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Background Lidocaine is a local anesthetic that wildly used in surgical treatment and postoperative medical care for lung cancers. We hypothesized that lidocaine at clinical plasma concentration can inhibit CXCL12/CXCR4 axis-regulated cytoskeletal remodeling thereby reduce the migration of Non-small-cell lung cancers (NSCLC) cells. Methods We determined the effect of lidocaine at clinical plasma concentration on CXCL12-induced cell viability, apoptosis, cell death, monolayer cell wound healing rate, individual cell migration indicators, expression of CXCR4, CD44, and ICAM-1, intracellular Ca2+ level, and filamentous actin level alteration of NSCLC cells A549 and CXCR4-knocked down A549 cells using CCK-8, Bcl-2 ELISA, Cell death ELISA, wound healing assay, chemotaxis assay, western blotting, QPCR, Fura-2-based intracellular Ca2+ assay, and Fluorescein Phalloidin staining respectively. Results Lidocaine did not affect cell viability, apoptosis, and cell death but inhibited CXCL12-induced migration, intracellular Ca2+ releasing, and filamentous actin increase. Lidocaine decreased expression of CXCR4, increased CD44, but had no effect on ICAM-1. CXCL12 induced the increase of CD44 and ICAM-1 but did not affect CD44 in the presence of lidocaine. The knockdown of CXCR4 eliminated all the effects of lidocaine. The overexpression of CXCR4 promoted migration but the migration was inhibited by lidocaine. Conclusion Lidocaine at clinical plasma concentrations inhibited CXCL12-induced CXCR4 activation, thereby reduced the intracellular Ca2+-dependent cytoskeleton remodeling, resulting in slower migration of A549 cells.

Objective: To verify the association between CD44 and CD133 expression levels and the prognosis of patients with lower grade gliomas (LGGs) and constructing radiomic models to predict those two genes’ expression levels before surgery. Materials & methods: Genomic data of patients with LGG and the corresponding T2-weighted fluid-attenuated inversion recovery images were downloaded from The Cancer Genome Atlas and The Cancer Imaging Archive, which were utilized for prognosis analysis, radiomic feature extraction and model construction, respectively. Results & conclusion: CD44 and CD133 expression levels in LGG can significantly affect the prognosis of patients with LGG. Based on the T2-weighted fluid-attenuated inversion recovery images, the radiomic features can effectively predict the expression levels of CD44 and CD133 before surgery.

Aberrant expression of SNX5 can contribute to tumorigenesis, invasion, and metastasis of several human cancers. However, the clinicopathological and biological significance of SNX5 in clear cell renal cell carcinoma (ccRCC) remain unclear. In this study, we found that SNX5 expression was downregulated and negatively correlated with tumor size, American Joint Committee on Cancer stage, tumor thrombus of inferior vena cava, and poor prognosis in human ccRCC. Ectopic expression of SNX5 inhibited ccRCC cell proliferation and metastasis, whereas knockdown of SNX5 increased these activities both in vitro and in vivo. Mechanistically, overexpression of SNX5 blocked internalization and intracellular trafficking of CD44 in ccRCC cells. Knockdown of SNX5 was associated with epithelial-to-mesenchymal transition (EMT) in ccRCC cells. Overexpression of SNX5 inhibited TGF-β-induced migration, invasion, and EMT in ccRCC cells. KLF9 directly bound to the SNX5 promoter and increased SNX5 transcription. Moreover, we found that the combination of SNX5 and CD44 or E-cadherin or KLF9 was a more powerful predictor of poor prognosis than either parameter alone. Collectively, our data reveal a mechanism that KLF9-mediated SNX5 expression was associated with poor prognosis via trafficking of CD44 and promoting EMT in ccRCC. SNX5 may be a potential prognostic biomarker and therapeutic target for patients with ccRCC., Graphical abstract, This study aimed at researching the function of SNX5 in clear cell renal cell carcinoma (ccRCC). The authors showed that SNX5 expression was downregulated by KLF9, leading to trafficking of CD44 and epithelial-to-mesenchymal transition and poor prognosis in ccRCC. SNX5 was a potential prognostic biomarker and therapeutic target in ccRCC.

Limited number of tumor types have been examined for Orthopedia Homeobox (OTP) expression. In pulmonary carcinoids, loss of expression is a strong indicator of poor prognosis. Here, we investigated OTP expression in 37 different tumor types, and the association between OTP expression and DNA methylation levels in lung neuroendocrine neoplasms. We analyzed publicly available multi-omics data (whole-exome-, whole-genome-, RNA sequencing and Epic 850K-methylation array) of 58 typical carcinoids, 27 atypical carcinoids, 69 large cell neuroendocrine carcinoma and 51 small cell lung cancer patients and TCGA (The Cancer Genome Atlas) data of 33 tumor types. 850K-methylation analysis was cross-validated using targeted pyrosequencing on 35 carcinoids. We report bimodality of OTP expression in carcinoids (OTPhigh vs OTPlow group, likelihood-ratio test P = 1.5 × 10−2), with the OTPhigh group specific to pulmonary carcinoids while absent from all other cohorts analyzed. Significantly different DNA methylation levels were observed between OTPhigh and OTPlow carcinoids in 12/34 OTP infinium probes (FDR < 0.05 and β-value effect size >.2). OTPlow carcinoids harbor high DNA methylation levels as compared to OTPhigh carcinoids. OTPlow carcinoids showed a significantly worse overall survival (log-rank test P =.0052). Gene set enrichment analysis for somatically mutated genes associated with hallmarks of cancer showed robust enrichment of three hallmarks in the OTPlow group, that is, sustaining proliferative signaling, evading growth suppressor and genome instability and mutation. Together our data suggest that high OTP expression is a unique feature of pulmonary carcinoids with a favorable prognosis and that in poor prognostic patients, OTP expression is lost, most likely due to changes in DNA methylation levels.

P53 and DNA damage-regulated gene1 (PDRG1) is overexpressed in diverse carcinomas. Here, we discover that PDRG1 is overexpressed in glioblastoma multiforme (GBM). However, the clinical significance, biological role, and underlying molecular mechanisms of PDRG1 in GBM remain unclear. PDRG1 was aberrantly overexpressed in glioma, especially prevalent in GBM, and correlated with poor clinicopathologic features of glioma. The risk score, operational feature curve analysis, Kaplan-Meier curve, and univariate and multivariate Cox regression analysis indicated that PDRG1 was an independent prognostic indicator and significantly correlates with disease progression of glioma. A prognostic nomogram was constructed to predict the survival risk of individual patients. The function and pathway enrichment analysis of PDRG1 in The Cancer Genome Atlas cohort was performed. PDRG1 knockdown significantly inhibited the migration and proliferation of GBM cells in vitro and in vivo. Transcriptome sequencing analysis of PDRG1 knockdown U-118 MG(U118) cells indicated that biological regulation adhesion, growth and death, cell motility, cell adhesion molecular and proteoglycans in cancer were significantly enriched. Importantly, we found that the expression of adhesion molecule cluster of differentiation 44 (CD44) was regulated by PDRG1 in GBM. We found that PDRG1 promoted the migration and proliferation of GBM cells via the mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinase (ERK)/CD44 pathway. Our findings provide proof that PDRG1 upregulation predicts progression and poor prognosis in human gliomas, especially in isocitrate dehydrogenase (IDH) wt glioma patients. The study provides new evidence that PDRG1 regulates the expression of CD44 in GBM cells and might promote the migration and proliferation via the MEK/ERK/CD44pathway. PDRG1 might be a novel diagnostic indicator and promising therapeutic target for GBM.

In the present study, we show that the inhibitor of the apoptosis-stimulating protein of p53 (iASPP) physically interacts with the hyaluronan receptor CD44 in normal and transformed cells. We noticed that the CD44 standard isoform (CD44s), but not the variant isoform (CD44v), bound to iASPP via the ankyrin-binding domain in CD44s. The formation of iASPP-CD44s complexes was promoted by hyaluronan stimulation in fibroblasts but not in epithelial cells. The cellular level of p53 affected the amount of the iASPP-CD44 complex. iASPP was required for hyaluronan-induced CD44-dependent migration and adhesion of fibroblasts. Of note, CD44 altered the sub-cellular localization of the iASPP-p53 complex; thus, ablation of CD44 promoted translocation of iASPP from the nucleus to the cytoplasm, resulting in increased formation of a cytoplasmic iASPP-p53 complex in fibroblasts. Overexpression of iASPP decreased, but CD44 increased the level of intracellular reactive oxygen species (ROS). Knock-down of CD44s, in the presence of p53, led to increased cell growth and cell density of fibroblasts by suppression of p27 and p53. Our observations suggest that the balance of iASPP-CD44 and iASPP-p53 complexes affect the survival and migration of fibroblasts. De två första författarna delar förstaförfattarskapet.

Glioblastoma (GBM) is a heterogeneous tumor of the brain with a poor prognosis due to recurrence and drug resistance following therapy. Genome-wide profiling has revealed the existence of distinct GBM molecular subtypes that respond differently to aggressive therapies. Despite this, molecular subtype does not predict recurrence or drug resistance and overall survival is similar across subtypes. One of the key features contributing to tumor recurrence and resistance to therapy is proposed to be an underlying subpopulation of resistant glioma stem cells (GSC). CD133 expression has been used as a marker of GSCs, however recent evidence suggests the relationship between CD133 expression, GSCs and molecular subtype is more complex than initially proposed. The expression of CD133, Olig2 and CD44 was investigated using patient derived glioma stem-like cells (PDGCs) in vitro and in vivo. Different PDGCs exhibited a characteristic equilibrium of distinct CD133+ and CD44+ subpopulations and the influence of environmental factors on the intra-tumor equilibrium of CD133+ and CD44+ cells in PDGCs was also investigated, with hypoxia inducing a CD44+ to CD133+ shift and chemo-radiotherapy inducing a CD133+ to CD44+ shift. These data suggest that surveillance and modulation of intra-tumor heterogeneity using molecular markers at initial surgery and surgery for recurrent GBM may be important for more effective management of GBM. ispartof: PLoS One vol:12 issue:2 pages:e0172791- ispartof: location:United States status: published

GC is a fatal disease with high heterogeneity and invasiveness. Recently, SPP1 has been reported to be involved in the tumor progression of multiple human cancers; however, the role of SPP1 in GC heterogeneity and whether it is associated with the invasiveness and mortality of GC remain unclear. Here, we combined multiple RNA sequencing approaches to evaluate the impact of SPP1 on GC. Through bulk RNA sequencing (bulk RNA-seq) and immunohistochemistry (IHC), we found that SPP1 was highly expressed in GC, and high levels of SPP1 were associated with macrophage infiltration, an advanced tumor stage, and higher mortality for advanced GC patients. Furthermore, through simultaneous single-cell and spatial analysis, we demonstrated that SPP1+ macrophages are tumor-specific macrophages unique to cancer and enriched in the deep layer of GC tissue. Cell—cell communication analysis revealed that SPP1/CD44 interactions between SPP1+ macrophages and their localized tumor epithelial cells could activate downstream target genes in epithelial cells to promote dynamic changes in intratumor heterogeneity. Moreover, these activated genes were found to be closely associated with poor clinical GC outcomes and with cancer-related pathways that promote GC progression, as shown by survival analysis and enrichment analysis, respectively. Collectively, our study reveals that tumor-specific SPP1+ macrophages drive the architecture of intratumor heterogeneity to evolve with tumor progression and that SPP1 may serve as a prognostic marker for advanced GC patients, as well as a potential therapeutic target for GC.

Background We have developed a novel imaging analysis procedure that is highly predictive of local failure after chemoradiation in head and neck cancer. In this study we investigated whether any pretreatment biomarkers correlated with key imaging parameters. Methods Pretreatment biopsy material was available for 28 patients entered into an institutional trial of adaptive radiotherapy in which FDG-PET images were collected weekly during treatment. The biopsies were immunohistochemically stained for CD44, EGFR, GLUT1, ALDH1, Ki-67 and p53 and quantified using image analysis. Expression levels were correlated with previously derived imaging parameters, the pretreatment SUVmax and the dose response matrix (DRM). Results The different parameters of the SUVmax and DRM did not correlate with each other. We observed a positive and highly significant (p = 0.0088) correlation between CD44 expression and volume of tumor with a DRM greater than 0.8. We found no correlation between any DRM parameter and GLUT1, p53, Ki-67 and EGFR or ALDH1. GLUT1 expression did correlate with the maximum SUV0 and the volume of tumor with an SUV0 greater than 20. Conclusions The pretreatment SUVmax and DRM are independent imaging parameters that combine to predict local recurrence. The significant correlation between CD44 expression, a known cancer stem cell (CSC) marker, and volume of tumor with a DRM greater than 0.8 is consistent with concept that specific foci of cells are responsible for tumor recurrence and that CSCs may be randomly distributed in tumors in specific niches. Dose painting these small areas may lead to improved tumor control.

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

Tumour heterogeneity in oral cancer is attributed to the presence of cancer stem cells (CSCs). CSCs are the most migratory and metastatic cellular subpopulation within tumours. Assessment of CSC markers as significant predictors of lymph node metastasis may prove valuable in the clinical setting. Furthermore, analysis of this panel of putative stem cell markers in oral dysplasia may additionally inform of the likelihood for oral potentially malignant disorders (OPMDs) to progress to oral squamous cell carcinoma (OSCC). The present study aims to assess the significance of CSC markers in the progression of OPMDs to OSCC and assessment of lymph node metastasis in OSCC. CD44 and ALDH1 were assessed immunohistochemically in 25 normal, 30 OPMDs, and 24 OSCCs. CD44 is a membranous marker and ALDH1 is a cytoplasmic marker. The immunohistochemical expression of these markers were compared between OPMDs with and without dysplasia, as well as between low-risk and high-risk dysplasias. Similarly, expression was compared between OSCC with and without lymph node metastasis and among grades of OSCC. Positive CD44 expression was seen in all normal mucosal tissues. The expression decreased from normal epithelium to OPMDs but increased in OSCC. CD44 expression was positive in 21 cases of OSCC (87.5%) and reduced from well-differentiated to poorly differentiated OSCC. CD44 staining index was higher in OSCC without lymph node metastasis (3.59) when compared with OSCC with lymph node metastasis (1.33). There was a statistically significant difference observed in the ALDH1 staining index among three groups (p

Although the guidelines recommended some biomarkers for detecting minimal residual disease (MRD) in multiple myeloma (MM) by multiparameter flow cytometry (MFC), there is still room for the exploration of the selection of biomarkers for this purpose. To seek a more effective combination of biomarkers to monitor MRD in MM. A comparison of the expression of each biomarker (CD19, etc.) by MFC in the bone marrow of the MM group (56 MM patients with MRD) in comparison with the biomarker expression in the control group (49 non-MM patients) was conducted, and an analysis of the abnormal expression of each biomarker was assessed in each patient by a semiquantitative assay. The CD44 expression and median fluorescence intensity in myeloma cells were significantly lower than that in the control group (P = 0.000), and CD44 may be an alternative myeloma-specific biomarker. Subsequently, we obtained the positive threshold for each biomarker (except for CD49d and CD184), and the median number 4 (2, 6) of abnormal biomarkers was significantly higher in the MM group than in the control group (0, 2) (P = 0.000). We defined greater than 2 abnormal biomarkers as the criterion for diagnosing MRD in MM, and the sensitivity of the novel biomarker combination (including CD44; 94.64% sensitivity) was enhanced from that of the previous biomarker combination (except for CD44; 83.93% sensitivity). CD44 can serve as an alternative myeloma-specific biomarker. The novel MFC biomarker combination can more effectively evaluate MRD in MM and could be considered for use in clinical practice.

Pancreatic cancer has an extremely poor prognosis because of its resistance to conventional therapies. Cancer stem cell (CSC)‐targeted therapy is considered a promising approach for this disease. Epithelial‐mesenchymal transition‐inducing transcription factors (EMT‐TFs) contribute to CSC properties in some solid tumors; however, this mechanism has not been fully elucidated in pancreatic cancer. Zinc finger protein, SNAIL2 (also known as SLUG), is a member of the SNAIL superfamily of EMT‐TFs and is commonly overexpressed in pancreatic cancer. Patients exhibiting high SNAIL2 expression have a poor prognosis. In this study, we showed that the suppression of SNAIL2 expression using RNA interference decreased tumorigenicity in vitro (sphere formation assay) and in vivo (xenograft assay) in 2 pancreatic cancer cell lines, KLM1 and KMP5. In addition, SNAIL2 suppression resulted in increased sensitivity to gemcitabine and reduced the expression of CD44, a pancreatic CSC marker. Moreover, experiments on tumor spheroids established from surgically resected pancreatic cancer tissues yielded similar results. A microarray analysis revealed that the mechanism was mediated by insulin‐like growth factor (IGF) binding protein 2. These results indicate that IGFBP2 regulated by SNAIL2 may represent an effective therapeutic target for pancreatic cancer., SNAIL2 knockdown reduces tumorigenicity and resistance to gemcitabine in tumor spheroid established from resected tissue of human pancreatic cancer as well as human pancreatic cancer cell lines.

Despite new combination therapies improving survival of breast cancer patients with estrogen receptor α (ER+) tumors, the molecular mechanisms for endocrine-resistant disease remain unresolved. Previously we demonstrated that expression of the RNA binding protein and N6-methyladenosine (m6A) reader HNRNPA2B1 (A2B1) is higher in LCC9 and LY2 tamoxifen (TAM)-resistant ERα breast cancer cells relative to parental TAM-sensitive MCF-7 cells. Here we report that A2B1 protein expression is higher in breast tumors than paired normal breast tissue. Modest stable overexpression of A2B1 in MCF-7 cells (MCF-7-A2B1 cells) resulted in TAM- and fulvestrant- resistance whereas knockdown of A2B1 in LCC9 and LY2 cells restored TAM and fulvestrant, endocrine-sensitivity. MCF-7-A2B1 cells gained hallmarks of TAM-resistant metastatic behavior: increased migration and invasion, clonogenicity, and soft agar colony size, which were attenuated by A2B1 knockdown in MCF-7-A2B1 and the TAM-resistant LCC9 and LY2 cells. MCF-7-A2B1, LCC9, and LY2 cells have a higher proportion of CD44+/CD24-/low cancer stem cells (CSC) compared to MCF-7 cells. MCF-7-A2B1 cells have increased ERα and reduced miR-222-3p that targets ERα. Like LCC9 cells, MCF-7-A2B1 have activated AKT and MAPK that depend on A2B1 expression and are growth inhibited by inhibitors of these pathways. These data support that targeting A2B1 could provide a complimentary therapeutic approach to reduce acquired endocrine resistance.

Accumulating evidence has presented that microRNA-148a/152 (miR-148a/152) acts as the tumor inhibitor in various cancers. In this article, we aimed to probe the inhibition of colon cancer stem cells by miR-148a/152 cluster via regulation of CCT6A. miR-148a/152 and CCT6A expression in colon cancer tissues and cells was detected. The relationship between miR-148a/152 expression and the clinicopathological features of patients with colon cancer was analyzed. Colon cancer stem cells (CD44+/CD133+) were selected and high/low expression of miR-148a/152 plasmids were synthesized to intervene CD44+/CD133+ colon cancer stem cells to investigate the function of miR-148a/152 in invasion, migration, proliferation, colony formation and apoptosis of cells. The growth status of nude mice was observed to verify the in-vitro results. The relationship between miR-148a/152 and CCT6A was analyzed. CCT6A upregulated and miR-148a/152 downregulated in colon cancer tissues. MiR-148a/152 expression was correlated with tumor node metastasis stage, lymph node metastasis and differentiation degree. Upregulated miR-148a/152 depressed CCT6A expression and restrained invasion and migration ability, colony formation and proliferation, induced cell apoptosis, depressed OCT4, Nanog and SOX2 mRNA expression of colon cancer stem cells, and descended tumor weight and volume in nude mice. CCT6A was a target gene of miR-148a/152. Overexpression of CCT6A protected colon cancer stem cells. Functional studies showed that upregulation of miR-148a/152 can suppress the migration, invasion and proliferation of CD44+/CD133+ colon cancer stem cells, advance its apoptosis via inhibition of CCT6A expression.

Ling Zhang, Bijay Khadka, Jieying Wu, Yashu Feng, Bing Long, Ruozhi Xiao, Jiajun Liu Department of Hematology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou City, Guangdong Province, 510630, People’s Republic of ChinaCorrespondence: Jiajun LiuDepartment of Hematology, The Third Affiliated Hospital of Sun Yat-sen University, No. 600 Tianhe Avenue, Guangzhou City, Guangdong Province, 510630, People’s Republic of ChinaEmail jiajunliu2000@163.comIntroduction: Acute myeloid leukemia (AML) is a predominant blood malignancy with high mortality and severe morbidity. AML is affected by microRNAs (miRNAs) loaded in exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs). MiR-425-5p has been reported to participate in different cancer models. However, the function of BM-MSCs-derived exosomal miR-425-5p in AML is unclear.Methods: The expression of miR-425-5p was measured by qRT-PCR in clinical AML samples. The immunophenotype of BM-MSCs was analyzed using antibodies against CD44, CD90, and CD105. The exosome was isolated from BM-MSCs. The effect of BM-MSCs-derived exosomal miR-425-5p on AML was analyzed by CCK-8 assay, Edu assay, transwell assay, flow cytometry in AML cells. qRT-PCR, luciferase reporter gene assay and Western blot analysis were also conducted in AML cells.Results: The expression levels of miR-425-5p were decreased in CD34 + CD38-AML cells from primary AML patients compared to that from the bone marrow of healthy cases, and were reduced in exosomes from AML patients compared that from healthy cases. Similarly, miR-425-5p was also down-regulated in AML cell lines compared with BM-MSCs. MiR-425-5p was able to express in exosomes from BM-MSCs. CCK-8, Edu, transwell assay and flow cytometry analysis revealed that BM-MSCs-derived exosomal miR-425-5p significantly inhibited cell viability, Edu positive cells, invasion and migration, and induced apoptosis of AML cells. Meanwhile, the expression levels of cleaved PARP and cleaved caspase3 were increased by BM-MSCs-derived exosomal miR-425-5p in cells. MiR-425-5p inhibited the expression of Wilms tumor 1-associated protein (WTAP). Moreover, overexpression of WTAP could reverse the miR-425-5p-induced inhibition effect on AML cell proliferation, apoptosis, migration and invasion.Conclusion: BM-MSCs-derived exosomal miR-425-5p inhibits proliferation, invasion and migration of AML cells and induced apoptosis of AML cells by targeting WTAP. Therapeutically, BM-MSCs-derived exosomal miR-425-5p may serve as a potential target for AML therapy.Keywords: AML, BM-MSCs, exosome, miR-425-5p, WTAP

(1) Background: Recently, cell division cycle associated 8 (CDCA8) was found to be overexpressed in pancreatic ductal adenocarcinoma (PDAC). Here, we aimed to explore the specific mechanism of action of CDCA8 in PDAC progression. (2) Methods: All human PDAC samples and clinical data were collected from Huashan Hospital, Fudan University. All experimental studies were carried out using many in vitro and in vivo assays, including lentiviral transfection, real-time quantitative polymerase chain reaction (qPCR), western blotting, co-immunoprecipitation (Co-IP), chromatin IP (ChIP)-qPCR, dual-luciferase reporter, and in vivo imaging assays. (3) Results: Clinical data analysis of human PDAC samples revealed that CDCA8 overexpression were positively and negatively associated with tumor grade (p = 0.007) and overall survival (p = 0.045), respectively. CDCA8 knockdown inhibited PDAC proliferation and invasion in in vitro and in vivo assays. CD44 was also up-regulated by CDCA8 during PDAC progression. CDCA8 could be combined with SNAI2 to form a CDCA8/SNAI2 complex to integrate with the CD44 promoter as indicated through ChIP-qPCR and dual-luciferase reporter assays. (4) Conclusion: We showed that CDCA8-CD44 axis plays a key role in the proliferation and invasion of PDAC, which provides a potential target for treatment.

Supplemental Digital Content is available in the text., In our previous studies, we found that T24 lung metastatic cancer cells showed high invasion and metastasis abilities and cancer stem cell characteristics compared with T24 primary cancer cells. By screening for the expression of CXC chemokines in both cell lines, we found that CXCL5 is highly expressed in T24-L cells. The aim of this study is to shed light on the relationship of CXCL5 with epithelial–mesenchymal transition (EMT) and cancer stem cells (CSCs). RNAi technology was used to decrease CXCL5 expression in the T24-L cell line, and the EMT and CSCs of the shCXCL5 group and the control group were compared. The CXCR2 inhibitor SB225002 was used to inhibit the receptor of CXCL5 to determine the effect of the CXCL5/CXCR2 axis. The knockdown of CXCL5 expression in T24-L cells reduced their EMT and CSC characteristics. RT-PCR and Western blot analyses revealed the downregulation of N-cadherin, Vimentin and CD44. In addition, when CD44 expression was knocked down, the EMT ability of the cells was also inhibited. This phenomenon was most pronounced when both CXCL5 and CD44 were knocked down. CXCL5 and CD44 can affect the EMT and stem cell capacity of T24-L cells through some interaction.

Introduction Distant recurrence, especially liver metastases, occurs in one-third of rectal cancer patients initially treated with curative therapy and is still an unsolved problem. The identification of patients at risk is crucial for enabling individualized treatment. Material and methods All patients undergoing curative resection for histologically confirmed rectal cancer after neoadjuvant radiochemotherapy between January 2001 and December 2015 were included. Sections were stained for Ki67, CD44, apoptosis and CD133. Patients were categorized based on whether they were found to have (CD44+/Ki67+) or not have (CD44+/Ki67+) still proliferating tumor cells. Results 218 patients who underwent R0 resection for stage I-III rectal cancer were selected. In 37 (17%) of these patients, CD44+/Ki67+ tumor cells were found. In multivariable Cox regression analysis, patients with CD44+/Ki67+ cells had significantly impaired overall (hazard ratio (HR): 3.84, 95% CI: 1.77–8.31, p = 0.001) and relative survival (HR 3.44, 95% CI: 1.46–8.09). The previous results were confirmed after propensity-score matching. In mediation-analysis, the presence of CD44+/Ki67+ cells was associated with a substantial direct effect on overall (HR 1.92, 95% CI: 1.09–9.28) and relative survival (HR 1.63, 95% CI: 1.31–6.38). Conclusions The presence of still proliferating CD44+/Ki67+ tumor cells after neoadjuvant radiochemotherapy was associated with impaired oncological long-term outcomes. Characterization of these cells should be performed.

Background Metaplastic breast cancer (MpBC) is a heterogeneous subtype of invasive mammary carcinoma associated with epithelial-mesenchymal transition (EMT) and cancer stem cell characteristics. Data regarding prognostic markers and potentially actionable targets for therapy are still limited. The present study aimed to characterize the immunohistochemical landscape of this rare malignancy and to identify potential prognostic factors and targets for therapy. Material and methods A total of 75 patients diagnosed with MpBC over a 15-year period were included in the study. We performed immunohistochemical analyses for Ki-67 (MIB-1), epidermal growth factor receptor (EGFR), cytokeratin 5/6, vimentin, CD44, and androgen receptor (AR) and correlated their expression with clinicopathologic features and clinical outcomes. The p-values for survival analyses were corrected for multiple testing (threshold 0.01). Results Most tumors expressed CK5/6 (73%), EGFR (59%), CD44 (81%), and vimentin (87%). Eighty-nine percent had a high Ki-67 index. Eighty-four percent were classified as basal-like (CK 5/6 or EGFR positive). AR was expressed in 21% of the tumors. The basal-like phenotype was significantly (p = 0.009) associated with inferior disease-free (DFS) and breast-cancer-specific overall survival (BCOS) with borderline significance (p = 0.01). In addition, a low Ki-67 index was associated with improved DFS (p = 0.033) and BCOS (p = 0.03). Conclusion Most MpBCs express basal markers (CK5/6, EGFR), epithelial-mesenchymal transition marker vimentin, and the stem cell marker CD44. Expression of basal-like markers was significantly related to inferior DFS. All the 11 patients with a lack of expression of basal markers survived without relapse.

Background/aim Cancer stem cells (CSCs) contribute to resistance against neoadjuvant chemotherapy (NAC) in esophageal squamous cell carcinoma (ESCC). We conducted a retrospective observational study for the relationship between the expression levels of CSC markers in biopsy specimens prior to 5-fluorouracil plus cisplatin (FP)-NAC and the pathological responses. Patients and methods We included 171 patients with ESCC who underwent the FP-NAC followed by radical resection. Biopsy specimens prior to the FP-NAC were obtained and immunochemically stained for CD44, CD133, and CD24. Results The biopsy specimens of the non-responders had the CD44high/CD24low expression at high levels, which was found as an independent predictor of not only FP-NAC resistance but also poor overall survival by multivariate analyses. Conclusion CD44high/CD24low expression in the biopsy specimens prior to FP-NAC may be a predictor of FP-NAC resistance and poor prognosis of ESCC patients.

Helper T lymphocytes (HTLs) play a central role in cancer immunity because they can not only help the induction and proliferation of cytotoxic T lymphocytes (CTLs) but also their differentiation into cytotoxic CD4+ T cells and directly kill the target cells.This study describes the identification of three novel mouse Th epitope peptides, WT135-52, WT186-102 and WT1294-312, derived from WT1 protein, which is the most potent tumor-associated antigen. Compared to immunization with WT1 CTL peptide alone, immunization with the addition of these WT1-specific Th peptides strongly induced WT1-specific CTLs, continued to maintain them, and efficiently rejected the challenge of WT1-expressing tumor cells. Importantly, the majority of WT1-specific CTLs induced by the co-immunization with WT1 CTL and the WT1-specific Th peptides were CD44+CD62L- effector memory CD8+ T cells, which played a central role in tumor rejection. Establishment of mouse models suitable for the analysis of the detailed mechanism of these functions of HTLs is very important. These results clearly showed that WT1-specific HTLs perform an essential function in WT1-specific tumor immunity. Therefore, the WT1-specific Th peptides identified here should make a major contribution to elucidation of the mutual roles of WT1-specific CTLs and HTLs in cancer immunity in in vivo mouse models.

Background Cervical cancer ranks the second female malignancy after breast cancer. Cancer stem cells (CSCs) are hard to be eradicated, so can recur. We aim to isolate and characterize CSCs from HeLa cells. Methods These cells express clusters of differentiation (CDs), 44 and 24, to be sorted by fluorescence-activated cell sorting (FACS). Results CD44 + CD24+ cells showed potential to form spheres, tumorigenicity, stemness genes and higher resistance to cisplatin, X-ray. Conclusion CD44+CD24+ HeLa cells hold characteristics of CSCs, in vitro, in vivo studies, suggested that targeting of them lead to screening of new anti-cancer therapies.

Cancer-associated fibroblasts (CAFs) are recruited to the tumor microenvironment (TME) and are critical drivers of breast cancer (BC) malignancy. Circulating tumor cells (CTCs) travel through hematogenous routes to establish metastases. CTCs circulate both individually and, more rarely, in clusters with other cell types. Clusters of CTCs have higher metastatic potential than single CTCs. Previously, we identified circulating CAFs (cCAFs) in patients with BC and found that while healthy donors had no CTCs or cCAFs, both were present in most Stage IV patients. cCAFs circulate individually, as cCAF–cCAF homotypic clusters, and in heterotypic clusters with CTCs. In this study, we evaluate CTCs, cCAFs, and heterotypic cCAF-CTC clusters in patients with stage I–IV BC. We evaluate the association of heterotypic clusters with BC disease progression and metastasis in a spontaneous mouse model. Using previously established primary BC and CAF cell lines, we examine the metastatic propensity of heterotypic cCAF-CTC clusters in orthotopic and tail vein xenograft mouse models of BC. Using an in vitro clustering assay, we determine factors that may be involved in clustering between CAF and BC cells. We report that the dissemination of CTCs, cCAFs, and clusters is an early event in BC progression, and we find these clusters in all clinical stages of BC. Furthermore, cCAFs-CTC heterotypic clusters have a higher metastatic potential than homotypic CTC clusters in vivo. We also demonstrate that the adhesion and stemness marker CD44, found on a subset of CTCs and CAF cells, is involved in heterotypic clustering of these cells. We identify a novel subset of circulating tumor cell clusters that are enriched with stromal CAF cells in BC patient blood and preclinical mouse models of BC metastasis. Our data suggest that clustering of CTCs with cCAFs augments their metastatic potential and that CD44 might be an important mediator of heterotypic clustering of cCAFs and BC cells.

Background Metastasis is the major cause of treatment failure and cancer-related deaths in prostate cancer (PCa) patients. Our previous study demonstrated that a CD44+ subpopulation isolated from PCa cells or tumours possesses both stem cell properties and metastatic potential, serving as metastatic prostate cancer stem cells (mPCSCs) in PCa metastasis. However, the underlying mechanisms remain unknown. Methods In this study, we established PCa models via the orthotopic and subcutaneous implantation of different human PCa cancer cell lines, and compared the metastatic efficacy, after which process function analysis of target genes was pinpointed. Results Several novel differentially expressed genes (DEGs) between orthotopic and ectopic tumours were identified. Among them, human homeobox B9 (HOXB9) transcription factor was found to be essential for PCa metastasis, as evidenced by the diminished number of lung metastatic foci derived from orthotopic implantation with HOXB9-deficient CWR22 cells, compared with the control. In addition, HOXB9 protein expression was upregulated in PCa tissues, compared with paracancer and benign prostate hyperplasia tissues. It was also positively correlated with Gleason scores. Gain- and loss-of-function assays showed that HOXB9 altered the expression of various tumour metastasis- and cancer stem cell (CSC) growth-related genes in a transforming growth factor beta (TGFβ)-dependent manner. Moreover, HOXB9 was overexpressed in an ALDH+CD44+CXCR4+CD24+ subpopulation of PCa cells that exhibited enhanced TGFβ-dependent tumorigenic and metastatic abilities, compared with other isogenic PCa cells. This suggests that HOXB9 may contribute to PCa tumorigenesis and metastasis via TGFβ signalling. Of note, ALDH+CD44+CXCR4+CD24+-PCa cells exhibited resistance to castration and antiandrogen therapy and were present in human PCa tissues. Conclusion Taken together, our study identified HOXB9 as a critical regulator of metastatic mPCSC behaviour. This occurs through altering the expression of a panel of CSC growth- and invasion/metastasis-related genes via TGFβ signalling. Thus, targeting HOXB9 is a potential novel therapeutic PCa treatment strategy.

Objective Long non-coding RNAs (lncRNAs) and microRNAs (miRs) are differentially expressed in ovarian cancer (OC) cells and influence OC progression. This study intended to explore the underlying roles of LINC00115 and miR-30a in OC. Methods Gene Expression Omnibus database was used to find OC microarray datasets and bioinformatics analysis predicted the potential molecular mechanism of OC. OC stem cells (OCSCs) surface marker was isolated from human OC cell line and identified. CD133+ OCSCs were transfected with LINC00115, miR-30a and SOX9 alone or together to detect sphere-forming ability and apoptosis of OCSCs. Caspase-3 activity and DNA damage in cell supernatant were detected. The levels of CD44, NANOG, POU5F1, LINC00115, CD133, miR-30a and SOX9 were measured. Then sh-LNC00115-treated OCSCs were added with Wnt/β-catenin activator SKL2001 to observe the changes of cell stemness and activity. Finally, animal models were established to evaluate the effect of LINC00115 on OCSC in vivo. Results LINC00115 and SOX9 were highly expressed in OC, while miR-30a was lowly expressed. After silencing LINC00115 or overexpressing miR-30a, the sphere-forming rate of CD133+ OCSC and levels of CD133, CD44, NANOG and POU5F1 decreased, while apoptotic rate, Caspase-3 activity and histone-related DNA damage increased. SOX9 reversed these trends. Additionally, LINC00115 could bind to miR-30a and miR-30a could target SOX9. SKL2001 partially reversed cell stemness and activity in sh-LNC00115-treated OCSCs. Finally, silencing LINC00115 could inhibit OCSCs growth in vivo. Conclusion LINC00115 promoted stemness and inhibited apoptosis of OCSCs by upregulating SOX9 and in activating the Wnt/β-catenin pathway through competitively binding to miR-30a.

Background Gastric adenocarcinoma with peritoneal carcinomatosis (PC) is therapy resistant and leads to poor survival. To study PC in depth, there is an urgent need to develop representative PC-derived cell lines and metastatic models to study molecular mechanisms of PC and for preclinical screening of new therapies. Methods PC cell lines were developed from patient-derived PC cells. The tumorigenicity and metastatic potential were investigated by subcutaneously (PDXs) and orthotopically. Karyotyping, whole-exome sequencing, RNA-sequencing, and functional studies were performed to molecularly define the cell lines and compare genomic and phenotypic features of PDX and donor PC cells. Results We established three PC cell lines (GA0518, GA0804, and GA0825) and characterized them in vitro. The doubling times were 22, 39, and 37 h for GA0518, GA0804, and GA0825, respectively. Expression of cancer stem cell markers (CD44, ALDH1, CD133 and YAP1) and activation of oncogenes varied among the cell lines. All three PC cell lines formed PDXs. Interestingly, all three PC cell lines formed tumors in the patient derived orthotopic (PDO) model and GA0518 cell line consistently produced PC in mice. Moreover, PDXs recapitulated transcriptomic and phenotypic features of the donor PC cells. Finally, these cell lines were suitable for preclinical testing of chemotherapy and target agents in vitro and in vivo. Conclusion We successfully established three patient-derived PC cell lines and an improved PDO model with high incidence of PC associated with malignant ascites. Thus, these cell lines and metastatic PDO model represent excellent resources for exploring metastatic mechanisms of PC in depth and for target drug screening and validation by interrogating GAC for translational studies.

Purpose The identification of prognostically and therapeutically relevant molecular markers is fundamental to the further development of personalised therapies in brain tumours. Current therapeutic options for the treatment of gliomas rely mainly on surgical resection and the inhibition of tumour cell proliferation by irradiation and chemotherapy. Glioma stem cells are a subpopulation of proliferating tumour cells that have self-renewal capacity and can give rise to heterogeneous cells that comprise the tumour and are thought to play a role in the resistance of gliomas to therapy. The aim of this study was to evaluate the expression of markers of glioma stem cells and differentiated glial cells in proliferating glioma cells in comparison to the overall expression of the respective markers in the tumour tissue. Methods Tissue microarrays were assembled from specimen of pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, and anaplastic ependymoma. These were immunohistochemically double stained with antibodies against the proliferation-associated antigen Ki67 and marker proteins for glioma stem cells (CD133, Nestin, Musashi, CD15, CD44), and differentiated glioma cells (GFAP, MAP2c). Results The expression of both glial and glioma stem cell markers differs between proliferating and non-proliferating glioma cells. Furthermore, the proliferating cells in the different glial tumour entities show a different expression profile. Conclusion Further analysis of marker expression in proliferating glioma cells and correlation with clinical outcome and susceptibility to irradiation and chemotherapy might help establish new biomarkers and therapies for glioma.

Background Pancreatic adenocarcinoma (PAAD) has a high rate of mortality. Unfortunately, it is difficult to diagnosis. This study aimed to develop a more in-depth understanding of the disease. Methods A total of 177 patients with PAAD were recruited from The Cancer Genome Atlas (TCGA) database. Microarray analysis was performed to identify differentially expressed genes (DEGs) in PAAD. The microarray data were adapted to the ingenuity pathway analysis (IPA) for annotation and visualization, followed by protein-protein interaction (PPI) network analysis. In vitro transwell migration assays were conducted to explore the molecular and functional characteristics of pancreatic adenocarcinoma cells (PANC-1) with stable low expression of G-protein signaling modulator 2 (GPSM2). Expression of GPSM2 and the associated hub genes were detected by reverse transcription-quantitative polymerase chain reaction (qPCR). Results The overexpression of GPSM2 was proved in PAAD, as compared with the healthy tissues, as well as its correlation with history of chronic pancreatitis, T stage, TNM stage and tumor grade. We described it as an independent prognostic factor and found that it could influence the infiltration of immune cells in the tumor microenvironment. Silencing of GPSM2 restrained the and migration of the cells. Microarray analysis identified 1,631 DEGs in PAAD cells. The PPI network analysis identified hub genes including CD44, ITGB1, ITGB5, ITGA2, ITGA5, AKT1, EGFR, NRAS and MAP2K1, and their relationship with GPSM2 was confirmed by qPCR. Conclusions GPSM2 is a novel prognostic factor and therapeutic target for PAAD. GPSM2 promoted the migration of pancreatic adenocarcinoma cells .Targeting GPSM2 and its downstream genes may prolong the survival time of patients with PAAD.

The metabolism in tumors is reprogrammed to meet its energetic and substrate demands. However, this metabolic reprogramming creates metabolic vulnerabilities, providing new opportunities for cancer therapy. Metabolic vulnerability as a therapeutic target in esophageal squamous cell carcinoma (ESCC) has not been adequately clarified. Here, we identified pyruvate dehydrogenase (PDH) component X (PDHX) as a metabolically essential gene for the cell growth of ESCC. PDHX expression was required for the maintenance of PDH activity and the production of ATP, and its knockdown inhibited the proliferation of cancer stem cells (CSCs) and in vivo tumor growth. PDHX was concurrently upregulated with the CD44 gene, a marker of CSCs, by co‐amplification at 11p13 in ESCC tumors and these genes coordinately functioned in cancer stemness. Furthermore, CPI‐613, a PDH inhibitor, inhibited the proliferation of CSCs in vitro and the growth of ESCC xenograft tumors in vivo. Thus, our study provides new insights related to the development of novel therapeutic strategies for ESCC by targeting the PDH complex‐associated metabolic vulnerability., Inoue et al. found that pyruvate dehydrogenase (PDH) component X expression is necessary for PDH activity and is involved in the cancer stemness of esophageal squamous cell carcinoma (ESCC) cells, thereby being metabolically essential for the tumor growth of ESCC. This study provides new insights related to the development of novel therapeutic strategies for ESCC by targeting the PDH complex‐associated metabolic vulnerability.

HER2+ breast leptomeningeal carcinomatosis (HER2+ LC) occurs when tumor cells spread to cerebrospinal fluid–containing leptomeninges surrounding the brain and spinal cord, a complication with a dire prognosis. HER2+ LC remains incurable, with few treatment options. Currently, much effort is devoted toward development of therapies that target mutations. However, targeting epigenetic or transcriptional states of HER2+ LC tumors might efficiently target HER2+ LC growth via inhibition of oncogenic signaling; this approach remains promising but is less explored. To test this possibility, we established primary HER2+ LC (Lepto) cell lines from nodular HER2+ LC tissues. These lines are phenotypically CD326+CD49f−, confirming that they are derived from HER2+ LC tumors, and express surface CD44+CD24−, a cancer stem cell (CSC) phenotype. Like CSCs, Lepto lines showed greater drug resistance and more aggressive behavior compared with other HER2+ breast cancer lines in vitro and in vivo. Interestingly, the three Lepto lines overexpressed Jumonji domain–containing histone lysine demethylases KDM4A/4C. Treatment with JIB04, a selective inhibitor of Jumonji demethylases, or genetic loss of function of KDM4A/4C induced apoptosis and cell-cycle arrest and reduced Lepto cell viability, tumorsphere formation, regrowth, and invasion in vitro. JIB04 treatment of patient-derived xenograft mouse models in vivo reduced HER2+ LC tumor growth and prolonged animal survival. Mechanistically, KDM4A/4C inhibition downregulated GMCSF expression and prevented GMCSF-dependent Lepto cell proliferation. Collectively, these results establish KDM4A/4C as a viable therapeutic target in HER2+ LC and spotlight the benefits of targeting the tumorigenic transcriptional network. Significance: HER2+ LC tumors overexpress KDM4A/4C and are sensitive to the Jumonji demethylase inhibitor JIB04, which reduces the viability of primary HER2+ LC cells and increases survival in mouse models.

Primary central nervous system lymphomas (PCNSLs) often present a unique histopathological feature of aggregative perivascular tumor cells (APVT). Our previous studies showed that patients of PCNSL with APVTs exhibited poor long-term outcomes and increased expression of the endoplasmic reticulum stress (ERS) factor X-box-binding protein (XBP1). However, very little is known about molecular mechanism of the APVT formation in PCNSLs. The aim of this study is to determine if hypoxia-induced ERS is related to the APVT formation in PCNSLs. In this study, cell culture was used to observe the interplay between diffuse large B cell lymphoma (DLBCL) tumor cells and human brain microvascular endothelial cells (HBMECs) in different oxygen conditions. The expression of XBP1, CXCR and CD44 was manipulated by molecular cloning and siRNA technology. Mouse in vivo experiments and clinical studies were conducted to confirm our hypothesis. Our results showed that activated B-cell type-DLBCL cells easily migrated and invaded, and expressed high levels of XBP1 and stromal molecules CXCR4 and CD44 during hypoxia-induced ERS and dithiothreitol unfolded protein response (UPR). The gene upregulation (using overexpression vector) and downregulation (siRNA gene knock-out) in cultured cells and in mouse models further confirmed a close relation of the expression of XBP1, CXCR4, and CD44 with APVT formation, which is coincided with our clinical observation that increased expression of XBP1, CXCR4, and CD44 in the APVT cells in PCNSLs were associated with poor clinical outcomes. The results suggest that hypoxia-induced ERS and UPR might be associated with APVTs formation in PCNSL and its poor clinical outcomes. The results will help us better understand the progression of PCNSL with APVTs feature in daily pathological work and could be valuable for future target treatment of PCNSLs.

Mining databases and data obtained from assays on human specimens had shown that Fzd7 is closely associated with Wnt7b, that Fzd7/Wnt7b expression is upregulated in pancreatic cancer tissues compared with normal tissues, and its expression is negatively correlated with survival. Fzd7/Wnt7b knockdown in Capan‐2 and Panc‐1 cells reduced the proliferative capacity of pancreatic cancer stem cells (PCSCs), reduced drug resistance, decreased the percentage of CD24+CD44+ subset of cells and the levels of ABCG2, inhibited cell‐sphere formation, and reduced gemcitabine (GEM) resistance. In contrast, Fzd7/Wnt7b overexpression increased the percentage of the CD24+CD44+ subset of cells, and increased the levels of ABCG2 detected in cell spheroids. The gem‐resistant cells exhibited higher levels of Fzd7/Wnt7b expression, an increased percentage of CD24+CD44+ cells, and higher levels of ABCG2 compared with the parental cells. Taken together, Fzd7/Wnt7b knockdown can reduce PDAC cell stemness and chemoresistance by reducing the percentage of CSCs. Mechanistically, Fzd7 binds with Wnt7b and modulates the levels of β‐catenin, and they may exert their role via modulation of the canonical Wnt pathway., This study aims to elucidate the regulatory mechanism of stemness and chemoresistance in pancreatic cancer, especially the role of Wnt signaling pathway on stemness and chemoresistance of pancreatic cancer. Fzd7/Wnt7b can regulate the proportion of CSC subset, so that they influence stemness and chemoresistance.

OBJECTIVES: Cancer stem cells (CSCs) have been implicated in disease progression of aggressive cancers including small cell lung carcinoma (SCLC). Here, we have examined the possible contribution of CSCs to SCLC progression and aggressiveness.MATERIALS AND METHODS: GLC-14, GLC-16 and GLC-19 SCLC cell lines derived from one patient, representing increasing progressive stages of disease were used. CSC marker expressions was determined by RT-qPCR and western blotting analyses, and heterogeneity was studied by CSC marker expression by immunofluorescence microscopy and flow cytometry. Colony formation assays were used to assess stem cell properties and therapy sensitivity.RESULTS: Increasing expression of stem cell markers MYC, SOX2 and particularly CD44 were found in association with advancing disease. Single and overlapping expression of these markers indicated the presence of different CSC populations. The accumulation of more homogeneous double- and triple-positive CSC populations evolved with disease progression. Functional characterization of CSC properties affirmed higher proficiency of colony forming ability and increased resistance to γ-irradiation in GLC-16 and GLC-19 compared to GLC-14. GLC-19 colony formation was significantly inhibited by a human anti-CD44 antibody.CONCLUSION: The progressive increase of MYC, SOX2 and particularly CD44 expression that was accompanied with enhanced colony forming capacity and resistance in the in vitro GLC disease progression model, supports the potential clinical relevance of CSC populations in malignancy and disease relapse of SCLC.

The androgen receptor (AR) is important in the development of both experimental and human bladder cancer. However, the role of AR in bladder cancer growth and progression is less clear, with literature indicating that more advanced stage and grade disease are associated with reduced AR expression. To determine the mechanisms underlying these relationships, we profiled AR-expressing human bladder cancer cells by AR chromatin immunoprecipitation sequencing and complementary transcriptomic approaches in response to in vitro stimulation by the synthetic androgen R1881. In vivo functional genomics consisting of pooled shRNA or pooled open reading frame libraries was employed to evaluate 97 genes that recapitulate the direction of expression associated with androgen stimulation. Interestingly, we identified CD44, the receptor for hyaluronic acid, a potent biomarker and driver of progressive disease in multiple tumor types, as significantly associated with androgen stimulation. CRISPR-based mutagenesis of androgen response elements associated with CD44 identified a novel silencer element leading to the direct transcriptional repression of CD44 expression. In human patients with bladder cancer, tumor AR and CD44 mRNA and protein expression were inversely correlated, suggesting a clinically relevant AR–CD44 axis. Collectively, our work describes a novel mechanism partly explaining the inverse relationship between AR and bladder cancer tumor progression and suggests that AR and CD44 expression may be useful for prognostication and therapeutic selection in primary bladder cancer. Significance: This study describes novel AREs that suppress CD44 and an expected inverse correlation of AR-CD44 expression observed in human bladder tumors.

Asporin (ASPN), a small leucine‐rich proteoglycan expressed predominantly by cancer associated fibroblasts (CAFs), plays a pivotal role in tumor progression. ASPN is also expressed by some cancer cells, but its biological significance is unclear. Here, we investigated the effects of ASPN expression in gastric cancer cells. Overexpression of ASPN in 2 gastric cancer cell lines, HSC‐43 and 44As3, led to increased migration and invasion capacity, accompanied by induction of CD44 expression and activation of Rac1 and MMP9. ASPN expression increased resistance of HSC‐43 cells to oxidative stress by reducing the amount of mitochondrial reactive oxygen species. ASPN induced expression of the transcription factor HIF1α and upregulated lactate dehydrogenase A (LDHA) and PDH‐E1α, suggesting that ASPN reprograms HSC‐43 cells to undergo anaerobic glycolysis and suppresses ROS generation in mitochondria, which has been observed in another cell line HSC‐44PE. By contrast, 44As3 cells expressed high levels of HIF1α in response to oxidant stress and escaped apoptosis regardless of ASPN expression. Examination of xenografts in the gastric wall of ASPN–/– mice revealed that growth of HSC‐43 tumors with increased micro blood vessel density was significantly accelerated by ASPN; however, ASPN increased the invasion depth of both HSC‐43 and 44As3 tumors. These results suggest that ASPN has 2 distinct effects on cancer cells: HIF1α‐mediated resistance to oxidative stress via reprogramming of glucose metabolism, and activation of CD44‐Rac1 and MMP9 to promote cell migration and invasion. Therefore, ASPN may be a new therapeutic target in tumor fibroblasts and cancer cells in some gastric carcinomas., We demonstrate the importance of ASPN‐mediated resistance of tumor cells to oxidative stress. ASPN expression increased the levels of HIF1a, which shifted glucose metabolism to anaerobic glycolysis and reduced the amount of mitochondrial reactive oxygen species (mtROS), thereby attenuating ROS‐induced apoptosis.

To evaluate the validity of CHAC1 for predicting the prognosis of kidney renal clear cell carcinoma (KIRC) and to explore its therapeutic potential for KIRC, we conducted several bioinformatic analyses using the sequencing data and clinical information derived from online databases. We found CHAC1 is down‐regulated in KIRC samples when compared with normal samples but up‐regulated in KIRC samples with relatively higher malignancy and later stages. Univariate cox analysis and multivariate cox regression analysis were conducted and the results revealed up‐regulated CHAC1 is an independent risk factor for poor prognosis of KIRC. Further, the nomogram model based on the result of multivariate cox regression analysis was constructed and effectively predicted patients' 1‐year, 3‐year and 5‐year survival respectively. The correlation analyses showed CHAC1 is associated with the immune pathway markers of memory B cell, natural killer cell and type1 T helper cell as well as the checkpoint genes like ADORA2A, CD200, CD44, CD70, HHLA2, NRP1, PDCD1LG2 and TNFRSF18. Furthermore, experiments in vitro indicated CHAC1 could induce cell death in KIRC cell lines but had limited influence on cell migration and cell invasion. In conclusion, CHAC1 is found a valid indicator for poor prognosis of kidney renal clear cell carcinoma.

Background. Gastric cancer is the second leading cause of cancer-related death due to advanced disease. A special role in the pathogenesis and metastasis of the tumor is assigned to tumor stem cells (TSC ), responsible for resistance to chemotherapy and radiotherapy and causing tumor progression.Objective: to determine the CD 44 and CD 133 markers of TSC in tumor tissues of non-metastatic and metastatic gastric cancer using the immunohistochemical method.Material and Methods. A prospective study of tumors in patients with gastric cancer was conducted: Group 1 – 20 people with T3–4aN0–3M0G2 tumor, average age 58.9 ± 9.7; Group 2 – 20 people with T3–4aN0–3M1G2 tumor, with metastases in the peritoneum, average age 53.4 ± 11.9. The expression of CD 44 and CD 133 in the tumor tissue was determined by immunohistochemistry.Results. Differences were found in the number of tumor cells expressing the CD 44 marker in the presence and absence of metastases in patients with gastric cancer – their number was 10.0 ± 3.08 and 6.0 ± 2.3, respectively. The CD 133 molecule was detected in 100 % of cases having metastases, while in cases having no metastases, the marker was detected only in 80 % of cases. The average percentage of CD 133 + cells was 21.3 ± 11.6 % in patients with metastatic gastric cancer and 10.0 ± 2.4 % in patients having no metastases.Conclusion. The degree of expression of the CD 44 and CD 133 markers had characteristic differences in patients with gastric cancer, which can be used further to explain the results of the treatment and the prognosis of the disease.

Background/aim Pancreatic ductal adenocarcinoma is one of the deadliest forms of human cancer. Since only a vast panel of cell lines can fully recapitulate disease heterogeneity, our aim was to establish a new pancreatic cancer cell line. Materials and methods Newly established pancreatic ductal adenocarcinoma cell line Capan-26 was characterized by assessing growth rate, tumor and stem cell marker expression, colony forming efficiency, mutations of KRAS and TP53 genes, karyotype and sensitivity to drug treatment. Results Cell doubling time was 74 h. We detected CA19-9, CEACAM6, CD44, OCT4 and ZEB1 expression in Capan-26 cell line. Cells formed colonies in soft agar, have a deletion of KRAS exon 3 and a point mutation V172F in TP53 exon 5. They are a mixed aneuploid/polyploid population with high sensitivity to gemcitabine. Conclusion Capan-26 is a unique cell line that may be used to study the mechanism of pancreatic cancer.

Antiangiogenic therapy with bevacizumab (Bev), a monoclonal antibody targeting vascular endothelial growth factor (VEGF), is a common treatment for recurrent glioblastoma (GBM), but its survival benefit is limited. Resistance to Bev is thought to be a major cause of ineffectiveness on Bev therapy. To optimize Bev therapy, identification of a predictive biomarker for responsiveness to Bev is required. Based on our previous study, we focused on the expression and functions of CD44 and VEGF in the Bev therapy. Here, we analyze a relationship between CD44 expression and responsiveness to Bev and elucidate the role of CD44 in anti‐VEGF therapy. CD44 and VEGF expression in the tumor core and periphery of 22 GBMs was examined, and the relationship between expression of these molecules and progression‐free time on Bev therapy was analyzed. The degree of CD44 expression in the tumor periphery was evaluated by the ratio of the mRNA expression in the tumor periphery to that in the tumor core (P/C ratio). VEGF expression was evaluated by the amount of the mRNA expression in the tumor periphery. To elucidate the roles of CD44 in the Bev therapy, in vitro and in vivo studies were performed using glioma stem‐like cells (GSCs) and a GSC‐transplanted mouse xenograft model, respectively. GBMs expressing high P/C ratio of CD44 were much more refractory to Bev than those expressing low P/C ratio of CD44, and the survival time of the former was much shorter than that of the latter. In vitro inhibition of VEGF with siRNA or Bev‐activated CD44 expression and increased invasion of GSCs. Bev showed no antitumor effects in mice transplanted with CD44‐overexpressing GSCs. The P/C ratio of CD44 expression may become a useful biomarker predicting responsiveness to Bev in GBM. CD44 reduces the antitumor effect of Bev, resulting in much more highly invasive tumors., In this research, we analyze a relationship between CD44 expression and responsiveness to Bev and elucidate the role of CD44 in anti‐VEGF therapy. These results indicate CD44 will be a useful biomarker for predicting responsiveness to bevacizumab and may serve as a therapeutic target in both primary and recurrent GBMs.

International audience; Previous work identified Tissue Factor (TF), a key activator of the coagulation cascade, as a gene induced in cellular contexts of Epithelial-Mesenchymal Transitions (EMTs), providing EMT+ Circulating Tumor Cells (CTCs) with coagulant properties that facilitate their metastatic seeding. Deciphering further molecular aspects of TF regulation in tumor cells, we report here that CD44 and TF coexpress in EMT contexts, and that CD44 acts as a regulator of TF expression supporting procoagulant properties and metastatic seeding. A transcriptional regulatory mechanism bridging CD44 to TF expression was further evidenced. Comparing different TF –promoter luciferase reporter constructs, we indeed found that the shortest -111 pb TF promoter fragment harboring three Specificity Protein 1 (Sp1) binding sites is still responsive to CD44 silencing. The observation that (i) mutation within Sp1 binding sites decreased the basal activity of the -111 pb TF promoter construct, (ii) CD44 silencing decreased Sp1 protein and mRNA levels and (iii) Sp1 silencing diminished TF expression further points to Sp1 as a key mediator linking CD44 to TF regulation. All together, these data thus report a transcriptional regulatory mechanism of TF expression by CD44 supporting procoagulant activity and metastatic competence of CTCs.

Factor de impacto: 6,244 Q2 It is widely accepted that the tumor microenvironment, particularly the extracellular matrix, plays an essential role in the development of tumors through the interaction with specific protein-membrane receptors. One of the most relevant proteins in this context is the transmembrane protein CD44. The role of CD44 in tumor progression, invasion, and metastasis has been well established in many cancers, although a comprehensive review concerning its role in sarcomas has not been published. CD44 is overexpressed in most sarcomas and several in vitro and in vivo experiments have shown a direct effect on tumor progression, dissemination, and drug resistance. Moreover, CD44 has been revealed as a useful marker for prognostic and diagnostic (CD44v6 isoform) in osteosarcoma. Besides, some innovative treatments such as HA-functionalized liposomes therapy have become an excellent CD44-mediated intracellular delivery system for osteosarcoma. Unfortunately, the reduced number of studies deciphering the prognostic/diagnostic value of CD44 in other sarcoma subgroups, neither than osteosarcoma, in addition to the low number of patients involved in those studies, have produced inconclusive results. In this review, we have gone through the information available on the role of CD44 in the development, maintenance, and progression of sarcomas, analyzing their implications at the prognostic, therapeutic, and mechanistic levels. Moreover, we illustrate how research involving the specific role of CD44 in the different sarcoma subgroups could suppose a chance to advance towards a more innovative perspective for novel therapies and future clinical trials. This review was funded by the Instituto de Salud Carlos III, grant numbers PI20CIII/00020, DTS18CIII/00005, Asociacion Pablo Ugarte, grant numbers TRPV205/18; Asociación Candela Riera, Asociación Todos Somos Iván & Fundación Sonrisa de Alex, grant numbers TVP333-19, TVP-1324/15;ASION, grant number TVP141/17. R.M.M-FdM is supported for a grant of the Spanish Center for Biomedical Network Research on Rare Diseases (CIBERER). Sí

Breast cancer remains one of the leading cancers among women and an estimated 90% of breast cancer deaths are due to metastasis. Cancer stem cells (CSCs) are a sub-population of cancer cells which are responsible for its initiation, progression and metastasis. CSCs are resistant to conventional chemo- and radio-therapies and therefore therapeutic strategies targeting CSCs hold great potential for novel advances in cancer treatment. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic which was shown to effectively target breast CSCs. A library of 17 novel SAL analogs was synthesized and screened to identify compounds with improved selectivity against breast cancer stem cells. SAL analogs were either single modified esters or amides in the C1 position or double-modified C20-oxo derivatives. Eight single- and two double-modified analogs were more potent (IC50 range of 1.13 ± 0.19 to 3.93 ± 0.39 µM) towards the breast cancer cell line MDA-MB-231 compared to parent SAL (IC50 of 4.90 ± 1.60 µM). These analogs induced DNA fragmentation suggestive of apoptotic cell death. Compounds 2 (butyl ester analog of SAL) and 17 (double-modified C20-oxosalinomycin with benzhydroxamic acid) have been chosen for follow-up screening due to their improved activity and selectivity versus parent SAL. This included clonogenic assays to assess the ability of the compounds to affect cell renewal, and wound healing assays to assess cell migratory properties. In both assays, compound 17 showed superior properties over SAL. Furthermore, analog 17 showed improved targeting of breast CSCs in both cell monolayer and organoid culture as assessed in assays measuring the CD44+/CD24- stem cell sub-population. Analogs and parent compound were further studied examining (ADP-ribose) polymerase (PARP) cleavage and Bcl-2 levels by immunoblotting. All three compounds induced loss of 116 kDa PARP expression within 48h, with the highest effect induced by analog 17. In addition, treatment with compound 17 caused a decrease in Bcl-2 expression as early as 24 h. Select analogs were next screened against the NCI-60 Human Tumor Cell Line Panel. The described above double-modified analog was found to be more potent than SAL towards all 6 breast cancer cell lines in the panel as well as other tumor types. The present findings highlight the therapeutic potential of SAL analogs towards breast stem cells and support further research and clinical development of these compounds. Funding: The present study was funded by grants (to AM and TCC) from the Arkansas Breast Cancer Research Program. Testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, http://dtp.cancer.gov. Citation Format: Alicja Joanna Urbaniak, Megan R. Reed, Michał Antoszczak, Michał Sulik, Adam Huczyński, Robert L. Eoff, Melanie C. MacNicol, Timothy C. Chambers, Angus M. MacNicol. Inhibition of breast cancer stem cells in 2- and 3-dimensional culture by novel salinomycin analogs [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-46.

Breast cancer stem cells (BCSCs) have been implicated as the root of tumorinitiation, therapeutic resistance, relapse, and metastasis due to their uniqueabilities of self-renew, differentiation potentials and resistance to mostconventional therapies.Signaling pathways of stemness markers, Notch-1, ALDH1 andmicroRNAs, a class of emerging small non-coding RNAs, are importantregulators of BCSC functions and self-renewal capacity by modulatingmultiple biological processes via the post-transcriptional regulation of geneexpression and cellular signaling pathways. Understanding the earlymolecular mechanisms and identifying the miRNAs that are involved inchemo-resistance, and their crosstalk with Notch-1 and ALDH1, could be apotential therapeutic option for breast cancer drug resistant.Previously, we showed that Trigonella foenum (Fenugreek) extract (FCE),a traditional herbal plant, has an anti-tumor activity against HepG2 cell line.In the present study, we evaluated and screened a panel of aberrant expressedmiRNAs (i.e. miR-142, let-7a, let-7b and miR-34a) that might have a crosstalkwith Notch-1 and ALDH1 in isolated (CD44+/CD24−/ALDH1+)subpopulation treated with or without FCE extract alone or in combinationwith Doxorubicin for different time intervals.Our results revealed that FCE treatment alone for 48 h showed a significantdecrease in cell viability, clonogenicity and invasion capacity of(CD44+/CD24−/ALDH1+) BCSCs subpopulation compared to untreatedcells. Furthermore, FCE-inhibited Notch-1 and ALDH1 expression that wasassociated with a significant decrease in miR-142 by 4.6-folds and an increasein expression levels of let-7a, let-7b and miR-34a by 4.5, 7.7 and 15.4 foldsrespectively. Moreover, ectopic miR-34a expression significantly reduce cellproliferation and increase apoptotic induction consistent with downregulationof Notch-1 and ALDH1 expression in treated cells extract alone or incombination with Doxorubicin for 24h, suggesting that miR-34a expressionmight play a role in the breast cancer potential.Furthermore, FCE treated (CD44+/CD24−/ALDH1+) subpopulationectopically expressing miR-34a showed an increase in chemosensitivity todoxorubicin at low doses after 24 h compared to the controls. In initiated(CD44+/CD24−/ALDH1+) BCSC tumor xenografts ectopically expressingmiR-34a, FCE/Doxorubicin combined treatment showed a significant regression in tumor size by 62% after 72h compared to untreated controls orDoxorubicin alone.All in all, the aberrant expression of selected miRNAs, such as of miR-34a, might regulate a crosstalk with stemness characteristic markers, Notch-1and ALDH1, that are involved in the pathogenesis of breast cancer. Citation Format: Rana H. Ateya, Ahmed S. Sultan. Trigonella foenum (fenugreek) extract and ectopic miR-34a inhibit stemness characteristics and enhance sensitivity of (CD44+/CD24−/ALDH1+) breast cancer stem cells subpopulation to doxorubicin by targeting notch1/ALDH1signaling pathways [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-26.

Background and Aims: Chemotherapy is the main treatment for metastatic triple negative breast cancer (mTNBC) but many patients develop resistance and progress to metastatic disease, with a 5-year survival rate of 11%. Therefore, more effective and less toxic treatments are required. 20(S)-ginsenoside Rg3 (SRg3) and 20(R)-ginsenoside Rg3 (RRg3) are two pharmacologically active enantiomers extracted from Panax ginseng. In our studies, we have shown that these two enantiomers have stereoselective activities in inhibition of proliferation, migration and invasion of triple-negative breast cancer (TNBC) cells, and hence, could be considered as two separate drugs that can be combined in a synergistic dose (S+R). Based on the inhibitory effects of single enantiomers on loop formation of human umbilical vein endothelial cells (HUVEC), we have optimized the concentrations of Rg3 enantiomers to be combined that have shown time- and dose-dependent inhibition of loop formation and migration of human and mouse endothelial cell lines. The aim of the current study was to show the mechanisms of S+R in inhibition of angiogenesis in vitro and its efficacy in inhibiting metastasis in vivo. Methodology: Molecular docking was used to study the Rg3- vascular endothelial growth factor receptor (VEGFR) 2 interaction, and the VEGF bioassay kit (Promega) was used to show this interaction in vitro. ELISA, quantitative-PCR and western blot (WB) were used to study the expression of transcript and protein levels of signalling molecules. Two TNBC cell lines (MDA-MB-231 and HCC1143) were cultured as mammospheres and the effects of S+R on formation of mammospheres, viability (trypan blue assay and flow cytometric analysis of apoptosis or cell cycle arrest) and expression of CD44+ cells (flow cytometry) were studied. Expression microarrays were used to determine the effect of the treatment on the PI3K signalling pathway in MDA-MB-231. Nod Scid gamma mice were used to develop a murine model of metastatic breast cancer model. Luciferase-tagged MDA-MB-231 cells were injected into the 4th mammary fat pad of the mice and upon primary tumour establishment, treatment started. Tumour growth and metastasis was monitored using IVIS Spectrum. Statistical analysis was performed using ANOVA (Prism v8). Results: According to molecular docking, binding score of Rg3-VEGFR2 was -9.0 cal/mol. The VEGF bioassay showed that Rg3 is an allosteric modulator of VEGFR2 receptor. In HUVEC, S+R treatment decreased the expression of VEGF (ELISA) and its receptor VEGFR2. It also decreased the expression of pAKT:AKT, aquaporin 1 (AQP1) and pFAK:FAK (WB). These proteins are involved in cell survival, migration, invasion and proliferation. In breast cancer cell lines, S+R decreased the expression of AQP1 and decreased mammosphere formation efficiency, not by decreasing cell viability but by decreasing the expression of CD44+ stem cell marker. In expression arrays, the treatment decreased the expression of BTK, a tyrosine kinase promoting cell death escape; increased the expression of both CASP9, promoting apoptosis, and INPPD5, favouring inhibition of metastasis. In vivo experiments showed that S+R lead to tumour shrinkage (p < .0001), decreased body burden of tumour (p < .0001) and decreased number of metastases (p = .002). Conclusion: These results support the anti-cancer effects of the combination of S+R for the treatment of mTNBC. Citation Format: Maryam Nakhjavani, Eric Smith, Timothy Price, Amanda R Townsend, Jennifer E Hardingham. Ginsenoside rg3 enantiomers in a defined ratio as a novel treatment for metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-38.

The membrane transporters activated in cancer stem cells (CSCs) are the target of novel cancer therapies for gastric cancer. The present study investigated ion channel expression profiles in gastric CSCs (GCSCs). Cells strongly expressing CD44 were separated from MKN74 cells, a human gastric cancer cell line, by fluorescence-activated cell sorting (FACS), and GCSCs were identified based on tumorsphere formation. Gene expression profiles in GCSCs were examined by a microarray analysis. Among MKN74 cells, CD44 messenger RNA levels were higher in CSCs than in non-CSCs. These CSCs also exhibited resistance to cisplatin. The microarray analysis revealed that the expression of several genes related to voltage-gated Ca2+ channels (VGCCs), including CACNA2D1 and CACNB4, was upregulated. The cytotoxicities of the CACNA2D1 inhibitor amlodipine and the CACNB4 inhibitor verapamil were greater at lower concentrations in CSCs than in non-CSCs, and markedly reduced tumorsphere numbers. Tumor volumes were significantly smaller in a xenograft nude mouse model treated with amlodipine or verapamil in combination with cisplatin than in that treated with cisplatin alone. The present results indicate that VGCCs play a role in maintaining CSCs, and demonstrated the potential of their specific inhibitors, amlodipine and verapamil, as targeted therapeutic agents against gastric cancer.

5‐Fluorouracil (5‐FU) is one of the most frequently used pharmacological agents in the treatment of colorectal cancer (CRC). Resistance to chemotherapy is a major cause of treatment failure of CRC, and it is a well known fact that cancer stem cells play a significant role in the acquisition of drug resistance. In this study, we focused on the KHDRBS3 gene that encodes KH RNA Binding Domain Containing, Signal Transduction Associated 3. We first clarified the relationship between KHDRBS3 and 5‐FU resistance. We then observed higher expression levels of KHDRBS3 in KRAS‐mutant organoids and cell lines in comparison with KRAS wild‐type organoids and cell lines. Immunohistochemical analysis using CRC cases revealed that the prognosis of KHDRBS3‐positive patients was significantly worse compared with that of KHDRBS3‐negative patients. Univariate and multivariate Cox proportional hazards analyses showed that KHDRBS3 was an independent prognostic factor in patients with CRC. We determined that KHDRBS3 might play a crucial role in the acquisition of stem cell properties, such as drug resistance and spheroid/organoid formation, by regulating CD44 variant expression and the Wnt signaling pathway. In an immunodeficient mouse model, KHDRBS3‐positive cells showed efficient tumor formation and formed metastatic lesions in the lungs. These results indicated that KHDRBS3 plays a crucial role in drug resistance and anchorage‐independent growth by maintaining stem cell‐like features in CRC cells. KHDRBS3 could be a promising candidate marker for predicting chemotherapeutic effect and prognosis in CRC patients., KHDRBS3 plays a crucial role in drug resistance and anchorage‐independent growth by maintaining stem cell‐like features in CRC cells. KHDRBS3 could be a promising candidate marker for predicting chemotherapeutic effect and prognosis in CRC patients.

Background Gastric cancer (GC) is considered one of the most lethal malignancies worldwide, which is accompanied by a poor prognosis. Although reports regarding the importance of cancer stem cell (CSC) markers in gastric cancer progression have rapidly developed over the last few decades, their clinicopathological and prognostic values in gastric cancer still remain inconclusive. Therefore, the current meta-analysis aimed to quantitatively re-evaluate the association of CSC markers expression, overall and individually, with GC patients’ clinical and survival outcomes. Methods Literature databases including PubMed, Scopus, ISI Web of Science, and Embase were searched to identify the eligible articles. Hazard ratios (HRs) or odds ratios (ORs) with 95% confidence intervals (CIs) were recorded or calculated to determine the relationships between CSC markers expression positivity and overall survival (OS), disease-free survival (DFS)/relapse-free survival (RFS), disease-specific survival (DSS)/ cancer-specific survival (CSS), and clinicopathological features. Results We initially retrieved 4,425 articles, of which a total of 66 articles with 89 studies were considered as eligible for this meta-analysis, comprising of 11,274 GC patients. Overall data analyses indicated that the overexpression of CSC markers is associated with TNM stage (OR = 2.19, 95% CI 1.84–2.61, P = 0.013), lymph node metastasis (OR = 1.76, 95% CI 1.54–2.02, P P P P P = 0.035). Sub-analysis demonstrated a significant positive relationship between most of the individual markers, specially Gli-1, Oct-4, CD44, CD44V6, and CD133, and clinical outcomes as well as the reduced survival, whereas overexpression of Lgr-5, Nanog, and sonic hedgehog (Shh) was not found to be related to the majority of clinical outcomes in GC patients. Conclusion The expression of CSC markers is mostly associated with worse outcomes in patients with GC, both overall and individual. The detection of a combined panel of CSC markers might be appropriate as a prognostic stratification marker to predict tumor aggressiveness and poor prognosis in patients with GC, which probably results in identifying novel potential targets for therapeutic approaches.