistance. The tumorigenicity and invasiveness of the cells were demonstrated in vivo using chicken chorioallantoic membrane grafts. CONCLUSIONS: We have developed a unique ascites-derived PDAC primary cell culture model. This model has the potential to study signalling pathways in PDAC progression and to evaluate targeted therapies for the individual patient expeditiously, thereby supporting personalised treatment decisions.

Localization of luteinizing hormone receptor protein in the human ovary.

The luteinizing hormone receptor (LHR) plays a pivotal role during follicular development. Consequently, its expression pattern is of major importance for research and has clinical implications. Despite the accumulated information regarding LHR expression patterns, our understanding of its expression in the human ovary, specifically at the protein level, is incomplete. Therefore, our aim was to determine the LHR protein localization and expression pattern in the human ovary. We examined the presence of LHR by immunohistochemical staining of human ovaries and western blots of mural granulosa and cumulus cells aspirated during IVF treatments. We were not able to detect LHR protein staining in primordial or primary follicles. We observed equivocal positive staining in granulosa cells and theca cells of secondary follicles. The first appearance of a clear signal of LHR protein was observed in granulosa cells and theca cells of small antral follicles, and there was evidence of increasing LHR production as the follicles mature to the pre-ovulatory stage. After ovulation, LHR protein was ubiquitously produced in the corpus luteum. To confirm the expression pattern in granulosa cells and cumulus cells, we performed western blots and found that LHR expression was stronger in granulosa cells than in cumulus cells, with the later demonstrating low, but still significant, amounts of LHR protein. In summary, we conclude that LHR protein starts to appear on granulosa cells and theca cells of early antral follicles, and low but significant expression of LHR exists also in the cumulus cells. These results may have implications for the future design of clinical protocols and culture mediums for in vitro fertilization and especially in vitro maturation of oocytes.

Efficacy of intravenous immunoglobulin (IVIG) affinity-purified anti-desmoglein anti-idiotypic antibodies in the treatment of an experimental model of pemphigus vulgaris.

Pemphigus vulgaris is a rare life-threatening autoimmune bullous disease caused by immunoglobulin G (IgG) autoantibodies directed against desmogleins 1 and 3. Previously, we showed that intravenous immunoglobulin (IVIG) ameliorates anti-desmoglein-induced experimental pemphigus vulgaris in newborn naive mice. The aim of this study was to examine the efficacy of anti-anti-desmoglein-specific IVIG in a similar model. Pemphigus-vulgaris-specific IVIG (PV-sIVIG) was affinity-purified from IVIG on a column of single-chain variable fragment (scFv) anti-desmogleins 1 and 3. The anti-idiotypic activity of PV-sIVIG was confirmed by enzyme-linked immunosorbent assay, inhibition assay. After induction of pemphigus by injection of anti-desmogleins 1 and 3 scFv to newborn mice, the animals were treated with PV-sIVIG, IVIG (low or high dose) or IgG from a healthy donor (n = 10 each). The skin was examined 24-48 h later, and samples of affected areas were analysed by histology and immunofluorescence. In vitro study showed that PV-sIVIG significantly inhibited anti-desmogleins 1 and 3 scFv binding to recombinant desmoglein-3 in a dose-dependent manner. Specificity was confirmed by inhibition assay. In vivo analysis revealed cutaneous lesions of pemphigus vulgaris in mice injected with normal IgG (nine of 10 mice) or low-dose IVIG (nine of 10 mice), but not in mice treated with PV-sIVIG (none of 10) or high-dose IVIG (none of 10). On immunopathological study, PV-sIVIG and regular IVIG prevented the formation of acantholysis and deposition of IgG in intercellular spaces. In conclusion, the PV-sIVIG preparation is more effective than native IVIG in inhibiting anti-desmoglein-induced pemphigus vulgaris in mice and might serve as a future therapy in patients with the clinical disease.

New chromogens for alkaline phosphatase histochemistry: salmon and magenta phosphate are useful for single- and double-label immunohistochemistry.

Two new substrate chromogens for alkaline phosphatase (ALP) detection have been recently synthesized for use in molecular biology research, salmon and magenta phosphate. We show here that these two chromogens have advantageous characteristics for immunocytochemistry as well. Their relatively delicate pink- and magenta-colored products do not mask the colors produced by other staining procedures. In addition, the reaction products of these substrates are insoluble in water, ethanol, and xylene, permitting the use of regressive hematoxylin staining procedures and coverslipping in permanent resin-based media. Most importantly, when these ALP substrates are used in double-label immunocytochemistry in combination with horseradish peroxidase-diaminobenzidine (HRP-DAB) and counterstained with hematoxylin, all three colors can be easily distinguished. An application using these substrates for simultaneous immunocytochemical detection of two monoclonal antibodies of different classes, in combination with hematoxylin staining, is illustrated.

Differential expression of Islet-1 in neural crest-derived ganglia: Islet-1 + dorsal root ganglion cells are post-mitotic and Islet-1 + sympathetic ganglion cells are still cycling.

The Islet-1 antigen is an early marker of differentiation of neural tube cells, and is expressed in many other embryonic cells as well. It had been reported that Islet-1 is expressed only in post-mitotic sympathetic neuroblasts in vitro, unlike other differentiation markers. We have double-labeled St. 23 chick embryos for bromodeoxyuridine (BrDU) and Islet-1 and found that neural tube and dorsal root ganglion (DRG) cells express Islet-1 after leaving the cell cycle, while sympathetic ganglion (SG) cells express Islet-1 while still dividing.

Mutant p53(R175H) upregulates Twist1 expression and promotes epithelial-mesenchymal transition in immortalized prostate cells.

A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 'gain of function' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53(R175H) mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53(R175H), was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial-mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53(R175H) mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53(R175H) mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.

Initial axial level-dependent differences in size of avian dorsal root ganglia are imposed by the sclerotome.

We have recently shown that there is very early variation in dorsal root ganglia (DRG) size based on their axial position. From the time of their condensation at Stage (St.) 20 (Embryonic Day 3), before the onset of apoptosis, the ganglia in brachial segments 14 and 15 are more than 80% larger on the average than those in cervical 5 and 6. This difference in volume is due to increased numbers of cells in the brachial DRG. In addition, the rostrocaudal length was found to be significantly greater for brachial ganglia, and the greater length of the brachial ganglia was found to be correlated with a greater length of brachial than cervical sclerotomes. It was therefore proposed that the difference in DRG size at the time of gangliogenesis is likely to arise from colonization by a larger initial number of neural crest cells of the longer adjacent rostral sclerotomes in brachial somites. In the present work, we have performed two types of experiments to test this hypothesis. First, we have performed heterotopic grafts of segmental plate mesoderm from cervical to brachial levels and vice versa. In all of these grafts, the sclerotomes developed with a rostrocaudal extent (length) corresponding to their level of origin in the donor embryo. DRG that formed in the grafted mesoderm attained a length appropriate to that of ganglia developing in segments of the axial level of the donor mesoderm, not that of the host. Second, we have estimated proliferation of DRG cells at St. 20 using HNK-1/bromodeoxyuridine double-stain immunocytochemistry. The percentage of cells in S-phase at both brachial and cervical levels of the neuraxis in newly formed ganglia was observed to be the same. These two lines of evidence strongly argue that the initial difference in size between DRG at different axial levels is not intrinsic, but rather is imposed by the mesodermal microenvironment in which they develop, as is the case for DRG segmentation. This is in contrast to what may occur in the hindbrain, where determination of rhombomere identity and neural crest segmentation are thought to be intrisic to the nervous system.

Isolation and characterization of normal and neoplastic colonic epithelial cell populations.

The aim of the present study was to characterize rat mucosal colonic cells harvested from the crypt continuum during differentiation and dimethylhydrazine-induced neoplasia. The collection of colonocytes was performed using a modified nonenzymatic isolation procedure based on Ca2+ chelation and gentle mechanical dissociation. Light and electron microscopy histomorphological examinations, [3H]thymidine incorporation studies, and activity gradients of alkaline phosphatase, thymidine kinase, and cytoskeleton-associated protein tyrosine kinase indicated that distinct cell populations were harvested from the various crypt regions in a temporal sequence mirroring their zonal and functional distribution in situ. After dimethylhydrazine administration, marked protein tyrosine kinase activity was noted in colonic cells harvested from upper crypt zones. The misplaced and sustained kinase activity preceded the actual polyp or tumor formation. This observation is consistent with the expansion of colonic proliferative compartments beyond allowable boundaries during the preneoplastic period. Companion studies in human colonic epithelial specimens corroborate the findings observed in normal and transformed murine colonocytes. It is believed that the characterization and manipulation of colonocytes using our in vitro model will provide important clues to the molecular events underlying the differentiation program and carcinogenic process in the colonic cell.

In vivo NGF treatment increases proliferation in the primary sympathetic ganglia of chick embryos.

Nerve growth factor (NGF) is considered to be a target-derived survival or differentiation factor for neural crest cells of the sympathoadrenal lineage. However, exogenous NGF was found to have a positive effect on the size of the primary sympathetic ganglia (PSG) of the chick embryo, well before sympathetic innervation of the periphery. We have determined the cellular mechanism of NGF's action on the PSG by quantifying both proliferation and apoptosis. The proportion of PSG cells in S-phase is nearly double in NGF-treated embryos compared to that in controls, strongly suggesting that NGF acts as a mitogenic factor. NGF reduced the low level of apoptosis at this stage as well. Since trkA has not been detected in the avian sympathetic ganglia until later in development, we suggest that these early effects of exogenous NGF may be mediated by the low-affinity neurotrophin receptor, p75, which is expressed from neural crest migration stages.

Growth, proliferation, and cell death in the ontogeny of transient DRG (Froriep's ganglia) of chick embryos.

A striking example of axial patterning in nervous system development is the unusual fate of dorsal root ganglia (DRG) that develop in the most rostral somites, the Froriep's ganglia. In amniotes, the DRG that develop adjacent to the occipital (cranial) and the first cervical segments of the CNS "disappear" early in embryonic development. In contrast, all other DRG are present throughout the animal's life. We here reexamine in greater detail the ontogeny of the longest surviving Froriep's ganglion of the chick embryo, DRG C-2. By 50 h of development (stage, st. 15), an anlagen of a DRG had formed in C-2 that was indistinguishable from those of adjacent "permanent" ganglia. At st. 18 [embryonic day (E) 2.5+], the C-2 DRG had the same shape and volume as permanent ganglia C-5 and C-6. C-2's development first diverged from that of normal DRG at st. 19 (E3-), when C-2 was observed to be half the size and shaped differently from its neighbors, and its peripheral nerve root began to degenerate. Two cellular mechanisms appear to contribute to the reduced size of C-2 compared to normal DRG at st. 20 at this early stage: lower proliferation and higher apoptosis rates. One-third fewer C-2 cells were found to be in the S phase when compared to neighboring ganglia, and apoptotic cells were more than three times more abundant in C-2 than in conventional DRG at this stage. The C-2 DRG continued to grow, but at a slower pace than neighboring ganglia through st. 32 (E7). At the height of the normal programmed DRG cell death in normal cervical DRG at st. 28 (E6), even more massive apoptosis occurred in C-2, which resulted in the absence of this ganglion in 80% of st. 36 (E10) embryos. A recent study demonstrated that the overexpression of a single Hox gene can "rescue" the C-2 DRG in transgenic mice. We speculate that Hox genes may produce the difference in fate between C-2 and normal DRG by modulating proliferation and apoptosis via modified neurotrophic factor and/or receptor expression.

Interleukin-1-mediated H2O2 production by hepatic sinusoidal endothelium in response to B16 melanoma cell adhesion.

We have examined H2O2 production by in vitro enriched hepatic sinusoidal endothelium (HSE) during interleukin-1 beta (IL-1 beta) stimulation and B16 melanoma cell adhesion. Production of H2O2 was quantified by flow cytometry and multiwell plate-scanning fluorimetry of intracellular 2', 7'-dichlorofluorescein (DCFH) oxidation in HSE. Under IL-1 beta treatment there was a 6-fold increase in endothelial cells producing H2O2 (67%) and a 4-fold augmentation in the Kupffer cell population (86%). The average H2O2 content per cell size unit significantly (P < 0.01) increased in endothelial cells (2.6-fold) and Kupffer cells (1.7-fold). In contrast to the homogeneity of Kupffer cells, H2O2 production intensity was largely heterogeneous in IL-1 beta-activated HSE. Enhancement of H2O2 production by IL-beta-treated HSE started at the 4th h and peaked 2-3 h later. The addition of increasing concentration of IL-1 beta to HSE for 4 h caused the progressive activation of H2O2 production by treated cells. The addition of 80 M excess of IL-1 receptor antagonist (IL-1 Ra) 10 min before IL-1 beta treatment abrogated IL-1 beta-mediated enhancement of H2O2. From the 2nd h of B16 melanoma adhesion to HSE there was significantly (P < 0.05) enhancement of H2O2 content in HSE. This activation increased 2.25-fold by the 3rd h of coculture and had reduced again by the 5th h. IL-1 Ra (80 ng/ml) given to HSE 10 min before melanoma cells abrogated the HSE response to melanoma cells. The addition of 1% paraformaldehyde (PFA)-fixed B16 melanoma cells to HSE did not affect H2O2 production response, indicating that HSE-activating agents were on the melanoma cell surface. Preincubation of B16 melanoma cells in the presence of 5 micrograms/ml anti-mouse IL-1 beta neutralizing antibody reduced the melanoma cell-induced HSE production of H2O2 by 80%. On the contrary, B16 melanoma cell-conditioned medium did not vary HSE production of H2O2 compared to control HSE. Western blot analysis of cytosolic and membrane sediments from B16 melanoma cells confirmed the presence of IL-1 beta (17.4 kDa) in both cell compartments. Thus, HSE responded to melanoma cell contact with a rapid production of H2O2. HSE activation was IL-1-dependent. This cytokine was directly provided to HSE by the cell surface of adhered melanoma cells.