Long-term human breast carcinoma cell lines of metastatic origin: Preliminary characterization

Summary Nineteen human breast carcinoma cell lines have been established as continous cultures during the past 6 years in our laboratory. This preliminary report is designed to list the lines by their designated code numbers (MDA-MB) and present a brief summary of their morphological, cytogenetic and biochemical characteristics. Sixteen of our lines were obtained from pleural effusions, two from brain metastases, and one from pericardial fluid. All lines have been shown to be distinct entities and are uncontaminated by HeLa cells or each other. A lq marker chromosome is present in all but one of the lines examined. This research was sponsored by the National Cancer Institute Contract NO1-CB-23869; Institutional Grant 5S 07 RR 5511-15 awarded by the Division of Research Resources, and a Kelsey-Leary Grant NO 974.     

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133⁺ cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.     

Absence of correlation between chemo- and radioresistance in a range of human tumour cell lines

The correlation between cellular resistance to radiation and to chemotherapeutic drugs has been investigated in a number of solid tumour cell lines, and preliminary results indicate no direct relationship. The acquisition of a multidrug resistance (MDR) profile by adriamycin-selected variants of a human squamous lung carcinoma, an ovarian carcinoma, a cervical carcinoma and by a colchicine-selected variant of a Chinese hamster ovarian carcinoma resulted in alterations to their radiosensitivity. However, the degree of change in the radiosensitivity of the MDR cell lines could not be predicted from their level of resistance to adriamycin. Clonal populations derived from DLKP-A, an adriamycin-selected MDR variant of the human lung carcinoma cell line DLKP, exhibited individual radiosensitivity profiles, which did not correlate with their chemoresistance. Exposure of DLKP to consecutive increasing doses of radiation did not confer cross-resistance to chemotherapeutic drugs.Abbreviations LD₅₀ Radiation dose which kills 50% of the cell population - IC₅₀ Chemotherapeutic drug concentration which kills 50% of the cell population - MDR Multidrug resistance - Gy Gray - EMS Ethyl methanesulfonate     

A set of stage-dependent embryonic antigens expressed in cell cultures of BALB/c mouse embryos and in transformed cell lines

A rabbit antiserum (A2) directed against the detergent-solubilized fraction of the simian virus 40-transformed mouse embryo fibroblast cell line VLM detects common antigens in primary cell cultures from BALB/c mouse embryos and in transformed cell lines from various species. Positively reacting cell cultures show a set of polypeptides with molecular weight species p86, p74, p68, p46, p42, p40, and p35. As tested by Western blotting procedures, all immunoprecipitated proteins carry immunologically reactive determinants. By analysis with two-dimensional gel electrophoresis, all precipitated polypeptides show charge heterogeneities. Concerning the two major members of the protein set, p40 consists of at least four subspecies with isoelectric points in the range of pH 6.2-6.8, whereas p35 is composed of two subspecies focusing between pH 6.4 and pH 7.2. By comparison of the two-dimensional patterns of p35 of various transformed cell lines, a basic (pH 6.6-7.2) and an acidic (6.4-6.6) charge type of p35 could be observed. Comparative analyses of primary cell cultures from 12-16-day mouse embryos show the immunoprecipitated set of polypeptides only in the 16-day embryo cell cultures. After six further propagations, these cells express the immunoreactive proteins as strongly as the primary cell cultures. In embryonic cell cultures of day 14 of gestation the expression of this set of antigens is induced only when cells are propagated at least six times. Under identical conditions these proteins could not be induced in cell cultures of 18-day-old mouse embryos. None of the polypeptides could be immunoprecipitated from primary mouse kidney cell cultures of 12-day-old mice even when the cultures were propagated at least 15 times. This set of polypeptides is also present in simian virus 40-transformed cells of hamster, rat, monkey, and human origin. These findings suggest that in simian virus 40-transformed mouse cells, in addition to p53, the synthesis of other embryonic antigens is reactivated. The presence of the described set of polypeptides in polyoma virus-transformed cells of rat and mouse origin and in cell lines derived from malignant human tumors might indicate common functions in metabolic patterns of transformed cells.     

In vitro proliferation and in vivo malignancy of cell lines simultaneously derived from a chemically-induced heterogeneous rat mammary tumor

Summary Identification of clones in primary tumors responsible for proliferation, invasion, and metastasis was carried out. Four different aneuploid established cell lines derived from a ductal infiltrating mammary rat tumor induced by 7,12-dimethylbenz[a]anthracene were studied for proliferative and growth features in vitro and for tumorigenic and metastatic potential in vivo in nude mice. Clones, named RM1, RM2, RM3, and RM4, were characterized by different proliferative activity. Clone RM1 showed the highest proliferative activity by both tritiated thymidine incorporation and S-phase flow cytometry, followed by clone RM4. Conversely, clones RM2 and RM3 showed a lower proliferation rate. Growth-promoting activity, tested on 3T3 Swiss cells, was high in all clones, although RM1 showed significantly lower growth factors—releasing activity. Nude mice tumorigenesis demonstrated a strong tumor induction of line RM1 (100% of the mice after 47±7 d) and a slightly lower tumor induction of line RM4 (70% of the mice after 69±9 d). Line RM3 showed tumor induction in 40% of the mice after 186±16 d. Lines RM2 showed no tumor induction. Metastasis occurred in mice treated with line RM1 only. Therefore, tumorigenesis and metastasis correlate with proliferation but not with the release of growth factors. In conclusion, flow cytometry monitoring of clones from heterogeneous primary tumors proved to be a suitable model for the study of in vivo malignancy and in vitro proliferation.     

NOB1在食管鳞状细胞癌中的表达及临床意义

目的研究NOB1基因在食管鳞状细胞癌（Esophageal squamous cell carcinoma,ESCC）中的表达及临床意义。方法利用免疫组织化学SP法检测59例ESCC及其相应（50例）的远端正常食管黏膜组织中NOB1的表达。结果 ESCC中NOB1的阳性率为71%,正常食管黏膜鳞状上皮中的阳性率为26%,两组比较,差异有统计学意义（P〈0.05）。NOB1的表达与ESCC的分化程度及淋巴结转移相关,与患者的性别,年龄以及肿瘤浸润深度无关。结论 NOB1在ESCC中表达升高,可能在ESCC发生发展过程中起重要作用。     

NUMB在口腔白斑和鳞癌中的表达变化及意义

目的:研究NUMB在口腔白斑和鳞癌中的表达及意义。方法:采用免疫组织化学法检测88例口腔正常黏膜、口腔白斑及口腔鳞癌石蜡包埋组织中NUMB蛋白的表达。结果:NUMB在口腔正常黏膜(95.7%)、口腔白斑(75%)及口腔鳞癌(4.7%)中均有阳性表达但是表达频率依次降低。NUMB在各个组中的阳性表达率具有统计学差异(P0.05)。结论NUMB阳性表达率随口腔组织恶性程度增高而减少的趋势提示该基因可能在口腔正常黏膜到口腔白斑再到口腔鳞癌的转化中起作用,NUMB有可能成为早期发现癌变的分子生物学标志之一。     

食管鳞癌染色体及基因组DNA畸变研究进展

食管鳞癌是我国常见的消化道恶性肿瘤,进展快且预后差。由于早期一般无明显症状,临床确诊的食管鳞癌大多已发展到了中晚期,治愈难度较大。越来越多的证据表明,在食管鳞癌发生发展过程中,染色体及基因组DNA畸变均是最常见的遗传学改变。文章就食管鳞癌染色体及基因组水平异常的研究进展作一综述。     

BIIB021, a novel Hsp90 inhibitor,sensitizes esophageal squamous cell carcinoma to radiation

BIIB021 is a novel, orally available inhibitor of heat shock protein 90 (Hsp90) that is currently in phase I/II clinical trials. BIIB021 induces the apoptosis of various types of tumor cells in vitro and in vivo. The aim of this study is to investigate the effect of BIIB021 on the radiosensitivity of esophageal squamous cell carcinoma (ESCC). The results indicated that BIIB021 exhibited strong antitumor activity in ESCC cell lines, either as a single agent or in combination with radiation. BIIB021 significantly downregulated radioresistant proteins including EGFR, Akt, Raf-1 of ESCC cell lines, increased apoptotic cells and enhanced G₂ arrest that is more radiosensitive cell cycle phase. These results suggest that this synthetic Hsp90 inhibitor simultaneously affects multiple pathways involved in tumor development and progression in the ESCC setting and may represent a better strategy for the treatment of ESCC patients, either as a monotherapy or a radiosensitizer.     

Characterization of VIP- and helodermin-preferring receptors on human small cell lung carcinoma cell lines

We investigated the molecular and pharmacologic characteristics of VIP receptors on two human SCLC cell lines: NCI-N592 and NCI-H345. With NCI-N592 cell, the order of potency of VIP-related peptides in inhibiting ¹²⁵I-VIP binding and in stimulating cAMP production was typical of the human VIP receptor. By covalent cross-linking, a polypeptide of Mr 62,300 was obtained. Conversely, the behavior of NCI-H345 cell line was totally different: helodermin was the most potent peptide, VIP and PHI were equipotent, while hGRF and secretin were totally ineffective. These results suggest that NCI-N592 cells possess a typical VIP receptor while NCI-H345 cells possess a helodermin-preferring receptor, and that the natural target of helodermin might not be the VIP receptor.     

血管生成拟态和E-cad在食管鳞状细胞癌中的表达及临床意义

目的 探讨血管生成拟态( vasculogenic mimicry,VM)与E-钙粘蛋白(E-cadherin,E-cad))在食管鳞癌(esophageal squamous cell carcinoma)组织中的表达及意义.方法 收集食管鳞状细胞癌术后标本100例和30例癌旁正常食管黏膜,应用免疫组化法和组织化学法检测食管鳞状细胞癌和正常食管黏膜组织中VM和E-cad的表达情况.结果 在食管鳞状细胞癌组织和正常食管黏膜组织中,VM和E-cad的阳性表达率分别为47.0％、48.0％和0％、70.0％,差异有统计学意义(P＜0.05);VM及E-cad的表达与食管鳞癌的组织学分级、临床分期及淋巴结转移(P＜0.05);VM与E-cad在食管鳞癌中的表达呈负相关(r=-0.865,P=0.000).多因素分析:PTNM分期、淋巴结转移、VM和E-cad的表达是影响食管鳞癌根治术后患者预后的独立因素(P＜0.05);VM阳性组与阴性组的5年生存率分别为4.3％和64.2％,,差异有统计学意义(P=0.000);E-cad阳性组与阴性组5年生存率分别为60.4％和15.4％,差异有统计学意义(P=0.000).结论 具有VM结构的食管鳞状细胞癌的分化程度低,恶性度高,预后差,VM和E-cad表达的程度与食管鳞状细胞癌的进展和预后密切相关.     

Focal adhesion complex proteins in epidermis and squamous cell carcinoma

Focal adhesions (FAs) are large, integrin-containing, multi-protein assemblies spanning the plasma membrane that link the cellular cytoskeleton to surrounding extracellular matrix. They play critical roles in adhesion and cell signaling and are major regulators of epithelial homeostasis, tissue response to injury, and tumorigenesis. Most integrin subunits and their associated FA proteins are expressed in skin, and murine genetic models have provided insight into the functional roles of FAs in normal and neoplastic epidermis. Here, we discuss the roles of these proteins in normal epidermal proliferation, adhesion, wound healing, and cancer. While many downstream signaling mechanisms remain unclear, the critically important roles of FAs are highlighted by the development of therapeutics targeting FAs for human cancer.     

MicroRNA and protein profiles in invasive versus non-invasive oral tongue squamous cell carcinoma cells in vitro

Complex molecular pathways regulate cancer invasion. This study overviewed proteins and microRNAs (miRNAs) involved in oral tongue squamous cell carcinoma (OTSCC) invasion. The human highly aggressive OTSCC cell line HSC-3 was examined in a 3D organotypic human leiomyoma model. Non-invasive and invasive cells were laser-captured and protein expression was analyzed using mass spectrometry-based proteomics and miRNA expression by microarray. In functional studies the 3D invasion assay was replicated after silencing candidate miRNAs, miR-498 and miR-940, in invasive OTSCC cell lines (HSC-3 and SCC-15). Cell migration, proliferation and viability were also studied in the silenced cells. In HSC-3 cells, 67 proteins and 53 miRNAs showed significant fold-changes between non-invasive vs. invasive cells. Pathway enrichment analyses allocated “Focal adhesion” and “ECM-receptor interaction” as most important for invasion. Significantly, in HSC-3 cells, miR-498 silencing decreased the invasion area and miR-940 silencing reduced invasion area and depth. Viability, proliferation and migration weren’t significantly affected. In SCC-15 cells, down-regulation of miR-498 significantly reduced invasion and migration. This study shows HSC-3 specific miRNA and protein expression in invasion, and suggests that miR-498 and miR-940 affect invasion in vitro, the process being more influenced by mir-940 silencing in aggressive HSC-3 cells than in the less invasive SCC-15.     

Deep-seated rectal/anal basaloid carcinoma: useful immunocytochemistry in rare squamous cell carcinoma variants

Objectives:  To report the cytological aspects of ano-rectal basaloid carcinoma (BC) variant in endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) conventional and liquid-based cytology (LBC), in a series of 10 cases of deep-seated squamous cell carcinomas (SCC), and to discuss the diagnostic difficulties in interpreting the morphology and immunocytochemical findings.
Methods:  Ten cases of EUS-FNA smears and LBC specimens of deep-seated pelvic masses were retrospectively collected from January 2001 to November 2006.
Results:  Ten EUS-FNA specimen cases were SCC, eight corresponding to usual SCC and two to BC-variant. Of these two cases, only one was correctly diagnosed by EUS-FNA specimen, whereas in the second case, the initial cytological diagnosis was poorly differentiated adenocarcinoma and the final diagnosis of basaloid carcinoma variant was established on surgical resection. Immunocytochemistry (ICC) using CK7, CK20 and CK34βe12 on FNA specimens confirmed the diagnosis retrospectively.
Conclusion:  The diagnosis of basaloid variant of SCC in a rectal location can be very difficult, both on account of the uncommon location and because of the low specificity of morphological aspects on EUS-FNA smears. The immunocytochemical technique, including a limited spectrum of keratins (CK7, CK20, CK34βe12, and p63) is necessary to avoid this diagnostic pitfall.     

Cytotoxic T cell responses to Streptococcus are associated with improved prognosis of oral squamous cell carcinoma

Several species of Streptococcus, such as S. salivarius, S. mitis, and S. anginosus, are found to extensively colonize the oral cavity and the upper respiratory tract, and have been shown to increase in patients with oral squamous cell carcinoma (OSCC). Accumulating evidence have revealed that commensal bacteria are involved in antitumor immunity via T cell-mediated mechanisms, but the role of Streptococcus enrichment in OSCC is yet unclear. In this study, we stimulated peripheral blood mononuclear cells from non-cancer controls (NCs) and OSCC patients with S. salivarius, S. mitis, and S. anginosus. We observed that compared to NC subjects, OSCC patients at earlier stages had higher frequencies of granzyme B-expressing CD8 T cells for all Streptococcus species tested, while OSCC patients at more advanced stages had higher frequencies of granzyme B-expressing CD8 T cells for S. anginosus but not other Streptococcus species. In OSCC patients, the Streptococcus-reactive CD8 T cells presented significantly lower levels of PD-1 and TIM-3 expression than Streptococcus-nonreactive CD8 T cells. The clinical outcomes of OSCC patients in our cohort were tracked for 24 months after the resection of the primary tumor. In patients that did not present tumor recurrence, the frequencies of S. salivarius-reactive and S. mitis-reactive CD8 T cells were significantly higher than that in patients that developed recurrent tumor. Furthermore, in patients with tumor recurrence, the duration between primary tumor resection and tumor recurrence was positively associated with the frequencies of S. salivarius-reactive and S. anginosus-reactive CD8 T cells. Together, we demonstrated that Streptococcus-reactive CD8 T cell responses might contribute to antitumor immunity in OSCC patients.     

maspin在食管鳞状细胞癌中的表达及其与微血管密度的关系

目的研究旨在寻找能预测食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)浸润、转移及术后生存率的生物因子。方法采用免疫组织化学ElivisionTM plus法检测100例ESCC和30例正常食管组织中maspin的表达和微血管密度(microvessel density,MVD)情况。结果在正常食管组织和ESCC组织中,maspin的阳性表达率分别为100%和43.0%,差异有显著性(P<0.01);maspin的表达水平与肿瘤的分化程度、浸润深度、淋巴结转移和临床分期有关(全部P<0.01);MVD计数与肿瘤的大小、分化程度、淋巴结转移以及临床分期有关(全部P<0.01);且maspin的表达与MVD计数呈负相关(P<0.01)。结论 maspin的表达和MVD计数与ESCC组织的分化程度、转移和预后等均有关;maspin和MVD联合检测对ESCC的进展及预后判断有重要意义。     

KITENIN increases invasion and migration of mouse squamous cancer cells and promotes pulmonary metastasis in a mouse squamous tumor model

KAI1 C-terminal interacting tetraspanin (KITENIN) is reported to promote metastasis in mouse colon cancer models. We investigated the role of KITENIN on the progression of squamous cell carcinoma (SCC). In a preliminary clinical study using resected tissues from head and neck SCC patients, KITENIN was highly expressed in tumors and metastatic lymph nodes, while KAI1 was more increased in adjacent mucosa than in tumor. KITENIN-transfected mouse squamous cancer (SCC VII/KITENIN) cells showed significantly higher invasion, migration, and proliferation than empty vector-transfected cells. In syngeneic mouse squamous tumor models, more increased tumor volume and enhanced lung metastasis were found in SCC VII/KITENIN cells-injected mice. Thus, KITENIN increases invasion and migration of squamous cancer cells and thereby promotes distant metastasis in mouse squamous tumor models.     

Growth of normal oral keratinocytes and squamous cell carcinoma cells in a novel protein-free defined medium

Summary A novel protein-free synthetic medium was developed for the culture of normal human oral keratinocytes. This medium, designated PFM-7, supports the serial cultivation of primary or secondary normal oral keratinocytes in protein-free, chemically defined conditions. Normal oral keratinocytes in PFM-7 exhibited nearly equal growth in mass culture without noticeable changes in morphology, response to added growth factors, or gene expression of growth factors and their receptors, compared to cells in Keratinocyte-SFM containing epidermal growth factor and bovine pituitary extract. Furthermore, PFM-7 supported the serial subcultivation of human squamous cell carcinoma cells and enabled both normal and malignant oral squamous cells derived from the same patient to grow under the same protein-free defined conditions. These results indicate that PFM-7 can be used for precise investigations of growth mechanisms, cell products, and gene expression associated with carcinogenesis of human epidermal cells.     

Over-expression of Oct4 in human esophageal squamous cell carcinoma

The Octamer 4 gene (Oct4) is a master pluripotency controller that has been detected in several types of tumors. Here, we examine the expression of Oct4 in human esophageal squamous cell carcinoma (ESCC). We found that punctate Oct4 protein was expressed in most (93.7%) ESCC samples but it was not observed in esophageal mucosa. Some ESCC cells had the capacity to form tumorospheres; those with an Oct4⁺-rich cell phenotype had increased proliferation and Oct4 mRNA levels compared to those of differentiated cells in culture or xenograft tumors. The over-expression of Oct4 in ESCCs suggests that it is a potential target for ESCC therapy. Oct4 could be a useful tumor marker in an immunohistochemical panel designed to differentiate between ESCC and esophageal mucosa. Expression of Oct4 in tumorospheres might indicate the presence of a population of ECSCs and its expression in xenograft tumors suggests that Oct4 is also associated with tumor metastasis.