Interleukin (IL)-17A in triple-negative breast cancer: a potent prognostic factor associated with intratumoral neutrophil infiltration.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized as highly immunogenic and lacks specific targeted therapies. Interleukin 17A (IL-17A) is a controversial cytokine and is known to have anti-tumor and pro-tumor roles depending on the tumor microenvironment. In addition, IL-17A has been recently implicated in the recruitments of neutrophil into tumor tissues. Although IL-17A is considered tumor-promoting in breast cancer, its significance in the possible regulation of neutrophil infiltration in TNBC is not clearly defined. MATERIALS AND METHODS: We immunolocalized IL-17A, CD66b (neutrophil marker), and chemokine (C-X-C motif) ligand 1 (CXCL1, neutrophil chemoattractant) in 108 TNBC specimens and assessed their correlation among each other. The correlation between these markers and clinicopathological parameters was also assessed. We subsequently performed in vitro study to address the possible regulation of CXCL1 by IL-17A using TNBC cell lines, MDA-MB-231 and HCC-38. RESULTS: It was revealed that IL-17A correlated significantly with CXCL1 and CD66b, also CD66b with CXCL1. Furthermore, IL-17A was significantly associated with shorter disease-free and overall survival, especially in a high density CD66b group of patients. In vitro results revealed that IL-17A upregulated CXCL1 mRNA expression in a dose and time dependent manner, and this induction was significantly suppressed by an Akt inhibitor. CONCLUSION: IL-17A was considered to contribute to neutrophil infiltration by inducing CXCL1 in TNBC tissues and educating neutrophils to promote tumor progression. IL-17A might therefore serve as a potent prognostic factor in TNBC.

Automatic breast carcinoma detection in histopathological micrographs based on Single Shot Multibox Detector.

Background: A diagnosis with histological classification by pathologists is very important for appropriate treatments to improve the prognosis of patients with breast cancer. However, the number of pathologists is limited, and assisting the pathological diagnosis by artificial intelligence becomes very important. Here, we presented an automatic breast lesions detection model using microscopic histopathological images based on a Single Shot Multibox Detector (SSD) for the first time and evaluated its significance in assisting the diagnosis. Methods: We built the data set and trained the SSD model with 1361 microscopic images and evaluated using 315 images. Pathologists and medical students diagnosed the images with or without the assistance of the model to investigate the significance of our model in assisting the diagnosis. Results: The model achieved 88.3% and 90.5% diagnostic accuracies in 3-class (benign, non-invasive carcinoma, or invasive carcinoma) or 2-class (benign or malignant) classification tasks, respectively, and the mean intersection over union was 0.59. Medical students achieved a remarkably higher diagnostic accuracy score (average 84.7%) with the assistance of the model compared to those without assistance (average 67.4%). Some people diagnosed images in a short time using the assistance of the model (shorten by average 6.4 min) while others required a longer time (extended by 7.2 min). Conclusion: We presented the automatic breast lesions detection method at high speed using histopathological micrographs. The present system may conveniently support the histological diagnosis by pathologists in laboratories.

Diverse role of androgen action in human breast cancer.

Breast cancer is a hormone-dependent cancer, and sex steroids play a pivotal role in breast cancer progression. Estrogens are strongly associated with breast cancers, and the estrogen receptor (estrogen receptor α; ERα) is expressed in 70-80% of human breast carcinoma tissues. Although antiestrogen therapies (endocrine therapies) have significantly improved clinical outcomes in ERα-positive breast cancer patients, some patients experience recurrence after treatment. In addition, patients with breast carcinoma lacking ERα expression do not benefit from endocrine therapy. The androgen receptor (AR) is also expressed in >70% of breast carcinoma tissues. Growing evidence supports this novel therapeutic target for the treatment of triple-negative breast cancers that lack ERα, progesterone receptor, and human EGF receptor 2, and ERα-positive breast cancers, which are resistant to conventional endocrine therapy. However, the clinical significance of AR expression is still controversial and the biological function of androgens in breast cancers is unclear. In this review, we focus on the recent findings concerning androgen action in breast cancers and the contributions of androgens to improved breast cancer therapy.

Forkhead Box I1 in Breast Carcinoma as a Potent Prognostic Factor.

Forkhead box (FOX) proteins are family of transcriptional factors and regulate cell growth and differentiation as well as embryogenesis and longevity. Previous studies have demonstrated that several FOX members regulate growth or metastasis of breast carcinoma, but clinical significance of total FOX members remains unclear. We first examined associations between expression of 40 FOX genes and TNM status of 19 breast carcinoma using microarray data. Subsequently, we immunolocalized FOXI1 in 140 breast carcinomas and evaluated its clinicopathological significance. In the microarray analysis, we newly identified that gene expression of FOXI1 was most pronouncedly linked to metastasis of the breast carcinoma among the FOX members examined. However, clinicopathological significance of FOXI1 has not been examined in the breast carcinoma. FOXI1 immunoreactivity was positive in 44 out of 140 (31%) of breast carcinomas, and it was significantly associated with stage, lymph node metastasis and distant metastasis. The FOXI1 status was significantly associated with worse prognosis of the breast cancer patients, and it turned out to be an independent prognostic factor for both distant disease-free survival and breast cancer-specific survival. These findings suggest that FOXI1 plays important roles in the metastasis of breast carcinoma and immunohistochemical FOXI1 status is a potent prognostic factor.

Androgens enhance the ability of intratumoral macrophages to promote breast cancer progression.

Androgens are produced locally in breast carcinoma tissues by androgen­producing enzymes such as 5α­reductase type 1 (5αRed1) and affect not only breast cancer cells but the tumor microenvironment as well. Tumor­associated macrophages (TAMs) are primary components of the tumor microenvironment and contribute to tumor progression. Although previous studies suggest that androgen/androgen receptor (AR) signaling in macrophages has important roles in human diseases, androgen action on TAMs has remained largely unknown. We immunolocalized macrophage marker CD163 as well as AR and 5αRed1 in 116 breast carcinomas and correlated them with clinicopathological parameters and clinical outcomes. Moreover, we examined the roles of androgens on macrophages in breast cancer progression using cell lines 4T1 (mouse breast cancer) and RAW264.7 (macrophage) in a tumor­bearing female BALB/c mouse model. Double immunohistochemistry revealed that AR was sporadically expressed in the macrophages in breast carcinoma tissues. Macrophage infiltration was significantly correlated with an aggressive phenotype of breast carcinomas and worse prognosis, especially in the 5αRed1­positive group. In a sphere­forming assay using 4T1 and RAW­AR cells, which stably express AR, the sphere size was significantly increased due to androgens when 4T1 cells were cocultured with RAW­AR cells. Furthermore, in vivo experiments revealed that tumor growth and Ki67, a cell proliferation marker, were increased when androgens were stably produced in breast cancer cells and AR was expressed in macrophages. In conclusion, AR is expressed in intratumoral macrophages and is associated with an aggressive phenotype of breast carcinomas, especially when breast cancer cells actively produce androgens. Thus, androgens may enhance the ability of macrophages to promote breast cancer progression.

D-2-hydroxyglutarate dehydrogenase in breast carcinoma as a potent prognostic marker associated with proliferation.

BACKGROUND: D-2-hydroxyglutarate dehydrogenase (D2HGDH) catalyzes D-2-hydroxyglutarate to α-ketoglutarate and is involved in the regulation of cellular energy and biosynthetic intermediates. Previously, D2HGDH was reported to decrease 2-hydroxyglutarate level in breast carcinoma cells, but no other report has examined D2HGDH in breast carcinoma, and its significance remains unknown. METHODS: We first immunolocalized D2HGDH in 224 invasive breast carcinomas and evaluated its clinicopathological significance. We next examined associations between gene expression of D2HGDH and α-ketoglutarate-dependent dioxygenases in 23 breast carcinoma tissues using the gene expression profile data. Finally, we examined the effects of D2HGDH on the proliferation in three breast carcinoma cells. RESULTS: D2HGDH immunoreactivity was detected in 49% of invasive breast carcinomas, and the immunohistochemical D2HGDH status was positively associated with histological grade, HER2 and Ki-67, while it was inversely associated with estrogen receptor. Moreover, it was significantly associated with worse prognosis of the breast cancer patients, and it turned out to be an independent prognostic factor for both the disease-free and breast cancer-specific survival in these patients. Gene expression profile data revealed that D2HGDH expression was positively associated with the expression of 6 α-ketoglutarate-dependent dioxygenases (KDM3A, PLOD1, EGLN2, ALKBH1, ASPH and ALKBH7). Consequent in vitro experiments demonstrated that D2HGDH overexpression significantly increased the cell proliferation activity of MCF-7, T47D and MDA-MB-231 cells. CONCLUSION: These results suggest that D2HGDH plays an important role in the growth of breast carcinoma, possibly through regulating functions of α-ketoglutarate-dependent dioxygenases, and that D2HGDH status is a potent worse prognostic factor in breast cancer patients.

Isoforms of IDH in breast carcinoma: IDH2 as a potent prognostic factor associated with proliferation in estrogen-receptor positive cases.

BACKGROUND: Isocitrate dehydrogenase (IDH) is an important enzyme that oxidatively decarboxylates isocitrate to α-ketoglutarate, and three isoforms (IDH1-3) have been identified. Overexpression and/or downregulation of IDH isoforms was reported in several human malignancies, suggesting importance of IDH in oncogenesis. However, significance of IDH isoforms remains largely unclear in the breast carcinoma. METHODS: We immunolocalized IDH1, IDH2 and IDH3α in 226 breast carcinomas and evaluated their clinical significance. Subsequently, we examined effects of IDH2 on proliferation in breast carcinoma cells. RESULTS: Immunoreactivity of IDH1-3α was detected in 53%, 38% and 41% of breast carcinomas, and the non-neoplastic epithelium was IDH1-positive, IDH2-negative and IDH3α-positive. IDH1 immunoreactivity was inversely associated with pathological T factor (pT) and Ki-67 in the breast carcinoma, while IDH3α immunoreactivity was not significantly associated with clinicopathological factors. IDH2 status was positively correlated with stage, pT, histological grade, HER2, Ki-67 and microvessel density. Moreover, IDH2 status was significantly associated with worse prognosis of the patients, and it turned out an independent prognostic factor for estrogen-receptor (ER) positive patients. These findings were more evident in the IDH1-negative / IDH2-positive/IDH3α-negative subgroup which is the opposite immunohistochemical IDH phenotype of normal mammary epithelium. In vitro studies demonstrated that RNA interference of IDH2 significantly decreased proliferation activity of T47D and SKBR-3 cells. CONCLUSION: These results suggest that IDH2 is associated with an aggressive phenotype of breast carcinoma through increasing cell proliferation, different from IDH1 and IDH3α, and immunohistochemical IDH2 status is a potent prognostic factor especially in ER-positive breast cancer patients.

Stromal CCL5 Promotes Breast Cancer Progression by Interacting with CCR3 in Tumor Cells.

Chemokines secreted from stromal cells have important roles for interactions with carcinoma cells and regulating tumor progression. C-C motif chemokine ligand (CCL) 5 is expressed in various types of stromal cells and associated with tumor progression, interacting with C-C chemokine receptor (CCR) 1, 3 and 5 expressed in tumor cells. However, the expression on CCL5 and its receptors have so far not been well-examined in human breast carcinoma tissues. We therefore immunolocalized CCL5, as well as CCR1, 3 and 5, in 111 human breast carcinoma tissues and correlated them with clinicopathological characteristics. Stromal CCL5 immunoreactivity was significantly correlated with the aggressive phenotype of breast carcinomas. Importantly, this tendency was observed especially in the CCR3-positive group. Furthermore, the risk of recurrence was significantly higher in the patients with breast carcinomas positive for CCL5 and CCR3 but negative for CCR1 and CCR5, as compared with other patients. In summary, the CCL5-CCR3 axis might contribute to a worse prognosis in breast cancer patients, and these findings will contribute to a better understanding of the significance of the CCL5/CCRs axis in breast carcinoma microenvironment.

Rac1 activation in human breast carcinoma as a prognostic factor associated with therapeutic resistance.

BACKGROUND: RAS-related C3 botulinus toxin substrate 1 (Rac1) is a molecular switch fluctuating between GDP-bound inactive form (Rac1-GDP) and GTP-bound active form (Rac1-GTP) and involved in diverse function in both normal and malignant cells such as breast carcinoma cells. Although several studies have demonstrated immunolocalization of Rac1 protein in human breast carcinoma tissues, activation status of Rac1 still remains to be elucidated. METHODS: We immunolocalized active form of Rac1 (Rac1-GTP) as well as total Rac1 using antibody specific for them in 115 invasive breast carcinoma tissues and correlated with clinicopathological parameters and clinical outcomes. RESULTS: Rac1-GTP was frequently immunolocalized in the cytoplasm or cell membrane of breast carcinoma cells and it was positively correlated with Ki-67 labeling index and total Rac1 while negatively correlated with progesterone receptor. On the other hand, immunohistochemical Rac1-GTP status was significantly correlated with increased risk of recurrence and breast cancer-specific mortality of breast cancer patients and multivariate analyses did demonstrate Rac1-GTP as an independent worse prognostic factor for both disease-free and breast cancer-specific survival. In addition, Rac1-GTP was still correlated with worse prognosis in the patients who had received adjuvant chemotherapy or endocrine therapy. CONCLUSION: These findings suggested Rac1 activation played pivotal roles in the progression and therapeutic resistance of breast cancers and Rac1 might be an important therapeutic target for improvement of the therapy for breast cancer patients.

Duodenal infusion of fatty acids differentially affects plasma glucagon-like peptide-1 and ghrelin concentrations in sheep.

The aim of this study was to investigate how intraduodenal infusions of fatty acids (FA) affect appetite-related gut peptides such as glucagon-like peptide-1 (GLP-1) and ghrelin in sheep. We hypothesized that these peptides can be highly reactive to unsaturated long-chain FA, because they are well known to decrease dry matter intake (DMI). Four ewes were fitted with a duodenal cannula and a jugular vein catheter for a 6-h duodenal infusion of the 9 FA (C8:0, C10:0, C12:0, C14:0, C16:0, C18:0, C18:1, C18:2, and C18:3) and water (control). The concentration of each FA was 1.6 g per metabolic body weight (BW), approximately corresponding to the amount of supplemented fat in a standard dairy cow diet. Each infusion was separated by at least 2 d. During the infusion period, blood samples were collected periodically to determine changes in plasma GLP-1, ghrelin, and metabolite concentrations. Duodenal infusions of C18:1, C18:2, and C18:3 led to higher plasma GLP-1 (P < 0.05) and lower glucose (P < 0.05) than control. Plasma ghrelin concentrations were greater in C18:1 and C18:3 infusions than control (P < 0.05). Plasma ketone bodies were higher in C8:0 and C10:0 infusions (P < 0.05), but plasma triglyceride concentrations were lower in C8:0, C10:0, C12:0, and C16:0 infusions (P < 0.05) than control. Fatty acid infusions except for C18:3 led to higher plasma NEFA concentrations than control (P < 0.05). These results confirmed that the hypophagic effect of dietary unsaturated long-chain FA is mediated by GLP-1 (an anorexigenic effect) secretion. However, we also observed higher plasma ghrelin (an orexigenic effect) partially by unsaturated long-chain FA. Thus, the gut peptide secretions when ruminant animals ingest FA supplements would complexly affect satiety and further studies are needed to determine their each impact on DMI.

Drosophila Peptide Hormones Allatostatin A and Diuretic Hormone 31 Exhibiting Complementary Gradient Distribution in Posterior Midgut Antagonistically Regulate Midgut Senescence and Adult Lifespan.

Enteroendocrine cells (EEs) are evolutionarily conserved gastrointestinal secretory cells that show scattered distribution in the intestinal epithelium. These cells classified into several subtypes based on the hormones they produce in both mammals and insects. In the fruit fly Drosophila, it has been suggested that nearly equal numbers of two subtypes of EEs (Allatostatin A: AstA and Diuretic hormone 31 : Dh31) are alternately produced from the intestinal stem cells in the posterior midgut. However, we found that these two subtypes are not always present in this manner, but are rather distributed in a complementary frequency gradient along the posterior midgut. We show that midgut-preferential RNA knockdown of the peptide hormones AstA or Dh31 respectively results in decreased or increased adult lifespan. This effect on longevity is apparently correlated with the midgut senescence phenotypes as a result of direct hormone action through both hormone receptors expressed in the enteroblasts or other midgut cell types. However, gut senescence does not appear to be the direct cause for longevity regulation, as knockdown of both hormone receptors did not affect adult lifespan. Furthermore, these senescence phenotypes appear to be independent of insulin signaling and manifest in an organ-specific manner. These results indicate that the two intestinal secretory peptides antagonistically regulate adult lifespan and intestinal senescence through multiple pathways, irrespective of insulin, which implicates a complementary gradient distribution of each of the hormone-producing EEs, consistent with local requirements for cell activity along the posterior midgut.

Effects of insulin-like growth factor-1 on the in vitro maturation of canine oocytes.

The maturation rate of canine oocytes during in vitro maturation (IVM) needs to be improved. The present study was designed to evaluate the effects of insulin-like growth factor-1 (IGF-1) on the IVM of canine oocytes. Ovaries were obtained by ovariohysterectomy and were sliced to release cumulus-oocyte complexes (COCs). In Experiment 1, the effects of different concentrations of IGF-1 on the nuclear maturation of oocytes was investigated. The COCs were cultured in a modified medium (mTCM199) with IGF-1 (0, 0.5, 5, 10, and 50 µg/ml). At the end of the 48 h culture, oocytes were fixed and stained to evaluate their nuclear stage. Supplementation with 50 µg/ml IGF-1 induced a significantly higher metaphase II (MII) rate (P < 0.05) compared to the 0 and 0.5 µg/ml IGF-1 groups. In Experiment 2, the expression levels of insulin receptor (INSR), IGF-1 receptor (IGF-1R), and IGF-2 receptor (IGF-2R) genes, localized to canine oocytes and cumulus cells, were investigated before and after IVM. The expression level of IGF-1R in cumulus cells after IVM was higher than that before IVM (P < 0.05). In Experiment 3, it was investigated whether an inhibitor of PTEN (phosphatase and tensin homolog), bpV, affects the nuclear maturation of oocytes. Regardless of bpV supplementation at a concentration of 0.2 to 200 µmol/l, there was no significant difference in the proportion of oocytes that reached the MII stage. These results indicated that IGF-1 has a favorable effect on the IVM of canine oocytes, possibly through the stimulation of the Ras/MAPK pathway via IGF-1R expressed in cumulus cells.

Histochemical properties of bovine and ovine mammary glands during fetal development.

In order to obtain more information on the development of bovine and ovine fetal mammary glands, a series of mammary glands from fetuses of different ages were analyzed. A total of 16 bovine fetuses with curved crown rump lengths ranging from 12 cm (80 days) to 75 cm (240 days) and 15 ovine fetuses ranging from 55 days to 131 days were examined. We used hematoxylin and eosin stain and Oil-Red-O stain to analyze the developmental and morphogenetic processes of mammary glands. In addition, we used immunohistochemical staining to determine the pattern of expression of cytokeratin 18 (CK18) during luminal epithelial differentiation, α-smooth-muscle actin (α-SMA) for myoepithelial differentiation, Ki-67 for cell proliferation, and estrogen receptor α (ERα). Our analyzes showed: (a) The primary mammary duct begin to proliferate in a lengthwise within the teat at 90 days in bovine fetuses and 63 days in ovine fetus; (b) luminal epithelial cells and myoepithelial cells appeared from 90 days in bovine fetuses and 63 days in ovine fetus; (c) proliferation of epithelial cells appeared to coincide with the development of the primary and secondary ducts; and (d) ERα was not found in the fetal mammary gland, but adipocytes showed the presence of ERα. Overall, these results indicate that the sequence of events in the prenatal development of the mammary gland of sheep is similar to that of cattle.

Influence of maternal remifentanil concentration on fetal-to-maternal ratio in pregnant ewes.

PURPOSE: Maternal remifentanil infusion is used for minimally invasive fetal surgery or ex-utero intrapartum treatment. The fetal-to-maternal (F/M) ratio of remifentanil concentration at various dosing regimens is useful to manage remifentanil effects. The aim of this study was to investigate the F/M ratio of remifentanil at various concentrations. METHODS: Five pregnant ewes received continuous remifentanil infusion under propofol anesthesia. The remifentanil infusion rate was increased by 0.4 µg/kg/min every 15 min. The response to tail clamping in fetuses was assessed immediately before the change of infusion rate. Arterial remifentanil concentrations in the mother and fetus were determined at each tail clamp. After observing a loss of response to tail clamping, remifentanil infusion was terminated and the concentrations were assessed. RESULTS: The median remifentanil maximum infusion rate and maternal concentration were 3.0 µg/kg/min (range 2.4-3.6) and 21.6 (range 18.0-29.9) ng/mL, respectively. During continuous infusion, the F/M ratio was 0.15 (0.07-0.17), and the slope of the linear regression for the F/M ratio versus infusion rate in each individual was -0.001 ± 0.012/µg kg min (P = 0.876 vs hypothetical value of 0). The F/M ratio at the first sampling point in the elimination phase [0.33 (0.07-0.65)] was higher (P = 0.033) than at the last sampling point during continuous infusion [0.15 (0.06-0.17)]. CONCLUSION: The F/M ratio was constant at a steady state regardless of the remifentanil concentration up to 29.9 ng/mL, and increased in the elimination phase in pregnant ewes.

Effect of increased feeding of dietary α-linolenic acid by grazing on formation of the cis9,trans11-18:2 isoform of conjugated linoleic acid in bovine milk.

Feeding systems such as grazing affect the fatty acid profile of bovine milk fat. In addition, milk fat is formed as the product of fatty acid metabolism in cow bodies before being secreted into milk. However, how grazing influences milk fatty acid profile through the metabolism has not been completely characterized. When fatty acid concentrations in Holstein milk were compared between grazing and non-grazing periods, α-linolenic acid was significantly higher in the grazing period than in the non-grazing period. This could be explained with an increase in α-linolenic acid feeding with grazing. α-linolenic acid had a linear positive correlation with conjugated linoleic acid (9c,11t-18:2) (CLA) and vaccenic acid (VA) during the grazing period, whereas CLA had higher correlation with linoleic acid rather than with α-linolenic acid during the non-grazing period. These data indicate that the high content of dietary α-linolenic acid affects CLA and VA formation in milk of grazing periods via α-linolenic acid metabolism into VA.

Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination.

The present study was conducted to evaluate whether supplementation of semen extender with glutathione (GSH) can maintain the quality of frozen-thawed canine spermatozoa. Eighteen ejaculates were obtained from 5 dogs and placed in extender (20% egg yolk, Tris, citric acid, lactose, raffinose, antibiotics and 6.5% glycerol) containing 0 (control), 2.5, 5, 7.5 or 10 mM GSH. The samples were cooled to 4 C and then frozen in liquid nitrogen vapor. Motility parameters of the sperm were evaluated at 0, 1, 2, 3, 4, 12 and 24 h after thawing. Sperm motility was higher in the 5 mM GSH group than in the control or 2.5 and 10 mM GSH groups; this effect was observed at 1 to 24 h after thawing (P < 0.05). The 5 mM GSH group had a higher sperm viability index at 12 and 24 h after thawing compared with the other groups (P < 0.05). Acrosome integrity, evaluated at 4 h after thawing, was greater in two of the GSH-treated groups (5 and 10 mM) compared with the control. Lipid peroxidation (LP) levels immediately after thawing were lower in the 5 and 10 mM GSH groups compared with the control, while those at 12 h after thawing did not differ significantly. Frozen-thawed semen in the 5 mM GSH group was used for transcervical insemination of 4 bitches, resulting in delivery of 5 puppies from 2 bitches. These results indicate that supplementation of semen extender with 5 mM GSH was effective in improving motility, longevity and acrosomal integrity and inhibiting LP levels in post-thaw canine spermatozoa, without any adverse impacts on full-term development after transcervical insemination.

Intrahepatic splenosis with severe iron deposition presenting with atypical magnetic resonance images.

An intrahepatic mass was incidentally found in a 41-year-old man with a history of a traffic accident injury which resulted in removal of a ruptured spleen. Hepatic splenosis was considered in the differential diagnosis but magnetic resonance imaging showed hypointensity on T2-weighted images, atypical for normal spleen. Histologically, the mass showed sinusoidal structures and lymphoid follicular aggregates. Immunohistochemical study showed that the phenotype of the vascular lining cells was CD8-positive, CD31-positive, and CD34 negative, the pattern diagnostic for ectopic spleen. In addition, severe iron deposition was histologically demonstrated, which was considered as the cause of the hypointense T2-weighted images.