Diagnosis of verruciform acanthotic vulvar intra-epithelial neoplasia (vaVIN) using CK17, SOX2 and GATA3 immunohistochemistry.

AIMS: Verruciform acanthotic vulvar intra-epithelial neoplasia (vaVIN) is an HPV-independent, p53 wild-type lesion with distinct morphology and documented risk of recurrence and cancer progression. vaVIN is rare, and prospective distinction from non-neoplastic hyperplastic lesions can be difficult. CK17, SOX2 and GATA3 immunohistochemistry has emerging value in the diagnosis of HPV-independent lesions, particularly differentiated VIN. We aimed to test the combined value of these markers in the diagnosis of vaVIN versus its non-neoplastic differentials in the vulva. METHODS AND RESULTS: CK17, SOX2 and GATA3 immunohistochemistry was evaluated on 16 vaVINs and 34 mimickers (verruciform xanthoma, lichen simplex chronicus, lichen sclerosus, psoriasis, pseudo-epitheliomatous hyperplasia). CK17 was scored as 3+ = full-thickness, 2+ = partial-thickness, 1+ = patchy, 0 = absent; SOX2 as 3+ = strong staining ≥ 10% cells, 2+ = moderate, 1 + =weak, 0 = staining in < 10% cells; and GATA3 as pattern 0 = loss in < 25% basal cells, 1 = loss in 25-75% basal cells, 2 = loss in > 75% basal cells. For analysis, results were recorded as positive (CK17 = 3+, SOX2 = 3+, GATA3 = patterns 1/2) or negative (CK17 = 2+/1+/0, SOX2 = 2+/1+/0, GATA3 = pattern 0). CK17, SOX2 and GATA3 positivity was documented in 81, 75 and 58% vaVINs, respectively, versus 32, 17 and 22% of non-neoplastic mimickers, respectively; ≥ 2 marker positivity conferred 83 sensitivity, 88 specificity and 86% accuracy in vaVIN diagnosis. Compared to vaVIN, SOX2 and GATA3 were differentially expressed in lichen sclerosus, lichen simplex chronicus and pseudo-epitheliomatous hyperplasia, whereas CK17 was differentially expressed in verruciform xanthoma and adjacent normal mucosa. CONCLUSIONS: CK17, SOX2 and GATA3 can be useful in the diagnosis of vaVIN and its distinction from hyperplastic non-neoplastic vulvar lesions. Although CK17 has higher sensitivity, SOX2 and GATA3 are more specific, and the combination of all markers shows optimal diagnostic accuracy.

The role of SOX family in cancer stem cell maintenance: With a focus on SOX2.

The role of cancer stem cells (CSCs) in cancer incidence, drug resistance, and relapse after chemotherapy has been discussed and it has been confirmed that CSCs are extremely important and so, are suitable for therapeutic targeting. Sox families play an important role in carcinogenesis and dis-regulation of SOXs molecules has been observed in different types of cancers. The members of this family have been shown to play an important role in the maintenance of CSCs. In this article, we have tried to evaluate the role of different family members in CSCs maintenance, review various studies in this field and provide a perspective view on this issue. Also, due to the important role and many studies in the field of SOX2 molecule in CSCs, we try to have more focus on this molecule and examine the potential of these molecules for therapeutic targeting.

SHH Expression Is Significantly Associated With Cancer Stem Cell Markers in Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is a highly invasive malignancy with poor prognosis. Recent reports suggest that Sonic Hedgehog (SHH) plays a key role in tumor progression and worsens the response to therapy, possibly through an association with a cancer stem cell (CSC) phenotype. The objective of our study was to investigate the relationship between SHH expression and CSC markers in OSCC. MATERIALS AND METHODS: A total of 67 OSCC specimens were immunostained for SHH and CSC markers using specific antibodies and expression was correlated with clinicopathological parameters. RESULTS: SHH expression was significantly correlated with CD133 (p=0.026, r=0.272) and SRY-box transcription factor 2 (SOX2; p<0.001, r=0.793). SHH and SOX2 expression were associated with worse survival in OSCC (p=0.003 and p=0.003, respectively). In multivariate analysis SHH and CD44 were independent prognostic biomarkers in patients with OSCC (p=0.001 and p=0.008, respectively). CONCLUSION: Our study revealed that SHH overexpression is closely associated with CSC markers, contributing to tumor progression and worse outcomes of patients with OSCC.

New insights into the role and origin of pituitary S100ß-positive cells.

In the anterior pituitary, S100ß protein (S100ß) has been assumed to be a marker of folliculo-stellate cells, which are one of the non-hormone-producing cells existing in the parenchyma of the adult anterior lobe and are composed of subpopulations with various functions. However, recent accumulating studies on S100ß-positive cells, including non-folliculo-stellate cells lining the marginal cell layer (MCL), have shown the novel aspect that most S100ß-positive cells in the MCL and parenchyma of the adult anterior lobe are positive for sex determining region Y-box 2 (SOX2), a marker of pituitary stem/progenitor cells. From the viewpoint of SOX2-positive cells, the majority of these cells in the MCL and in the parenchyma are positive for S100ß, suggesting that S100ß plays a role in the large population of stem/progenitor cells in the anterior lobe of the adult pituitary. Reportedly, S100ß/SOX2-double positive cells are able to differentiate into hormone-producing cells and various types of non-hormone-producing cells. Intriguingly, it has been demonstrated that extra-pituitary lineage cells invade the pituitary gland during prenatal pituitary organogenesis. Among them, two S100ß-positive populations have been identified: one is SOX2-positive population which invades at the late embryonic period through the pituitary stalk and another is a SOX2-negative population that invades at the middle embryonic period through Atwell's recess. These two populations are likely the substantive origin of S100ß-positive cells in the postnatal anterior pituitary, while S100ß-positive cells emerging from oral ectoderm-derived cells remain unclear.

Prognostic and predictive value of ALDH1, SOX2 and SSEA-4 in bladder cancer.

Transurethral resection of bladder tumor (TUR-BT) and radical cystectomy (RC) are standard treatment options for bladder cancer (BC). Neoadjuvant chemotherapy (NAC) prior to RC improves outcome of some patients but currently there are no valid biomarkers to identify patients who benefit from NAC. Presence of cancer stem cells (CSC) has been associated with poor outcome and resistance to chemotherapy in various cancers. Here we studied the expression of stem cell markers ALDH1, SOX2 and SSEA-4 with immunohistochemistry in tissue microarray material consisting of 195 BC patients treated with RC and 74 patients treated with TUR-BT followed by NAC and RC. Post-operative follow-up data of up to 22 years was used. Negative to weak cytoplasmic SOX2 staining was associated with lymphovascular invasion and non-organ confined disease. It was also associated with shortened cancer-specific survival, but the finding was not statistically significant. Contrary to previous reports, none of the other tested biomarkers were associated with cancer-specific mortality or clinicopathological characteristics. Neither were they associated with response to NAC. Despite the promising results of previously published studies, our results suggest that CSC markers ALDH1, SOX2 and SSEA-4 have little if any prognostic or predictive value in BC treated with RC.

Simultaneous and quantitative monitoring transcription factors in human embryonic stem cell differentiation using mass spectrometry-based targeted proteomics.

Human embryonic stem cells (hESCs) can be self-propagated indefinitely in culture while holding the capacity to generate almost all cell types. Although this powerful differentiation ability of hESCs has become a potential source of cell replacement therapies, application of stem cells in clinical practice relies heavily on the exquisite control of their developmental fate. In general, an essential first step in differentiation is to exit the pluripotent state, which is precariously balanced and depends on a variety of factors, mainly centering on the core transcriptional mechanism. To date, much evidence has indicated that transcription factors such as Sox2, Oct4, and Nanog control the self-renewal and pluripotency of hESCs. Their expression displays a restricted spatial-temporal pattern and their small changes in level can significantly affect directed differentiation and the cell type derived. So far, few assays have been developed to monitor this process. Herein, we provided a mass spectrometry (MS)-based approach for simultaneous and quantitative monitoring of these transcription factors, in an attempt to provide insight into their contributions in hESC differentiation.

Colon adenocarcinoma-derived cells that express induced-pluripotent stem cell markers possess stem cell function.

AIMS: Much work has been done to find markers of cancer stem cells (CSCs) that distinguish them from the tumor bulk cells and normal cells. Recent CSC research has applied the induced pluripotent stem cell (iPSC) concept. In this study, we investigated the expression of a panel of iPSC markers in primary colon adenocarcinoma (CA)-derived cell lines. MATERIALS AND METHODS: Expression of iPSC markers by CA-derived primary cell lines was interrogated using immunocytochemistry, western blotting and RT-qPCR. The stem cell function of these cells was then assessed in vitro using differentiation and tumorsphere assays. RESULTS: Expression of iPSC markers OCT4, SOX2, NANOG, KLF4 and c-MYC was more widespread in high-grade CA (HGCA) cell lines than low-grade CA (LGCA) cell lines, as demonstrated by western blotting and RT-qPCR. These cells could be induced to differentiate down the three embryonic lineages. Cells derived from HGCA were more capable of forming tumorspheres than those derived from LGCA. EpCAM sorting revealed that a population enriched for EpCAMHigh cells formed larger tumorspheres than EpCAMLow cells. Pluripotency markers, SSEA4 and TRA-1-60, were co-expressed by a small subpopulation of cells that also co-expressed SOX2 in 75% and OCT4 in 50% of the cell lines. CONCLUSIONS: CA-derived primary cell lines contain tumorsphere-forming cells which express key pluripotency genes and can differentiate down 3 embryonic lineages, suggesting a pluripotent CSC-like phenotype. There appear to be two iPSC-like subpopulations, one with high EpCAM expression which forms larger tumorspheres than another with low EpCAM expression. Furthermore, these cells can be characterized based on iPSC marker expression, as we have previously demonstrated in the original CA tumor tissues.

SOX2 and BCL-2 Expressions in Odontogenic Keratocyst and Ameloblastoma

BACKGROUND: The purpose of this experimental study was to compare the immunohistochemical expression of SOX2 and BCL-2 in Odontogenic Keratocyst (OKC) and Ameloblastoma (AB) specimens, and to identify a possible correlation in their expression.MATERIAL AND METHODS: Immunohistochemical analysis was performed to evaluate SOX2 and BCL-2 expression in OKC (n = 20) and AB (n = 20). The immunoexpression was analyzed by a quantitative and qualitative scoring system. The comparison between the immunoexpression of SOX 2 and BCL-2 was assessed by the Mann-Whitney U-test. Spearman's correlation coefficient evaluated the correlation between SOX2 and BCL-2 expressions.RESULTS: SOX2 and BCL-2 expression was observed in all specimens of OKC in the full thickness of the epithelium lining. SOX2 immunostaining was higher in OKC, in comparison with AB samples (P<0.05). BCL-2 immunostaining between OKC and AB was not statistically significant. There was no significant correlation between SOX2 and BCL-2 in OKC and AB specimens.CONCLUSIONS: SOX2 and BCL-2 expressions in OKC may suggest their relationship with the biological behavior of this lesion, and the higher expression of SOX2 might be an upstream influence on the Hh signaling pathway

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Association of SOX2, OCT4 and WNT5A Expression in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma: An Immunohistochemical Study.

The cancer stem cells deliver uncontrolled proliferative capacity within the tumor imparting to increasing size while epithelial mesenchymal transition adds to the invasive potential. Studies using specific markers elucidating the role of these phenomena may bring advancement in the targeted therapy of tumor. SOX2 and OCT4 are two among few stem cell markers indicative of proliferative potential and WNT5A is an epithelial mesenchymal transition marker indicative of invasive potential. We aimed to determine the association between expression of SOX2, OCT4 and WNT5A in oral epithelial dysplasia, oral squamous cell carcinoma and normal oral mucosa. 20 cases of oral squamous cell carcinoma, 20 cases of oral epithelial dysplasia (leukoplakia with dysplasia) and 25 normal oral mucosa tissues specimens were immunohistochemically stained to assess SOX2, OCT4 and WNT5A expression. SOX2 expression was higher in oral squamous cell carcinoma than in oral epithelial dysplasia and very low in normal oral mucosa. OCT4 was very low in oral squamous cell carcinoma and oral epithelial dysplasia when compared to SOX2, while negative in normal tissues. Co-expression of SOX2 and OCT4 showed statistically non-significant difference for tumor proliferation. WNT5A expression was found to be increasing from normal oral mucosa to oral epithelial dysplasia and oral squamous cell carcinoma. In conformity with present study, SOX2 itself can act as a potential marker for proliferation in tumor cells while OCT4 has non-significant role in regulation of tumor behavior in oral squamous cell carcinoma as well as in oral epithelial dysplasia. WNT5A can be a putative marker in studying invasive potential of oral squamous cell carcinoma.

Dynamic Changes in the Localization of Neuronatin-Positive Cells during Neurogenesis in the Embryonic Rat Brain.

Neuronatin (NNAT) was first identified as a gene selectively and abundantly expressed in the cytoplasm of the newborn mouse brain, and involved in neonatal neurogenesis. However, the particular roles of NNAT in the developing prenatal brain have not been identified, especially in mid to late stages. In this study, we performed immunohistochemical analyses of NNAT and SOX2 proteins, a nuclear transcription factor and neural stem/progenitor marker, in the rat brain on embryonic days 13.5, E16.5, and E20.5. NNAT signals were broadly observed across the developing brain on E13.5 and gradually more localized in later stages, eventually concentrated in the alar and basal parts of the terminal hypothalamus, the alar plate of prosomere 2 of the thalamus, and the choroid plexus in the lateral and fourth ventricles on E20.5. In particular, the mammillary body in the basal part of the terminal hypothalamus, a region with a high number of SOX2-positive cells, evidenced intense NNAT signals on E20.5. The intracellular localization of NNAT showed diverse profiles, suggesting that NNAT was involved in various cellular functions, such as cell differentiation and functional maintenance, during prenatal neurogenesis in the rat brain. Thus, the present observations suggested diverse and active roles of the NNAT protein in neurogenesis. Determining the function of this molecule may assist in the elucidation of the mechanisms involved in brain development.

Association of the co-expression of SOX2 and Podoplanin in the progression of oral squamous cell carcinomas - an immunohistochemical study.

SOX2 is a transcription factor related to the maintenance of stem cells in a pluripotent state. Podoplanin is a type of transmembrane sialoglycoprotein, which plays an important role in tumor progression and metastasis. This study aims to determine association of SOX2 and podoplanin expression in the progression of oral squamous cell carcinomas and to elucidate the association between two proteins. Label="METHODOLOGY">The immunohistochemical expression of SOX2 and podoplanin were evaluated in 60 cases of primary oral squamous cell carcinomas. The correlation between the SOX2 and podoplanin expression and the clinicopathological features of the tumors and the patient outcomes were assessed. RESULTS The expression of SOX2 was seen in 38/60 (63%) of the cases and the expression for podoplanin was seen in 45/60 (75%) cases. There was a significant inverse correlation between the expression of SOX2 and podoplanin with the tumor grade (p=0.002 and p=0.017, respectively). There was a high expression of SOX2 in 9/13 cases that presented with disease free survival. Survival analysis showed that a high expression of SOX2 correlated positively (p=0.043) with the disease-free survival. There was a significant positive association between the pattern of SOX2 and podoplanin expression (p=0.002). CONCLUSION A high expression of SOX2 was associated with better disease-free survival. The expression of podoplanin was associated with the degree of differentiation of the tumors. Analysis of these biomarkers can aid in the prognosis and treatment of oral squamous cell carcinomas.

Comprehensive in situ analysis of ALDH1 and SOX2 reveals increased expression of stem cell markers in high-grade serous carcinomas compared to low-grade serous carcinomas and atypical proliferative serous tumors.

Recent studies have shown that re-expression of stem cell factors contribute to pathogenesis, therapy resistance, and recurrent disease in ovarian carcinomas. In this study, we compare the expression and co-expression of stem cell markers ALDH1 and SOX2 in different types of serous ovarian tumors. A total of 215 serous ovarian tumors (161 high-grade serous carcinomas (HGSC), 17 low-grade serous carcinomas (LGSC), 37 atypical proliferative serous tumors (APST)), and 10 cases of serous tubal intraepithelial carcinoma (STIC) were analyzed. Double immunostaining experiments addressed the association of cell proliferation (Ki67) with ALDH1 and the potential co-expression of SOX2 and ALDH1. The prognostic effect was analyzed in the cohort of HGSC. Expression of ALDH1and/or SOX2 was detected with increased frequency in HGSC (88.8%), compared with LGSC (70.5%) and APST (36.4%), while ALDH1 alone was significantly more frequently expressed in LGSC. The majority of all tumor types showed expression of ALDH1 and SOX2 in different cells. Only a minority of HGSC (4.6%) and STIC (20%) showed SOX2/ALDH1 co-expression in > 10% of tumor cells. Double staining also revealed that ALDH1 is associated with the non-proliferating Ki67-negative fraction consistent with a stem cell phenotype. Co-expression of ALDH1 and SOX2 or ALDH1 and Ki67 has no effect on survival. Expression of stem cell factors ALDH1 and/or SOX2 shows increased frequency in high-grade serous ovarian carcinomas compared to low-grade carcinomas and borderline tumors, supporting the concept that stem cell markers play different biological roles in low-grade versus high-grade serous neoplasia of the ovary.

Diabetes impairs the angiogenic capacity of human adipose-derived stem cells by reducing the CD271+ subpopulation in adipose tissue.

Type 2 diabetes mellitus is an important risk factor for cardiovascular diseases (CVDs). Therapeutic angiogenesis using adipose-derived stem cells (ADSCs) is attractive for CVD therapy. However, although it would be critical for ADSC application on CVD therapy, whether and how diabetes impairs human ADSC therapeutic potential is still uncertain. In this study, we aimed to investigate the impact of diabetes on the angiogenic potential of ADSCs in patients with CVDs, with special focus on stemness-related genes and cellular alteration of ADSCs. We established cultured ADSCs from diabetic (DM-ADSCs) and non-diabetic patients (nonDM-ADSCs) with CVDs. DM-ADSCs demonstrated limited proliferative capacity and reduced paracrine capacity of VEGF, with lower expression of the stemness gene SOX2. Angiogenic capacity and ADSC engraftment were assessed using xenograft experiments in a hindlimb ischemia model of athymic nude mice. Consistent with the results of in vitro assays, DM-ADSCs did not rescue limb ischemia. In contrast, nonDM-ADSCs induced neovascularization with enhanced engraftment. To elucidate the mechanism underlying these ADSC changes, we compared the surface marker profiles of freshly isolated ADSCs obtained from diabetic and non-diabetic patients by flow cytometry. Among studied subsets, the CD34+CD31-CD271+ subpopulation was reduced in the adipose tissues of diabetic patients. In addition, SOX2 expression and proliferative capacity were considerably reduced in nonDM-ADSCs derived from the stromal vascular fraction (SVF) with depletion of CD271+ cells (p < 0.01). Our observations elucidated that reduced CD271+ subpopulation is critical for the impairment of ADSCs in diabetic patients. Further investigations on the CD271+ subset of ADSCs might provide novel insights into the mechanisms and solutions for diabetes-related ADSC dysfunction in cell therapy.

Correlation between SOX2 and Survivin clinical features in patients with salivary adenoid cystic carcinoma.

OBJECTIVE: In this study, expression of cancer stem cells (CSCs)-related factor-Sex-determining region of Y chromosome-related high-mobility-group box 2 (SOX2) and anti-apoptotic specific factor- Survivin in salivary adenoid cystic carcinoma (SACC) was detected to provide important clues for effective SACC prevention and treatment by combining clinical pathological parameters analysis. METHODS: Paraffin and fresh specimens were collected from SACC patients who underwent surgery at the Oral and Maxillofacial Surgery Department of Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital. The experimental group was designed as SACC tissue, and the control group normal paracancerous normal gland tissue. (1) SOX2 and Survivin expression were detected using immunohistochemistry and analyzed by comnining clinical pathological parameter analysis. (2) mRNA and protein expression levels of SOX2 and Survivin were detected using RT-PCR, Western Blot. RESULTS: 1. Immunohistochemistry: (1) SOX2 was mainly expressed on the nucleus. The SOX2 positive rate was 28.57% in clinical stage I-II, and 76.92% in stage III-IV. (2) Survivin was mainly expressed in the cytoplasm. The Survivin positive rate was 61.90% in clinical stage I-II, and 76.92% in stage III-IV. (3) There was a clear correlation between SOX2 and Survivin. 2. RT-PCR and Western Blot: The mRNA and protein expression levels of SOX2 and Survivin were significantly higher in the experimental group than in the control group (P < 0.01). CONCLUSION: (1) The mRNA and protein expression level of SOX2 and Survivin was significantly higher in SACC tissues than in paracancerous normal salivary gland tissues, indicating that both of the two are tissue-specific and may become SACC oncogenes. (2) SOX2 and Survivin are significantly correlated in expression, which may coorinatively participate in SACC incidence and development.

Neural stem cells promote glioblastoma formation in nude mice.

PURPOSE: Neural stem cells (NSCs) have been characterized with the ability of self-renewal and neurogenesis, which has inspired lots of studies to clarify the functions of NSCs in neural injury, ischemic stroke, brain inflammation and neurodegenerative diseases. We focused on the relationship of NSCs with glioblastoma, since we have discovered that recurrent glioblastomas were inclined to be derived from subventricular zone (SVZ), where NSCs reside. We want to clarify whether NSCs are involved in glioblastoma relapse. METHODS: Immunocytochemistry was used to confirm the stemness of NSCs. The Cell Counting Kit-8 was used to measure the proliferation of cells. Migration abilities were examined by wound healing and transwell assays, and tumor formation abilities were confirmed in nude mice. RESULTS: We found in experiments that NSCs promoted proliferation of a glioblastoma cell line-Ln229, the migration ability of Ln229 cells was motivated by co-cultured with NSCs. Tumor formation of Ln229 cells was also accelerated in nude mice when co-transplanted with NSCs. In immunohistochemistry, we found that the Sox2- and Ki67-positive cells were much higher in co-transplanted groups than that of control groups. CONCLUSIONS: These results imply the potential role that NSCs play in speeding up tumor formation in the process of glioblastoma relapse, providing the basis for dealing with newly diagnosed glioblastoma patients, which may help postpone the recurrence of glioblastoma as far as possible through preprocessing the tumor-adjacent SVZ tissue.

Homeostatic and tumourigenic activity of SOX2+ pituitary stem cells is controlled by the LATS/YAP/TAZ cascade.

SOX2 positive pituitary stem cells (PSCs) are specified embryonically and persist throughout life, giving rise to all pituitary endocrine lineages. We have previously shown the activation of the STK/LATS/YAP/TAZ signalling cascade in the developing and postnatal mammalian pituitary. Here, we investigate the function of this pathway during pituitary development and in the regulation of the SOX2 cell compartment. Through loss- and gain-of-function genetic approaches, we reveal that restricting YAP/TAZ activation during development is essential for normal organ size and specification from SOX2+ PSCs. Postnatal deletion of LATS kinases and subsequent upregulation of YAP/TAZ leads to uncontrolled clonal expansion of the SOX2+ PSCs and disruption of their differentiation, causing the formation of non-secreting, aggressive pituitary tumours. In contrast, sustained expression of YAP alone results in expansion of SOX2+ PSCs capable of differentiation and devoid of tumourigenic potential. Our findings identify the LATS/YAP/TAZ signalling cascade as an essential component of PSC regulation in normal pituitary physiology and tumourigenesis.

High-Throughput Automated Single-Cell Imaging Analysis Reveals Dynamics of Glioblastoma Stem Cell Population During State Transition.

Cancer stem cells (CSCs) are a heterogeneous and dynamic self-renewing population that stands at the top of tumor cellular hierarchy and contribute to tumor recurrence and therapeutic resistance. As methods of CSC isolation and functional interrogation advance, there is a need for a reliable and accessible quantitative approach to assess heterogeneity and state transition dynamics in CSCs. We developed a high-throughput automated single cell imaging analysis (HASCIA) approach for the quantitative assessment of protein expression with single-cell resolution and applied the method to investigate spatiotemporal factors that influence CSC state transition using glioblastoma (GBM) CSCs (GSCs) as a model system. We were able to validate the quantitative nature of this approach through comparison of the protein expression levels determined by HASCIA to those determined by immunoblotting. A virtue of HASCIA was exemplified by detection of a subpopulation of SOX2-low cells, which expanded in fraction size during state transition. HASCIA also revealed that GSCs were committed to loose stem cell state at an earlier time point than the average SOX2 level decreased. Functional assessment of stem cell frequency in combination with the quantification of SOX2 expression by HASCIA defined a stable cutoff of SOX2 expression level for stem cell state. We also developed an approach to assess local cell density and found that denser monolayer areas possess higher average levels of SOX2, higher cell diversity, and a presence of a sub-population of slowly proliferating SOX2-low GSCs. HASCIA is an open source software that facilitates understanding the dynamics of heterogeneous cell population such as that of GSCs and their progeny. It is a powerful and easy-to-use image analysis and statistical analysis tool available at https://hascia.lerner.ccf.org. © 2019 International Society for Advancement of Cytometry.

Chronic pain induces nociceptive neurogenesis in dorsal root ganglia from Sox2-positive satellite cells.

Chronic pain is one of the most prevalent chronic diseases in the world. The plastic changes of sensory neurons in dorsal root ganglia (DRG) have been extensively studied as the underlying periphery mechanism. Recent studies revealed that satellite cells, the major glial cells in DRG, also played important roles in the development/modulation of chronic pain. Whether DRG satellite glial cells generate new neurons as their counterparts in enteric nerve ganglia and carotid body do under pathological conditions remains poorly investigated. Here, we report that chronic pain induces proliferation and upregulation of progenitor markers in the sex-determining region Y-box 2 (Sox2)- and platelet-derived growth factor receptor alpha (PDGFRα)-positive satellite glial cells. BrdU incorporation assay revealed the generation of IB4- and CGRP-positive neurons, but not NF200-positive neurons in DRG ipsilateral to injury. Genetic fate tracings showed that PDGFRα-positive cells did not generate neurons, whereas Sox2-positive cells produced both IB4- and CGRP-positive neurons. Interestingly, glial fibrillary acidic protein-positive cells, a subpopulation of Sox2-positive satellites, only gave birth to IB4-positive neurons. Local persistent delivery of tetrodotoxin to the sciatic nerve trunk significantly reduced the pain-induced neurogenesis. Furthermore, patch-clamp studies demonstrated that these glia-derived new neurons could fire action potentials and respond to capsaicin. Taken together, our data demonstrated a chronic pain-induced nociceptive neurogenesis in DRG from Sox2-positive satellite cells, indicating a possible contribution of DRG neurogenesis to the pathology of chronic pain.

Gynecologic Serous Carcinoma: An Immunohistochemical Analysis of Malignant Body Fluid Specimens.

CONTEXT.­: Evaluation of fluid specimens involved by serous carcinoma might potentially include PAX8, GATA3, Uroplakin II, SOX2, and SALL4 antibodies. Those markers are commonly employed for diagnosing carcinomas of various types, including urothelial malignancies and germ cell tumors. There have been no comprehensive immunohistochemical studies, to our knowledge, for those markers on fluid specimens involved by serous carcinoma. OBJECTIVE.­: To evaluate immunohistochemical markers PAX8, GATA3, SOX2, uroplakin II, and SALL4 in the diagnosis of high-grade serous carcinoma in fluid specimens. DESIGN.­: We examined 113 fluids (96 ascites specimens and 17 pleural fluid specimens) that were positive for carcinoma. Most (94 cases; 83.2%) consisted of high-grade serous carcinoma of Müllerian origin. Nineteen cases of non-high-grade serous carcinoma (including one case of low-grade serous carcinoma) of gynecologic origin were also included as anecdotal data. RESULTS.­: In 113 fluid specimens with positive results for carcinoma, including nonserous types, 99 (87.6%) had positive results for PAX8, 19 (16.8%) for GATA3; 19 (16.8%) for SOX2, 23 (20.4%) for uroplakin II, and 8 (7.1%) for SALL4. Of 94 fluids (83.2%) involved with high-grade serous carcinoma, 84 (89.4%) had positive results for PAX8, 18 (19.1%) for GATA3, 17 (18.1%) for SOX2, 22 (23.4%) for uroplakin II, and 8 (8.5%) for SALL4. Some of these specimens showed reactivity for more than one immunohistochemical marker. CONCLUSIONS.­: Most fluids involving high-grade serous carcinoma showed positive results for PAX8, and some cases expressed GATA3, SOX2, uroplakin II, and SALL4. Serous carcinoma in fluids may be positive for immunohistochemical markers not thought of traditionally as associated with gynecologic malignancy, an important consideration in avoiding misdiagnosis.

Cancer Stem Cells, CD44, and Outcomes Following Chemoradiation in Locally Advanced Cervical Cancer: Results From a Prospective Study.

PURPOSE: Although cancer stem cells (CSCs) have been reported across solid tumors, there is a dearth of data regarding CSC and its impact on outcomes of cervical cancer. METHODS AND MATERIALS: From October 2013 to December 2015, patients with squamous cancer of the cervix (stage IB2-IVA) were included. Pretreatment and posttreatment biopsy was obtained and immunohistochemistry was performed for SOX-2, OCT-4, Nanog, CD44, and Podoplanin. All patients received concurrent radiation and brachytherapy to an equivalent dose of 80 to 84 Gy to point A with concurrent weekly cisplatin. Correlation of CSC expression was performed with known prognostic factors. The effect of stem cell expression on disease outcomes was tested within multivariate analysis. RESULTS: One hundred fifty patients were included. The median dose to point A was 83 Gy (46-89 Gy) and a median of 4 cycles (range, 0-6 cycles) of chemotherapy was administered. At baseline, moderate to strong immunohistochemical expression of SOX-2, OCT-4, Nanog, CD44, and Podoplanin was observed in 12.8%, 4.8%, 24.4%, 15.5%, and 1.3% of patients, respectively. At median follow-up of 30 months (range, 3-51 months), locoregional and distant relapse was observed in 12.2% and 23.1% of patients, of whom 4.7% had both local and distant relapse. The 3-year disease-free survival rate was 87%. On multivariate analysis, moderate to high CSC expression and CD44 low status (hazard ratio [HR] = 8.8; 95% confidence interval [CI], 1.0-77.2; P < .04) independently predicted for locoregional relapse-free survival. International Federation of Gynecology and Obstetrics stage (HR = 2.6; 95% CI, 1.3-5.4; P = .004) and presence of residual tumor after external radiation (HR = 3.5; 95% CI, 1.8-6.5; P = .0001) predicted for a detriment in disease-free survival. CONCLUSIONS: The presence of stem cell proteins and loss of CD44 independently predicts for reduced locoregional control in locally advanced cervical cancer. Further investigation into the interaction of stem cell and CD44 biology is warranted.