Serial propagation of adult human prostatic epithelial cells with cholera toxin

Summary Reproducible subculture of adult human prostatic epithelial cells from normal, benign hyperplastic and malignant tissue has been achieved. Cholera toxin is the key component in the culture system, but use of an optimal basal medium (PFMR-4) supplemented with a high level of serum in collagen-coated dishes also improves growth and serial propagation. Editor’s statement The critical first step in the development of an optimized culture system for any cell type is to obtain enough growth to perform detailed growth-response studies. Multiplication of human prostatic epithelial cells in primary culture has been possible for several years, but scarcity of tissue specimens and inability to subculture have severely limited the usefulness of such cultures. The procedures described by Peehl and Stamey in this communication make it possible to expand the original inoculum substantially and to use subcultures to perform precisely controlled replicate experiments. This will open the way for rapid progress in the development of practical cell culture model systems for research on normal, hypertrophic, and malignant human prostatic cells. Richard G. Ham     

Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.     

Tissue-specific expression of Na,K-ATPase beta-subunit. Does beta 2 expression correlate with tumorigenesis?

We have found a substantial decrease in the level of Na,K-ATPase beta 2-subunit mRNA in xenografts of human renal, lung hepatocellular carcinomas in nude mice as compared with corresponding normal tissues, as well as in the neuroblastoma cell line as compared with the neuron primary cell culture. The level of beta 1 mRNA is decreased in kidney and lung tumor cells, but is unchanged in hepatocellular carcinoma. In the neuroblastoma cell line the level of beta 1 subunit mRNA was found to be higher then in neuron primary cell culture. The level of alpha 1 mRNA in investigated tumors was the same as in normal tissues. These results may give evidence of the involvement of beta 2-subunit in the process of tumorigenesis as was shown for some other adhesion molecules.     

3D printing to construct in vitro multicellular models of melanoma

Currently, there is a lack of suitable models for in-vitro studies of malignant melanoma and traditional single cell culture models no longer reproduce tumor structure and physiological complexity well. The tumor microenvironment is closely related to carcinogenesis and it is particularly important to understand how tumor cells interact and communicate with surrounding nonmalignant cells. Three-dimensional (3D) in vitro multicellular culture models can better simulate the tumor microenvironment due to their excellent physicochemical properties. In this study, 3D composite hydrogel scaffolds were prepared from gelatin methacrylate and polyethylene glycol diacrylate hydrogels by 3D printing and light curing techniques, and 3D multicellular in vitro tumor culture models were established by inoculating human melanoma cells (A375) and human fibroblasts cells on them. The cell proliferation, migration, invasion, and drug resistance of the 3D multicellular in vitro model was evaluated. Compared with the single-cell model, the cells in the multicellular model had higher proliferation activity and migration ability, and were easy to form dense structures. Several tumor cell markers, such as matrix metalloproteinase-9 (MMP-9), MMP-2, and vascular endothelial growth factor, were highly expressed in the multicellular culture model, which were more favorable for tumor development. In addition, higher cell survival rate was observed after exposure to luteolin. The anticancer drug resistance result of the malignant melanoma cells in the 3D bioprinted construct demonstrated physiological properties, suggesting the promising potential of current 3D printed tumor model in the development of personalized therapy, especially for discovery of more conducive targeted drugs.     

Integrated analysis of miRNA and mRNA expression in childhood medulloblastoma compared with neural stem cells

Medulloblastoma (MB) is the most common malignant brain tumor in children and a leading cause of cancer-related mortality and morbidity. Several molecular sub-types of MB have been identified, suggesting they may arise from distinct cells of origin. Data from animal models indicate that some MB sub-types arise from multipotent cerebellar neural stem cells (NSCs). Hence, microRNA (miRNA) expression profiles of primary MB samples were compared to CD133+ NSCs, aiming to identify deregulated miRNAs involved in MB pathogenesis. Expression profiling of 662 miRNAs in primary MB specimens, MB cell lines, and human CD133+ NSCs and CD133- neural progenitor cells was performed by qRT-PCR. Clustering analysis identified two distinct sub-types of MB primary specimens, reminiscent of sub-types obtained from their mRNA profiles. 21 significantly up-regulated and 12 significantly down-regulated miRNAs were identified in MB primary specimens relative to CD133+ NSCs (p<0.01). The majority of up-regulated miRNAs mapped to chromosomal regions 14q32 and 17q. Integration of the predicted targets of deregulated miRNAs with mRNA expression data from the same specimens revealed enrichment of pathways regulating neuronal migration, nervous system development and cell proliferation. Transient over-expression of a down-regulated miRNA, miR-935, resulted in significant down-regulation of three of the seven predicted miR-935 target genes at the mRNA level in a MB cell line, confirming the validity of this approach. This study represents the first integrated analysis of MB miRNA and mRNA expression profiles and is the first to compare MB miRNA expression profiles to those of CD133+ NSCs. We identified several differentially expressed miRNAs that potentially target networks of genes and signaling pathways that may be involved in the transformation of normal NSCs to brain tumor stem cells. Based on this integrative approach, our data provide an important platform for future investigations aimed at characterizing the role of specific miRNAs in MB pathogenesis.     

A novel preclinical model of human malignant melanoma utilizing bioreactor rotating-wall vessels

Malignant melanoma poses a serious health risk which is becoming more crucial as the incidence of this disease steadily increases. The development of appropriate in vitro models that reflect the in vivo tumor environment is a key factor for the study of this malignancy. The local tumor microenvironment plays a critical role in the ability of tumor cells to proliferate and metastasize. While interactions among various cell types are known to be important for tumor growth, most in vitro models utilize only tumor cells, ignoring the importance of tumor-stroma interactions, as well as the contribution of immune cells, which may be important for potential therapies. In addition, the cellular architecture found in vivo, known to be involved in changes in gene expression, is not reflected in standard two-dimensional culture systems. In this study, we have utilized rotating-vessel bioreactors to culture minced human melanoma specimens, allowing the culture of three-dimensional structures which reflect the cellular architecture and heterogeneous composition of the tumor site in vivo. The viability of the pieces in culture can be maintained for 1-2 wk. Immunohistochemical analysis shows multiple cellular types similar to the in vivo situation. Therefore, this system provides a unique model of human melanoma that mimics the in vivo tumor environment much more closely than current culture methods. This novel system may be utilized to determine the mechanism of action of current therapy protocols, as well as to develop new treatment regimens.     

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.     

Lack of Ephrin Receptor A1 Is a Favorable Independent Prognostic Factor in Clear Cell Renal Cell Carcinoma

The EPH receptor tyrosine kinases and their cell-bound ligands, the ephrins, have been shown to be associated with cancer development and progression. In this study, mRNA and protein expression of the receptors EPHA1 and EPHA2 as well as of their ligand EFNA1 and their prognostic relevance in clear cell renal cell carcinoma was evaluated. Gene expression was measured in 75 cryo-preserved primary tumors and matched non-malignant renal specimens by quantitative PCR. Protein expression was analyzed by immunohistochemistry on tissue microarrays comprising non-malignant, primary tumors and metastatic renal tissues of 241 patients. Gene and protein expression of all three factors was altered in tumor specimens with EPHA1 and EPHA2 being generally diminished in tumors compared to normal renal tissue, whereas EFNA1 was commonly elevated. A positive EPHA1 and EPHA2 protein staining as well as a low EFNA1 protein level were significantly linked to more aggressive tumor features, but only a positive EPHA1 immunoreactivity was significantly associated with poor survival. In subgroup analyses, EPHA1 and EPHA2 protein levels were significantly higher in metastatic than in primary lesions. Patients with EPHA1/EPHA2-positive tumors or with tumors with positive EPHA1 and low EFNA1 immunoreactivity had the shortest survival rates compared to the respective other combinations. In a multivariate model, EPHA1 was an independent prognostic marker for different survival endpoints. In conclusion, an impaired EPH-ephrin signaling could contribute to the pathogenesis and progression of clear cell renal cell carcinoma.     

Increased expression of geminin stimulates the growth of mammary epithelial cells and is a frequent event in human tumors

Geminin is a potent inhibitor of origin assembly and re-replication in multicellular eukaryotes and is a negative regulator of DNA replication during the cell cycle. Thus, it was proposed as an inhibitor of cell proliferation and as a potential tumor suppressor gene. However, the protein was found specifically expressed in proliferating lymphocytes and epithelial cells and up-regulated in several malignancies. Therefore, geminin is now regarded as an oncogene but its role in tumor development remains unknown. In this study, we evaluated by Western blot analysis the expression of geminin in a series of human cancer cell lines of various histogenetic origin and in a series of human primary colon, rectal, and breast cancers. Expression of geminin was variable in different cell lines and not related to the expression level of the corresponding mRNA. Moreover, geminin was expressed at higher level in 56% and 58% of colon and rectal cancers, respectively, compared with the corresponding adjacent normal mucosa. A high expression of geminin was also detected by immunohistochemistry in 60% of human primary breast cancers. We also transfected a full-length geminin cDNA in a human non-tumorigenic and a cancer breast cell lines and obtained derivatives expressing high levels of the protein. Geminin overexpression stimulated cell cycle progression and proliferation in both normal and cancer cells and increased the anchorage--independent growth of breast cancer cells. These results demonstrate that expression of geminin is frequently deregulated in tumor cells and might play an important role in the regulation of cell growth in both normal and malignant cells.     

Gene expression and immunohistochemical localization of basic fibroblast growth factor in renal cell carcinoma.

Renal cell carcinoma is known as a neoplastic condition of renal tubular cells and usually shows a hypervascular tumor in angiographic examination. We examined the presence of basic fibroblast growth factor (bFGF) in human renal cell carcinoma. To determine if alterations in bFGF gene expression are present in human renal cell carcinoma, paired samples of normal and neoplastic renal tissue from 6 patients were analyzed for bFGF mRNA content by Northern blot hybridization. In 4 out of 6 patients, tumor tissue expressed bFGF mRNA 2 to 4 times greater than corresponding normal tissue. Two patients showed minimal elevation of tumor bFGF mRNA. The localization of bFGF in the renal cell carcinoma tissue was also examined using immunohistochemical staining, and it was found that bFGF was positively stained at the nuclei of tumor cells and the cell surface. These results suggest that increased expression of bFGF may be associated with neoplastic growth in renal tubular epithelial cells and neovascularization.     

Cytogenetic diversity in primary human tumors

Cytogenetic patterns from primary short-term culture of breast cancer, renal carcinoma, and tumors of the central nervous system are presented to illustrate the range of karyotypic diversity of human solid tumors as well as their biologic differences in culture systems that support their growth. These studies have illustrated several major issues. 1) Results vary with the tissue of origin: primary cultures from breast are almost uniformly diploid, while renal tumors are near-diploid, mosaic, and show clonal aberrations; and CNS tumors are heterogeneous: some diploid, some near-diploid and some highly aneuploid. 2) Results after short-term culture are selective, representing subpopulations from the heterogeneous cells that are detected on direct analysis of fresh tumors by cytogenetics or flow cytometry (FCM). It is not yet clear whether prognosis depends on the dominant population of the primary tumor or alternatively should be influenced by detection of small aneuploid subpopulations. 3) Evidence from all three tumor types supports the interpretation that cytogenetically normal diploid cells constitute part of some tumor populations, and may be better adapted to routine growth in culture than aneuploid subpopulations from the same primary tumors. These cells may also compose a major portion of the viable population of tumors in vivo and, therefore, could represent a useful model for studies of tumorigenesis and therapeutic regimens.     

Preclinical Evaluation of Engineered Oncolytic Herpes Simplex Virus for the Treatment of Pediatric Solid Tumors

Recently, investigators showed that mice with syngeneic murine gliomas that were treated with a neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ₁34.5 gene, enabling replication in tumor cells but precluding infection of normal cells. Previous studies have shown antitumor effects of other oHSV against a number of adult tumors including hepatocellular carcinoma and renal cell carcinoma. The purpose of the current study was to investigate the oncolytic potential of M002 against difficult to treat pediatric liver and kidney tumors. We showed that the oHSV, M002, infected, replicated, and decreased cell survival in hepatoblastoma, malignant rhabdoid kidney tumor, and renal sarcoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly increased survival and decreased tumor growth. Finally, these studies showed that the primary entry protein for oHSV, CD111 (nectin-1) was present in human hepatoblastoma and malignant rhabdoid kidney tumor specimens. We concluded that M002 effectively targeted these rare aggressive tumor types and that M002 may have potential for use in children with unresponsive or relapsed pediatric solid tumors.     

Critical role of TRPC6 channels in the development of human renal cell carcinoma

Renal cell carcinoma (RCC) is the most common tumor arising from the cells in the lining of tubules in the kidney. Some members of the Ca²⁺-permeable transient receptor potential canonical (TRPC) family of channel proteins have demonstrated a role in the proliferation of some types of cancer cells. In this study, we investigated the role of TRPC6 in the development of human RCC. RT-PCR and Western blotting were used to investigate TRPC6 expression in 1932 and ACHN cells. Immunohistochemical techniques were applied to study TRPC6 expression in 60 cases of RCC primary tissue samples and 10 cases of corresponding normal renal tissues. To inhibit TRPC6 activity or expression, RNA interference was used. The effects of TRPC6 channels on RCC cell viability and cell cycle progression were investigated by MTT and flow cytometry. TRPC6 was expressed in 1932 and ACHN cells. TRPC6 protein was detected in 73.3 % of RCC samples, and there was a significant difference compared with the normal renal samples (30 %) (p < 0.05). Moreover the level of TRPC6 expression was associated with RCC Fuhrman grade (p < 0.01). Blockade of TRPC6 channels in ACHN cells suppressed basal cell proliferation and partially inhibited HGF-induced cell proliferation. Furthermore, inhibition of TRPC6 channels expression prolonged the transition through G₂/M phase in ACHN cells. In summary, expression of TRPC6 is markedly increased in RCC specimens and associated with RCC histological grade. TRPC6 plays an important role in ACHN cells proliferation.     

VEGF和Survivin在肾癌中的表达及其相关性研究

目的：探讨肾癌组织中血管内皮生长因子VEGF与凋亡抑制蛋白Survivin表达的相关性及其之间的关系,研究Survivin和VEGF在肾癌发生发展中的作用机制。方法：应用免疫组织化学方法检测70例肾癌组织和70例癌旁正常肾脏组织中VEGF和Survivin的表达,并将检测结果与临床病理特征进行综合分析。结果：VEGF和Survivin在肾癌中表达均高于癌旁正常肾脏组织;Survivin和VEGF在肾癌中的阳性表达率分别为75.71%（53/70）和72.86%（51/70）,在癌旁肾脏组织中的表达率分别为0%（0/70）、17.14%（12/70）,差异均有显著性意义（P〈0.05）;VEGF和Survivin的表达与患者的性别、年龄、肿瘤大小、病理分级均无相关性;VEGF和Survivin表达呈正相关性。结论：VEGF和Survivin在肾癌组织中表达率较高,为肾癌的分子靶向治疗提供了新的靶点。Survivin和VEGF在RCC中的表达关系密切,测定RCC中Survivin、VEGF蛋白的表达,有助于临床判断病人预后。     

Reprogramming strategies for the establishment of novel human cancer models

Cancer comprises heterogeneous cells, ranging from highly proliferative immature precursors to more differentiated cell lineages. The emergence of the “cancer stem cell” (CSC) hypothesis that they are the cells responsible for resistance, metastasis and secondary tumor appearance identifies these populations as novel obligatory targets for the treatment of cancer. CSCs, like their normal tissue-specific stem cell counterparts, are multipotent, partially differentiated, self-sustaining, yet transformed cells. To date, most studies on CSC biology have relied on the use of murine models and primary human material. In spite of much progress, the use of primary material presents several limitations that limit our understanding of the mechanisms underlying CSC formation, the similarities between normal stem cells and CSCs and ultimately, the possibility for developing targeted therapies. Recently, different strategies for controlling cell fate have been applied to the modeling of human cancer initiation and for the generation of human CSC models. Here we will summarize recent developments in the establishment and application of reprogramming strategies for the modeling of human cancer initiation and CSC formation.     

The expression and regulation of DFNA5 in human hepatocellular carcinoma DFNA5 in hepatocellular carcinoma

Hepatocellular carcinoma is a primary malignancy of hepatocytes which accounts for 80 % of all primary liver cancers. DFNA5 has been identified as a tumor suppressor gene with an important role in several frequent forms of cancers, while little is known about its role in hepatocellular carcinoma. Through comparison of the DFNA5 protein expression in hepatocellular carcinoma cells (HepG2) with human fetal lung fibroblast cells (MRC5), we found that the DFNA5 protein expression in hepatocellular carcinoma cells was significantly lower than that in normal cells. The transfection of DFNA5 gene into HepG2 cells could increase DFNA5 protein expression, which subsequently led to inhibition of cell proliferation. Underlying mechanism study revealed that decreased proliferation was due to increased apoptosis and cell cycle arrest. In view of the important role of DFNA5 gene in carcinogenesis, these findings are expected to provide new understanding on development and treatment of human hepatocellular carcinoma.     

Bat Airway Epithelial Cells: A Novel Tool for the Study of Zoonotic Viruses

Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba''s short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells.     

Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue

Mouse melanoma B16-BL6 cells are useful cells for cancer metastatic studies. To understand the metastatic principle at molecular levels, it is necessary to carry out experiments in which cancer cells and their normal counterparts are compared. However, unlike normal human melanocytes, preparation of normal mouse melanocytes is quite difficult due to the lack of marketing and insufficient information on an established protocol for primary culture of mouse melanocytes. In this study, we aimed to establish a convenient method for primary culture of mouse melanocytes on the basis of the protocol for human melanocytes. The main obstacles to preparing pure mouse melanocytes are how to digest mouse skin tissue and how to reduce the contamination of keratinocytes and fibroblasts. The obstacles were overcome by collagenase digestion for skin specimens, short time trypsinization for separating melanocytes and keratinocytes, and use of 12-O-Tetradecanoylphorbol 13-acetate (TPA) and cholera toxin in the culture medium. These supplements act to prevent the proliferation of keratinocytes and fibroblasts, respectively. The convenient procedure enabled us to prepare a pure culture of normal mouse melanocytes. Using enriched normal mouse melanocytes and cancerous B16-BL6 cells, we compared the expression levels of melanoma cell adhesion molecule (MCAM), an important membrane protein for melanoma metastasis, in the cells. The results showed markedly higher expression of MCAM in B16-BL6 cells than in normal mouse melanocytes.     

JQ1, a BET‐bromodomain inhibitor,inhibits human cancer growth and suppresses PD‐L1 expression

Most traditional cytotoxic chemotherapeutic agents have poor aqueous solubility and significant toxicity. Hence, there is a need to develop molecule‐targeted drugs. Programmed death‐ligand 1 (PD‐L1) is associated with the prognosis of several cancer types, and blockade of PD‐1/PD‐L1 signaling increases the amplitude of anti‐tumor immunity. In the present study, we investigated the effects of JQ1, a bromodomain and extraterminal‐bromodomain inhibitor, on cell growth, and messenger RNA (mRNA) and protein levels of PD‐L1 in renal cell carcinoma primary culture cells, and prostate, liver, and lung cancer cell lines. The results of the cell counting kit‐8 assay suggested that JQ1 inhibits cell growth in a dose‐dependent manner. The mRNA and protein levels of PD‐L1 decreased in the primary culture of JQ1‐treated renal carcinoma, prostate cancer, liver cancer, and lung cancer cell lines. In addition, the mRNA level of PD‐L2 also decreased in the JQ1‐treated cells. Overall, JQ1 might be a potential anti‐tumor agent.