High CD44 Expression Research Articles

Polychromatic Flow Cytometric Analysis of Stromal Vascular Fraction from Lipoaspirate and Microfragmented Counterparts Reveals Sex-Related Immunophenotype Differences.

Mesenchymal stem/stromal cells or medicinal signaling cells (MSC)-based therapy holds promise as a beneficial strategy for treating knee OA (osteoarthritis), but there is no standardized protocols nor mechanistic understanding. In order to gain a better insight into the human MSC from adipose tissue applied for autologous OA treatment, we performed extensive comparative immunophenotyping of the stromal vascular fraction from lipoaspirate or microfragmented lipoaspirates by polychromatic flow cytometry and investigated the cellular components considered responsible for cartilage regeneration. We found an enrichment of the regenerative cellular niche of the clinically applied microfragmented stromal vascular fraction. Sex-related differences were observed in the MSC marker expression and the ratio of the progenitor cells from fresh lipoaspirate, which, in female patients, contained a higher expression of CD90 on the three progenitor cell types including pericytes, a higher expression of CD105 and CD146 on CD31highCD34high endothelial progenitors as well as of CD73 on supra-adventitialadipose stromal cells. Some of these MSC-expression differences were present after microfragmentation and indicated a differential phenotype pattern of the applied MSC mixture in female and male patients. Our results provide a better insight into the heterogeneity of the adipose MSC subpopulations serving as OA therapeutics, with an emphasis on interesting differences between women and men.

CD44: A Multifunctional Mediator of Cancer Progression.

CD44, a non-kinase cell surface transmembrane glycoprotein, has been widely implicated as a cancer stem cell (CSC) marker in several cancers. Cells overexpressing CD44 possess several CSC traits, such as self-renewal and epithelial-mesenchymal transition (EMT) capability, as well as a resistance to chemo- and radiotherapy. The CD44 gene regularly undergoes alternative splicing, resulting in the standard (CD44s) and variant (CD44v) isoforms. The interaction of such isoforms with ligands, particularly hyaluronic acid (HA), osteopontin (OPN) and matrix metalloproteinases (MMPs), drive numerous cancer-associated signalling. However, there are contradictory results regarding whether high or low CD44 expression is associated with worsening clinicopathological features, such as a higher tumour histological grade, advanced tumour stage and poorer survival rates. Nonetheless, high CD44 expression significantly contributes to enhanced tumourigenic mechanisms, such as cell proliferation, metastasis, invasion, migration and stemness; hence, CD44 is an important clinical target. This review summarises current research regarding the different CD44 isoform structures and their roles and functions in supporting tumourigenesis and discusses CD44 expression regulation, CD44-signalling pathways and interactions involved in cancer development. The clinical significance and prognostic value of CD44 and the potential of CD44 as a therapeutic target in cancer are also addressed.

Differentiation of cancer stem cells into erythroblasts in the presence of CoCl2

Cancer stem cells (CSCs) are subpopulations in the malignant tumors that show self-renewal and multilineage differentiation into tumor microenvironment components that drive tumor growth and heterogeneity. In previous studies, our group succeeded in producing a CSC model by treating mouse induced pluripotent stem cells. In the current study, we investigated the potential of CSC differentiation into blood cells under chemical hypoxic conditions using CoCl2. CSCs and miPS-LLCcm cells were cultured for 1 to 7 days in the presence of CoCl2, and the expression of VEGFR1/2, Runx1, c-kit, CD31, CD34, and TER-119 was assessed by RT-qPCR, Western blotting and flow cytometry together with Wright-Giemsa staining and immunocytochemistry. CoCl2 induced significant accumulation of HIF-1α changing the morphology of miPS-LLCcm cells while the morphological change was apparently not related to differentiation. The expression of VEGFR2 and CD31 was suppressed while Runx1 expression was upregulated. The population with hematopoietic markers CD34+ and c-kit+ was immunologically detected in the presence of CoCl2. Additionally, high expression of CD34 and, a marker for erythroblasts, TER-119, was observed. Therefore, CSCs were suggested to differentiate into erythroblasts and erythrocytes under hypoxia. This differentiation potential of CSCs could provide new insight into the tumor microenvironment elucidating tumor heterogenicity.

Isolation and phenotyping of cardiac-derived progenitor cells from neonatal mice

Dysfunctions of resident progenitor cells play a significant role in the pathogenesis of decreased myocardial contractility in heart failure, so the most promising approaches for the treatment of heart disease are cardiac-derived stem/progenitor cells (CSCs). Materials and methods. Protocols for progenitor cell cultures from different parts of the heart of newborn FVB/N mice have been developed and their proliferative potential has been characterized. Comparative analysis of the expression of CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD117, CD309 and troponin I by cells from native myocardial biopsies and in the obtained cultures was performed by flow cytometric immunophenotyping. Results. The expression of mesenchymal markers CD44 and CD90 in the absence of the hematopoietic marker CD45 was demonstrated in early passages in mouse myocardial progenitor cell cultures. Relatively high expression of CD34 and CD31 was found. The presence of a minor population of CD44+117+ cells which correspond to the phenotype of cardiac progenitor cells, was detected. Expression of troponin I as one of the key markers of cardiomyocytes as well as the vascular endothelial growth factor receptor has been confirmed in terminally differentiated cultures of cells with contractile activity. Conclusions. It was found that newborn mice in the myocardial tissue contain more cells with the expression of markers of cardiac progenitors than in adult animals. The relative content of such cells is higher in the atria than in the ventricles. Cardiac progenitor cells in neonatal mice derived from the atrial appendages have better proliferative potential than cell cultures isolated from the ventricles.

18F-fluorodeoxyglucose uptake in advanced gastric cancer correlates with histopathological subtypes and volume of tumor stroma

PurposeThe aim of this study was to investigate the correlation between preoperative 18F-fluorodeoxyglucose (FDG) uptake and histological subtypes, amount of tumor stroma in advanced gastric cancer (GC), and clinical outcomes. MethodsWe evaluated 56 patients (male/female, 42:14; mean age, 69 years) with advanced GC who underwent surgical resection at our institution and positron emission tomography–computed tomography with 18F-FDG prior to surgery. We used the maximum standardized uptake value (SUVmax) of the tumor and the tumor-to-liver ratio (TLR) of the SUVmax for the analysis. The SUVmax and TLR correlated with histological subtypes, immunohistochemistry (IHC) for CD34, and recurrence-free survival (RFS). Tumor stroma in GC was evaluated by CD34 expression. GCs were classified according to the Lauren and World Health Organization (WHO) classifications. ResultsThe average FDG uptakes (SUVmax) were 4.17% and 14.04% in diffuse and intestinal type GCs, respectively, according to the Lauren classification, and 4.17%, 13.87%, 7.70%, 9.71%, and 19.45% in the poorly cohesive, tubular, mucinous, and papillary adenocarcinomas, respectively, according to the WHO classification. The FDG uptake in diffuse type was significantly lower than that in the intestinal type (p = 0.000). The SUVmax and TLR of the CD34(+) group (mean SUVmax, 5.50; TLR, 1.56) were significantly lower than those of the CD34(−) group (mean SUVmax, 14.09; TLR, 4.09). RFS was not associated with TLR or CD34 expression. ConclusionGC, which has abundant tumor stroma characterized by high CD34 expression on IHC, shows low FDG uptake.

STEM-03. Oct4/Sox2 DRIVE AN IMMUNOSUPPRESSIVE GSC PHENOTYPE BY INDUCING T-REG EFFECTOR GENES VIA TGFBR2 SIGNALING

Abstract Solid malignancies, including GBM, contain small subsets of cells that display stem-like properties (i.e. glioma stem cells or GSCs) and act as key determinants of therapeutic resistance and tumor recurrence. Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. GBM cells escape antitumor immunity by modifying the tumor microenvironment by secreting immune-suppressive factors and recruiting anti-inflammatory/pro-oncogenic cells (e.g. T-regulatory cells, M2-like macrophages). However, whether subsets of GSCs can mimic T-reg-like functions by expressing proteins associated with T-reg immune-suppression remains unknown. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3- cell subpopulation with high expression of TGFb1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with T-reg cells and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with CD44 and TGFb type II receptor (TGFBR2) expression in both clinical GBM and primary GSCs. Mechanistically, Oct4 and Sox2 were found to directly induce TGFBR2 transcription concurrent with chromatin architectural changes (i.e. heterochromatin-to-euchromatin transition) involving the TGFBR2 gene. Transgenic TGFBR2 expression increased neurosphere cell growth capacity, upregulated expression of T-reg-like effector genes and enriched for CD44+ GSCs. Functionally, neurospheres expressing transgenic Oct4/Sox2 or transgenic TGFBR2 possess increased immunosuppressive capacity, and pharmacological inhibition of TGFBR2 depleted CD44+ GSCs and reduced immunosuppression in Jurkat cell co-cultures. Taken together, we show that reprogramming events initiated by Oct4 and Sox2 induce a CD44+/FOX3P- GSC cell subset with a T-reg-like immunosuppressive transcriptional profile via a TGFBR2-dependent mechanism. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies.

Transcriptional Profile Investigation Identified Aberrant CD34 Isoforms and Apela As a Hub Gene in IGH-DUX4 Subtype B-Cell Precursor Acute Lymphoblastic Leukemia

BackgroundCD34, a transmembrane sialoglycoprotein, is expressed in hematopoietic stem and progenitor cells, endothelial cells, and bone marrow stromal cells. Therefore, in the clinical diagnosis and classification of leukemia, high expression of CD34 is customized as a marker for the immature phenotype. Although alternative splicing is a common oncogenic mechanism in various cancers, abnormal splicing of CD34 has not been revealed in hematological malignancies.MethodsHere, we investigated the transcriptional profile of CD34, including expression level and alternative splicing, by RNA sequencing (RNA-seq) analysis from hematological malignancies and normal bone marrow samples. The raw sequencing reads were aligned to human reference genome hg38 using HISAT2, followed by featureCounts quantification, and co-expressed and differentially expressed genes (DEGs) were detected by WGCNA and DESeq2, respectively. Alternative splicing events were calculated by rMATS, and further validated by reverse transcriptase PCR (RT-PCR).ResultsThe transcriptome characteristics of hematological malignancies, including AML, B-ALL, T-ALL, and MPAL, were elaborately investigated. We found that two accompanying novel in-frame splicing isoforms of CD34 were exclusively detected in B-ALL. Furthermore, we focused on B-ALL (n = 504) to systematically explore the transcriptional profile of CD34. The two novel splicing isoforms share a common first exon at the 5' untranslated region of CD34 (Figure 1B), suggesting an alternative promoter that mediated the splicing.We further observe that the novel aberrant CD34 isoforms are mainly accompanied by IGH-DUX4 gene fusion, which has been reported characterized by high expression of CD34 and intragenic ERG deletion. All IGH-DUX4 B-ALL cases (n = 20) in our cohort were positive with the novel aberrant CD34 isoforms and validated by RT-PCR (Figure 1A). We also validated the negative result for the aberrant CD34 isoforms in ZNF384-r (n = 32) B-ALL in our cohort, which is also characterized by high CD34 expression.According to the RNA-seq based pseudo-time analysis of our B-ALL cohort, IGH-DUX4 cases mostly tended to be common-B ALL, while ZNF384-r cases presented with more Pro-B ALL, which is also in accordance with the previous report based on immunophenotype analysis. To further discover the discrepancy between DUX4-r and ZNF384-r B-ALL subtypes, we compared DEGs and functional enrichment analysis between these two subtypes with B-other ALL as control, respectively (Figure 2). Activation of JAK-STAT signaling and downregulation of SOCS2, which acts as a negative controller of JAK-STAT signaling, were observed in both subtypes (Figure 2). The hematopoietic cell lineage gene set was enriched in ZNF384-r, consistent with its more immature phenotype. The co-expression gene network for IGH-DUX4 was constructed, and the hub gene APELA was emphasized among the closely related genes (Figure 3).DiscussionIn this study, we firstly identified two novel in-frame CD34 isoforms that share a common alternative first exon at its 5'UTR and are highly enriched in the IGH-DUX4 subtype B-ALL. It has been reported that intragenic focal ERG deletion is characteristically co-occurred with IGH-DUX4, and ERG targets CD34 through regulates its super-enhancer to affect the transcription program. It needs further investigation whether ERG deletion contributes to the aberrant transcription of CD34 in IGH-DUX4 subtype B-ALL. We also emphasized APELA as a hub gene in the transcription regulation network of IGH-DUX4. As it currently lacks effective targeted therapy for IGH-DUX4 subtype B-ALL, the potential therapeutic significance of the closely accompanied aberrant CD34 isoforms and APELA as a hub gene deserves further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis.

Background: The AT-rich interaction domain 1A (ARID1A) is thought to be a tumor suppressive gene, and most of its mutations result in loss of expression of ARID1A protein. Combined with SIRPα on the surface of macrophages, CD47 on the surface of cancer cells can send an antiphagocytic “Don’t eat me” signal to the immune system that helps to avoid immune surveillance. However, the relationship between ARID1A and CD47 expression and their prognostic value in gastric cancer (GC) are still unknown.Methods: In this study, we evaluated ARID1A and CD47 expression in 154 GC patients’ tissues using tissue microarray. Expressions of ARID1A and CD47 in GC cell lines were determined by western blot and quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) techniques, and cell membranous CD47 expression was quantified by flow cytometry. In addition, chromatin immunoprecipitation (ChIP)–qPCR was used to determine the aspects of regulation of CD47 by ARID1A. The proportions of tumor-infiltrating immune cells were estimated on The Cancer Genome Atlas (TCGA) data set by using quanTIseq and EPIC algorithms. The infiltration of M1-polarized macrophages, M2-polarized macrophages, and regulatory T cells (Tregs) in GC tissues was determined by multispectral immunofluorescence.Results: A significant correlation was found between loss of ARID1A and high expression of CD47 at protein level in GC. By integrating 375 bulk RNA sequencing samples from TCGA data set, we found that mutated ARID1A correlated with high CD47 expression. In GC cell lines, knockdown of ARID1A significantly increased CD47 expression both at protein and mRNA levels as measured by western blot, qRT-PCR, and flow cytometry. Moreover, ChIP-qPCR revealed that CD47 was a direct downstream target gene of ARID1A in GC. Utilizing univariate and multivariate survival analyses, we found that patients with ARID1AlossCD47high expression had a worse prognosis. Estimation of infiltrating immune cells on TCGA data set showed that a higher infiltration proportion of M2 macrophages and Tregs was found in ARID1AmutatedCD47high expression subgroup. Furthermore, application of multispectral immunofluorescence revealed a higher infiltration proportion of M2 macrophages and Tregs in ARID1AlossCD47high GC tissues.Conclusion: Loss of ARID1A is strongly correlated with high CD47 expression in GC, and combination of ARID1A and CD47 is a promising prognosis factor in GC.

Identification and characterization of murine adipose tissue-derived somatic stem cells of Shenque (CV8) acupoint.

Background:Shenque (CV8) acupoint is located on the navel and has been therapeutically used for more than 2000 years in Traditional Chinese Medicine (TCM). However, clinical research on the underlying therapeutic molecular mechanisms of the CV8 acupoint lags far behind. This study aimed to study the mechanisms of umbilical acupoint therapy by using stem cells.Methods:The morphological characteristics of CV8 acupoint were detected under a stereomicroscope using hematoxylin and eosin (H&E) staining. Oil Red, Masson, and immunohistochemical staining on multi-layered slices were used to identify the type of cells at the CV8 acupoint. Cell proliferation was measured by a cell counting kit-8 (CCK-8) method. Flow cytometry and immunohistochemistry were used for cell identification. Induced differentiation was used to compare the differentiation of cells derived from CV8 acupoint and non-acupoint somatic stem cells into other cell types, such as osteogenic, adipogenic, and neural stem cell-like cells.Results:Morphological observations showed that adipose tissues at the linea alba of the CV8 acupoint in mice had a mass-like distribution. Immunohistochemical staining confirmed the distribution of stem cell antigen-1 (Sca-1) positive cells in the multi-layered slices of CV8 acupoint tissues. Cells isolated from adipose tissues at the CV8 acupoint exhibited high expression of Sca-1 and CD44 and low expression of CD31 and CD34, and these cells possessed osteogenic, adipogenic, and neurogenic stem cell-like cell differentiation ability. The cell proliferation (day 4: 0.5138 ± 0.0111 vs. 0.4107 ± 0.0180, t = 8.447, P = 0.0011; day 5: 0.6890 ± 0.0070 vs. 0.5520 ± 0.0118, t = 17.310, P < 0.0001; day 6: 0.7320 ± 0.0090 vs. 0.6157 ± 0.0123, t = 13.190, P = 0.0002; and day 7: 0.7550 ± 0.0050 vs. 0.6313 ± 0.0051, t = 42.560, P < 0.0001), adipogenic ([9.224 ± 0.345]% vs. [3.933 ± 1.800]%, t = 5.000, P = 0.0075), and neurogenic stem cell-like cell differentiation (diameter < 50 μm: 7.2000 ± 1.3040 vs. 2.6000 ± 0.5477, t = 7.273, P < 0.0001; diameter 50–100 μm: 2.6000 ± 0.5477 vs. 1.0000 ± 0.7071, t = 4.000, P = 0.0039; and diameter >100 μm: 2.6000 ± 0.5477 vs. 0.8000 ± 0.8367, t = 4.025, P = 0.0038) were significantly enhanced in somatic stem cells derived from the CV8 acupoint compared to somatic stem cells from the groin non-acupoint. However, cells possessed significantly weaker osteogenicity ([2.697 ± 0.627]% vs. [7.254 ± 0.958]%, t = 6.893, P = 0.0023) in the CV8 acupoint group.Conclusions:Our study showed that CV8 acupoint was rich with adipose tissues that contained abundant somatic stem cells. The biological examination of somatic stem cells derived from the CV8 acupoint provided novel insights for future research on the mechanisms of umbilical therapy.

K-RAS Acts as a Critical Regulator of CD44 to Promote the Invasiveness and Stemness of GBM in Response to Ionizing Radiation.

Radiation therapy is a current standard-of-care treatment and is used widely for GBM patients. However, radiation therapy still remains a significant barrier to getting a successful outcome due to the therapeutic resistance and tumor recurrence. Understanding the underlying mechanisms of this resistance and recurrence would provide an efficient approach for improving the therapy for GBM treatment. Here, we identified a regulatory mechanism of CD44 which induces infiltration and mesenchymal shift of GBM. Ionizing radiation (IR)-induced K-RAS/ERK signaling activation elevates CD44 expression through downregulation of miR-202 and miR-185 expression. High expression of CD44 promotes SRC activation to induce cancer stemness and EMT features of GBM cells. In this study, we demonstrate that the K-RAS/ERK/CD44 axis is a key mechanism in regulating mesenchymal shift of GBM cells after irradiation. These findings suggest that blocking the K-RAS activation or CD44 expression could provide an efficient way for GBM treatment.

Alcohol Metabolism Enriches Squamous Cell Carcinoma Cancer Stem Cells That Survive Oxidative Stress via Autophagy.

Background: Alcohol (ethanol) consumption is a major risk factor for head and neck and esophageal squamous cell carcinomas (SCCs). However, how ethanol (EtOH) affects SCC homeostasis is incompletely understood. Methods: We utilized three-dimensional (3D) organoids and xenograft tumor transplantation models to investigate how EtOH exposure influences intratumoral SCC cell populations including putative cancer stem cells defined by high CD44 expression (CD44H cells). Results: Using 3D organoids generated from SCC cell lines, patient-derived xenograft tumors, and patient biopsies, we found that EtOH is metabolized via alcohol dehydrogenases to induce oxidative stress associated with mitochondrial superoxide generation and mitochondrial depolarization, resulting in apoptosis of the majority of SCC cells within organoids. However, CD44H cells underwent autophagy to negate EtOH-induced mitochondrial dysfunction and apoptosis and were subsequently enriched in organoids and xenograft tumors when exposed to EtOH. Importantly, inhibition of autophagy increased EtOH-mediated apoptosis and reduced CD44H cell enrichment, xenograft tumor growth, and organoid formation rate. Conclusions: This study provides mechanistic insights into how EtOH may influence SCC cells and establishes autophagy as a potential therapeutic target for the treatment of EtOH-associated SCC.

P68.12 Inter-Tumor Heterogeneity of CD44 Expression in Non-Small Cell Lung Cancers / EGFR Mutated Lung Adenocarcinomas

Epithelial to mesenchymal transition (EMT) is one of mechanisms of acquired resistance (AR) to EGFR-TKIs in EGFR-mutated lung cancers. In our previous study, we observed that homogeneous high CD44 expression will predict emergence of EMT-mediated AR to EGFR-TKIs in lung cancer cell lines with EGFR mutation as represented by HCC4006 and H1975 cells (PMID:30049789). In this study, we examined inter-tumor heterogeneity of CD44 status to evaluate if CD44 can be a potential biomarker with less sampling variation in the clinical setting.

CD44 Is Involved in Sunitinib Resistance and Poor Progression-free Survival After Sunitinib Treatment of Renal Cell Carcinoma.

Sunitinib continues to be administered as a first-line therapeutic agent in metastatic renal cell carcinoma (mRCC). This study aimed to examine the role of CD44 in sunitinib resistance and as a predictive marker in mRCC. We analyzed the effect of CD44 knockdown on sunitinib resistance in RCC cell lines using WST-1 assays. CD44 expression in mRCC patients treated with first-line sunitinib was determined by immunohistochemistry. We validated the findings of this study by in silico analysis. CD44 knockdown increased sensitivity to sunitinib. Immunohistochemical analysis revealed that 19 (34.5%) of 55 mRCC cases were positive for CD44. CD44-positive cases were associated with poor progression-free survival (PFS) after first-line sunitinib treatment. In the JAVELIN 101 study, high CD44 expression was significantly associated with poor PFS after sunitinib but not after avelumab + axitinib therapy. CD44 is involved in sunitinib resistance and may be a promising marker for sunitinib treatment in mRCC.

Abstract MP04: Single Cell Profiling Identifies IgM To MDA-LDL Producing Human B Cells And Atheroprotective Role Of CD24

Murine B-1 cells produce IgMs that inactivate oxidation-specific epitopes and protect against atherosclerosis. Despite compelling data that IgM to MDA-LDL (IgM MDA-LDL ) is inversely associated with coronary artery disease (CAD) and cardiovascular (CV) events in human, the human B cell subtype that produces IgM MDA-LDL is unknown. Here, we utilized mass cytometry to identify the 11 circulating human B cell subtypes and discovered that only the frequency of CD20 + CD27 + IgM + cells was associated with IgM MDA-LDL levels. Notably, high expression of CD24, a GPI-anchored sialoglycoprotein, was associated with IgM MDA-LDL (r = 0.57, p = 0.002) and inversely associated with CAD severity (r = -0.58, p=0.001). Adoptive transfer (AT) of sorted CD20 + CD27 + IgM + CD24hi (B 27+IgM+CD24hi ) and CD24lo/- (B 27+IgM+CD24lo/- ) human B cells into humanized mice demonstrated that CD24 is critical for trafficking to the spleen and IgM production. Bulk RNAseq coupled with pathway analysis of B 27+IgM+CD24hi and B 27+IgM+CD24lo/- cells revealed enhanced BCR and CCR6 signaling in B 27+IgM+CD24hi . Imaging flow cytometry indicated colocalization between CD24 and CCR6 which was abrogated by CD24 blocking antibody (CD24mAb). Trans-well migration and internalization assays demonstrated that CD24 blockade increased CCL20-induced CCR6 internalization (p &lt; 0.05, n =4), and reduced CCL20-induced migration of B 27+IgM+CD24hi cells (p &lt; 0.05, n =4). CD24mAb treatment of B 27+IgM+CD24hi cells prior to AT into humanized mice also reduced the amount of surface CCR6 on B 27+IgM+CD24hi cells in vivo (p &lt; 0.05, n = 4). Both CCR6 knockout and CD24 inhibition of B 27+IgM+CD24hi cells reduced migration to the spleen (p &lt; 0.05, n = 4), and plasma level of human IgM (p = 0.057, n = 4). Lastly, single cell multi-omics sequencing of PBMCs from 60 CAD subjects revealed elevated CCR6 and IgM signaling in B 27+IgM+CD24hi cells in subjects with less severe CAD. Results provide the first evidence for a role for CD24 in regulating CCR6-mediated B cell homing to the spleen, IgM production and CAD in humans. Findings may have important implications for the new CD24 immunotherapy entering clinical trials and raise the possibility that augmenting CD24 and/or CCR6 on B 27+IgM+ cells may be an effective atheroprotective strategy.

CD73 Promotes Tumor Progression in Patients with Esophageal Squamous Cell Carcinoma

Simple SummaryThe immune system plays important roles in antitumor activities. However, increasing evidence shows that tumor cells develop several mechanisms to escape the immune attack, resulting in immunosuppression. One of the most important immunosuppressive pathways is the CD73-adenosinergic pathway. In addition, this pathway participates in the development of cancer, including tumor cell proliferation, angiogenesis, and anti-inflammation mechanisms. Moreover, CD73 can mediate the invasion and metastasis of tumor cells via the regulation of cell interactions with the extracellular matrix components. Therefore, overcoming immunosuppression to restore the antitumor functions of T cells may be explored as a potential treatment strategy. Overexpression of CD73 promotes the malignant properties of cancers and is associated with specific clinical characteristics and worse prognosis in many types of cancers. The current study is the first to investigate the role of CD73 in determining the clinical outcomes of patients with esophageal squamous cell carcinoma.Cluster of differentiation (CD)-73 plays pivotal roles in the regulation of immune reactions via the production of extracellular adenosine, and the overexpression of CD73 is associated with worse outcomes in several types of cancers. Here, we identified 167 esophageal squamous cell carcinoma (ESCC) patients who underwent esophagectomy, including 64 and 103 patients with high and low expression levels of CD73, respectively. Univariate and multivariate analyses showed high expression of CD73 was an independent prognostic factor for worse disease-free survival and overall survival. In addition, we selected another cohort consisting of 38 ESCC patients receiving nivolumab or pembrolizumab and found that treatment response and survival benefit to immunotherapy were strongly correlated with the expression levels of CD73/programmed death ligand 1. Moreover, the transwell assay revealed knockdown of CD73 in two ESCC cell lines, TE1 and KYSE30, exhibited significantly reduced abilities of cell invasion and migration. CD73 silencing also showed that the protein expression levels of CD73, vimentin, and snail were downregulated, while those of E-cadherin were upregulated in Western blotting. The findings of our study indicate CD73 may be an independent prognostic factor for ESCC patients who underwent esophagectomy. Furthermore, it may be associated with the patient responses to immunotherapy.

SPL-108 mediates metastasis and chemoresistance in high-grade serous tubo-ovarian carcinoma

Objectives: Overcoming chemoresistance will improve outcomes for all women with tubo-ovarian carcinomas. Mechanisms of chemoresistance are multifactorial and heterogeneous. ABCB1 (MDR1) represents one common resistance mechanism in tubo-ovarian carcinomas and is under the regulation of CD44. In the present study we examined the effects of a novel CD44 modulator, SPL-108, in a panel of ovarian cancer cell lines. Methods: We assessed chemosensitivity and migration effects of SPL-108 in a panel of ovarian cancer cell lines with (OVCAR5, OVCAR8, OVCAR3) and without (OVCAR4, CAOV3) MDR1 expression. In vitro experiments (cell viability assay, Western blot analysis, Calcein AM fluorescence assay, and migration assay) were carried out to determine the functional effects of SPL-108. Results: Higher expression of CD44 was seen in OVCAR5 and OVCAR8 on Western Blot analysis. In transwell migration assay, treatment with SPL-108 decreased the number of migrating cells compared to control in OVCAR8 (mean ± SEM, 149.1 ± 22.8 vs 89.0 ± 16.5 cells, p=0.04), OVCAR5 (177.7 ± 15.4 vs 134.5 ± 14.7 cells, p=0.04) and OVCAR3 (111.2 ± 12.4 vs 50.5 ± 9.4 cells, p Conclusions: SPL-108 treatment has anti-metastatic properties and suppresses chemoresistance in preclinical models of ovarian cancer. Ongoing studies in animal models will help to guide the further clinical development of SPL-108. Download : Download high-res image (262KB) Download : Download full-size image

Slug and CD133 expression are associated with peritoneal carcinomatosis and survival in gastric cancer.

Slug is an activating transcription factor involved in epithelial-mesenchymal transition, and CD133 is a cancer stem cell marker found in various cancers, including gastric cancer (GC). We investigated the relationship between Slug and CD133 and the occurrence of peritoneal carcinomatosis and survival in patients with GC. This retrospective study included 196 patients with stage 2 or 3 GC who underwent curative surgery with D2 lymph node dissection and adjuvant chemotherapy between 2001 and 2009. We analyzed the expression of Slug, CD133, ABCG2, E-cadherin, vimentin, NEDD9, and SMAD4 in surgical tissue specimens using immunohistochemical analysis to determine their prognostic value. Among the 196 patients, expression of Slug was elevated in 157 tumors (81%) while the expression of CD133 was high in 153 tumors (81%). The expression of Slug and CD133 in combination significantly predicted peritoneal relapse (P=0.002). High Slug and high CD133 expression were significantly associated with poor peritoneal relapse-free survival [hazard ratio (HR), 7.239; P=0.007] and overall survival (HR, 1.682; P=0.027) in multivariate Cox analysis. Our study shows that a high Slug and high CD133 expression status is predictive of peritoneal recurrence in high-risk resected GC patients. They are also a poor prognostic factor for peritoneal relapse-free survival and overall survival.

Activation of CD44-Lipoprotein lipase axis in breast cancer stem cells promotes tumorigenesis

Breast cancer stem cells (CSCs) are distinct CD44+-subpopulations that are involved in metastasis and chemoresistance. However, the underlying molecular mechanism of CD44 in breast CSCs-mediated tumorigenesis remains elusive. We observed high CD44 expression in advanced-stage clinical breast tumor samples. CD44 activation in breast CSCs sorted from various triple negative breast cancer (TNBC) cell lines induced proliferation, migration, invasion, mammosphere formation that were reversed in presence of inhibitor, 4-methyl umbelliferone or CD44 silencing. CD44 activation in breast CSCs induced Src, Akt, and nuclear translocation of pSTAT3. PCR arrays revealed differential expression of a metabolic gene, Lipoprotein lipase (LPL), and transcription factor, SNAI3. Differential transcriptional regulation of LPL by pSTAT3 and SNAI3 was confirmed by promoter-reporter and chromatin immunoprecipitation analysis. Orthotopic xenograft murine breast tumor model revealed high tumorigenicity of CD24−/CD44+-breast CSCs as compared with CD24+-breast cancer cells. Furthermore, stable breast CSCs-CD44 shRNA and/or intratumoral administration of Tetrahydrolipstatin (LPL inhibitor) abrogated tumor progression and neoangiogenesis. Thus, LPL serves as a potential target for an efficacious therapeutics against aggressive breast cancer.

Integration of RNA-Seq and proteomics data identifies glioblastoma multiforme surfaceome signature

BackgroundGlioblastoma multiforme (GBM) is a highly lethal, stage IV brain tumour with a prevalence of approximately 2 per 10,000 people globally. The cell surface proteins or surfaceome serve as information gateway in many oncogenic signalling pathways and are important in modulating cancer phenotypes. Dysregulation in surfaceome expression and activity have been shown to promote tumorigenesis. The expression of GBM surfaceome is a case in point; OMICS screening in a cell-based system identified that this sub-proteome is largely perturbed in GBM. Additionally, since these cell surface proteins have ‘direct’ access to drugs, they are appealing targets for cancer therapy. However, a comprehensive GBM surfaceome landscape has not been fully defined yet. Thus, this study aimed to define GBM-associated surfaceome genes and identify key cell-surface genes that could potentially be developed as novel GBM biomarkers for therapeutic purposes.MethodsWe integrated the RNA-Seq data from TCGA GBM (n = 166) and GTEx normal brain cortex (n = 408) databases to identify the significantly dysregulated surfaceome in GBM. This was followed by an integrative analysis that combines transcriptomics, proteomics and protein-protein interaction network data to prioritize the high-confidence GBM surfaceome signature.ResultsOf the 2381 significantly dysregulated genes in GBM, 395 genes were classified as surfaceome. Via the integrative analysis, we identified 6 high-confidence GBM molecular signature, HLA-DRA, CD44, SLC1A5, EGFR, ITGB2, PTPRJ, which were significantly upregulated in GBM. The expression of these genes was validated in an independent transcriptomics database, which confirmed their upregulated expression in GBM. Importantly, high expression of CD44, PTPRJ and HLA-DRA is significantly associated with poor disease-free survival. Last, using the Drugbank database, we identified several clinically-approved drugs targeting the GBM molecular signature suggesting potential drug repurposing.ConclusionsIn summary, we identified and highlighted the key GBM surface-enriched repertoires that could be biologically relevant in supporting GBM pathogenesis. These genes could be further interrogated experimentally in future studies that could lead to efficient diagnostic/prognostic markers or potential treatment options for GBM.

Selective Adhesion and Switchable Release of Breast Cancer Cells via Hyaluronic Acid Functionalized Dual Stimuli-Responsive Microgel Films.

The detection of tumor cells from liquid biopsy samples is of critical importance for early cancer diagnosis, malignancy assessment, and treatment. In this work, coatings of hyaluronic acid (HA)-functionalized dual-stimuli responsive poly(N-isopropylacrylamide) (PNIPAM) microgels are used to study the specificity of breast cancer cell binding and to assess cell friendly release mechanisms for further diagnostic procedures. The microgels are established by straightforward precipitation polymerization with amine bearing comonomers and postfunctionalization with a UV-labile linker that covalently binds HA to the microgel network. Well-defined microgel coatings for cell binding are established via simple physisorption and annealing. The HA-presenting PNIPAM microgel films are shown to specifically adhere CD44 expressing breast cancer cell lines (MDA-MB-231 and MCF-7), where an increase in adhesion correlates with higher CD44 expression and HA functionalization. Upon cooling below the lower critical solution temperature of PNIPAM microgels, the cells could be released; however, 10-30% of the cells still remained on the surface even after prolonged cooling and mild mechanical agitation. A complete cell release is achieved after applying the light stimulus by short UV treatment cleaving HA units from the microgels. Owing to the comparatively straightforward preparation procedures, such dual-responsive microgel films could be considered for the effective capture, release, and diagnostics of tumor cells.