A unique pathway in the homing of murine multiple myeloma cells: CD44v10 mediates binding to bone marrow endothelium

Our group recently reported that multiple myeloma (MM) cells preferentially adhere to bone marrow (BM) endothelial cells and selectively home to the BM, suggesting the involvement of specific adhesive interactions in this process. The highly regulated expression of CD44 variant isoforms (CD44v) on the MM cells makes them good candidate adhesion molecules involved in this homing. We addressed this in the 5T experimental mouse model of myeloma. Fluorescence-activated cell sorting analysis demonstrated expression of CD44v6, CD44v7, and CD44v10 on the in vivo growing 5T2MM and 5T33MM myeloma lines. Antibody blocking experiments revealed the involvement of CD44v10 in the adhesion of 5T2MM and 5T33MM cells to BM endothelial cells. Coinjection of anti-CD44v10 antibodies with the myeloma cells into syngeneic mice demonstrated a selective blocking of their BM homing which resulted in a decreased BM tumor load and serum paraprotein at the end stage of the disease. The highly restricted expression of CD44v10 on MM cells, the blocking of MM adhesion to BM endothelial cells and of homing to BM by anti-CD44v10, and the decreased BM tumor load suggest that myeloma cells home to the BM via interactions mediated by this specific region of the adhesion molecule CD44.     

CD44 variant isoforms are involved in plasma cell adhesion to bone marrow stromal cells.

Expression of CD44v9-containing isoforms (CD44v9) on myeloma plasma cells correlates with unfavorable prognosis, suggesting that CD44 variant molecules are involved in the disease process. In this study, the presence of CD44v on B cell lines from different stages of development was analyzed by flow cytometry and a role in adhesion to stromal cells from different tissues was evaluated in in vitro binding assays. CD44v3, v6 and v9 isoforms were exclusively expressed on plasma cell lines and CD44v9 expression correlated with IL-6-dependent plasma cell growth. Binding studies using CD44 isoform- specific reagents showed that CD44v6 and CD44v9 were involved in binding to bone marrow stromal cells, but not to in vitro synthesized ECM or hyaluronic acid. CD44v9-mediated plasma cell binding resulted in a significant induction of IL-6 secretion by bone marrow stromal cells. Large differences in quantitative plasma cell binding to stromal cells from different tissues were observed. These, however, could not be related to a differential use of CD44v in these binding processes. The role of CD44v9 in adhesion induced IL-6 secretion and its preferential expression on IL-6-dependent plasma cell lines may explain the previously observed correlation between CD44v9 expression and adverse prognosis in multiple myeloma.     

Acceleration of lung metastasis by up-regulation of CD44 expression in osteosarcoma-derived cell transplanted mice

The effect of CD44-phenotypic expression on metastasis to the lung was studied using a spontaneous murine osteosarcoma-derived cell line, POS-1, stimulated with lipopolysaccharide (LPS). POS-1 cells were inoculated into the hind paws of 20 C3H/HeJ mice and produced a visible mass in all mice in 5 weeks, and these transplanted tumors resulted in lung metastasis in all mice. The number of metastatic foci in the lungs was 12.0±2.1 (mean±SD) with LPS-stimulated cells, which was significantly higher than that of unstimulated cells (5.8±1.4; N=10 for each; P<0.05). Hyaluronate (HA), a ligand of CD44, inhibited a number of lung metastases in a dose-dependent manner (0.5% HA, 3.0±1.1; 0.005% HA, 5.1±1.5; without HA, 8.6±1.7; N=10 for each; P<0.05, each group with HA versus the group without HA). Adhesion assay by coculturing POS-1 cells and lung microvascular endothelial cells on culture plate showed that the adhesion was significantly lower in HA treated POS-1 than those without HA (1.18±0.12 and 2.74±0.17, respectively, P<0.05). These results suggest that lung metastasis was accelerated by up-regulation of CD44.     

The prognostic value of CD44 isoform expression in endometrial cancer.

Isoforms of the transmembrane glycoprotein CD44 have been implicated in tumour cell adhesion, tumour differentiation and metastatic spread in various human malignancies. We investigated the expression of CD44 isoforms containing variant exons v3, v5, v6 and v7-8 in 156 human endometrium cancer specimens by means of immunohistochemistry. CD44 isoforms CD44v3, CD44v5, CD44v6 and CD44v7-8 were detected in 26% (41 out of 156), 31% (48 out of 156), 22% (35 out of 156) and 15% (23 out of 156) of the tumour samples respectively. The expression of CD44 isoforms CD44v3, CD44v5 and CD44v7-8 showed no prognostic impact. In the univariate analysis, the expression of CD44v6 showed an association with shortened overall survival (log-rank test, P = 0.06). Multivariate analysis correcting for the confounding variable histological grading revealed CD44v6 not to be a prognostic factor in endometrial cancer (log-rank test, P = 0.06). Comparing the expression of CD44 isoforms CD44v3, CD44v5, CD44v6 and CD44v7-8 in 45 specimens of normal endometrial tissue, we found an up-regulation of all investigated CD44 isoforms in the secretory phase compared with the proliferative phase of the menstrual cycle. Our data indicate that the expression of CD44 isoforms, while obviously playing a role in the functional changes of normal endometrium, is not an adverse predictive factor in endometrial cancer.     

In vivo induction of insulin-like growth factor-I receptor and CD44v6 confers homing and adhesion to murine multiple myeloma cells

One of the main characteristics of multiple myeloma (MM) cells is their specific homing and growth in the bone marrow (BM). Differences between stroma-dependent and -independent MM cell lines may reveal key molecules that play important roles in their homing to the BM. We addressed this topic with a murine MM model, including the in vivo 5T33MM (5T33MMvv) stroma-dependent cell line and its in vitro stroma-independent variant (5T33MMvt). Fluorescence-activated cell-sorting analysis showed expression of insulin-like growth factor (IGF)-I receptor and CD44v6 on all 5T33MMvv cells but not on 5T33MMvt cells. Checkerboard analysis and adhesion assays revealed IGF-I-dependent chemotaxis toward BM-conditioned medium and involvement of CD44v6 in the adhesion to BM stroma of only 5T33MMvv cells. However, when 5T33MMvt cells were injected in vivo (5T33MMvt-vv), after 18 h the MM cells harvested from BM were IGF-I receptor and CD44v6 positive. This up-regulation was confirmed in 5T33MMvt-vv cells isolated from terminally diseased animals. These ST33MMvt-vv cells exhibited IGF-I-dependent chemotaxis and CD44v6-dependent adhesion to BM stroma. In vitro culture of the 5T33MMvt-vv cells could completely down-regulate IGF-I receptor and CD44v6. In fact, we could show that direct contact of 5T33MMvt cells with BM endothelial cells is a prerequisite for IGF-I receptor and CD44v6 up-regulation. These data indicate that the BM microenvironment is capable of up-regulating molecules such as IGF-I receptor and CD44v6, which facilitate homing of MM cells to the BM and support their adhesion to BM stroma.     

Expression of CD44 variant isoforms and association to the benign form of endocrine pancreatic tumours

Background:The expression of CD44 and its isoforms have beenshown in many neoplastic tissues to serve as prognostic indicators, therefore,the feasibility of using these as prognostic markers in endocrine pancreatictumour patients was examined. Patients and methods:Immunohistochemistry (IHC) was performed on26 tumour samples (5 gastrinomas, 3 glucagonomas, 10 non-functioning tumours,6 insulinomas, 2 mixed insulinoma and glucagonomas) with monoclonal antibodiesagainst CD44s (standard form) and variant isoforms (v4, v5, v6, v7,v7–8, v9, v10). Staining was correlated to the tumour proliferation,malignancy, metastasis and patients survival. Results:There was variable expression of CD44s. All tumoursshowed complex expression of many isoforms. CD44v6 and CD44v9 were downregulated in malignant tumours. There was statistical significance of CD44v6expression in benign tumours (P < 0.05) compared to malignanttumours and near significance in CD44v9 expression (P = 0.0574).Survival of the patients with CD44v6 positive staining was higher than thosewho were negative (P = 0.0822). Moreover, the expression was wellcorrelated to the patients without any distant metastases (CD44v6, p<0.001;CD44v9, P < 0.01). Tumour proliferation (Ki67 index) correlateddirectly to the malignancy (P < 0.05) and there was inversecorrelation between Ki67 index and CD44v6 (P < 0.05) as well asv9 (P < 0.05). Conclusions:Endocrine pancreatic tumours express CD44s andisoforms differentially. Expression of the two isoforms of CD44, namely v6 andv9 seem to be related more to benign form of the tumour and could serve as apredictor of good prognosis.     

CD44v3 and v6 variant isoform expression correlates with poor prognosis in early-stage vulvar cancer.

Expression of alternatively spliced CD44 isoforms has been reported to correlate with poor prognosis in human squamous cell cancers, i.e. squamous cell cancer of the lung and cervix. The aim of this study was to evaluate whether CD44 isoform expression is a prognostic factor in early-stage squamous cell cancer of the vulva. Seventy cases of squamous cell carcinoma of the vulva International Federation of Gynaecology and Obstetrics (FIGO) stage I were examined immunohistochemically for expression of CD44 isoforms. We used four different variant exon sequence-specific murine monoclonal antibodies to epitopes encoded by exons v3, v5, v6 and v7-8 of human variant CD44. The correlation of CD44 expression with histological grade and disease-free and overall survival was investigated. CD44 isoforms CD44v3, CD44v5, CD44v6 and CD44v7-8 were detected in 28% (20/70), 47% (33/70), 33% (23/70) and 17% (12/70) of the tumour samples respectively. Patients suffering from tumours expressing CD44v6 had a poorer relapse-free (log-rank test, P = 0.02) and overall survival (log-rank test, P = 0.03). Likewise, patients suffering from tumours expressing CD44v3 had a poorer relapse-free (log-rank test, P = 0.04) and overall survival (log-rank test, P = 0.01). Expression of CD44v5 and CD44v7-8 did not compromise the patients'' outcome. Histological grade did not correlate with CD44 isoform expression. Immunohistochemically detected expression of CD44 isoforms containing variant exon v6 or v3 is correlated with a poor relapse-free and overall survival in FIGO stage I vulvar cancer patients.     

C-kit receptor (CD117) expression on plasma cells in monoclonal gammopathies

The surface expression of CD117 antigen (c-kit) on plasma cells from 158 multiple myeloma (MM), 12 plasma cell leukemia (PCL), 7 MGUS, 7 IgM lymphoplasmacytic lymphoma patients and 10 healthy subjects has been analyzed by flow cytometry using triple staining with the monoclonal antibodies CD138, CD117 and CD38. The antigen expression intensity was calculated as relative fluorescence intensity (RFI) and for direct quantitative analysis the QuantiBRITE test (Becton Dickinson) was applied. Antibody bounding capacity (ABC) was calculated using QuantiCALC software. CD117 antigen was present in 49/158 MM, 5/12 PCL and 5/7 MGUS patients. The RFI values ranged from 0.2 to 20.2 in particular MM patients (mean: 11.0 ± 5.3; median 11.5) while the number of CD117 binding sites (ABC) on MM plasma cells ranged from 637 to 6217 (mean: 3029 ± 1568; median 2946) (r = 0.8328). In responsive to chemotherapy c-kit positive MM patients the percentage of CD117⁺ plasma cells in the bone marrow decreased significantly while in c-kit negative MM patients the percentage of CD117⁺ cells in bone marrow did not change and remained in the normal limits. When comparing the clinical and biological disease characteristics (monoclonal protein isotype, albumin, β₂-microglobulin, lactate dehydrogenase, stage of disease, response to chemotherapy, survival time) of c-kit positive and c-kit negative cases, no significant differences were found. In CD117 positive PCL cases expression of CD117 was detected in bone marrow plasma cells as well as in peripheral blood plasma cells. Normal plasma cells and those in IgM lymphoplasmacytic lymphoma did not show reactivity for the CD117 antigen. We conclude that it may be rationale to consider usefulness of therapy with tyrosine kinase inhibitors in the management of c-kit positive plasma cell proliferations. In one third of MM and PCL patients c-kit antigen could be considered as a "tumor associated marker" and together with CD38 and CD138 it may be of value for the identification of the malignant clone in minimal residual disease as it was first suggested by Spanish authors.     

Expression of CD44 Isoforms in Infiltrating Lobular Carcinoma of the Breast

Expression of CD44 has been shown to correlate with the progression and prognosis of some malignant tumors. The aim of this study was to evaluate the expression of CD44 isoforms in infiltrating lobular carcinomas and analyse their potential as prognostic indicators. A panel of 39 tumors were examined for their expression of membranous and cytoplasmic CD44s, v3, v5, v6, v7 and v3-10 in the infiltrating cells, by immunohistochemical staining. The protein positive tumors showed membranous and/or cytoplasmic staining with all antibodies used except for CD44v7, which only displayed cytoplasmic staining. Cytoplasmic expression of CD44v3 (P=0.014) and membranous expression of v6 (P=0.039) were significantly associated with alveolar, classical/alveolar carcinomas and mucinous/alveolar carcinomas. Furthermore, in alveolar, classical/alveolar and mucinous/alveolar carcinomas, cytoplasmic staining of CD44v5 was correlated with lymph node negative patients (P=0.048), whereas membranous v5 was correlated with lymph positive patients (P=0.048). In classical, classical/trabecular and trabecular carcinomas expression of membranous CD44s was significantly correlated with lymph node status (P=0.042).     

Osteopontin-dependent CD44v6 expression and cell adhesion in HepG2 cells

The interaction of osteopontin (OPN) with CD44 and alphavbeta3-integrin has been implicated in numerous signal transduction pathways that may promote cancer metastasis. CD44v6 is a splice variant of CD44 which has been identified as a marker of cancer progression. In this study, immortalized liver carcinoma cells (HepG2) were used to examine the effect of OPN on two isoforms of CD44: CD44 standard (CD44 s) and CD44v6. Western blots demonstrated that OPN up-regulated plasma membrane CD44v6 protein expression in a concentration- and time-dependent fashion. CD44v6 levels returned to control levels when OPN-alphavbeta3-integrin binding was blocked by an RGD peptide or tyrosine kinase activity was inhibited. OPN significantly increased CD44v6 protein synthesis, while simultaneously decreasing protein degradation. Steady-state mRNA levels of both CD44s and CD44v6 were unaltered in the presence of OPN stimulation. OPN increased HepG2 in vitro adhesion to hyaluronate (HA); excess soluble HA extinguished OPN-mediated HepG2 adhesion, indicating CD44 dependence. In conclusion, OPN binds to the alphavbeta3-integrin to increase plasma membrane CD44v6 expression and augment in vitro adhesion to HA. This may contribute to the mechanism by which OPN enhances metastatic behavior in hepatocellular cancer cells.     

CD44 expression in human gliomas.

BACKGROUND AND OBJECTIVES: Glioma invasiveness involves the attachment of tumor cells to the brain extracellular matrix, rich in hyaluronic acid (HA). CD44, the principal receptor for HA is found as a standard molecule (CD44s) or as variants (CD44v). We undertook a retrospective study to evaluate the expression pattern of CD44s, the isoforms CD44v3, v4/5 and v6 and to correlate their expression with clinical-anatomopathological parameters and survival rate. METHODS: The expression of these molecules was evaluated immunohistochemically in 84 gliomas. RESULTS: No expression of CD44v was detected in any tumors. CD44s staining of tumor cells was found in 70 of 84 (83.3%) of the gliomas. In 23 of 39 (59.0%) of the GBM more than the 70% of the cells were stained, while only 2 of 21 (9.5%) of LGA showed high expression. The association between CD44 and histological grade remained when the prognostic variables were considered in a multivariate analysis. Higher expression of CD44 was associated with worse overall survival rate; however, the Cox analysis indicated that survival was not associated with CD44. CONCLUSIONS: Our results suggest that overexpression of CD44s could be relevant in determining the highly invasive behaviour of gliomas, though it does not behave as an independent prognostic factor for survival.     

Serum CD44 splice variants in cervical cancer patients

Aberrant expression of the cell adhesion molecule CD44 has been detected in human tumours and has been shown to be associated with metastasis and poor prognosis in human malignancies. We evaluated serum levels of different soluble CD44 molecules (CD44 standard form and CD44 splice variants v5 and v6) in cervical cancer patients stage IB to IIIB. Two-hundred three serum samples were analysed. Serum levels of CD44st and CD44v5 showed no significant correlation with the presence or absence of cervical cancer. The splice variant CD44v6 showed a mean concentration of 227.3 ± 90.9 (minimum 71.4, maximum 543.9) ng/ml when tumour was present and a mean concentration of 198.7 ± 135.4 (minimum 67.2, maximum 696.3) ng/ml in cases of complete remission (P-value = 0.0001). However, in this preliminary study the sensitivity/specificity characteristic of CD44v6 was poor.     

CD44 is an independent prognostic factor in early-stage cervical cancer

The expression of specific cell-adhesion molecule CD44 isoforms (splice variants) is associated with metastatic spread and poor prognosis in human malignancies. The aim of this study was to evaluate whether CD44 isoform expression is a prognostic factor in early-stage cervical cancer. We used 4 different variant exon sequence-specific murine monoclonal antibodies to the CD44 isoforms CD44v3, CD44v5, CD44v6 and CD44v7-8 to study the prognostic value of CD44 splice variants in 200 cases of International Federation of Gynecology and Obstetrics (FIGO) stage-IB cervical cancer by immunohistochemistry. In the univariate analysis, the expression of CD44v3 (log-rank test, p = 0.03) and CD44v6 (log-rank test, p = 0.03) was correlated with poor overall survival. In the subgroup of patients without metastatic disease in the pelvic lymph nodes, expression of CD44v6 was correlated with poor disease-free and overall survival (log-rank test, p = 0.04 and p = 0.01, respectively). Multivariate analysis, correcting for the confounding variables pelvic lymph-node involvement, depth of invasion and histologic grading, revealed CD44v6 to be an independent prognostic factor for overall survival of patients with early-stage cervical cancer. The results of this study indicate that CD44v6 is an additional prognostic marker in surgically treated cervical cancer. The assessment of CD44 isoform expression could be of clinical value in deciding upon adjuvant therapy, resulting in a more individualized management of therapy. Int. J. Cancer 74:185–188, 1997. © 1997 Wiley-Liss, Inc.     

Cytosol concentrations of CD44 isoforms in breast cancer tissue

The role of the adhesion molecule CD44 in the natural history of breast cancer is controversial. We investigated the CD44 isoform CD44v5 and CD44v6 concentrations in the cytosol of 90 breast cancer specimens, 9 fibroadenomas and 22 normal breast tissue specimens by means of ELISA. CD44v5 and CD44v6 cytosol concentrations were statistically significantly higher in breast cancer compared with fibroadenoma and normal breast tissue (Mann-Whitney U-test, p = 0.009 and p < 0.001, respectively). When CD44 isoforms were correlated with lymph node involvement, histological grading, histological type, tumor stage and age at diagnosis, we found no statistically significant correlation with any of the investigated clinico-pathological parameters. In univariate and multivariate analyses, CD44v5 and CD44v6 were of no prognostic relevance regarding disease-free survival in breast cancer patients (log-rank test, p = 0.16 and p = 0.08, respectively). Our results indicate that CD44 isoforms are increased in samples from tumors relative to normal tissue. Our data show that CD44v5 and CD44v6 isoform expression, although up-regulated by malignant transformation, is not required to acquire a metastatic phenotype in breast cancer. Furthermore, our data support the assumption that cytosolic CD44 isoforms are of no prognostic relevance for disease-free survival of breast cancer patients. Int. J. Cancer (Pred. Oncol.) 79:541–545, 1998.© 1998 Wiley-Liss, Inc.     

CD44变异型在支气管粘膜上皮癌变过程中的表达及其意义

目的　探讨CD44变异型 (以下简称CD44v)表达在肺鳞癌发生中的作用。方法　应用免疫组织化学方法检测 3 4例肺鳞癌及其癌前病变标本中CD44v的表达 ,分析CD44v在肺鳞癌发生过程中出现表达的时间和频率。结果　癌旁正常支气管上皮细胞未见CD44v表达 ,在鳞状化生的支气管上皮细胞、轻→中→重度不典型增生、原位癌、浸润癌中 ,CD44v表达的阳性率逐渐升高。结论　本研究结果表明 ,CD44v可作为判断肺鳞癌发生可能性的标记物 ,在肺鳞癌的早期诊断中具有重要意义。     

多发性骨髓瘤患者OPN、CD44v6的表达与病情进展的关系

背景与目的:骨桥蛋白(osteopontin,OPN)与其受体--CD44受体和整合素受体结合后,参与骨重建、骨质重吸收、血管重塑、癌细胞浸润和转移等过程.为此本研究检测了多发性骨髓瘤(multiple myeloma,MM)患者的OPN和变异的CD44受体(CD44v6)水平,以探讨其与MM病情进展的关系.方法:32例MM患者,分为A组(包括初发和复发病例)和B组(化疗后病情稳定)2组,同时收集外伤骨折或非肿瘤良性贫血患者15例作为对照.采用酶链免疫吸附试验(ELISA)分别检测了患者和对照组的单个核细胞及骨髓基质细胞(bone marrow mononuclear eells,BMSCs)培养上清液的0PN、CD44v6水平并进行相关分析.结果:A组(19例)患者的0PN表达水平显著高于B组(13例)和对照组患者(P<0.05).对A组中的14例患者和B组中的10例患者进行了CD44v6水平检测,其结果A组的CD44v6表达水平显著高于B组和对照组患者(P<0.05),B组的CD44v6水平显著高于对照组(P<0.05);0PN水平与CD44v6(r=0.52,P=0.000)、恶性浆细胞数(r=0.74,P=0.000)、M蛋白水平(r=0.53,P=0.014)、血β2-MG水平(r=0.62,P=0.002)均呈正相关.结论:0PN和CD44v6水平升高与删的发生和病情进展有关;可能与MM的肿瘤负荷、疾病阶段和肿瘤浸润有关.     

CD44 expression in dysplastic epithelium and squamous-cell carcinoma of the esophagus

The molecular events involved in the development of esophageal dysplasia and carcinoma are poorly understood. We examined the expression of CD44, a cell-adhesion molecule, in normal and dysplastic epithelia and in squamous-cell carcinoma (SCC) of the esophagus. A monoclonal antibody (MAb) which recognized all CD44 isoforms and 2 MAbs specific to the CD44v3 and CD44v6 splice variants were used to detect CD44 isoforms in 50 archival specimens. A semi-quantitative scoring system based on the extent and intensity of the immunostaining was used to quantify CD44 expression. In normal epithelium, expression of CD44 was strongest in the basal-cell layer and weak or absent in surface cells. Expression of CD44 was increased in dysplastic epithelium as compared with normal epithelium. The extent of this increase correlated directly with the severity of dysplasia. CD44 was expressed in all SCCs, but the extent and intensity of immunostaining varied with areas of tumor differentiation. The well-differentiated components showed greater CD44 expression than the moderately and poorly differentiated components. The patterns of expression of CD44v3 and CD44v6 were strikingly similar to that of total CD44 in normal, dysplastic and malignant esophageal epithelia. Thus, changes in expression of these splice variants likely account to some extent for the changes in total CD44 expression observed in the dysplastic and malignant transformation of the esophagus. Our results suggest that changes in the expression of CD44 may be involved in the development of esophageal dysplasia as well as SCC. © 1996 Wiley-Liss, Inc.     

Reduced expression of CD44 v3 and v6 is related to invasion in lung adenocarcinoma

We have previously reported that the histological pattern of invasion is correlated with the prognosis of surgically treated patients of lung adenocarcinoma. On the other hand, several clinicopathologic studies have shown that CD44 variant isoforms are associated with invasion and metastasis in human malignant tumors. The expression of CD44 variant isoforms v3 and v6 was analyzed in 93 Japanese lung adenocarcinoma patients by immunostaining to study the relationship between their expression and the invasion in lung adenocarcinoma. The specimens were histologically categorized into three groups. Both the invasive lesion and the noninvasive lesion were observed in 49 out of 93 cases (group I). Twenty cases were noninvasive carcinoma growing mainly in a lepidic pattern (group II). Twenty-three cases were invasive carcinoma which showed no frankly noninvasive lesion growing in a lepidic pattern (group III). The significant reduced expression of CD44 v3 and v6 was observed in the invasive lesion compared with the noninvasive lesion in adenocarcinoma of group I (P < 0.05). Although reduced expression of CD44 v3 and v6 was observed in the invasive carcinoma of group III compared with the noninvasive carcinoma of group II, it was not significant (P = 0.0693 for v3, P = 0.0827 for v6). The pattern of expression of CD44 v3 was significantly concordant with that of CD44 v6 (P < 0.0001). Our results suggest that reduced expression of CD44 v3 and v6 is associated with the invasion in the lung adenocarcinoma.