Evidence for preferential adhesion of ovarian epithelial carcinoma cells to type I collagen mediated by the αA2β1 integrin

Epithelial ovarian carcinoma, the leading cause of gynecologic cancer death, is characterized by widespread intra-abdominal metastases mediated primarily by surface shedding of tumor cells and peritoneal implantation. Whereas hematogenous metastasis is known to involve cellular adhesion, extracellular matrix proteolysis and cell migration, the role of these processes in the intraperitoneal dissemination of ovarian cancer remains unclear. To analyze further the role of adhesion and proteolysis in ovarian carcinoma dissemination, we have characterized the adhesive profiles of 4 primary cultures of ovarian carcinoma cells and 5 ovarian carcinoma cell lines. Our data demonstrate preferential adhesion of ovarian carcinoma cells to interstitial type I collagen. Analysis of adhesion molecule expression demonstrated the presence of the α2 and β1 integrin subunits by cell surface ELISA, immunoprecipitation and immunohistochemistry. Furthermore, antibodies directed against the α2 and β subunits inhibited adhesion of ovarian carcinoma cells to type 1 collagen by 56% and 95%, respectively. Plasminogen activator and matrix metalloproteinase production by adherent cells was not altered as a consequence of adhesion to individual extracellular matrix proteins; however, adhesion to an extracellular matrix comprised primarily of interstitial collagen increased plasminogen activator activity in 5 of 5 cell lines. Since the ovarian carcinoma micro-environment is rich in type 1 collagen, our data suggest that preferential adhesion to type 1 collagen followed by secretion of serine and metalloproteinases may represent a biochemical mechanism by which the intraperitoneal dissemination of ovarian carcinoma is mediated. © 1996 Wiley-Liss, Inc.     

Phenotypic characterization and prognostic impact of circulating γδ and αβ T‐cells in metastatic malignant melanoma

Human T cells carrying γδ T‐cell receptors (TCRs) represent a minor population relative to those with αβ TCRs. There has been much interest recently in the possibility of using these γδ T‐cells in cancer therapy because they can kill tumor cells in vitro in an MHC‐unrestricted manner, and possess potential regulatory capability and antigen‐presenting capacity. The presence of γδ T‐cells in late‐stage melanoma patients and their relationship with survival has not been extensively explored, although relatively lower percentages of total γδ T‐cells and Vδ2+ cells have been reported. Here, we present a detailed analysis of associations of γδ T‐cell subsets and differentiation stages with survival in Stage IV patients, compared with CD4+ and CD8+ αβ T‐cells. We found an increased Vδ1:Vδ2‐ratio and a decreased CD4:CD8‐ratio in patients compared to healthy controls, on the basis both of relative frequencies and absolute cell counts per μL blood. Nonetheless, Kaplan–Meier analyses showed that a higher than median frequency of Vδ1+ cells was negatively associated with survival, whereas there were no positive or negative associations with frequencies of Vδ2+ cells. Correlations of cell differentiation status with survival revealed a negative association of early‐differentiated Vδ1+ T cells with survival, both on the basis of relative frequencies and absolute counts. There was also a positive correlation between the frequencies of early‐differentiated CD8+ αβ T‐cells and survival. Our findings suggest peripheral blood frequencies of Vδ1+ T‐cells as a potential prognostic marker in melanoma. The mechanisms by which higher abundance of Vδ1+ cells are associated with poorer survival require determination.     

CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

Cadherin 6 (CDH6) is significantly overexpressed in advanced ovarian and renal cancers. However, the role of CDH6 in cancer metastasis is largely unclear. Here, we investigated the impact of CDH6 expression on integrin‐mediated metastatic progression. CDH6 preferentially bound to αIIbβ3 integrin, a platelet receptor scarcely expressed in cancer cells, and this interaction was mediated through the cadherin Arginine–glycine–aspartic acid (RGD) motif. Furthermore, CDH6 and CDH17 were found to interact with α2β1 in αIIbβ3^low cells. Transient silencing of CDH6, ITGA2B, or ITGB3 genes caused a significant loss of proliferation, adhesion, invasion, and lung colonization through the downregulation of SRC, FAK, AKT, and ERK signaling. In ovarian and renal cancer cells, integrin αIIbβ3 activation appears to be a prerequisite for proper α2β1 activation. Interaction of αIIbβ3 with CDH6, and subsequent αIIbβ3 activation, promoted activation of α2β1 and cell adhesion in ovarian and renal cancer cells. Additionally, monoclonal antibodies specific to the cadherin RGD motif and clinically approved αIIbβ3 inhibitors could block pro‐metastatic activity in ovarian and renal tumors. In summary, the interaction between CDH6 and αIIbβ3 regulates α2β1‐mediated adhesion and invasion of ovarian and renal cancer metastatic cells and constitutes a therapeutic target of broad potential for treating metastatic progression.     

β1-integrins dominate cell traffic of leukemic cells in human bone-marrow stroma

Migration patterns of leukemic cells in bone marrow are largely regulated by cell contacts between leukemic cells and stromal cells or extra-cellular matrix. The mechanism of this interaction with bone-marrow stromal cells was studied in a human in vitro model. Migration behavior of erythroleukemia cell line K562, derived from a patient with chronic myeloid leukemia, was compared with that of the erythroleukemia cell line HEL92.1.7 and the promyelocytic leukemia cell line HL60 from acute leukemias. Interaction varied between low binding affinity (K562) to intensive cell interaction (HEL92.1.7) followed by invasion into the stromal cell monolayer. Some of the HL60 cells adhered to stromal cells, while the remainder migrated into the stromal cell monolayer. The role of adhesion molecules in these cell interactions was determined. Distinct expression of β₁-integrins ICAM-I, CD44 and VCAM-I was detected on the different cell lines. Inhibition studies pointed to a dominant role of VLA-4- and VLA-5-mediated interactions. K562 lacked VLA-4 and a low affinity of the VLA-5 on these cells resulted in an absence of binding to the bone-marrow stroma. These results indicate the VLA-5/fibronectin, VLA-4/fibronectin and the VLA-4/VCAM-I interaction pathways between leukemic cells and bone-marrow stroma. © 1996 Wiley-Liss, Inc.     

Expression of β1 integrin receptors in transformed mouse epidermal keratinocytes: Upregulation of α5β1 in spindle carcinoma cells

The adhesive properties and the expression of extracellular matrix receptors of the β1-integrin subfamily were analyzed in transformed epidermal keratinocyte cell lines of different stages of mouse skin carcinogenesis. One- and two-dimensional analyses of the immunoprecipitates obtained with anti-β1- and specific anti-α-integrin subunits showed qualitative and quantitative changes in the expression of β1 integrins by the different cell lines. The polyvalent α3β1 integrin was expressed by all analyzed cell lines, although the levels detected in undifferentiated spindle CarC cells were lower than those present in the rest of keratinocyte cell lines. In contrast, spindle cells expressed high levels of the specific fibronectin receptor α5β1, whereas this integrin was absent or expressed at very reduced levels in the other epithelial cell lines. Expression of α5β1 integrin in spindle cells appeared organized in cell-substratum contact areas on spread cells. In addition, high and homogenous expression of α5β1 was detected in fully undifferentiated tumors induced in nude mice by three independent spindle cell lines. These results suggest that the expression of α5β1 integrin is upregulated during the development of spindle cell carcinomas that occur in the last stages of mouse skin carcinogenesis and can be associated with the acquisition of the fibroblastoid phenotype of spindle cells. On the other hand, expression of the collagen receptor α2β1 was demonstrated in a transformed cell line (PDV), and it was apparently also expressed in two other malignant keratinocyte cell lines (PDVC57 and HaCa4). The expression of α2β1 was correlated with the increased adhesion to collagen type I and Collagen type IV exhibited by the tumorigenic cell lines. © 1995 Wiley-Liss Inc.     

Integrin expression in cutaneous malignant melanoma: Association of the α3/β1 heterodimer with tumor progression

The cell-surface heterodimers of the integrin family of molecules, which mediate cell-cell and cell-substratum interactions, are likely to be functionally relevant in local and metastatic tumor growth. In the present study we have analyzed whether the α₃/β₁ receptor for collagen, laminin and fibronectin undergoes changes in expression during tumor progression in cutaneous malignant melanoma (CMM). The results of this study have demonstrated that, while low levels of VLA₃ expression are detectable in benign lesions, in primary melanomas the heterodimer undergoes progressive increase in expression which correlates with the degree of dermal invasiveness. Metastatic lesions were found VLA₃ positive in 82% of cases. Furthermore, the heterodimer is homogeneously expressed in multiple autologous metastases. The presence of VLA₃ correlates with detection of at least one of the ligands in 45% of the cases studied. These findings provide additional evidence that tumor progression in CMM is associated with changes in integrin phenotypes which include the α₃/β₁ heterodimer.     

Integrin β1 is an essential factor in vasculogenic mimicry of human cancer cells

Vasculogenic mimicry (VM) formation by cancer cells is known to play a crucial role in tumor progression, but its detailed mechanism is unclear. In the present study, we focused on integrin β1 (ITGB1) and assessed the role of ITGB1 in VM formation. We used in vitro methods to seed cancer cells on Matrigel to evaluate the capability of VM formation. We carried out ITGB1 gene deletion using the CRISPR/Cas9 system, and these ITGB1‐knockout cells did not show a VM‐like network formation. Further, reintroduction of ITGB1 rescued VM‐like network formation in ITGB1‐knockout cells. In conclusion, ITGB1 is a critical factor in VM of human cancer cells, and inhibition of ITGB1 may be a novel therapeutic approach for malignant cancer.     

Estrogen receptor α and β in esophageal squamous cell carcinoma

A gender difference has been reported in the morbidity of esophageal squamous cell carcinoma (ESCC). Estrogens have been proposed to play a role in this difference but the details have not yet been clarified. Therefore, in the present study, we examined the status of estrogen receptor (ER)α and ERβ in 90 Japanese ESCC patients. ERα and ERβ immunoreactivity was detected in the nuclei of ESCC cells (41.1 and 97.8%, respectively). There was a significant positive association between the ERβ H score and histological differentiation (P = 0.0403), TNM‐pM (LYM) (P = 0.00164) and Ki67/MIB1 LI of carcinoma cells (P = 0.0497, r = 0.207). In addition, the ERβ status of carcinoma cells was significantly correlated with unfavorable clinical outcome of the patients. Multivariate analysis further revealed the ERβ status in carcinoma cells as an independent unfavorable prognostic factor of these patients. We further examined the effects of estrogen treatment on ESCC cell line (ECGI‐10) transfected with ERα or ERβ in vitro. The number of ECGI‐10 transfected with ERβ was increased by estradiol or ERβ specific agonist but estradiol did not exert any effect upon the cell number of ECGI‐10 transfected with ERα. In summary, the results of the present study clearly demonstrate that the status of ERβ in ESCC was closely associated with the unfavorable prognosis, possibly through altering cell proliferation of carcinoma cells. (Cancer Sci 2012; 103: 1348–1355)     

影响T1、T2乳腺癌淋巴结转移率的单因素分析

目的：回顾性分析乳腺癌患者的临床资料,探讨影响T1、T2乳腺癌淋巴结转移率的临床因素。方法：对中国人民解放军第八二医院2003年到2013年确诊的134例乳腺癌患者的临床资料进行回顾性分析。结果：全组均为女性患者,年龄27-74岁,中位年龄为48岁。单因素分析结果显示,患者年龄、肿瘤大小、肿瘤位置、病理类型、ER、PR、HER-2与T1、T2乳腺癌腋窝淋巴结转移率有统计学意义。P值分别为0．012、0．001、0．015、0．048、0．002、0．001、0．001。结论：老年组、T1组、其他象限组、特殊类型乳腺癌组、ER（＋）组、PR（＋）组、HER-2（-）组淋巴结转移率水平较低,更有可能选择前哨淋巴结活检术进一步检测。     

Phenotypic properties of cultured tumor cells: Integrin αIIbβ3 expression,tumor-cell-induced platelet aggregation,and tumor-cell adhesion to endothelium as important parameters of experimental metastasis

The present study was undertaken to investigate the factors involved in determining the metastatic potential of cultured cells derived from solid tumors. We first investigated the effects of cell source and culture conditions on lung colony formation by i.v. injected B16a (B16 amelanotic melanoma) cells and inhibition of tumor colony formation by the thromboxane A2 syn-thase inhibitor, CGS14854. Prolonged culture resulted in a 10-fold decrease in the incidence of B16a lung colonies, whereas passage in vivo for 150 days did not affect lung colony formation by tumor cells isolated from enzymatic dispersates by centrifugal elutriation. Cultured BI6a cells maintained at low density (LD) and harvested at low passage (LP) formed significantly more lung colonies than B16a cells harvested at high densities (HD) or high passage (HP). Over-confluent tumor cells produced even lower number of lung colonies. Lung colony formation by elutriated BI6a cells (i.e., cells freshly isolated from tumor tissue) was consistently inhibited by CGS14854, whereas inhibition of lung colony formation by cultured B16a cells was dependent upon culture conditions. CGS 14854 was ineffective or less effective against HD/HP B16a cells. The differences in lung colony formation between LD, HD and elutriated B16a cells were not due to differential cell-cycle distribution. Mechanistic studies indicated that LD/LP tumor cells induced aggregation of homologous platelets, whereas HD/HP BI6a cells failed to induce significant platelet aggregation. Aggregation of homologous platelets correlated positively with lung-colonizing ability. Additionally, LD/LP cells demonstrated higher adhesion to endothelium than HD/HP BI6a cells. Finally, LD/LP BI6a cells expressed higher levels of α_IIbβ₃ integrins than HD/HP tumor cells, as determined by flow cytometry and immunofluorescence.     

Small extracellular vesicles deliver TGF‐β1 and promote adriamycin resistance in breast cancer cells

Chemotherapeutic resistance is a major obstacle in the control of advanced breast cancer (BCa). We have previously shown that small extracellular vesicles (sEVs) can transmit adriamycin resistance between BCa cells. Here, we describe that sEV‐mediated TGF‐β1 intercellular transfer is involved in the drug‐resistant transmission. sEVs were isolated and characterized from both sensitive and resistant cells. sEVs derived from the resistant cells were incubated with the sensitive cells and resulted in transmitting the resistant phenotype to the recipient cells. Cytokine antibody microarray revealed that most metastasis‐associated cytokines present at the high levels in sEVs from the resistant cells compared with their levels in sEVs from the sensitive cells, particularly TGF‐β1 is enriched in sEVs from the resistant cells. The sEV‐mediated TGF‐β1 intercellular transfer led to increasing Smad2 phosphorylation and improving cell survival by suppressing apoptosis and enhancing cell mobility. Furthermore, sEV‐mediated drug‐resistant transmission by delivering TGF‐β1 was validated using a zebrafish xenograft tumor model. These results elaborated that sEV‐mediated TGF‐β1 intercellular transfer contributes to adriamycin resistance in BCa.     

Implication of the αvβ3 integrin in the adhesion of the ovarian-adenocarcinoma cell line IGROV1

Accumulating evidence suggests that integrins, which participate in many complex cellular processes, are important for tumor progression and metastasis. In order to understand the role of these cell-surface receptors and of their ligands in the biological behavior of ovarian tumor cells, we have examined the expression of integrins in the human ovarian-adenocarcinoma cell line IGROVI. These cells expressed the αv sub-unit and used it to attach on vitronectin (Vn). A monoclonal antibody (MAb) (69-6-5) specific to αv blocked the attachment of IGROVI cells on Vn and fibrinogen (Fg), but not on fibronectin (FN) and other adhesive ligands. Immunoprecipitation of surface biotinylated cells followed by Western blotting showed that the αv sub-unit was associated with β3, but not with β1, β5 or β6. When cells were cultivated on glass coverslips or on Vn sub-stratum in serum-free medium, immunofluorescence staining with MAb 69-6-5 revealed the presence of αv at cell-cell contacts and at focal contacts, supporting its active participation in adhesion as part of a functional heterodimer. Furthermore, immunofluorescence data showed the presence of Vn as a fibrillar network in IGROVI cells cultivated on FN-coated slides. These results suggest that αvβ3 and its Vn ligand may play a important role in the behavior of ovarian epithelial tumors.     

Expression of phospholipases γ1, β1, and δ1 in primary human colon carcinomas and colon carcinoma cell lines

The levels of expression of phosphoinositide-specific phospholipase Cs (PLCs) were examined in a series of primary human colon carcinomas and in eight colon carcinoma cell lines by using monoclonal antibodies and cDNA probes for PLCγ1, PLCβ1, and PLCδ1. Western and northern blot analyses of PLCγ1 revealed elevated expression of this isozyme at both the protein and mRNA levels in most tumors when compared with paired adjacent normal mucosa samples (in 11 of 13 pairs in the western blots and 8 of 9 pairs in the northern blots). On the other hand, decreased levels of the PLCδ1 protein were seen in most colon carcinomas (12 of 13 paired samples). The levels of PLCβ1 protein were too low to detect possible differences between the carcinoma and normal mucosa samples. Relatively high expression of PLCγ1 was found in almost all of the eight human colon carcinoma cell lines at both the protein and mRNA levels. Only weak expression of PLCβ1 was detected in these cell lines, by both western and northern blot analyses, and PLCδ1 protein was not detected in any of the carcinoma cell lines. These findings provide evidence that colon carcinomas display altered expression of individual isoforms of PLCs and suggest that increased expression of PLCγ1 may play an important role in colon carcinogenesis. © 1995 Wiley-Liss Inc.     

Expression of retinoic acid α and β receptor genes in liver and hepatocellular carcinoma

cDNA probes for human retinoic acid receptors α and β (RARα and RARβ) were modified for use as specific hybridization probes to study hepatocellular carcinomas (HCC) and cell lines, liver regeneration, and fetal development. RARβ mRNA was detected at low levels in adult liver and rose markedly during the early phase of liver regeneration. RARβ mRNA was present at very low levels in HCC and was not detected in fetal liver. In contrast, RARα mRNA was present at low levels in normal liver, but showed a marked elevation in several HCCs and cell lines. Growth of cell lines was altered by retinoic acid (RA), but the effects could not be predicted by the levels of either RARα or RARβ mRNA. However, the response correlated with cell phenotype. Three cell lines with an adult phenotype (high albumin and low α-fetoprotein) were inhibited by RA, two undifferentiated lines showed moderate growth stimulation, and two of three cell lines that had high levels of α-fetoprotein were markedly stimulated by RA.