H̱olon, Israel
H̱olon, Israel

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Amsterdam A.,Weizmann Institute of Science | Raanan C.,Weizmann Institute of Science | Schreiber L.,Wolfson Hospital | Freyhan O.,Weizmann Institute of Science | And 2 more authors.
Acta Histochemica | Year: 2013

LGR5 and Nanog were recently characterized as stem cell markers in various embryonic, adult and cancer stem cells. However, there are no data on their precise localization in the normal adult ovary, which may be important for the initial steps of development of ovarian cancer, the most lethal gynecological cancer. We analyzed by immunocytochemistry the precise localization of these markers in normal ovary (11 specimens, age range 43-76), in borderline specimens (12 specimens), and in serous ovarian cancer (12 specimens of stage II) which comprises the vast majority (80%) of all ovarian cancer. Surprisingly, we revealed that both Nanog and LGR5 are clearly localized in the epithelial cells of the normal ovary. However, in 5 of 12 ovaries there was no labeling at all, while in 3 ovaries staining of Nanog was more prominent with only weak labeling of LGR5. In addition, we found in 3 of 11 ovaries clear labeling in foci of both LGR5 and Nanog antibodies, with partial overlapping. Occasionally, we also found in the stroma foci labeled by either Nanog or LGR5 antibodies. In general, the stroma area of tissue sections labeled with LGR5 was much greater than that labeled with Nanog. In borderline tumors a significant portion of the specimens (7 of 12) was labeled exclusively with Nanog and not with LGR5. In ovarian carcinomas almost 100% of the cells were exclusively labeled only with Nanog (6 of 12 of the specimens) with no labeling of LGR5. These data may suggest the potential of ovaries from postmenopausal women, which express Nanog, to undergo transformation, since Nanog was shown to be oncogenic. We conclude that Nanog, which probably plays an important role in ovarian embryonic development, may be partially silenced in fertile and post-menopausal women, but is re-expressed in ovarian cancer, probably by epigenetic activation of Nanog gene expression. Expression of Nanog and LGR5 in normal ovaries and in borderline tumors may assist in the early detection and improved prognosis of ovarian cancer. Moreover, targeting of Nanog by inhibitory miRNA or other means may assist in treating this disease. © 2012 Elsevier GmbH.


Amsterdam A.,Weizmann Institute of Science | Shezen E.,Weizmann Institute of Science | Raanan C.,Weizmann Institute of Science | Schreiber L.,Wolfson Hospital | And 5 more authors.
Oncology Reports | Year: 2012

Synuclein α, β and γ are proteins usually found in neurodegenerative diseases. However, interestingly synucleins are expressed in cancer cells of several organs including ovary, mammary gland and colon. By immunocytochemistry using specific antibodies to γ synuclein (SNCG), we examined the distribution of this protein in poorly differentiated, compared to highly differentiated colon cancer cells. In poorly differentiated cancer cells tumors were very frequently stained intensely with antibodies to SNGG, suggesting high expression of this protein. In contrast, in highly differentiated cells, there was no labeling. Labeled cells could be found only at the edges or in between the lobules of the differentiated tumor cells. However, in moderately differentiated tumors, a weak cytoplasmic staining of SNCG was evident. Interestingly in cancer patients (stage II-IV) both poorly and highly differentiated tumor cells were often present in the same patient. Labeled cancer cells with SNCG were evident also in lymph nodes, around the wall of blood vessels and in fat tissue, where only poorly differentiated cancer cells were exclusively present. Since cancer cells with poor differentiation are believed to be aggressive with metastases formation it is suggested that SNCG can serve as a marker for the potential of the tumor cell for the rapid spreading and metastazing of the non-differentiated tumors.


Schreiber L.,Wolfson Hospital | Raanan C.,Weizmann Institute of Science | Amsterdam A.,Weizmann Institute of Science
Acta Histochemica | Year: 2014

Ovarian cancer is the most lethal gynecological cancer. There is a general debate whether ovarian cancer is an intrinsic or an imported disease. We investigated whether in normal morphological appearance and in early stages of ovarian tumorgenesis typical cancer cell markers such as CD24 and Nanog are expressed. In 25% of normal appearing ovaries of post-menopausal women there was co-localization of CD24 and Nanog in the walls of the ovarian cysts, leaving the epithelial cells on the surface of these ovaries free of Nanog or CD24 expression. In benign ovarian tumors 37% of specimens were positive to CD24 and Nanog labeling while 26% of them were localized in the cyst walls. In contrast, in serous borderline tumors 79% specimens were labeled with CD24, 42% of them were localized in cysts and in 32% of them showed co-localization with CD24 and Nanog was evident: the rest were labeled in the ovarian epithelial cells. In serous ovarian carcinomas 81% specimens were labeled with CD24 antibodies. In 45% of them co-localization with Nanog was evident in the bulk of the cancerous tissue. In mucinous carcinomas no labeling with CD24 or Nanog was evident. In view of the synergistic effect of CD24 and Nanog expressed in malignant cancer development in other systems, it is suggested that such an analysis can be valuable for early detection of ovarian cancer. Moreover, the abundance of these markers in cysts in the development of ovarian cancer may suggest that they present an intrinsic source of the development of the highly malignant disease. Finally, since CD24 is exposed on the surface of the cancer cells, it may be highly beneficial to target these cells with antibodies to CD24 conjugated to cytotoxic drugs for more efficient treatment of this malignant disease. © 2013 Elsevier GmbH.


Amsterdam A.,Weizmann Institute of Science | Raanan C.,Weizmann Institute of Science | Schreiber L.,Wolfson Hospital | Polin N.,Weizmann Institute of Science | Givol D.,Weizmann Institute of Science
Biochemical and Biophysical Research Communications | Year: 2013

Pancreas cancer, is the fourth leading cause of cancer death but its cell of origin is controversial. We compared the localization of stem cells in normal and cancerous pancreas using antibodies to the stem cell markers Nanog and LGR5. Here we show, for the first time, that LGR5 is expressed in normal pancreas, exclusively in the islets of Langerhans and it is co-localized, surprisingly, with Nanog and insulin in clusters of beta cells. In cancerous pancreas Nanog and LGR5 are expressed in the remaining islets and in all ductal cancer cells. We observed insulin staining among the ductal cancer cells, but not in metastases. This indicates that the islet's beta cells, expressing LGR5 and Nanog markers are the initiating cells of pancreas cancer, which migrated from the islets to form the ductal cancerous tissue, probably after mutation and de-differentiation. This discovery may facilitate treatment of this devastating cancer. © 2013 Elsevier Inc.


Amsterdam A.,Weizmann Institute of Science | Shezen E.,Weizmann Institute of Science | Raanan C.,Weizmann Institute of Science | Slilat Y.,Weizmann Institute of Science | And 3 more authors.
International Journal of Oncology | Year: 2011

Epiregulin (Ep) was found to be produced in non-cancer ovarian cells in response to gonadotropin stimulation as well in ovarian cancer cells in an autonomous manner. However, there were no systematic follow-up studies of Ep expression in the development of different stages of ovarian cancer. Using specific antibodies to Ep and the indirect immunocytochemistry methods, we found that in normal ovary the staining for Ep was mainly confined to the epithelial cells, while the stromal cells were only occasionally and moderately stained. In contrast in benign serous and mucinous tumors most of the tumor cells showed a clear staining in the cytoplasm. In borderline serous and mucinous tumors the staining was much more intensive, and appear occasionally in aggregated form. In serous, mucinous and endometrioid carcinomas labeling remain high, with more frequent aggregated form. It is suggested that follow-up of the expression of Ep can serve as a reliable early indication of the development of ovarian cancer. Moreover, the cytoplasmic aggregation of Ep may suggest a specific mechanism of the release of this growth factor to the extracellular space in order to exert its autocrine and paracrine effect on the family of the EGF receptors.


Amsterdam A.,Weizmann Institute of Science | Raanan C.,Weizmann Institute of Science | Polin N.,Weizmann Institute of Science | Melzer E.,Kaplan Medical Center | And 2 more authors.
Acta Histochemica | Year: 2014

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers because of late symptoms and resistance to chemotherapy and radiation therapy. We have investigated the appearance of c-kit, a stem cell marker, in both normal adult pancreatic tissue and in cancerous tissue. Apart from some very pale staining of islets of Langerhans, normal pancreas was devoid of staining with antibodies to c-kit. In contrast, in cancerous tissue that still preserves the overall integrity of the pancreatic tissue, there was a clear labeling in islets of Langerhans, which seemed to be co-localized with insulin containing β cells. In other cases, where the pancreatic tissue was completely deteriorated, intensive labeling was clearly evident in remnants of both the exocrine and the endocrine tissues. The duct cells of the adenocarcinoma were moderately but clearly labeled with antibodies to c-kit. In contrast, in metastasis of PDAC, very intensive labeling of c-kit was evident. The location of KRAS, which is strongly associated with PDAC, was also analyzed at the initial stages of the disease, when islets of Langerhans still preserve their integrity to a large extent. KRAS was found exclusively in islets of Langerhans and overlapped in its location with insulin and c-kit expressing cells. It is suggested that the modulation of the expression of c-kit, visualized by antibodies to the oncogene molecule, may play an important role in the formation and progression of PDAC. The absence of c-kit in normal pancreas and its appearance in PDAC is probably due to a mutational event, which probably allows conversion of the β cells into cancer stem cells (CSC). Co-expression of both c-kit and KRAS, typical markers for CSC with overlapping with insulin in islets of Langerhans, strongly support the notion that β-cells play a central role in the development of PDAC. The use of specific drugs that can attenuate the kinase activity of c-kit or target KRAS expressing cancer cells should be tested in order to attenuate the progression of this lethal disease. © 2013.


Amsterdam A.,Weizmann Institute of Science | Raanan C.,Weizmann Institute of Science | Schreiber L.,Wolfson Hospital | Freyhan O.,Weizmann Institute of Science | And 2 more authors.
International Journal of Oncology | Year: 2012

In this study, we used LGR5, γ-synuclein, p53, KRAS and epiregulin antibodies to localize stem cells by indirect immunocytochemistry in paraffin sections of normal and cancerous colon tissues. In the normal colon tissue, no staining of cells with LGR5, γ-synuclein, p53 and KRAS antibodies was observed, apart from a few scattered cells in between the colon villi that were faintly stained with antibodies to LGR5. Staining of highly differentiated cancer tissue with LGR5 antibodies revealed single cells or clusters of up to 4 cells in the interior space of the carcinoma cell layers. Staining of poorly differentiated cancer tissues (stage I-IV) revealed 9-81 clustered stem cells. The number of clustered stem cells increased significantly with the tumor stage, when comparing stage II to stage IV (p<00048). Occasionally, the clustered stem cells appeared in the interphase between the colon stroma and the tumor tissue. Surprisingly, antibodies to p53 clearly stained the clusters of stem cells both in the nuclei and the cytoplasm. The staining of the nuclei of other cells in the undifferentiated tumors was in general weaker, and no staining was found in the cytoplasm. Antibodies to γ-synuclein heavily stained the endothelial cells of the blood vessels and some other scattered cells in the highly differentiated tumors. Antibodies to γ-synuclein heavily stained the stem cells in both the cytoplasm and the nuclei of poorly differentiated tumors. Antibodies to KRAS stained the cytoplasm and the nuclei of stem cells in poorly differentiated tumors and also stained the cytoplasm of some scattered cells. Antibodies to epiregulin stained the cytoplasm of normal colon tissue cells in the crypt-villus axis. The antibodies weakly stained the highly differentiated tumor cells and moderately stained the moderately differentiated tumor cells. Of note, the antibodies intensively stained the clustered stem cells of the poorly differentiated tumor cells. These antibodies also clearly stained the clustered stem cells of poorly differentiated tumors but were not specific as they clearly stained cells in the crypt-villus axis of the normal colon wall. Our results show that LGR5 antibodies can serve as a reliable marker for colon cancer stem cells. Once the colon stem cells are identified, the targeting of specific drugs to kill these cells should be attempted in the future in order to cure this disease. Moreover, the fact that we did not find any stained cells with antibodies to LGR5 in normal tissues apart from a few scattered cells, suggests that the normal colon stem cells differ from the tumor stem cells at least as regards the expression of this protein. In addition, antibodies to γ-synuclein, p53 and KRAS only stained the tumor stem cells and not the normal tissue. Thus, they can serve as multiple biomarkers for the localization of colon cancer stem cells by indirect immunofluorescence.


Amsterdam A.,Weizmann Institute of Science | Shezen E.,Weizmann Institute of Science | Raanan C.,Weizmann Institute of Science | Schreiber L.,Wolfson Hospital | And 4 more authors.
International Journal of Oncology | Year: 2011

We examined the possibility that the localization of phosphorylated ERK1 and ERK2 (pERK1/2) can serve as a marker for the development of benign and borderline tumors as well as carcinoma of the ovary by an immunohistochemical method on ovarian paraffin sections, obtained from women aged 41-83 years. In normal tissue, 28.3% of nuclei were labeled, mainly confined to the epithelial cells at the surface of the ovary. In benign serous tumors, the label rose to 55.0%, while the intensity of the staining was weak. In contrast, in borderline serous tumors and in ovarian serous carcinoma (stage II) 52.1% and 70.3% of nuclei, respectively, were labeled with a high intensity. In mucinous benign tumors, the number of labeled nuclei was as in the control, but in addition, 49.4% of the cells demonstrated high concentration of pERK1/2 in aggregated form that was evident in the cytoplasm of the cells. In the mucinous and endometrioid ovarian carcinomas (stage II) very intensive labeling was found in 60% and 77.3% of cells, respectively. It is, therefore, suggested that since nuclear pERK1/2 can be mitogenic, it can serve as a reliable marker for the progression of ovarian cancer. Interestingly, the intense labeling of pERK1/2 was mainly confined to the peripheral areas of ovarian endometrioid carcinoma (stage II). In addition, all tumor cells in this class of cancer were positively stained with mutated p53. It seems, therefore, that immunohistochemical staining of normal and ovarian tumor cells with anti-pERK1/2 is a reliable marker for early detection of the cancer, which may assist in the early diagnosis and prognosis of this lethal disease.


Amsterdam A.,Weizmann Institute of Science | Raanan C.,Weizmann Institute of Science | Shpigner L.,Weizmann Institute of Science | Shwiqi A.,Kaplan Medical Center | And 2 more authors.
Acta Histochemica | Year: 2014

TADG-12 is a serine protease that was characterized as expressed in ovarian and gastric carcinomas. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and its late detection results in poor prognosis. Therefore, we decided to examine whether TADG-12 appears early in PDAC development. In normal pancreas, pale to moderate immunostaining is present in islets of Langerhans, while exocrine tissue and ducts are free from labeling. In contrast, in cancer patients, who still preserve the integrity of the exocrine and the endocrine tissues, a pronounced immunolabelling of TADG-12 was evident mainly located in the insulin containing β cells. In a more progressive stage of the disease TADG-12 was also evident in the deteriorated exocrine tissue. TADG-12 was also heavily labeled in islets of Langerhans, which were embedded in the stroma of the residual pancreatic tissue. Again, there was a considerable overlap between the labeling of insulin and TADG-12 in these islets. Close correlation between insulin and TADG-12 was also evident in islets of Langerhans surrounded by adipose cells. The TADG-12 labeled was confined to the cytoplasm and the membrane of the cells. In the progressive stage of PDAC, the cancerous ducts were clearly labeled with TADG-12 with no labeling of insulin. At high magnification the TADG-12 clearly labeled the cytoplasm and the cell wall membrane of duct cells, while the nuclei remained unstained upon incubation with antibodies to TADG-12. The present findings may assist in early detection of PDAC as well as targeting of TADG-12 in order to attenuate the rapid progression of the disease. © 2014 Elsevier GmbH.


Amsterdam A.,Weizmann Institute of Science | Raanan C.,Weizmann Institute of Science | Schreiber L.,Wolfson Hospital | Freyhan O.,Weizmann Institute of Science | And 3 more authors.
Acta Histochemica | Year: 2013

One paradigm of cancer development claims that cancer emerges at the niche of tissue stem cells and these cells continue to proliferate in the tumor as cancer stem cells. LGR5, a membrane receptor, was recently found to be a marker of normal colon stem cells in colon polyps and is also expressed in colon cancer stem cells. Nanog, an embryonic stem cell nuclear factor, is expressed in several embryonic tissues, but Nanog expression is not well documented in cancerous stem cells. Our aim was to examine whether both LGR5 and Nanog are expressed in the same clusters of colon stem cells or cancer stem cells, using immunocytochemistry with specific antibodies to each antigen. We analyzed this aspect using paraffin embedded tumor tissue sections obtained from 18 polyps and 36 colon cancer specimens at stages I-IV. Antibodies to LGR5 revealed membrane and cytoplasm immunostaining of scattered labeled cells in normal crypts, with no labeling of Nanog. However, in close proximity to the tumors, staining to LGR5 was much more intensive in the crypts, including that of the epithelial cells. In cancer tissue, positive LGR5 clusters of stem cells were observed mainly in poorly differentiated tumors and in only a few scattered cells in the highly differentiated tumors. In contrast, antibodies to Nanog mainly stained the growing edges of carcinoma cells, leaving the poorly differentiated tumor cells unlabeled, including the clustered stem cells that could be detected even by direct morphological examination. In polyp tissues, scattered labeled cells were immunostained with antibodies to Nanog and to a much lesser extent with antibodies to LGR5. We conclude that expression of LGR5 is probably specific to stem cells of poorly differentiated tumors, whereas Nanog is mainly expressed at the edges of highly differentiated tumors. However, some of the cell layers adjacent to the carcinoma cell layers that still remained undifferentiated, expressed mainly Nanog with only a few cells labeled with antibodies to LGR5. Considering the different sites and pattern of expression in the tumor, our data imply that targeting the clustered stem cells expressing LGR5 in poorly differentiated colon cancer may require different strategies than targeting the stem cells expressing Nanog in the highly differentiated tumors. Alternatively, combined application of specific inhibitory miRNAs to Nanog and to LGR5 expression may assist therapeutically. © 2012 Elsevier GmbH.

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