Graded Alcohol Series Research Articles

FRI390 The Effects Of Chronic Asprosin Administration On Ovarian Tissue

Abstract Disclosure: H. Kelestimur: None. Z. Oz: None. M. Ozdede: None. I. Serhatlioglu: None. N. Kaya Tektemur: None. E. Kacar: None. Purpose: Asprosin is a novel glucogenic and orexigenic hormone produced by the fibrillin 1 (FBN1) gene that is secreted by white adipose tissue during fasting. Fibrillins (FBNs) are structural and signalling proteins found mainly in the ovarian stroma, tunica albuginea and theca layers. The aim of this study was to determine the effects of asprosin on ovarian tissue in the female rats. Materials and methods: Twenty-four female Sprague-Dawley rats were randomly divided into 2 groups (n=12) as control and asprosin. Asprosin and control groups were given intraperitoneal saline at doses of 500 ng/kg and 1 ml/kg, respectively, at 14.00 every day for eight weeks. After the application, decapitation was performed, and the rapidly removed ovarian tissues were placed in a 10% formaldehyde solution for fixation. Formalin-fixed ovaries were dehydrated in a graded alcohol series and graded ethanol, respectively After that, all tissues were embedded in paraffin wax. The ovaries were cut into 5-µm sections and stained with hematoxylin and eosin (H&E) and also Masson trichrome staining. Ovarian follicular reserve and fibrosis were evaluated by light microscopy. The follicles were histologically classified as primordial, primary, secondary, and Graaf and all follicular structures were counted in each section. The fibrosis was evaluated with Masson’s trichrome staining and scored from 0 to 3 semi-quantitatively (0 = no fibrosis, 1 = low fibrosis, 2 = intermediate fibrosis, 3 = severe fibrosis). Results: In the asprosin group, unlike the control group, occasional congestion was observed in the vessels located in the ovarian medulla. Apart from this, no histopathological changes were found. An increase in fibrosis was detected in the ovarian sections of the asprosin group when compared to the control group (p = 0,035). In the light microscopic evaluation of the ovarian sections of the rats in the asprosin group, the primary and secondary follicle numbers were significantly higher than those in the control group (p = 0,008 and p = 0,047 respectively). However, the difference between the control and asprosin groups in terms of the primordial follicle, Graafian follicle and corpus luteum numbers were not statistically significant. Conclusion: Our results have shown that chronic asprosin administration causes an increase in the number of primary and secondary follicles, suggesting that changes in ovarian steroidogenesis and folliculogenesis depending on the application may lead to positive effects on female fertility. Keywords: Asprosin, adipokine, ovarian follicle. Acknowledgement: This study was supported by TUBITAK (Project: 220S744). Presentation: Friday, June 16, 2023

Immunohistological localisation of growth factors in stroma and interstitial gland tissue of goat (Capra hircus) ovary

The growth factors platelet derived growth factor (PDGF), transforming growth factor alpha (TGF-α) and transforming growth factor beta (TGF-β) have been demonstrated to stimulate the in vitro prolife­ration of theca and granulosa cells in different animals. The present study was conducted to localise the growth factors PDGF, TGF-α and TGF-β in different types of interstitial cells and stromal cells of normal cycling goat ovaries. Tissue fixed in formalin was processed through a graded series of alcohols and embedded in paraffin wax. The sections were immunohistochemically stained with antibo­dies against PDGF, TGF-α and TGF-β. The binding affinity of interstitial cells and stromal cells were observed and photographed. The staining pattern of PDGF, TGF-α and TGF-β was mild to strong in stromal cells. The primary and secondary interstitial cells exhibited varied staining patterns for all studied growth factors. These findings in goat suggests that PDGF, TGF-α, TGF-β were potentially an important autocrine regulator of different cell functions and possibly a paracrine regulator of ovarian cell function at various development stages.

Microstructure of Quills in Sunda Porcupine Hystrix javanica (F. Cuvier, 1823)

The purpose of this study was to identify the quills type, cuticle pattern, cross-section feature, and medulla structure of the quills in Sunda porcupine (Hystrix javanica). The second aim was to determine the nutritional content of the quills in Sunda porcupine. The specimens fixed in cacodylate buffer and glutaraldehyde, then dehydrated through graded series of alcohol, and freeze-dried. The specimens attached to the stubs by sticky tape, coated with gold and observed with Scanning Electron Microscope (SEM). Determination of cuticle pattern and medulla structure base on to the mammal's hair identification key by Teerink (1991). Nutritional content analysis using proximate. The study result showed that H. javanica has four types of quills; true, transitional, flat spine, and rattle quills. The SEM micrograph of the cuticle pattern showed characteristic variation in the shaft and base region, except the flat spine has no scaly feature in the base region. The cuticle of anogenital quill and hair in H. javanica has no specific feature or osmetrichia. The cross-sectional images of the three type quills of H. javanica revealed circular and alveolar arrangement. Only the flat spine has a quadriconcave feature. The true quills have multicellular and reverse cloisonné structure in the medulla, compare to the other three quills have no pattern. Nutritional content of the quills were water (89.93%), crude protein (93.66%), crude fat (0.44%), phosphorous (0.034%), calcium (0.2%), magnesium (0.01%), and sulphur (2,01%).

Morphologic and Histologic Observation of Red-Legged Partridge's (Alectoris Chukar) Liver

This study was aimed to investigate the liver of red-legged partridge in regards of morphologic and histologic features. In this study, the liver tissues of ten red-legged partridges were removed and fixed into 10% formaldehyde solution for 72 hours. After fixation, the liver tissues were dehydrated through a graded alcohol series to xylene and embedded in paraffin blocks. Obtained sections 5-7 µm thickness of sections from paraffin blocks were stained with Crossman Modified Triple staining and examined for histologic structures. In morphologic examining, the liver of red-legged partridges consisted of two lobs. In addition, histologic analysis showed that liver tissue was covered with a thick connective tissue and this connective tissue made up of many smaller units of liver cells called lobules. Hepatocytes were seen radially round and located around central vena, which consists of remark cords. The squamous cells and Kupffer cells were observed in the sinusoidal lining. In conclusion; the results from this study indicated that there were some structural differences from other bird species, but not functionally.

Conditional Alox12b Knockout: Degradation of the Corneocyte Lipid Envelope in a Mouse Model of Autosomal Recessive Congenital Ichthyoses

Conditional Alox12b Knockout: Degradation of the Corneocyte Lipid Envelope in a Mouse Model of Autosomal Recessive Congenital Ichthyoses

Probiotic supplementation affects the glycan composition of mucins secreted by Brunner’s glands of the pig duodenum

The effect of a dietary probiotic blend on the carbohydrate composition of mucins secreted by the Brunner’s glands in the duodenum of growing-finishing pigs was investigated by means of conventional (periodic acid-Schiff, Alcian Blue pH 2.5, high iron diamine staining) and lectin (15 lectins) histochemistry. Pigs were assigned to two dietary treatments: a control basal diet without the probiotic blend (No-Pro) and a test diet that included the probiotic blend (Pro). Duodenal tissue fragments were fixed in 4% phosphate-buffered-saline-buffered paraformaldehyde, dehydrated through a graded alcohol series, and embedded in paraffin wax. The secretory cells of the Brunner’s glands from No-Pro pigs primarily produced neutral glycoproteins and a small amount of acidic non-sulphated mucins. This glycan pattern was opposite that of the Brunner’s glands from Pro animals. A comparison of lectin-binding profiles of the secretory cells of Brunner’s glands in these two groups showed that in Pro pigs, there was (i) a decrease in N-linked glycans containing α1,2-linked fucose (Con A, UEA I); (ii) a loss of complex types of N-glycans (PHA-L, PHA-E) terminating with lactosamine (RCA120), α1,6- and α1,3-linked fucose (LTA), and α-galactose (GSA I-B4), as well as of O-glycans with terminal Galβ1,3GalNAc (PNA); and (iii) an increase in O-glycans containing GalNAc HPA. No-Pro and Pro samples showed no change in the expression of α2,6 sialoglycans and terminal GlcNAc residues and no affinity for MAL II, DBA, and SBA. These results indicate that probiotic supplementation affects the glycan composition of mucins produced in the Brunner’s glands of growing-finishing pigs. These changes could effectively act on the gastrointestinal function and health status of these animals because the probiotic blend induced higher growth performance and meat quality in the test probiotic group than it did in the control basal diet group (Tufarelli et al., 2017).

Antigen Masking During Fixation and Embedding, Dissected.

Antigen masking in routinely processed tissue is a poorly understood process caused by multiple factors. We sought to dissect the effect on antigenicity of each step of processing by using frozen sections as proxies of the whole tissue. An equivalent extent of antigen masking occurs across variable fixation times at room temperature. Most antigens benefit from longer fixation times (>24 hr) for optimal detection after antigen retrieval (AR; for example, Ki-67, bcl-2, ER). The transfer to a graded alcohol series results in an enhanced staining effect, reproduced by treating the sections with detergents, possibly because of a better access of the polymeric immunohistochemical detection system to tissue structures. A second round of masking occurs upon entering the clearing agent, mostly at the paraffin embedding step. This may depend on the non-freezable water removal. AR fully reverses the masking due both to the fixation time and the paraffin embedding. AR itself destroys some epitopes which do not survive routine processing. Processed frozen sections are a tool to investigate fixation and processing requirements for antigens in routine specimens.

HAART induces cell death in a cervical cancer cell line, HCS-2: A Scanning Electron Microscopy study

Apoptosis is a tightly programmed cell suicide which occurs in multiple physiologic and pathological conditions where it plays an important role in tissue development and homeostasis by eliminating unwanted and damaged cells. Appropriate apoptosis signalling is crucial in maintaining the fine balance between cell death and cell survival in cancer. In response to death stimuli the morphology of the cell undergoes unique changes. The aim of this study was to examine and compare the changes in the cell surface morphology using scanning electron microscopy in HCS-2 cells, following 24 hour treatment with components of highly active antiretroviral therapy (HAART) at their clinical plasma concentrations. The cells were fixed in 2.5% Glutaraldehyde and post-fixed in 1% osmium tetroxide. The cells were then dehydrated through a graded series of alcohol and treated with hexamethyldisilazane, then coated with a double layer of carbon. The cells were viewed under a Zeiss Ultra FEG Scanning Electron Microscope and a one way ANOVA and Tukey Kramer Post Hoc test was conducted based on the scoring of surface morphology of the cells using JMP 11 statistical software. The drugs used in this study induced morphological features which are known to be characteristic of apoptotic cell death. The drug combinations (ATP and LPV/r) were seemingly more effective than individual treatments in inducing cell death because morphological features observed were more advanced than those observed in individual treatments. However, LPV/r was more potent than ATP. In conclusion, HAART showed anticancer properties by inducing cell death through apoptosis.

Performance of domestic dogs on an olfactory discrimination of a homologous series of alcohols

Dogs are deployed for the detection of a wide variety of chemical stimuli. Despite their wide use, little basic research has explored canine olfactory generalization and discrimination. In the present study, we assessed canine odor discrimination amongst a series of chemically-related aliphatic alcohols. Domestic dogs were trained to discriminate 1-pentanol from air in a two-choice operant discrimination procedure until reaching an 85% accuracy criterion. In a series of transfer tasks, we assessed dogs’ generalization and discrimination between related odorants by replacing the S− stimulus with an alcohol related to pentanol, differing only in the length of the carbon chain. Dogs showed an increase in discrimination performance with an increase in the difference in the number of carbon atoms between pentanol and the comparison alcohol (p<0.001). These results indicate that this graded series of alcohols may be a useful stimulus set for studying olfactory generalization and discrimination processes in dogs, and that dogs show the same relationship between chemical similarity and discrimination performance as has been observed with humans, monkeys, honeybees, elephants, and rats.

A non-instrument-based method for the analysis of formalin-fixed paraffin-embedded human spinal cord via matrix-assisted laser desorption/ionisation imaging mass spectrometry.

This paper, in conjunction with a work published earlier this year by O'Rourke et al., aims to provide a comprehensive set of protocols for the analysis of formalin-fixed paraffin-embedded (FFPE) tissue via matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) in a low-cost and highly repeatable and robust way, thereby allowing other research teams to begin their own IMS-centered avenues of research. Samples of FFPE tissue were sectioned at 5 µm, water float mounted to specially prepared ITO glass slides and then dilipidated in a graded alcohol series. Tissue sections were then antigen retrieved under pressure in 20 mmol Tris-HCl (pH 8.8), coated with trypsin and digested O/N at 37°C. Samples were then sublimated with matrix to a final coverage of 0.2 mg/cm(2), recrystallised at 37°C with 50:50 acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) for 1 h and analysed with a MALDI TOF/TOF mass spectrometer. Serial sections were imaged, revealing little to no variation with regards to image quality and corresponding spectra. We have also attempted to describe the processes that govern the various aspects of this protocol with respect to each step necessary to ensure reproducibility. We are confident that this protocol in conjunction with the work published earlier by O'Rourke et al. provides the basis for a repeatable and robust protocol for the analysis of tissues from various sources via MALDI-IMS. The descriptions of key steps within allows for easy adoption of the protocol while allowing for desired modifications to be performed with minimal yet intuitive adjustment.

A versatile cost-effective method for the analysis of fresh frozen tissue sections via matrix-assisted laser desorption/ionisation imaging mass spectrometry.

There are currently multiple methods available for the preparation of fresh frozen tissue samples for analysis via matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) imaging mass spectrometry (IMS). Although these methods report excellent results, many are expensive automated approaches. With no published attempt to standardise less expensive manual processes, our work aims to provide a robust and repeatable method of sample preparation for MALDI-TOF-IMS that is applicable to a variety of tissue types, well explained, simple and cost effective. Fresh frozen tissue was sectioned at 12 µm and mounted onto liquid nitrocellulose coated slides, washed in a graded alcohol series and then mounted into a modified sublimation apparatus. Matrix is deposited onto the slide to achieve a desired coating of 0.2 mg/cm(2). Once coated, the slide is mounted into a custom-built vapor chamber and recrystallised with 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) for 1 h at 37°C. The slide is then analysed using MALDI-IMS. We have successfully implemented this method for a host of tissue samples, including brain, liver, kidney and heart, with no variation in relative spectra or processing method required. When the protocol is followed correctly, sublimations and recrystallisations are highly predictable with limited variation between samples and a very low failure rate. Additional apparatuses can be easily constructed by following the included instructions, that perform as per specifications with no variation. We believe that we have described a complete protocol for MALDI-IMS that is easy to use and highly reproducible. The lack of expensive commercially available equipment makes this process very cheap with a relatively low initial outlay and our hope is that more laboratories will begin IMS-based avenues of research based on the work we have performed.

The Selective A3AR Antagonist LJ-1888 Ameliorates UUO-Induced Tubulointerstitial Fibrosis

Adenosine in the normal kidney significantly elevates in response to cellular damage. The renal A3 adenosine receptor (A3AR) is up-regulated under stress, but the therapeutic effects of A3AR antagonists on chronic kidney disease are not fully understood. The present study examined the effect of LJ-1888 [(2R,3R,4S)-2-[2-chloro-6-(3-iodobenzylamino)-9H-purine-9-yl]-tetrahydrothiophene-3,4-diol], a newly developed potent, selective, species-independent, and orally active A3AR antagonist, on unilateral ureteral obstruction (UUO)-induced renal fibrosis. Pretreatment with LJ-1888 inhibited UUO-induced fibronectin and collagen I up-regulation in a dose-dependent manner. Masson's trichrome staining confirmed that LJ-1888 treatment effectively reduced UUO-induced interstitial collagen accumulation. Furthermore, delayed administration of LJ-1888 showed an equivalent therapeutic effect on tubulointerstitial fibrosis to that of losartan. Small-interfering A3AR transfection effectively inhibited transforming growth factor-β1 (TGF-β1)-induced fibronectin and collagen I up-regulation in proximal tubular cells similar to LJ-1888, confirming that the renoprotective effect of LJ-1888 resulted from A3AR blockade. UUO- or TGF-β1-induced c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation decreased significantly after LJ-1888 administration. A3AR blockade reduced UUO- or TGF-β1-induced up-regulation of lysyl oxidase, which induces cross-linking of extracellular matrix, suggesting that LJ-1888 may also regulate extracellular matrix accumulation via post-translational regulation. In conclusion, the present data demonstrate that the A3AR antagonist, LJ-1888, blocked the development and attenuated the progression of renal fibrosis, and they suggest that LJ-1888 may become a new therapeutic modality for renal interstitial fibrosis.

The first report of new species: Trichuris landak n. sp

ObjectiveTo study nematode parasites morphology of Hystrix javanica (H. javanica), both through the feces and internal organs. MethodsFeces were observed by direct smear method, internal organs were observed after dissecting the host. Specimens for light microscopy examination were fixed with 70% warm alcohol, cleared and mounted in lactophenol for wet mounting. Specimens for SEM examination were postfixed in cacodylate buffer and glutaraldehyde, dehydrated through a graded series of alcohol and freeze dried. The specimens were attached to stubs with double cello-tape, coated with gold and observed with a JSM5310 LV electron microscope. Figures were made with the aid of a drawing tube attached to Olympus compound microscope, other figures were photographs of scanning electron microscope images. Measurements were given in micrometers as the mean followed by the range in parentheses, unless otherwise stated. ResultsThe nematode species found in the intestine of H. javanica are Gireterakis girardi and a new species, Trihuris landak. The new species differs with previously reported species from Hystrix because of having stylet and short cervical alae. The pattern of bacillary band is closed to Trichuris trichiurus, the species that infect human, but differs because the surface of its vulva is not covered with densely spine. ConclusionsThe species of nematodes found on H. javanica were Gireterakis girardi and a new species Trichuris landak n.sp. Those two species are newly recorded in Indonesia.

Involvement of IL-1 and Oncostatin M in Acanthosis Associated With Hypertensive Leg Ulcer

Hypertensive leg ulcer (HLU) is an inflammatory disease characterized by intense pain, alteration of vascularization, and skin necrosis. The optimal treatment relies on surgical removal of necrotic tissues covered by a split-skin graft. We studied the histomorphology of the lesions and investigated the involvement of inflammatory cells and cytokines to further define the physiopathology of HLU. We report epidermis acanthosis and a preferential occlusion of the precapillary arterioles with infiltration of neutrophils, macrophages, and T lymphocytes in the dermis. OSM, IL-1β, and IL-6 were overexpressed in the ulcer, whereas the Th17-derived cytokines were not. In vitro, the addition of IL-1β and OSM promoted acanthosis and destructuring of reconstructed epidermis. Exogenous IL-1β and OSM synergistically induced epidermal acanthosis in mice. These data show that OSM and IL-1β are not only a biological characteristic signature of HLU, but these cytokines reflect a specific inflammatory state, directly involved in the pathogenesis. We suggest that anti-cytokine biotherapies could be an alternative strategy to surgery to treat HLU.

Histology of ovine placenta during gestation periods

Placetomes were collected from 93 pregnant slaughtered Ewes at different gestation periods , from AL.Falluja slaughter house during the periods from 2, Jully 2009 to 30, December ,2010 . Tissue specimens for microscopic examination were taken from the centers of the sampled placentomes .Immediately following collection ,the samples were fixed in 10% bufferd neutral formalin for 24 h .Tissue specimens were dehydrated in a graded series of alcohol , cleared by xylol and embded in paraffin .Histologic section were cut at 3-4 µm thickness ,Stained with hematoxylin and eosin(H&amp;E) (6).The period of gestation were measured according to Richardson (7) with aquation of x =2.1(Y+17) as x= gestation period in day and Y=the crown Rump Histologic examination during early pregnancy (30 – 40 d) Showed a pronounced BNC with anumbe of nuclei with in each cellular boundary in the uterine epithelium indicates that possible fusions are restricted , It is also there is an increase in blood vascularity. At 40 – 50 d of pregnancy, there was a further increase in caruncular vascularity by 2-fold characterized by increase capillary number and 2 to 3 – fold increase in capillary diameter. Endometrial gland hyperplasia showed during this period . Then placentomes showed grow in number and size until 8oth day. It was shown that the BNCS of the trophoblast increase in size, in polarity and in the number of their cytoplasmic granules as pregnancy advanced.

Histology of ovine placenta during gestation periods

Placetomes were collected from 93 pregnant slaughtered Ewes at different gestation periods , from AL.Falluja slaughter house during the periods from 2, Jully 2009 to 30, December ,2010 . Tissue specimens for microscopic examination were taken from the centers of the sampled placentomes .Immediately following collection ,the samples were fixed in 10% bufferd neutral formalin for 24 h .Tissue specimens were dehydrated in a graded series of alcohol , cleared by xylol and embded in paraffin .Histologic section were cut at 3-4 µm thickness ,Stained with hematoxylin and eosin(H&amp;E) (6).The period of gestation were measured according to Richardson (7) with aquation of x =2.1(Y+17) as x= gestation period in day and Y=the crown Rump. Histologic examination during early pregnancy (30 – 40 d) Showed a pronounced BNC with anumbe of nuclei with in each cellular boundary in the uterine epithelium indicates that possible fusions are restricted , It is also there is an increase in blood vascularity. At 40 – 50 d of pregnancy, there was a further increase in caruncular vascularity by 2-fold characterized by increase capillary number and 2 to 3 – fold increase in capillary diameter. Endometrial gland hyperplasia showed during this period . Then placentomes showed grow in number and size until 8oth day. It was shown that the BNCS of the trophoblast increase in size, in polarity and in the number of their cytoplasmic granules as pregnancy advanced.

Human skeletal muscle glycogen utilization in exhaustive exercise: role of subcellular localization and fibre type

Although glycogen is known to be heterogeneously distributed within skeletal muscle cells, there is presently little information available about the role of fibre types, utilization and resynthesis during and after exercise with respect to glycogen localization. Here, we tested the hypothesis that utilization of glycogen with different subcellular localizations during exhaustive arm and leg exercise differs and examined the influence of fibre type and carbohydrate availability on its subsequent resynthesis. When 10 elite endurance athletes (22 ± 1 years, VO2 max = 68 ± 5 ml kg-1 min-1, mean ± SD) performed one hour of exhaustive arm and leg exercise, transmission electron microscopy revealed more pronounced depletion of intramyofibrillar than of intermyofibrillar and subsarcolemmal glycogen. This phenomenon was the same for type I and II fibres, although at rest prior to exercise, the former contained more intramyofibrillar and subsarcolemmal glycogen than the latter. In highly glycogen-depleted fibres, the remaining small intermyofibrillar and subsarcolemmal glycogen particles were often found to cluster in groupings. In the recovery period, when the athletes received either a carbohydrate-rich meal or only water the impaired resynthesis of glycogen with water alone was associated primarily with intramyofibrillar glycogen. In conclusion, after prolonged high-intensity exercise the depletion of glycogen is dependent on subcellular localization. In addition, the localization of glycogen appears to be influenced by fibre type prior to exercise, as well as carbohydrate availability during the subsequent period of recovery. These findings provide insight into the significance of fibre type-specific compartmentalization of glycogen metabolism in skeletal muscle during exercise and subsequent recovery. .

Phase Advance of the Light-Dark Cycle Perturbs Diurnal Rhythms of Brain-derived Neurotrophic Factor and Neurotrophin-3 Protein Levels, Which Reduces Synaptophysin-positive Presynaptic Terminals in the Cortex of Juvenile Rats

In adult rat brains, brain-derived neurotrophic factor (BDNF) rhythmically oscillates according to the light-dark cycle and exhibits unique functions in particular brain regions. However, little is known of this subject in juvenile rats. Here, we examined diurnal variation in BDNF and neurotrophin-3 (NT-3) levels in 14-day-old rats. BDNF levels were high in the dark phase and low in the light phase in a majority of brain regions. In contrast, NT-3 levels demonstrated an inverse phase relationship that was limited to the cerebral neocortex, including the visual cortex, and was most prominent on postnatal day 14. An 8-h phase advance of the light-dark cycle and sleep deprivation induced an increase in BDNF levels and a decrease in NT-3 levels in the neocortex, and the former treatment reduced synaptophysin expression and the numbers of synaptophysin-positive presynaptic terminals in cortical layer IV and caused abnormal BDNF and NT-3 rhythms 1 week after treatment. A similar reduction of synaptophysin expression was observed in the cortices of Bdnf gene-deficient mice and Ca(2+)-dependent activator protein for secretion 2 gene-deficient mice with abnormal free-running rhythm and autistic-like phenotypes. In the latter mice, no diurnal variation in BDNF levels was observed. These results indicate that regular rhythms of BDNF and NT-3 are essential for correct cortical network formation in juvenile rodents.

Selection of Brain Metastasis-Initiating Breast Cancer Cells Determined by Growth on Hard Agar

An approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential is highly desirable. Here, we demonstrate that plating GI-101A human breast cancer cells on hard (0.9%) agar selects for the subpopulation of metastasis-initiating cells. The agar-selected cells, designated GI-AGR, were homogeneous for CD44(+) and CD133(+) and five times more invasive than the parental GI-101A cells. Moreover, mice injected with GI-AGR cells had significantly more experimental brain metastases and shorter overall survival than did mice injected with GI-101A cells. Comparative gene expression analysis revealed that GI-AGR cells were markedly distinct from the parental cells but shared an overlapping pattern of gene expression with the GI-101A subline GI-BRN, which was generated by repeated in vivo recycling of GI-101A cells in an experimental brain metastasis model. Data mining on 216 genes shared between GI-AGR and GI-BRN breast cancer cells suggested that the molecular phenotype of these cells is consistent with that of cancer stem cells and the aggressive basal subtype of breast cancer. Collectively, these results demonstrate that analysis of cell growth in a hard agar assay is a powerful tool for selecting metastasis-initiating cells in a heterogeneous population of breast cancer cells, and that such selected cells have properties similar to those of tumor cells that are selected based on their potential to form metastases in mice.

Coordinate Expression of Colony-Stimulating Factor-1 and Colony-Stimulating Factor-1-Related Proteins Is Associated with Poor Prognosis in Gynecological and Nongynecological Leiomyosarcoma

Previously, we showed that the presence of high numbers of macrophages correlates with poor prognosis in nongynecological leiomyosarcoma (LMS). In gynecological LMS, a similar trend was noted but did not reach statistical significance. Colony-stimulating factor-1 (CSF1) is a major chemoattractant for macrophages. Here we show that in a subset of LMS cases, CSF1 is expressed by the malignant cells. Previously, we found that CSF1 is translocated and highly expressed in tenosynovial giant cell tumors (TGCTs), and this observation allowed us to identify genes that showed a coordinate expression with CSF1. Here, we evaluated the expression of CSF1 and TGCT-associated proteins in 149 cases of LMS. The coordinate expression of CSF1 and three TGCT-associated proteins (CD163, FCGR3a, and CTSL1) identified cases with poor prognosis in both gynecological LMS (P = 0.00006) and nongynecological LMS (P = 0.03). In gynecological LMS, the coordinate expression of these four markers was the only independent prognosticator in multivariate analysis (hazard ratio, 4.2; 95% CI, 1.12 to 16; P = 0.03). Our findings indicate that CSF1 may play an important role in the clinical behavior of LMS that may open a window for new therapeutic reagents.