Cancer Stem Cell Markers CD44 Research Articles

Abstract B047: Advanced glycation end products promote prostate tumor growth and are a potential biologic consequence of lifestyle factors contributing to cancer disparity

Abstract Advanced glycation end-products (AGEs), are reactive metabolites produced endogenously as a consequence of glucose uptake during glycolysis. AGEs accumulate in tissues and organs as we grow older to promote multiple chronic disease phenotypes. AGE pathogenic effects are mediated through modification of protein function, genetic fidelity, stress responses and cellular signaling pathways. Critically, cancer disparity factors such as a sedentary lifestyle, obesity and an unhealthy diet are external influences that also contribute to the AGE accumulation pool in the body. This research group examined circulating and tumor AGE levels in clinical specimens of prostate cancer and identified a race specific, tumor-dependent pattern of accumulation. AGE levels were highest in aggressive tumors, especially those from men with African ancestry. Increased AGE levels correlated with an upregulation in the receptor for advanced glycation end products (RAGE) and activated NFkB. In a syngeneic subcutaneous prostate cancer mouse model, chronic consumption of AGE resulted in a 3-fold increase in tumor growth. Strikingly, dietary-AGE mediated increases in tumor growth were accompanied by a cytoplasmic accumulation of AR, elevation in MYC, RAGE, and AGE as well as increased cell proliferation. Cytoplasmic accumulation of AR is elevated in CRPC tissue and is an independent predictor of biochemical recurrence. Our data show that treatment of prostate cancer cells with AGEs induces a neuroendocrine differentiated phenotype by promoting a more mesenchymal morphology and increasing the expression of associated marker genes including ENO2, MYC and SYP and the prostate cancer stem cell marker CD44 as well as the downregulation of AR. The aberrant activation and recruitment of immune cells is a major pathogenic consequence of AGE accumulation, and a series of studies have highlighted the tumor-associated immune response as a critical pathway contributing to cancer disparity. Using patient-derived primary tumor cells, the investigators found that AGEs released into the extracellular matrix may recapitulate the tumor-associated immune response observed in ancestry-specific prostate tumor tissues. Further preliminary studies indicate that AGE treatment of prostate cancer cells can alter how cancer cells metabolize glucose to promote an aggressive phenotype. The investigators' studies support the concept that AGE metabolites represent a biologic consequence of the socioeconomic and environmental factors that promote cancer health disparity. As our understanding of tumor biology advances, it is becoming increasingly clear that these inter-related lifestyle factors have distinct molecular consequences on the biologic makeup of tumors, altering cell signaling events and gene expression profiles to contribute to cancer disparity outcomes such as its earlier development or its progression to more aggressive disease. Citation Format: Pamela Woods, Bradley A. Krisanits, Dion Foster, Lourdes M. Nogueira, Laura Spruill, Marvella E. Ford, Michael B. Lilly, Victoria J. Findlay, David P. Turner. Advanced glycation end products promote prostate tumor growth and are a potential biologic consequence of lifestyle factors contributing to cancer disparity [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B047.

MicroRNA-93 targets WASF3 and functions as a metastasis suppressor in breast cancer.

Cancer cells with cancer stem cell (CSC) properties initiate both primary tumor formation and metastases at distant sites. Acquisition of CSC properties is highly associated with epigenetic alterations, including those mediated by microRNAs (miRNAs). We have previously established the breast cancer patient‐derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, we found that the expression levels of 3 miRNAs (miR‐25, miR‐93, and miR‐106b) in the miR‐106b‐25 cluster were much lower in the CD44+ human cancer cells metastasized to the liver than those at the primary site. Constitutive overexpression of miR‐93 suppressed invasive ability and 3D‐organoid formation capacity of breast cancer cells in vitro and significantly suppressed their metastatic ability to the liver in vivo. Wiskott‐Aldrich syndrome protein family member 3 (WASF3), a regulator of both cytoskeleton remodeling and CSC properties, was identified as a functional target of miR‐93: overexpression of miR‐93 reduced the protein level of WASF3 in breast cancer cells and WASF3 rescued the miR‐93‐mediated suppression of breast cancer cell invasion. These findings suggest that miR‐93 functions as a metastasis suppressor by suppressing both invasion ability and CSC properties in breast cancers.

Expression of cancer stem cells markers in urinary bladder urothelial carcinoma and its precursor lesions

Cancer stem cells (CSC) and their role in tumorigenesis of various solid tumors have been studied in past decades. Urothelial CSC were first identified 10 years ago and subsequent studies have been performed with the aim to identify reliable markers of CSC. So far, a few studies have investigated a relationship between CSC markers expression in urothelial carcinoma tissue and histopathological characteristics of the tumor. In our study, we evaluated an immunoexpression of the CSC markers CD24, CD44, CD66 and CD133 in tissue sections of urothelial carcinoma (all tumor grades and stages were included), urothelial carcinoma in situ and non-neoplastic urothelium, totally 218 specimens were enrolled. All studied molecules were expressed either in tumor tissue and non-neoplastic urothelium. Urothelial carcinomas of higher tumor grade and stage expressed molecules CD24 and CD133 significantly more frequently whereas molecules CD44 and CD66 did not show significant association with tumor histopathological features. Our results showed that studied molecules are not suitable for direct detection of CSC in urothelial carcinoma tissue sections, but an expression of molecules CD24 and CD133 is significantly related to urothelial carcinoma grade and stage, which are both important prognostic indicators and therefore an expression of these markers might have a potential prognostic value.

The Role of Cancer Associated Fibroblasts in Maintaining Cancer Stem Cells

Breast cancer is one of the leading causes of cancer related deaths in females. Current treatment options for breast cancer ultimately fail, resulting in cancer relapse. One explanation for recurrence after treatment is the presence of cancer stem cells (CSCs). CSCs have high proliferative potential and are able to initiate tumor growth. In order to target CSCs, it is important to understand how CSCs are maintained. Recent studies have shown that cancer‐associated fibroblasts (CAFs) aid in the maintenance of CSCs. CAFs are an abundant cell type in the tumor microenvironment and have been found to promote tumor growth and confer resistance to anti‐cancer drugs by supporting cancer stem cells. We hypothesized that CAFs promote and maintain the growth of cancer stem cells in a novel tumor model (MCaP). Mammary carcinoma cells (MCaP) were propagated in culture from a tumor that developed spontaneously in an aged mouse. Cancer cells, along with CAFs were expanded from the isolated tumor. Cancer cells were grown as a mixed culture with CAFs or alone after enrichment. A colony growth assay was used to assess the formation of CSCs, which grow as colonies. Cultures were fixed and stained with cytokeratin and colonies were counted manually. Colony size was quantified using ImageJ. Flow cytometry was performed to detect and quantify surface expression of CSC markers CD44 and CD24 in mixed cultures and isolated cancer cells. To investigate whether CAFs enable cancer cell resistance to chemotherapy, we measured the dose response to cyclophosphamide or paclitaxel. Immunofluorescence was performed on tissue sections of metastatic lymph node and primary tumor. Tissues were stained with cytokeratin, aldehyde dehydrogenase (ALDH), and alpha‐sma (SMA). From the colony growth assay we noticed that colony formation and the size of colonies were both increased when MCaP cells were cultured with CAFs. Flow cytometry showed that MCaP cancer cells grown with CAFs resulted in the appearance of a CD44 high/CD24low population. This CSC population was not seen in cancer cells grown alone. After treating cells with paclitaxel or cyclophosphamide, we saw no notable difference in the viability of cancer cells in the presence or absence of CAFs. Immunofluorescence staining showed ALDH was expressed in SMA‐positive CAFs, but not in cytokeratin‐positive cancer cells. Although we did not find that CAFs protected cancer cells from chemotherapy, CAFs appear to have a positive effect on the size and quantity of CSC colonies. In addition, we detected a CD44 high, CD24 low CSC population in mixed cultures. CAFs were found to express the CSC marker ALDH, with expression increasing as the size of the tumor increased. This finding suggests that CAFs may provide a niche for CSCs. In conclusion, these results suggest that CAFs play an important role in maintaining CSCs in the MCaP mouse model.Support or Funding InformationThe University of the Virgin Islands MARC grant (GM008422)and The Jones lab ACS Pilot grant (K22CA230315)

DAXX inhibits cancer stemness and epithelial\u2013mesenchymal transition in gastric cancer

BackgroundDAXX is a transcription repressor that has been implicated in several types of cancers, but its role in the development of gastric cancer remains unknown.MethodsWe analysed the expression of DAXX in 83 pairs of gastric cancer samples, including neoplastic and adjacent tissues, and correlated the expression levels with clinical stages. We also investigated the molecular mechanisms by which DAXX downregulation promotes cancer growth using both in vitro and in vivo models.ResultsDAXX was downregulated in advanced gastric cancer samples. The expression of DAXX inversely correlates with that of cancer stem cell markers CD44 and Oct4 in gastric cancer lines. DAXX overexpression in gastric cancer cells inhibited migration, invasion and epithelial– mesenchymal transition (EMT). The inhibition of EMT was achieved through the repression of SNAI3, a key inducer of EMT, by recruiting HDAC-1 into the nucleus. Using a xenograft mouse model, we demonstrated that the MKN45 cells formed smaller tumours when DAXX was overexpressed. Wild-type AGS cells were not able to form tumours in nude mice, but in contrast, formed visible tumours when DAXX was silenced in the cells.ConclusionWe for the first time demonstrated that DAXX functions as a tumour suppressor in gastric cancer by inhibiting stem cell growth and EMT.

Analysis of Dual Class I Histone Deacetylase and Lysine Demethylase Inhibitor Domatinostat (4SC-202) on Growth and Cellular and Genomic Landscape of Atypical Teratoid/Rhabdoid.

Central nervous system atypical teratoid/rhabdoid tumors (ATRTs) are rare and aggressive tumors with a very poor prognosis. Current treatments for ATRT include resection of the tumor, followed by systemic chemotherapy and radiation therapy, which have toxic side effects for young children. Gene expression analyses of human ATRTs and normal brain samples indicate that ATRTs have aberrant expression of epigenetic markers including class I histone deacetylases (HDAC’s) and lysine demethylase (LSD1). Here, we investigate the effect of a small molecule epigenetic modulator known as Domatinostat (4SC-202), which inhibits both class I HDAC’s and Lysine Demethylase (LSD1), on ATRT cell survival and single cell heterogeneity. Our findings suggest that 4SC-202 is both cytotoxic and cytostatic to ATRT in 2D and 3D scaffold cell culture models and may target cancer stem cells. Single-cell RNA sequencing data from ATRT-06 spheroids treated with 4SC-202 have a reduced population of cells overexpressing stem cell-related genes, including SOX2. Flow cytometry and immunofluorescence on 3D ATRT-06 scaffold models support these results suggesting that 4SC-202 reduces expression of cancer stem cell markers SOX2, CD133, and FOXM1. Drug-induced changes to the systems biology landscape are also explored by multi-omics enrichment analyses. In summary, our data indicate that 4SC-202 has both cytotoxic and cytostatic effects on ATRT, targets specific cell sub-populations, including those with cancer stem-like features, and is an important potential cancer therapeutic to be investigated in vivo.

Metformin treatment decreases the expression of cancer stem cell marker CD44 and stemness related gene expression in primary oral cancer cells

ObjectiveMetformin, a common drug for diabetes treatment has shown promising characteristics against wide types of cancer cells in vitro as well as in vivo in the context of halted growth of cancer. But, it was unclear whether cancer stem cells are affected by the metformin treatment. Here, we attempt to find out the effect of metformin on cancer stem cell marker CD44 and stemness related transcription factors including OCT4, SOX2, NANOG, c-Myc and KLF4. Materials and MethodsWe prepared single-cell suspension from primary oral tumors and subjected the cells to grow in vitro. Gene expression of transcription factors was assessed by real-time PCR. Further, the expression of CD44 was checked by flow Cytometry. ResultsMetformin showed downregulation in the gene expressions of stemness related transcription factors OCT4, SOX2, NANOG, c-Myc, and KLF4 in a dose-dependent as well as time-dependent manner. Also, the most effective concentration of metformin at 25 μM was found to decrease the expression of CD44 in the primary tumor cells in a time-dependent manner. ConclusionContinuous treatment of lower concentrations of metformin decreases the expression of cancer stem cell markers at the transcription level and cancer stem cell-surface marker CD44 in primary oral cancer cells.

NEAT1 is Required for the Expression of the Liver Cancer Stem Cell Marker CD44

CD44, a cancer stem cell (CSC) marker, is required for maintaining CSC properties in hepatocellular carcinoma (HCC). Nuclear enriched abundant transcript 1 (NEAT1), a long noncoding RNA (lncRNA), is an oncogenic driver in HCC. In the present study, we investigated the significance of the NEAT1 gene in association with CD44 expression in liver CSCs of human HCC cell lines. The CSC properties were evaluated by spheroid culture, CSC marker expression, and sensitivity to anti-cancer drugs. The expression of both NEAT1 variant 1 (NEAT1v1) and variant 2 (NEAT1v2) as well as CD44 was significantly increased in the spheroid culture, compared with that in monolayer culture. Overexpression of Neat1v1, but not Neat1v2, enhanced the CSC properties, while knockout of the NEAT1 gene suppressed them. CD44 expression was increased by the overexpression of Neat1v1 and abrogated by NEAT1 knockout. The overexpression of NEAT1v1 restored the CSC properties and CD44 expression in NEAT1-knockout cells. NEAT1v1 expression in HCC tissues was correlated with poor prognosis and CD44 expression. These results suggest that NEAT1v1 is required for CD44 expression. To our surprise, NEAT1v1 also restored the CSC properties even in CD44-deficient cells, suggesting that NEAT1v1 maintains the properties of CSCs in a CD44-independent manner.

Induction of heparanase via IL-10 correlates with a high infiltration of CD163+ M2-type tumor-associated macrophages in inflammatory breast carcinomas

Inflammatory breast cancer (IBC) is the most aggressive and lethal form of breast cancer, characterized by a high infiltration of tumor-associated macrophages and poor prognosis. To identify new biomarkers and to elucidate the molecular mechanisms underlying IBC pathogenesis, we investigated the expression pattern of heparanase (HPSE) and its activator cathepsin L (CTSL). First, we quantitated the HPSE and CTSL mRNA levels in a cohort of breast cancer patients after curative surgery (20 IBC and 20-non-IBC). We discovered that both HPSE and CTSL mRNA levels were significantly induced in IBC tissue vis-à-vis non-IBC patients (p <0 .05 and p <0 .001, respectively). According to the molecular subtypes, HPSE mRNA levels were significantly higher in carcinoma tissues of triple negative (TN)-IBC as compared to TN-non-IBC (p <0 .05). Mechanistically, we discovered that pharmacological inhibition of HPSE activity resulted in a significant reduction of invasiveness in the IBC SUM149 cell line. Moreover, siRNA-mediated HPSE knockdown significantly downregulated the expression of the metastasis-related gene MMP2 and the cancer stem cell marker CD44. We also found that IBC tumors revealed robust heparanase immune-reactivity and CD163+ M2-type tumor-associated macrophages, with a positive correlation of both markers. Moreover, the secretome of axillary tributaries blood IBC CD14+ monocytes and the cytokine IL-10 significantly upregulated HPSE mRNA and protein expression in SUM149 cells. Intriguingly, massively elevated IL-10 mRNA expression with a trend of positive correlation with HPSE mRNA expression was detected in carcinoma tissue of IBC. Our findings highlight a possible role played by CD14+ monocytes and CD163+ M2-type tumor-associated macrophages in regulating HPSE expression possibly via IL-10. Overall, we suggest that heparanase, cathepsin L and CD14+ monocytes-derived IL-10 may play an important role in the pathogenesis of IBC and their targeting could have therapeutic implications.

MAOA-mediated reprogramming of stromal fibroblasts promotes prostate tumorigenesis and cancer stemness.

The tumor microenvironment plays a critical role in prostate cancer (PC) development and progression. Inappropriate activation of the stroma potentiates the growth and transformation of epithelial tumor cells. Here, we show that upregulation of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines, in stromal cells elevates production of reactive oxygen species, triggers an inflammatory response including activation of IL-6, and promotes tumorigenesis in vitro and in vivo. Mechanistically, MAOA enhances IL-6 transcription through direct Twist1 binding to a conserved E-box element at the IL-6 promoter. MAOA in stromal fibroblasts provides tumor cell growth advantages through paracrine IL-6/STAT3 signaling. Tissue microarray analysis revealed co-expression correlations between individual pairs of proteins of the stromal MAOA-induced Twist1/IL-6/STAT3 pathway in clinical specimens. Downstream of stromal MAOA, STAT3 also promotes cell stemness and transcriptionally activates expression of cancer stem cell marker CD44 in PC cells. MAOA inhibitor treatment effectively suppressed prostate tumor growth in mice in a stroma-specific targeted manner. Collectively, these findings characterize the contribution of MAOA to stromal activation in PC pathogenesis and provide a rationale for targeting MAOA in stromal cells to treat PC.

Relationship between Cancer Stem Cell Marker CD133 and Cancer Germline Antigen Genes in NCI-H292 Lung Cancer Cells.

BackgroundPrevious studies have shown that lung cancer stem cells express CD133 and that certain cancer stem cells express cancer germline antigens (CGAs). The transcriptional regulation of CD133 is complicated and poorly understood. We investigated CD133 and CGA expression in a non-small cell lung cancer cell line.MethodsThe expression levels of CD133 and CGAs (MAGE-6, GAGE, SSX, and TRAG-3) were measured in an NCI-H292 lung cancer cell line. The methylation status of the CD133 gene promoter region was analyzed. The expression levels and promoter methylation statuses of CD133 and CGAs were confirmed by treatment with the demethylating agent 5-aza-2′-deoxycytidine (ADC).ResultsAfter treatment with ADC, CD133 expression was no longer detected. MAGE-6 and TRAG-3 were detected before ADC treatment, while GAGE and SSX were not detected. ADC treatment upregulated MAGE-6 and TRAG-3 expression, while GAGE expression was still undetected after treatment, and only weak SSX expression was observed. GAGE expression was not correlated with expression of CD133, while the levels of expression of MAGE-6, TRAG-3, and SSX were inversely correlated with CD133 expression.ConclusionThese results showed that CD133 expression can be regulated by methylation. Thus, the demethylation of the CD133 promoter may compromise the treatment of lung cancer by inactivating cancer stem cells and/or activating CGAs.

Inhibition of Y Box Binding Protein 1 Suppresses Cell Growth and Motility in Colorectal Cancer.

Although chemo- or radiotherapy is usually performed in patients with colorectal cancer, the response is highly variable in locally rectal cancer. Therefore, additional studies are needed on predictable markers and the molecular mechanisms of chemo- and radiotherapy. Y box binding protein 1 (YB1) is an oncoprotein that is aberrantly expressed in many cancers, including colorectal cancer. However, to date there are no targeting agents or strategies to inhibit YB1 expression. Here, we investigate the oncogenic function of YB1 in colorectal cancer and methods to control its expression. We observed that YB1 expression level is correlated with colorectal cancer survival rate. Moreover, YB1 overexpression was associated with colorectal cancer lymph node metastasis and invasion. We also found that radiation exposure increased YB1 expression, which led to radioresistant colorectal cancer, mediated through the activation of cancer stem cell marker CD44 and PI3K/AKT/mTOR signaling. This study revealed, by both in vitro and in vivo assays, that depletion of YB1 could reduce cell proliferation and motility in colorectal cancer. We further demonstrated that the PI3K/mTOR inhibitor BEZ235 suppressed YB1 expression and enhanced the cytotoxicity of radiation. In addition, combined treatment with BEZ235 and radiation showed a significant antitumor response in an in vivo mouse xenograft model. Taken together, our results provide evidence that the activation of YB1 is a major factor in radioresistance and suggest that targeting YB1-mediated signaling is a promising therapeutic strategy for colorectal cancer.

Slug overexpression and association with clinicopathological features in gastric cancer.

426 Background: Slug is a suppressive transcriptional factor of E-cadherin, acting as an activator of epithelial-mesenchymal transition(EMT). Its clinical relevance in gastric cancer(GC) is not fully known. Methods: Our study evaluated the expression patterns of EMT and cancer stem cell markers in GC patients who had clinical stage 2-3, underwent gastrectomy, D2 lymph node dissection (LND), adjuvant chemotherapy. Immunohistochemistry of E-cadherin, vimentin, CD133, ABCG2, NEDD9, SMAD4, XB130, Slug, Snail were investigated from 210 gastric cancer samples using tissue microarrays. The correlation between each markers expressed and the association with clinicopathological factors were analyzed. Results: Slug expression was more frequent in stage 3 than stage 2 (p=0.000), advanced T (p=0.007) and N stage (p=0.001), while histologic type did not make difference. Slug expression correlated with the expression of cancer stem cell marker CD133 (r=0.180, p=0.015) and CD133 expression was also related with ABCG2 (r=0.412, p=0.000). High Slug group showed shorter overall survival, compared to low Slug group (median OS 134 vs 124 months, p=0.044). The 2-year and 5-year disease-free (DF) rate for patients with high Slug and low Slug was 87.1% and 79.8%, 68.1% and 79.8%, respectively(p=0.038). The DFS curve reached an earlier plateau at 11-month in low Slug group, while in high Slug group took as long as 99 months. A multivariate analysis using the Cox proportional hazards regression model demonstrated Slug to be an independent prognostic factor for overall survival; hazard ratio 0.504 [95% CI 0.278-0.916] (p=0.025). Conclusions: In stage 2-3 GC patients who underwent gastrectomy with D2 LND and adjuvant chemotherapy, high Slug expression is associated with better disease-free and overall survival. Patients may benefit by testing Slug immunohistochemistry to predict prognosis after gastrectomy.

Suppressing STAT5 signaling affects osteosarcoma growth and stemness

Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and induced sub G0/G1 cell cycle arrest and apoptosis. There was a reduction in cyclin D1 and CDK2 expression and Rb phosphorylation, and activation of Caspase-3 and PARP cleavage. In addition, pimozide suppressed the formation of 3-dimensional osteospheres and growth of the cells in the Tumor in a Dish lung organoid system. Furthermore, there was a reduction in expression of cancer stem cell marker proteins DCLK1, CD44, CD133, Oct-4, and ABCG2. More importantly, it was the short form of DCLK1 that was upregulated in osteospheres, which was suppressed in response to pimozide. We further confirmed by flow cytometry a reduction in DCLK1+ cells. Moreover, pimozide inhibits the phosphorylation of STAT5, STAT3, and ERK in OS cells. Molecular docking studies suggest that pimozide interacts with STAT5A and STAT5B with binding energies of −8.4 and −6.4 Kcal/mol, respectively. Binding was confirmed by cellular thermal shift assay. To further understand the role of STAT5, we knocked down the two isoforms using specific siRNAs. While knockdown of the proteins did not affect the cells, knockdown of STAT5B reduced pimozide-induced necrosis and further enhanced late apoptosis. To determine the effect of pimozide on tumor growth in vivo, we administered pimozide intraperitoneally at a dose of 10 mg/kg BW every day for 21 days in mice carrying KHOS/NP tumor xenografts. Pimozide treatment significantly suppressed xenograft growth. Western blot and immunohistochemistry analyses also demonstrated significant inhibition of stem cell marker proteins. Together, these data suggest that pimozide treatment suppresses OS growth by targeting both proliferating cells and stem cells at least in part by inhibiting the STAT5 signaling pathway.

Adipose-derived stem cells and cancer cells fuse to generate cancer stem cell-like cells with increased tumorigenicity.

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells isolated from adipose tissue and have the ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Despite their great therapeutic potentials, previous studies showed that ADSCs could enhance the proliferation and metastatic potential of breast cancer cells (BCCs). In this study, we found that ADSCs fused with BCCs spontaneously, while breast cancer stem cell (CSC) markers CD44+ CD24-/low EpCAM+ were enriched in this fusion population. We further assessed the fusion hybrid by multicolor DNA FISH and mouse xenograft assays. Only single nucleus was observed in the fusion hybrid, confirming that it was a synkaryon. In vivo mouse xenograft assay indicated that the tumorigenic potential of the fusion hybrid was significantly higher than that of the parent tumorigenic triple-negative BCC line MDA-MB-231. We had compared the fusion efficiency between two BCC lines, the CD44-rich MDA-MB-231 and the CD44-poor MCF-7, with ADSCs. Interestingly, we found that the fusion efficiency was much higher between MDA-MB-231 and ADSCs, suggesting that a potential mechanism of cell fusion may lie in the dissimilarity between these two cell lines. The cell fusion efficiency was hampered by knocking down the CD44. Altogether, our findings suggest that CD44-mediated cell fusion could be a potential mechanism for generating CSCs.

L1 Cell Adhesion Molecule Confers Radioresistance to Ovarian Cancer and Defines a New Cancer Stem Cell Population

Many solid tumors, including ovarian cancer, contain small populations of cancer stem cells (CSCs). These cells are usually resistant against conventional cancer therapies and play a role in disease recurrence. We demonstrated that the L1 cell adhesion molecule (L1CAM) is a new CSC target in ovarian cancer, triggering radioresistance. Using fluorescence-activated cell sorting, specific cell populations expressing L1CAM alone or in combination with the established CSC marker CD133 were isolated from three ovarian cancer cell lines. Double-positive L1CAM+/CD133+ cells displayed higher spherogenic and clonogenic properties in comparison to L1CAM−/CD133− cells. Furthermore, L1CAM+/CD133+ cells retained highest clonogenic capacity after irradiation and exhibited up-regulation of some CSC-specific genes, enhanced tumor-initiating capacity, self-renewal and higher tumor take rate in nude mice when compared with other cell populations. Superior radioresistance by L1CAM expression was confirmed by deletion of L1CAM using CRISPR-Cas9 technology. Moreover, we found expression signatures associated with epithelial-to-mesenchymal transition phenotype in L1CAM deleted cells. These results indicate that L1CAM in combination with CD133 defines a new cancer cell population of ovarian tumor-initiating cells with the implication of targeting L1CAM as a novel therapeutic approach for ovarian CSCs.

The clinicopathological and prognostic value of CD44 expression in bladder cancer: a study based on meta-analysis and TCGA data

ABSTRACT CD44 is reported to be involved in tumor invasion and metastasis. However, the role of cancer stem cell marker CD44 in bladder cancer still remains controversial. Hence, the correlations between CD44 expression and the clinicopathological features and the prognosis of bladder cancer were investigated. Publications using immunohistochemical methods were identified. The Cancer Genome Atlas (TCGA) data were also analyzed. The odds ratios (ORs) or hazard ratios (HRs) with their 95% confidence intervals (95% CIs) were calculated. 14 studies involving 1107 tissue samples were included. CD44 expression in bladder cancer was lower than in non-tumor tissue samples (OR = 0.14, P = 0.005), which was consistent with TCGA data. CD44 expression was correlated with advanced T stage (OR = 1.76, P = 0.029) and lymph node metastasis (OR = 4.09, P < 0.001). Multivariate survival analysis showed that CD44 expression was not linked to tumor-specific survival, overall survival, and recurrence/relapse-free survival, but was associated with disease failure (HR = 2.912, 95% CI = 1.51–5.61). No relationships of CD44 expression with the clinicopathological features and overall survival were found from TCGA data. Our finding suggested that CD44 expression may be correlated with progression, metastasis, and disease failure of bladder cancer. However, further large-scale studies are needed. Abbreviations: CD44: Cluster of Differentiation 44; CIs: Confidence Intervals; CSCs: Cancer Stem Cells; EMT: Epithelial-mesenchymal Transition; HRs: Hazard Ratios; ORs: Odds Ratios; TCGA: The Cancer Genome Atlas

Binding of Targeted Semiconducting Photothermal Polymer Nanoparticles for Intraperitoneal Detection and Treatment of Colorectal Cancer

Nanoparticles offer many promising advantages for improving current surgical regimens through their ability to detect and treat disseminated colorectal cancer (CRC). Hybrid Donor-Acceptor Polymer Particles (HDAPPs) have recently been shown to fluorescently detect and thermally ablate tumors in a murine model. Here, HDAPPS were functionalized with hyaluronic acid (HA) to improve their binding specificity to CT26 mouse CRC cells using HA to target the cancer stem cell marker CD44. In this work, we compared the binding of HA functionalized HDAPPs (HA-HDAPPs) in in vitro, ex vivo, and in vivo environments. The HA-HDAPPs bound to CT26 cells 2-fold more in vitro and 2.3-fold higher than un-functionalized HDAPPs ex vivo. Compared to intraoperative abdominal perfusion, intraperitoneal injection prior to laser stimulation for nanoparticle heat generation provides a superior modality of HA-HDAPPs delivery for CRC tumor selectivity. Photothermal treatment of disseminated CRC showed that only HA-HDAPPs delivered via intraperitoneal injection had a reduction in the tumor burden, and these nanoparticles also remained in the abdomen following resolution of the tumor. The results of this work confirm that HA-HDAPPs selectively bind to disseminated CRC, with ex vivo tumors having bound HA-HDAPPs capable of photothermal ablation. HA-HDAPPs demonstrated superior binding to tumor regions compared to HDAPPs. Overall, this study displays the theranostic potential of HDAPPs, emphasizing their capacity to detect and photothermally treat disseminated CRC tumors.

Clinical Significance of Cancer Stem Cell Markers CD133 and CXCR4 in Osteosarcomas.

Objective:Osteosarcomas (OS) is one the most common primary bone malignancy in humans with the lungs metastasis in most cases. Metastasis and recurrence of OS is attributed to cancer stem cells (CSCs). Our study aimed to evaluate the clinical significance of CD133 and C-X-C chemokine receptor type 4 (CXCR4) as the frequently applied markers for CSCs in OS patients. Methods:In this cross-sectional, a total of 50 tissue samples from the patients with primary OS were immunohistochemically examined to detect the expression of CD133 and CXCR4. The associations of the relative expression and clinical significance of each marker were also evaluated. Results:High level expression of CD133 was detected in 26% of OS patient tissues. Of the 12 patients who showed lung metastasis, 5 cases showed high expression of CD133 with marginal trend correlation (P=0.06). No significant correlation was observed between CD133 expression and clinicopathological factors. Only 36% of cases showed CXCR4 expression which was not significantly correlated with gender, age, tumor size, necrosis, stage and metastasis (P>0.05). Clinically, patients with concomitant CD133/CXCR4 expression had significant association with lung metastasis (P=0.05). Conclusion:Our findings showed that concomitant expression of CSC markers CD133/CXCR4 might had a synergistic effect on the OS poor prognosis. These markers could be considered as potential therapeutic candidates of OS targeted therapy.

Depressive Disorder promotes Hepatocellular Carcinoma metastasis via upregulation of ABCG2 gene expression and maintenance of self-renewal.

Depressive disorder (DD) is the leading cause of disability worldwide and is the most prevalent mood disorder. Accumulative evidence from epidemiological studies has shown that DD is a risk factor for cancer. However, the role and molecular mechanism of DD in hepatocellular carcinoma (HCC) are still unknown. In this study, 30 mice were randomly divided into two groups: the HCC group and the HCC-DD group. The DD mouse model of HCC was established by induction with reserpine every other day and with monthly doses of diethylnitrosamine (DEN). All of the molecular studies were based on primary cell culture, and the effects of DD on HCC cell proliferation and migration and cancer stem cell (CSC) self-renewal were determined by colony formation, wound healing, and sphere culture assays. We found that the CSC markers ABCG2 and CD133 were upregulated in HCC-DD primary cells compared with HCC primary cells. Moreover, HCC-DD primary cells were more aggressive in terms of metastasis and self-renewal than HCC primary cells. Further study revealed that DD promoted tumor growth and metastasis by activating the AKT signaling pathway followed by an increased ABCG2 expression. Taken together, our novel findings indicate that DD promotes proliferation, self-renewal, and metastasis by upregulating ABCG2 in the AKT pathway.