Unveiling heterogeneity in MSCs: exploring marker-based strategies for defining MSC subpopulations.

Mesenchymal stem/stromal cells (MSCs) represent a heterogeneous cell population distributed throughout various tissues, demonstrating remarkable adaptability to microenvironmental cues and holding immense promise for disease treatment. However, the inherent diversity within MSCs often leads to variability in therapeutic outcomes, posing challenges for clinical applications. To address this heterogeneity, purification of MSC subpopulations through marker-based isolation has emerged as a promising approach to ensure consistent therapeutic efficacy. In this review, we discussed the reported markers of MSCs, encompassing those developed through candidate marker strategies and high-throughput approaches, with the aim of explore viable strategies for addressing the heterogeneity of MSCs and illuminate prospective research directions in this field.

Isolation and characterization of rabbit limbal niche cells.

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs. Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRß) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRß and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRß in rabbit-LNCs P4. In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRß+).

Expression pattern of germ cell markers in cryptorchid stallion testes.

Cryptorchidism affects spermatogenesis and testis development, often resulting in stallion subfertility/infertility. This study aims to identify the specific germ cells impacted by cryptorchism in stallions. In a previous study, we found that PGP9.5 and VASA are molecular markers expressed in different germ cells within stallions. Herein, we assessed the heat stress-induced response of spermatogonial stem cells (SSCs) in the seminiferous tubules (ST) of cryptorchid stallion testes (CST) and normal stallion testes (NST). This goal was accomplished by comparing PGP9.5 and VASA expression patterns through reverse transcription quantitative PCR and immunofluorescence assays. We also compared the cross-sectional ST area between groups. Six post-pubertal Thoroughbred unilateral cryptorchid stallions were used. The relative abundance of the mRNA transcripts of PGP9.5 and VASA was significantly upregulated in the NST group than in the CST group. Additionally, the cross-sectional ST area and localization of PGP9.5 and VASA in germ cells were significantly higher in the NST group than in the CST group. Regarding Leydig cells, PGP9.5 staining was observed in both groups. Spermatogonia, primary spermatocytes and secondary spermatocytes were immunostained with VASA in the NST group, while immunostaining was only observed in spermatogonia in the CST group. These results indicate long-term exposure to heat stress conditions, such as cryptorchidism, directly impacts germ cell proliferation and differentiation, leading to impaired spermatogenesis and compromised fertility in stallions.

Comparative Characterization of Human Meibomian Glands, Free Sebaceous Glands, and Hair-Associated Sebaceous Glands Based on Biomarkers, Analysis of Secretion Composition, and Gland Morphology.

Meibomian gland dysfunction (MGD) is one of the main causes of dry eye disease. To better understand the physiological functions of human meibomian glands (MGs), the present study compared MGs with free sebaceous glands (SGs) and hair-associated SGs of humans using morphological, immunohistochemical, and liquid chromatography-mass spectrometry (LCMS)-based lipidomic approaches. Eyelids with MGs, nostrils, lips, and external auditory canals with free SGs, and scalp with hair-associated SGs of body donors were probed with antibodies against cytokeratins (CK) 1, 8, 10, and 14, stem cell markers keratin 15 and N-cadherin, cell-cell contact markers desmoglein 1 (Dsg1), desmocollin 3 (Dsc3), desmoplakin (Dp), plakoglobin (Pg), and E-cadherin, and the tight junction protein claudin 5. In addition, Oil Red O staining (ORO) was performed in cryosections. Secretions of MGs as well as of SGs of nostrils, external auditory canals, and scalps were collected from healthy volunteers, analyzed by LCMS, and the data were processed using various multivariate statistical analysis approaches. Serial sections of MGs, free SGs, and hair-associated SGs were 3D reconstructed and compared. CK1 was expressed differently in hair-associated SGs than in MGs and other free SGs. The expression levels of CK8, CK10, and CK14 in MGs were different from those in hair-associated SGs and other free SGs. KRT15 was expressed differently in hair-associated SGs, whereas N-cadherin was expressed equally in all types of glands. The cell-cell contact markers Dsg1, Dp, Dsc3, Pg, and E-cadherin revealed no differences. ORO staining showed that lipids in MGs were more highly dispersed and had larger lipid droplets than lipids in other free SGs. Hair-associated SGs had a smaller number of lipid droplets. LCMS revealed that the lipid composition of meibum was distinctively different from that of the sebum of the nostrils, external auditory canals, and scalp. The 3D reconstructions of the different glands revealed different morphologies of the SGs compared with MGs which are by far the largest type of glands. In humans, MGs differ in their morphology and secretory composition and show major differences from free and hair-associated SGs. The composition of meibum differs significantly from that of sebum from free SGs and from hair-associated SGs. Therefore, the MG can be considered as a highly specialized type of holocrine gland that exhibits all the histological characteristics of SGs, but is significantly different from them in terms of morphology and lipid composition.

Targeting stemness of cancer stem cells to fight colorectal cancers.

Cancer initiating/ stem cells (CSCs) undergo self-renewal and differentiation that contributes to tumor initiation, recurrence and metastasis in colorectal cancer (CRC). Targeting of colorectal cancer stem cells (CCSCs) holds significant promise in eradicating cancer cells and ultimately curing patients with cancer. In this review, we will introduce the current progress of CCSC studies, including the specific surface markers of CCSCs, the intrinsic signaling pathways that regulate the stemness and differentiation characteristics of CCSCs, and the tumor organoid model for CCSC research. We will focus on how these studies will lead to the progress in targeting specific surface markers or signaling pathways on CCSCs by monoclonal antibodies, or by natural or synthetic compounds, or by immunotherapy. As CSCs are highly heterogeneous and plastic, we suggest that combinatory approaches that target the stemness network may represent an important strategy for eradicating cancers.

PD-L1 is highly expressed in ovarian cancer and associated with cancer stem cells populations expressing CD44 and other stem cell markers.

BACKGROUND: Immune checkpoint inhibitors, including PD-L1 (programmed death ligand-1) inhibitors have well documented anticancer therapeutic effect in most types of cancers but its use in the treatment of ovarian cancer is not yet proven. The aim of our study is to explore the predictive biomarkers in ovarian cancer and its association with the outcomes. We have investigated the role of PD-L1 expressions in the tumor microenvironment cells including immune cells and cancer stem cells in different types of ovarian cancer. METHODS: A total of 119 surgical archived ovarian cancer samples were collected from the pathology department at King Fahad Specialist Hospital, Dammam, Saudi Arabia that included serous carcinomas, clear cell carcinomas, mucinous carcinomas, endometrioid carcinomas, and granulosa cell tumors. Immunohistochemistry (IHC) staining was performed using (i) PD-L1 antibodies to detect PD-L1 expressions; (ii) CD8 and CD4 to detect Tumor Infiltrating Lymphocytes (TILs); and (iii) CD44, LGR5, and ALDH2 to detect stem cell markers. The clinicopathological data were collected from patients' medical record to investigate the association with PD-L1, TILs, and stem cells expressions. RESULTS: We report high PD-L1 expressions in 47.8% of ovarian cancer samples. PD-L1 expressions were detected in different types of epithelial ovarian cancer and were not associated with poor prognosis of ovarian cancer. However, determining the expression levels of TILs in the ovarian cancer tissues found that 81% (n = 97) of ovarian cancer samples have TILs that express both of CD8 and CD4 and significantly associated with high PD-L1 expressions. Interestingly, we have found that ovarian cancer tissues with high expressions of PD-L1 were associated with high expressions of stem cells expressing CD44 and LGR5. CONCLUSIONS: PD-L1 is highly expressed in the serous type of ovarian carcinomas and the overall expression of PD-L1 is not associated with poor survival rate. Furthermore, PD-L1 expressions are strongly associated with TILs and stem cell markers in ovarian cancer. Inhibiting the PD-L1 using immune checkpoint inhibitors might downregulate stem cell population that known to be associated with cancer recurrence.

Establishment of an in vitro model from the vulnerable fish species Coregonus maraena (maraena whitefish): Optimization of growth conditions and characterization of the cell line.

In this study, a cell line of the fish species Coregonus maraena was produced for the first time. C. maraena is an endangered species, and studies indicate that this fish species will be affected by further population declines due to climate change. This cell line, designated CMAfin1, has been maintained in Leibovitz L-15 supplemented with 10% fetal bovine serum over 3 years. Both subculturing and storage (short-term storage at -80°C and long-term storage in liquid nitrogen) was successful. Cell morphology and growth rate were consistent from passage 10 onwards. Immunocytochemical examination of cellular proteins and matrix components confirmed the mechanical stability of the cells. Actin, fibronectin, vinculin, vimentin, and tubulin are present in the cells and form a network. In addition, the transport of molecules is ensured by the necessary proteins. Gene expression analysis showed a shift in the expressions of stem cell markers between younger and higher passages. While SOX2 and IGF1 were more highly expressed in the seventh passage, SOX9 and IGF2 expressions were significantly increased in higher passages. Therefore, the stable cell culture CMAfin1 can be used for applied analysis to further understand the cell physiology of C. maranea.

Muscarinic Acetylcholine Receptor M3 Expression and Survival in Human Colorectal Carcinoma-An Unexpected Correlation to Guide Future Treatment?

Muscarinic acetylcholine receptor M3 (M3R) has repeatedly been shown to be prominently expressed in human colorectal cancer (CRC), playing roles in proliferation and cell invasion. Its therapeutic targetability has been suggested in vitro and in animal models. We aimed to investigate the clinical role of MR3 expression in CRC for human survival. Surgical tissue samples from 754 CRC patients were analyzed for high or low immunohistochemical M3R expression on a clinically annotated tissue microarray (TMA). Immunohistochemical analysis was performed for established immune cell markers (CD8, TIA-1, FOXP3, IL 17, CD16 and OX 40). We used Kaplan-Meier curves to evaluate patients' survival and multivariate Cox regression analysis to evaluate prognostic significance. High M3R expression was associated with increased survival in multivariate (hazard ratio (HR) = 0.52; 95% CI = 0.35-0.78; p = 0.001) analysis, as was TIA-1 expression (HR = 0.99; 95% CI = 0.94-0.99; p = 0.014). Tumors with high M3R expression were significantly more likely to be grade 2 compared to tumors with low M3R expression (85.7% vs. 67.1%, p = 0.002). The 5-year survival analysis showed a trend of a higher survival rate in patients with high M3R expression (46%) than patients with low M3R expression CRC (42%) (p = 0.073). In contrast to previous in vitro and animal model findings, this study demonstrates an increased survival for CRC patients with high M3R expression. This evidence is highly relevant for translation of basic research findings into clinically efficient treatments.

S100B protein: general characteristics and pathophysiological implications in the Central Nervous System.

BACKGROUND: Calcium-binding proteins are heterogeneous proteins that act binding this ion in specific domains, performing numerous functions. OBJECTIVE: In the present review, we aim to gather principal information about S100B protein in the Central Nervous System (CNS), highlighting its particularities, mapping, functionalities, and consequences on CNS dysfunction. METHODS: The research was carried out by searching Pubmed, Medline, Science Direct, Lilacs, the Cochrane Library, and Web of Science databases using the following descriptors: S100 protein; Central Nervous System; Nervous Lesions, as well as their corresponding terms in Portuguese and Spanish. The terms were first searched separately, then together. RESULTS: Due to its ability to bind with calcium, S100B is involved in the regulation of several intra- and extracellular physiological processes. As well as being multifunctional, this protein can be considered both a "marker" and "signaling" since it is capable of triggering functions of detection of and protection in situations of injury to the CNS. CONCLUSIONS: In-depth studies are necessary to discover the innumerable actions of this protein which are still unknown. It is expected that these can bring varied benefits by elucidating its therapeutic potential in preclinical and clinical situations.

Knee and Peri-Knee Tissues of Post Mortem Donors Are Strategic Sources of Mesenchymal Stem/Stromal Cells for Regenerative Procedures.

Tissues of post mortem donors represent valuable alternative sources for the isolation of primary cells with mesenchymal stem/stromal cell (MSC)-like properties. However, the properties of primary cells derived from different tissues and at different post mortem times are poorly recognized. Here, we aim to identify the optimal tissue source between three knee and peri-knee tissues for the isolation of primary cells with MSC-like properties, and to define the influence of the time post mortem on the properties of these cells. We harvested tissues from subchondral bone marrow, synovium and periosteum from 32 donors at various post mortem times. Primary cells were evaluated using detailed in vitro analyses, including colony formation, trilineage differentiation, immunophenotyping and skeletal stem cell marker-gene expression profiling. These data show that the primary cells with MSC-like properties isolated from these three tissues show no differences in their properties, except for higher expression of CD146 in bone-marrow cells. The success rate of the primary cell isolation is dependent on the post mortem time. However, synovium and periosteum cells isolated more than 48 h post mortem show improved osteogenic and chondrogenic potential. This study suggests that knee and peri-knee tissues from donors even 3 days post mortem are strategic sources of MSCs for regenerative procedures.

Search for Novel Plasma Membrane Proteins as Potential Biomarkers in Human Mesenchymal Stem Cells Derived from Dental Pulp, Adipose Tissue, Bone Marrow, and Hair Follicle.

One of the drawbacks preventing the use of mesenchymal stem cells (MSCs) in clinical practice is the heterogeneous nature of their cultures. MSC cultures are not homogeneously formed by the MSCs and may contain non-mesenchymal cell types. Therefore, prior to use in clinics or research, complete characterization of MSCs should be performed to demonstrate the existence or absence of proper stem cell markers, many of which are happened to be cell-surface proteins. Unfortunately, the success of MSC characterization studies is limited due to the low specificity of the currently available cell-surface markers. Therefore, in this study, we aimed to investigate the plasma membrane (PM) proteins of MSCs isolated from human dental pulp (DP), adipose tissue (AT), bone marrow (BM), and hair follicle (HF) with the hope of proposing novel putative specific MSC markers. Differential-velocity centrifugation was used to enrich PM proteins. The isolated proteins were then identified by nLC-MS/MS and subjected to bioinformatics analysis. Proteins that were unique to each MSC type (CD9, CD10, CD63 for DP-MSCs; CD26, CD81, CD201, CD364 for AT-MSCs; Cd49a, CD49d for HF-MSCs; CD49e, CD56, CD92, CD97, CD156b, CD156c, CD220, CD221, CD298, CD315 for BM-MSCs) and common to all four MSC types (CD13, CD29, CD44, CD51, CD59, CD73, CD90) were identified. Uncharacterized proteins that have transmembrane (TM) domains were also detected. Some of the proteins identified in this study were the putative cell-surface markers that might be used for characterization of MSCs.

Reconnaissance of tumor immune microenvironment spatial heterogeneity in metastatic renal cell carcinoma and correlation with immunotherapy response.

A clearer understanding of the tumor immune microenvironment (TIME) in metastatic clear cell renal cell carcinoma (ccRCC) may help to inform precision treatment strategies. We sought to identify clinically meaningful TIME signatures in ccRCC. We studied tumors from 39 patients with metastatic ccRCC using quantitative multiplexed immunofluorescence and relevant immune marker panels. Cell densities were analyzed in three regions of interest (ROIs): tumor core, tumor-stroma interface and stroma. Patients were stratified into low- and high-marker density groups using median values as thresholds. Log-rank and Cox regression analyses while controlling for clinical variables were used to compare survival outcomes to patterns of immune cell distributions. There were significant associations with increased macrophage (CD68+ CD163+ CD206+ ) density and poor outcomes across multiple ROIs in primary and metastatic tumors. In primary tumors, T-bet+ T helper type 1 (Th1) cell density was highest at the tumor-stromal interface (P = 0·0021), and increased co-expression of CD3 and T-bet was associated with improved overall survival (P = 0·015) and survival after immunotherapy (P = 0·014). In metastatic tumor samples, decreased forkhead box protein 3 (FoxP3)+ T regulatory cell density correlated with improved survival after immunotherapy (P = 0·016). Increased macrophage markers and decreased Th1 T cell markers within the TIME correlated with poor overall survival and treatment outcomes. Immune markers such as FoxP3 showed consistent levels across the TIME, whereas others, such as T-bet, demonstrated significant variance across the distinct ROIs. These findings suggest that TIME profiling outside the tumor core may identify clinically relevant associations for patients with metastatic ccRCC.

In vitro study on the immunomodulatory effects of differently functionalized silver nanoparticles on human peripheral blood mononuclear cells.

The interaction of silver nanoparticles (AgNPs) with the immune system has not yet been sufficiently elucidated even though they belong to the most investigated and exploited group of nanomaterials. This study aimed to evaluate immunomodulatory effect of four different AgNPs on human peripheral blood mononuclear cells (hPBMCs). Fresh hPBMCs were exposed to the small sized (~ 10 nm) AgNPs immediately after isolation from the whole blood of healthy volunteers. The study considered coating-, time- and dose-dependent response of hPBMSc and stimulation of both early and intermediate activation of lymphocytes and monocytes using flow cytometry. The AgNPs differed in surface charge and were stabilised with polyvinyl pyrrolidone (PVP), poly-L-lysine (PLL), bis(2-ethylhexyl) sulfosuccinate sodium (AOT) or blood serum albumin (BSA). Response of hPBMCs to coating agents and ionic Ag form was evaluated to distinguish their effect from the AgNPs action as they may be released from the nanosurface. There was no significant effect of any tested AgNPs on relative count of hPBMCs subpopulations. The T-cells and monocytes were not activated after treatment with AgNPs, but the highest concentration of PLL- and BSA-AgNPs decreased density of CD4 and CD8 markers on T-helper and T-cytotoxic cells, respectively. The same AgNPs activated B- and NK-cells. Ionic Ag activated T-, B- and NK-cells, but at very higher concentration, whereas only PLL exhibited immunomodulatory activity. This study evidenced immunomodulatory activity of AgNPs that may be fine-tuned by the design of their surface functionalization.

The myeloid cell biomarker EMR1 is ectopically expressed in colon cancer.

OBJECTIVE: The microenvironment of colon cancer (CC) is heterogeneous including cells of myeloid lineage affecting tumor growth and metastasis. Two functional subtypes of myeloid cells have been identified; one (M1) is tumor-inhibitory and the other one (M2) is tumor-promoting. Whether the three myeloid markers EMR1, CD206 and CD86 are expressed only in the infiltrating myeloid cells or also in the tumor cells was investigated. METHODS: Expression of the myeloid markers was investigated in CC at the mRNA and protein levels in primary tumors and lymph nodes. mRNA expression was also determined in 5 CC cell lines. Protein expression was investigated by two-color immunofluorescence and consecutive-sections-immune-staining combined with morphometry using specific antibodies for the myeloid cell markers and the epithelial cell markers CEACAM5 and EpCAM. RESULTS: EMR1 and CD86, but not CD206, mRNA levels were significantly higher in CC primary tumors compared to apparently normal colon tissue (P <  0.0001). EMR1 mRNA levels were significantly higher in both hematoxylin-eosin positive (H&E(+)) and H&E(-) lymph nodes of CC patients compared to control nodes (P = 0.03 and P = 0.01, respectively). EMR1 and CD206 mRNAs were expressed in 4/5 and 5/5 CC cell lines, respectively, while CD86 mRNA was not expressed. Immuno-morphometry revealed that about 20% of the tumor cells expressed EMR1 and CD206. Positive cells were tumor cells as revealed by anti-CEACAM5 and anti-EpCAM staining. The number of EMR1, CD206 and CD86 positive cells were significantly increased in CC primary tumors compared to normal colon tissue (P <  0.0001). However, CD206 was also expressed in normal colonocytes. Only EMR1 showed significantly increased numbers of positive tumor cells in H&E(+) nodes compared to H&E(-) nodes (P = 0.001). EMR1 expression in CC tumor cells correlated with CXCL17 expressing tumor cells. CONCLUSION: EMR1, like the chemokine CXCL17, is ectopically expressed in colon cancer possibly in the same cancer cells.

Differential expression of ABCB1, ABCG2, and KLF4 as putative indicators for paclitaxel resistance in human epithelial type 2 cells.

Laryngeal squamous cell carcinoma (LSCC) is the second most common malignancy of the head and neck region in the USA with a declining 5-year survival rate. Paclitaxel resistance of tumors including LSCC still stands as a vital cause for poor clinical outcome in patients. In the current study, our aim was to explore the expressions of ATP-binding cassette transporters and stemness associated genes in human epithelial type 2 (Hep-2) cells with paclitaxel resistance. Resistant cells were developed via treatment with increasing doses of paclitaxel to acquire four sub-lines resistant to one-, two-, four-, and eightfold concentrations of paclitaxel (1×, 2×, 4×, 8×). Then, we profiled the expressions of ten selected ABC transporters (ABCA5, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC5, ABCC10, ABCF2, and ABCG2) and four stem cell markers (SOX2, OCT4, KLF, and CXCR4) using quantitative real time polymerase chain reaction in paclitaxel resistant cells to look for a link between these markers and chemoresistance. We demonstrated that ABCB1 and ABCG2 expressions gradually elevated and reached a maximum level in Taxol 8× cells. Considering stem cell markers, KLF4 expression elevated significantly, as soon as parental cells acquired resistance to the lowest dose of paclitaxel and its expression elevated stepwise. Expression levels of other tested ATP-binding cassette transporters and stem cell markers also elevated, although at different steps of paclitaxel resistance acquisition. Our findings suggest that higher expressions of ABCB1, ABCG2, and KLF4 might be considered as putative indicators for paclitaxel resistance in LSCC patients.

Conventional and Emerging Markers in Stem Cell Isolation and Characterization.

Stem cells have emerged as a promising source of cell-based therapy in regenerative medicine with several stem cell-based products currently in clinical trials. Despite the immense therapeutic potential, their isolation from some of the emerging sources and their characterization has been naïve owing to the lack of standard markers for the same. Some biomarkers have now been well established for the isolation and characterization of stem cells. However, there are emerging markers that can be used in addition to these conventional markers or independent of them to establish the identity of the stem cells. In this review, an attempt has been made to describe a few conventionally used markers and emerging markers for the identification, isolation and characterization of stem cells from various niches across the three germ layer origins.

Clinicopathological correlation of stem cell markers expression in oral squamous cell carcinoma; relation to patients` outcome.

Background: Squamous cell carcinoma (OSCC) is the commonest oral malignancy.The overall 5 year survival of OSCC has remained at 50%, largely unchanged for 40 years. CSCs are important within the development, invasion, drug resistance, and prediction of carcinomas treatment outcome. ALDH1 and CD44 are commonly used epithelial tumors cancer stem-like cells surface markers. Materials: Our study aimed to judge CD44 and ALDH1 immunohistochemical expressions in 44 cases of OSCC and relates the expression to patients' survival. Results: High CD44 & ALDH1 expressions were significantly expressed in variable histologic grades of OSCCs, large sized carcinomas, presence lymph vascular invasion, presence of nodal and distant metastasis, advanced TNM clinical stage, recurrence and death during follow up period (P ≤ 0.05). Reduced DFS and three years overall survival were significantly recorded in cases with high CD44 expression, and high ALDH1 expression (p < 0.05). CD44 & ALDH1 expressions, histologic grade, tumor size were the independent predictors of DFS and three years OS. Conclusion: CD44 and ALDH1 expressions are valuable prognostic factors in OSCC and could be well considered predictors for patients' 3 years OS and DFS.

Cancer stem cell markers in adenocarcinoma of the salivary glands - reliable prognostic markers?

PURPOSE: Adenocarcinoma of the salivary glands is of low incidence and a broad range of histopathological subtypes. Cancer stem cell markers (CSC) might serve as novel prognostic parameters. To date, only a few studies examined the expression of CSC in adenocarcinoma of the salivary glands with diverging results. To further investigate the reliability in terms of prognostic value, a histopathological analysis of CSCs on a cohort of patients with adenocarcinomas of the major salivary glands was performed. METHODS: Tumor samples of 40 consecutive patients with adenocarcinoma of the major salivary gland treated with curative intend at one tertiary center were stained with the CSCs ALDH1, BMI-1, CD44, Nanog, and SOX2. Expression of these markers was correlated with clinicopathological parameters and survival estimates. RESULTS: Correlation of high expression of ALDH1 with higher grading (p < 0.001) and high expression of CD44 with the localization of the neoplasm (p = 0.05), larger tumor size (p = 0.006), positive pN-category (p = 0.023), and advanced UICC stage (p = 0.002) was found. Furthermore, high expression of SOX2 correlated with a negative perineural invasion (p = 0.02). No significant correlation of any investigated marker with survival estimates was observed. CONCLUSION: In conclusion, our study did not find a significant correlation of the investigated CSCs with survival estimates in adenocarcinoma of the major salivary glands. Recapitulating the results of our study in conjunction with data in the literature, the CSCs ALDH1, BMI-1, CD44, Nanog, and SOX2 do not seem to serve as reliable prognostic parameters in the treatment of adenocarcinoma of the salivary glands.

Pilot study supporting the existence of novel lymphatic channels within the canine anterior uveal tract using Lyve-1 and CD31.

OBJECTIVE: To demonstrate the existence of lymphatics in the canine anterior uvea using lymphatic-specific markers Lyve-1, Prox-1, and podoplanin, the endothelial cell marker CD31, and basement membrane matrix marker collagen IV. DESIGN: Prospective Study. ANIMALS: Eight normal globes from animals euthanized for unrelated health problems. PROCEDURES: Sagittally cut serial sections of six normal canine eyes were immunofluorescence double-stained with Lyve-1 and CD31 and single-stained with colorimetric Prox-1 and collagen IV. Three serial sections from 2 additional eyes were cut in the coronal plane at the level of the ciliary body and immunofluorescence double-stained with Lyve-1 and CD31 to map lymphatic channel distribution. Lymphatics from normal canine lymph nodes were used for validation of podoplanin. RESULTS: Four of 6 of the sagitally sectioned eyes had Lyve-1-positive lymphatic-like structures that were distinct from CD31-positive blood vessels in the iris base and ciliary body. Both of the coronally sectioned globes had Lyve-1-positive lymphatic-like structures in the ciliary body. The location of these structures was evaluated and found to be diffusely present circumferentially around the ciliary body. CONCLUSION AND CLINICAL RELEVANCE: These results support the existence of lymphatic channels in the anterior uveal tract of the canine eye. This could indicate the presence of a novel uveolymphatic outflow pathway, which may play a role in aqueous humor outflow. Future studies are needed to confirm the existence and elucidate the role of this proposed uveolymphatic outflow pathway and potentially develop novel treatment options for managing glaucoma.

Clinical Significance of Stem Cell Biomarkers EpCAM, LGR5 and LGR4 mRNA Levels in Lymph Nodes of Colon Cancer Patients.

The significance of cancer stem cells (CSCs) in initiation and progression of colon cancer (CC) has been established. In this study, we investigated the utility of measuring mRNA expression levels of CSC markers EpCAM, LGR5 and LGR4 for predicting survival outcome in surgically treated CC patients. Expression levels were determined in 5 CC cell lines, 66 primary CC tumors and 382 regional lymph nodes of 121 CC patients. Prognostic relevance was determined using Kaplan-Meier survival and Cox regression analyses. CC patients with lymph nodes expressing high levels of EpCAM, LGR5 or LGR4 (higher than a clinical cutoff of 0.07, 0.06 and 2.558 mRNA copies/18S rRNA unit, respectively) had a decreased mean survival time of 32 months for EpCAM and 42 months for both LGR5 and LGR4 at a 12-year follow-up (p = 0.022, p = 0.005 and p = 0.011, respectively). Additional patients at risk for recurrence were detected when LGR5 was combined with the biomarkers CXCL17 or CEA plus CXCL16. In conclusion, the study underscores LGR5 as a particularly useful prognostic biomarker and illustrates the strength of combining biomarkers detecting different subpopulations of cancer cells and/or cells in the tumor microenvironment for predicting recurrence.