Growth of human breast carcinomas in nude mice and subsequent establishment in tissue culture

Thirty-two malignant human breast tumors were implanted s.c. in female nude mice. Seven tumors survived for two passages, and four were established into permanent transplantable tumor lines. The transplantable tumors have retained the histopathology of the original tumor throughout passaging in the nude mice. In addition, two of the transplantable tumors have low concentrations of estrogen receptor. Tissue culture of the original tumor specimens upon receipt resulted in epithelial outgrowth in 15 of 32 primary cultures. However, no permanent cell lines were established. Attempts to culture 23 tumors frozen with dimethyl sulfoxide upon receipt were unsuccessful. In contrast, establishment of cell strains was successful with tumor specimens cultured following passage in the nude mice; three cell strains were initiated from two of the transplantable tumors.     

Differential behavior of human bronchial carcinoma cells in culture

A feeder layer culture system suited to grow carcinoma cells derived from solid human lung tumors was developed. This report deals with culturing of the four main histological types of lung carcinomas observed in 37 patients: 19 squamous cell, 6 adenocarcinomas, 7 small cell, and 5 large cell carcinomas. The cultures were initiated from 24 fresh human surgical specimens and from 14 human lung tumors grown as xenografts in nude mice. Three different patterns of behavior in culture were found to be characteristic for squamous cell, adenocarcinomas, and small cell carcinomas, respectively. The culture pattern presented by the primary cultures did not appreciably change after passaging in vitro for periods of up to 2 years, even after infinite cell lines were established. Cultures of large cell carcinoma showed one or more of these patterns. From these patterns cells could be cloned and subsequently cultured as separate stable lines. The system described facilitates the identification of specific types of human lung carcinomas almost immediately (within 1 h) after plating (Phase I) as well as during culture.     

Murine colon adenocarcinomas: methods for selective culture in vitro.

Two murine colon adenocarcinoma cell lines were established from primary cultures. The MCA-38 cell line was begun by treatment of the primary culture with trypsin to remove the fibroblastoid elements. The MCA-36 epithelial cells were sensitive to trypsin; therefore, the growth medium of MCA-36 primary cultures was augmented with collagenase to release the tumor-cell elements from the fibroblast network. These tumor elements were dissociated with trypsin and placed in tissue culture. Each cell line was cultured for at least 10 passages in vitro and gave rise to tumors when reimplanted in vivo.     

Down-regulation of E-cadherin expression in Madin Darby canine kidney (MDCK) cells inside tumors of nude mice

The 120-kDa cell-cell adhesion molecule E-cadherin is localized at the epithelial junctional complex and participates in the organization and maintenance of epithelia. The Madin Darby canine kidney (MDCK) cell line expresses E-cadherin in a stable way and forms polarized epitheloid structures in vitro. Harvey-murine-sarcoma-virus-transformed derivatives (MDCK-ras) produce malignant (i.e., invasive and metastatic) tumors in nude mice. We obtained evidence that E-cadherin is down-regulated in nude mouse tumors and that this down-regulation is reversible. MDCK-ras-e cell lines were cloned in vitro from MDCK-ras cell cultures. They showed an epithelioid morphotype and expressed E-cadherin at homogeneously high level. This characteristic has been conserved for at least 60 passages in vitro. MDCK-ras-e cells were not invasive in vitro. When injected into nude mice, however, they produced invasive and metastatic tumors. Primary tumors as well as large metastases were heterogeneous, showing E-cadherin-positive well differentiated epithelial structures and E-cadherin-negative undifferentiated areas. Metastasis-derived cell cultures contained both E-cadherin-positive and E-cadherin-negative MDCK-ras-e cells during early passages in vitro. During further culture, however, they regained the homogeneous E-cadherin-positive characteristic of the original MDCK-ras-e cell line. The behavior of MDCK-ras-e cells in vitro, as compared with its in vivo behavior, points to the existence of host factors which are able to down-regulate E-cadherin expression. We hypothesize that this down-regulation plays a basic role in invasion.     

Cytokeratins expressed in experimental rat bronchial carcinomas

Cytokeratin expression in rat lung tumors was studied using polypeptide-specific monoclonal antibodies (MAbs) to human cytokeratins 4, 5, 7, 8, 10, 13, 14, 18 and 19. Experiments were performed on tumor fragments derived from 5 experimental rat squamous-cell lung tumors and one adenocarcinoma, as well as on cell lines obtained from the same tumors. The aims of this study were to investigate the differentiation profile of the rat tumor tissue and established tumor cell lines based on light and electron microscopical features and on cytokeratin phenotype, to characterize the tumor type and degree of differentiation of the lung tumors maintained during passaging in experimental animals, and to compare the cytokeratin expression pattern in transplanted tumors with that of the cultures derived from these tumors. Our results indicate that, in general, the antibodies used cross-react with rat cytokeratins and that these MAbs can be used to phenotype rat lung carcinomas. Both the tumor fragments and the cultured cells revealed a similar pattern of cytokeratin expression. In addition, the degree of differentiation was maintained upon prolonged culturing in vitro. MAbs to cytokeratin sub-types can therefore be used to distinguish the main sub-types of rat lung tumors and can give an indication about the degree of differentiation.     

Transforming growth factor-alpha secretion by epidermal growth factor-dependent human tumor cell lines

Pairs of cell lines from spontaneous human tumors (cervical adenocarcinoma, melanoma, and synovial sarcoma) were established using serum-free culture conditions with and without exogenous epidermal growth factor (EGF). EGF-adapted cultures of melanoma and cervical adenocarcinoma origin secreted higher levels of bioactive transforming growth factor alpha (TGF-alpha) when compared to cultures maintained in the absence of EGF. Depletion of EGF for these EGF-adapted cultures resulted in growth arrest. In contrast, the sarcoma cell lines did not secrete TGF-alpha regardless of the culture conditions but EGF significantly stimulated proliferation of these cells in short-term assays. We show that exogenous EGF induces TGF-alpha production and supports proliferation of tumor cells of various tissue origin but is not essential for in vitro growth factor-deprived conditions.     

Expression of Human Choriogonadotropin-Like Material Correlates with Metastatic Phenotype of R3230 AC Rat Adenocarcinoma

Human choriogonadotropin (hCG)-like material has been found in variable amounts on the surface of cells of human and animal tumors. Intravenous injection ofR3230 AC rat adenocarcinoma cells, one of the models investigated, results in multiple lung foci seeding. We analyzed the phenotypic diversity of this tumor by cloning and culturing two distinct cell subpopulations from a cell culture of this tumor, hereafter called OR or original cell culture. One was obtained after repeated exposure of the OR to increasing concentrations of concanavalin A and wheat germ agglutinin. A single clone was isolated and was named lectin-resistant (LR) cell line. The LR cells did not metastasize but maintained stable tumorigenicity and morphology over at least 10 passages. A second cell line was obtained by repeated passage and injection of cells from a single metastatic node. After repeating the process five times, a single clone of cells was selected from the final variant and was called lung metastatic (LM) cell line. The LM cultured cells maintained stable tumorigenicity, morphology, and metastatic properties for no more than 10 passages. OR, LR, and LM cells were assessed by their doubling time (DT), chromosome counts, and hCG immunocytochemistry. The results demonstrated that the LM cell line had a higher chromosome count than the LR and the OR cell lines, and its DTwas the shortest. Immunocytochemistry of the transplanted OR neoplasm showed scattered expression of the hCG-like material. By the same techniques a complete lack of reactivity of the LR cells was found. However, almost all cells of the LM line were strongly positive for hCG-like material. After a few passages, the great majority of the LM cells also became unreactive. Our data demonstrate: (i) the existence of marked heterogeneity of the expression ofhCG-like material in the primary tumor cell population; (ii) that the expression ofhCG-like material correlates with the metastasizing capacity of the cells; and (Hi) that there is a phenotypic instability for the expression of hCG-like material by tumor cells when maintained in vitro.     

CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells

CD133 is a cell surface marker expressed on progenitors of haematopoietic and endothelial cell lineages. Moreover, several studies have identified CD133 as a marker of brain tumor-initiating cells. In this study, human glioblastoma multiforme biopsies were engrafted intracerebrally into nude rats. The resulting tumors were serially passaged in vivo, and monitored by magnetic resonance imaging. CD133 expression was analyzed at various passages. Tumors initiated directly from the biopsies expressed little or no CD133, and showed no contrast enhancement suggesting an intact blood-brain barrier. During passaging, the tumors gradually displayed more contrast enhancement, increased angiogenesis and a shorter survival. Real-time qPCR and immunoblots showed that this was accompanied by increased CD133 expression. Primary biopsy spheroids and xenograft tumors were subsequently dissociated and flow sorted into CD133 negative and CD133 positive cell populations. Both populations incorporated BrdU in cell culture, and expressed the neural precursor marker nestin. Notably, CD133 negative cells derived from 6 different patients were tumorgenic when implanted into the rat brains. For 3 of these patients, analysis showed that the resulting tumors contained CD133 positive cells. In conclusion, we show that CD133 negative glioma cells are tumorgenic in nude rats, and that CD133 positive cells can be obtained from these tumors. Upon passaging of the tumors in vivo, CD133 expression is upregulated, coinciding with the onset of angiogenesis and a shorter survival. Thus, our findings do not suggest that CD133 expression is required for brain tumor initiation, but that it may be involved during brain tumor progression.     

Isolation and growth characteristics of continuous cell lines from small-cell carcinoma of the lung

Sixteen continuous tumor-cell cultures have been isolated from 91 tissue specimens from patients with small-cell carcinoma of the lung. Biopsy and autopsy specimens of primary and metastatic tumors have been utilized. The developing cell lines were recognized by proliferation of tumor cells in the culture from one to 14 weeks after explanation and have been maintained for up to four years. Primary lung tumor, bone marrow aspirations, pleural effusions and other metastases have all been productive explant material for the development of cell lines. Their human origin has been demonstrated by chromosome and/or isoenzyme analysis. Dense core vesicles, characteristically found in small-cell tumor cells were observed by electron microscopic examination of cultured cells. Growth rates in vitro have been measured and the in vitro cycle time in tumors of one cell line (DMS 79) has been compared with in vivo cycle time in tumors arising from DMS 79 cells in nude athymic mice.     

In vitro transformation of human B-cell follicular lymphoma cells by Epstein-Barr virus

Human low-grade B-cell lymphoma cells cannot be readily maintained in long-term tissue culture. In an effort to obtain low-grade B-cell lymphoma cell lines for in vitro study, we used Epstein-Barr virus (EBV) as a transforming agent. T-cells were removed prior to EBV transformation by rosetting with sheep erythrocytes, followed by treatment with anti-T11 monoclonal antibody plus complement. The resulting cell population was cocultured with EBV prepared from tissue culture supernatants of marmoset cell line B95-8. Identical immunoglobulin gene rearrangements of tumor cells and EBV-transformed cells were the criteria used to determine that the transformed cells were of tumor origin. DNA was prepared from both biopsy tissue and EBV cell lines and digested with restriction endonucleases, and Southern blots were prepared by standard methods. B-cells isolated from biopsies of four low-grade B-cell lymphomas of follicular, small cleaved cell type and one of follicular, mixed cell type were transformed by EBV into rapidly growing in vitro tissue culture lines. Two of the five transformed cell lines had immunoglobulin heavy chain and light chain gene rearrangements which were present in cells from the original tumor biopsy, indicating that these EBV-transformed cell lines are of tumor origin.     

An in vivo tumor model expressing green fluorescent protein for the investigation of metastasis

Although strong evidence is available suggesting that microenvironmental parameters play a role in lymphogenic or hematogenic metastasis, the underlying mechanisms are still unclear and further investigations of this topic are needed. For such a study however, an appropriate model of metastasis for in vivo analysis of this process would be required. An in vivo model of a solid tumor (rat DS sarcoma) has therefore been established to enable monitoring of the steps involved in tumor metastasis. Rat DS sarcoma cells were transfected with the pTracer-SV40 plasmid, containing the super-GFP and zeocin resistance genes. DS sarcoma cells showing high and stable expression of GFP (DSGFP cells) were selected by cell sorting and in vitro culturing with zeocin. To establish in vivo growth, DSGFP cells were subsequently injected intraperitoneally (i.p.) without additional selection by zeocin and GFP expression was monitored by flow cytometry. Using DSGFP ascites cells, solid tumors were implanted subcutaneously into the hind foot dorsum of rats. The expression of GFP was assayed by fluorescence microscopy. The detection of circulating DSGFP sarcoma cells in the blood was performed using the PCR technique. GFP expression in vitro was stable for more than 40 passages. Cell sorting, however, did not enable selection of a DSGFP cell population with a higher long-term stable GFP expression. After i.p. cell implantation, GFP expression in DSGFP ascites cells was maintained over at least 19 passages. Solid tumors implanted by injection of DSGFP ascites cells showed stable GFP expression. The growth rate of solid DSGFP sarcomas was slightly slower compared to that of non-transfected cell lines. The detection limit for circulating DS sarcoma cells in blood was 100 DSGFP cells/ml whole rat blood. Micrometastases in loco-regional lymph nodes, lung and liver were detectable by immunohistology and real-time PCR. This in vivo model showing stable expression of GFP could be useful for analyzing the mechanisms of metastasis, particularly where micrometastases or circulating tumor cells are to be identified.     

Cultured, AIDS-related Kaposi's sarcoma cells express endothelial cell markers and are weakly malignant in vitro

Up to 30% of patients with acquired immunodeficiency syndrome (AIDS) suffer from Kaposi's sarcoma (AIDS-KS). The histogenesis and neoplastic nature of this tumor is still controversial. We have established cell cultures of KS biopsies from 7 patients with AIDS. All donors were seropositive for the human immunodeficiency virus I (HIV-I), cytomegalovirus (CMV) and hepatitis B virus (HBV). The tumors were histologically shown to be KS. Cell cultures derived from these tumors all expressed the endothelial cell marker BMA 120 antigen. Most of these cultures were positive for acetylated low-density lipoprotein (acLDL) uptake and alkaline phosphatase (AP) expression, and negative for factor-VIII-related antigen (FVIII-RAg). The staining pattern was heterogeneous with respect to number of endothelial cell markers expressed in each culture. We conclude from subcloning experiments that the cultured cells cease to express acLDL receptor and AP, but not the antigen detected by the monoclonal antibody (MAb) BMA 120. The cells grew well in culture up to 50 passages and showed a fibroblast-like morphology. Assays performed to investigate their degree of malignancy revealed a significantly increased passage number under reduced serum conditions as compared to normal fibroblasts but no tumor formation in nude mice. Neither HIV, HBV nor CMV sequences were found in any of the cell lines tested. We conclude that AIDS-KS is an endothelial-cell-derived neoplasm of low malignancy and that HIV, HBV and CMV are not directly involved in its genesis.     

In vitro proliferation of primary human head and neck squamous cell carcinomas evaluated by flow cytometry

In vitro proliferation of primary human head and neck squamous cell carcinomas was investigated using single cell suspensions and tissue explants of primary specimens and xenografts from 20 tumor specimens. The evaluations of the cells emerging in culture were performed with flow cytometry. Epithelial-like cells proliferated in serum-free medium, while no fibroblast-like cells were observed in culture. The epithelial-like cells could be subcultured several passages before senescence occurred. Conditioned medium or serum supplementation was necessary for a sustained outgrowth of malignant squamous cells as documented by flow cytometry. From a tumor line established in nude mice slowly proliferating tumor cells emerged. After 4-5 months in culture tumor cells seemed to be adapted to the culture conditions used. This resulted in a more consistent tumor cell proliferation. Early passage cultures from primary human head and neck squamous cell carcinomas are clearly difficult to obtain either from primary human specimens or from tumor lines established in nude mice.     

The rate of homozygous CDKN2A/p16 deletions in glioma cell lines and in primary tumors.

The rate of homozygous deletions of CDKN2A/p16 is variable between different tumor entities, and in addition it is higher in established cell lines in comparison with primary tumors. Such incongruencies may reflect statistical sampling errors, true differences depending on tissue derivatisation and CDKN2A/p16 loss under selective pressure in tissue culture. Clarification of these issues is warranted in the context of defining tumor suppressor genes such as CDKN2A/p16 as targets for gene replacement therapies. We therefore compared established cell lines derived from human glioblastomas and their corresponding primary tumors by multiplex PCR methodology. Archival early passages were included to determine the time point at which the p16 status of a cell line changes if it is different from the original tumor. It was found that in 2 of 11 cases (18%) the primary tumor had no p16 alteration whereas the corresponding cell lines had a homozygous p16 deletion. Tracking the in vitro evolution of these two cell lines we found that CDKN2A/p16 was lost already in the earliest passages. This suggests a clonal outgrowth advantage of a subpopulation of p16 deleted tumor cells rather than instability of the CDKN2A/p16 genotype in vitro. Including 20 additional glioblastoma-derived cell lines we detected that in 19 of the total 31 lines at least one exon was lost bringing the rate of p16 loss in the whole panel to 61%. This compares to a rate of 49% which was found in original glioma tissue from 47 unselected other patients. It is concluded, that in cell culture selective pressure favours the outgrowth of pre-existing CDKN2A/p16 negative clones, which account for the difference of CDKN2A/p16 status between cell lines and tumors. In no case did we see a change of the CDKN2A/p16 status during prolonged tissue culture periods of up to 8 years.     

Biologic and biochemical differences between in vitro and in vivo passaged Friend erythroleukemia cells. I. Tumorigenicity and capacity to metastasize

Cloned interferon-sensitive (745) and interferon-resistant (3Cl-8) Friend erythroleukemia cells (FLC) passaged in vitro, are not very tumorigenic when first injected intraperitoneally (i.p.) into syngeneic DBA/2 mice although they do form solid tumors when injected subcutaneously (s.c.). By serially passaging FLC (either 745 or 3Cl-8 cells) i.p. in DBA/2 mice, we obtained two different FLC lines capable of growing i.p. and inducing tumor ascites. The s.c. injection of DBA/2 mice with these in vivo passaged FLC resulted in tumor metastases in the liver and spleen, whereas metastases were not observed in mice inoculated s.c. with in vitro passaged FLC. The capacity of in vivo passaged FLC to metastasize was acquired after several i.p. passages. This highly malignant behavior was a stable characteristic of these cells. All the clones derived from in vivo passaged FLC and passaged more than 14 times in vitro induced hemorrhagic ascites when injected i.p., and metastasized to the liver and spleen when injected s.c. The phenotype of sensitivity or resistance to the inhibitory effect of alpha/beta mouse interferon on virus replication and cell multiplication was conserved during serial i.p. passages and maintained in the clones derived from in vivo passaged cells. These FLC showed a decreased capacity to differentiate in vitro upon treatment with dimethylsulfoxide (DMSO) and a reduced production of Friend leukemia virus with respect to the original clones passaged in vitro.     

Improved Technique for Establishing Short Term Human Brain Tumor Cultures

Culturing human central nervous system tumors has been difficult compared to other neoplasms. We report improved success rates for establishing short term human brain tumor cultures using a modified tissue processing technique. Eighty-seven brain tumor specimens (56 glioblastomas, 8 mid grade astrocytomas, 8 oligodendrogliomas, 15 other) were obtained from June 1988 to March 1997. The first twenty-three samples were processed by dissection, partial enzyme dissociation, and filtration through a tissue culture sieve. Subsequent samples were processed identically except tumor cells were centrifuged on a density gradient prior to plating. Successful cultures were defined as those surviving greater than three passages in tissue culture and growing to sufficient numbers (>10⁶ cells) to allow freezing. Success rate was 42% (10/23) using standard processing methods and 86% (55/64) with the addition of density gradient centrifugation. Glial fibrillary acidic protein (GFAP) and vimentin staining, karyotypes, and growth curves were obtained for representative glioma cultures. All cultures tested were positive for vimentin (29/29) while 62% (18/29) were positive for GFAP. Of four cultures karyotyped (two glioblastomas, two oligodendrogliomas), all but one oligodendroglioma culture exhibited clonal cytogenetic abnormalities. These immunohistochemical and karyotypic results are consistent with the malignant glial origin of these cells. Of note, low passage human glioma cultures grew slower and exhibited more contact inhibition than immortalized human glioblastoma cell lines. Nevertheless, this simple method for establishing short term human brain tumor cultures should aid in further developing human brain tumor pre-clinical models as well as enhancing clinical applications dependent on in vitro human brain tumor cell growth adjust.     

Flow cytometric and morphological studies of ovarian carcinoma cell lines and xenografts

Human ovarian cancers of four different histological types have been cultured in vitro and in nude mice. Nineteen tumor specimens (11 solid tumors and eight malignant effusions) were obtained from 14 patients. Tumor lines from ten of these patients were established after several subpassages, and six xenograft lines have been grown, all of them from tumors of which a cell line exists in vitro. In all, 14 lines have been successfully cultured from the 19 tumor specimens. The morphology (studied by light and electron microscopy) of the established lines in vitro and in vivo resembles that of the original tumors in all cases. Flow cytometric studies of DNA content of the original tumor specimens and the cell culture and xenograft lines were performed. In all lines in both culture systems, aneuploid cells became predominant after the first to fourth passages, despite an aneuploid peak having been evident in only nine of the 19 initial specimens. Four of the original tumor specimens contained measurable estrogen receptors and five progesterone receptors, but none of the established cell lines expressed these hormone receptors. These results indicate that, while morphological features are similar in the initial tumor specimens and in the established lines in vitro and in vivo, flow cytometric and steroid hormone receptor data suggest selection of aneuploid and receptor-negative cells.     

Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity

Glioblastoma (GBM) is the most aggressive primary brain tumor and is resistant to all therapeutic regimens. Relapse occurs regularly and might be caused by a poorly characterized tumor stem cell (TSC) subpopulation escaping therapy. We suggest aldehyde dehydrogenase 1 (ALDH1) as a novel stem cell marker in human GBM. Using the neurosphere formation assay as a functional method to identify brain TSCs, we show that high protein levels of ALDH1 facilitate neurosphere formation in established GBM cell lines. Even single ALDH1 positive cells give rise to colonies and neurospheres. Consequently, the inhibition of ALDH1 in vitro decreases both the number of neurospheres and their size. Cell lines without expression of ALDH1 do not form tumor spheroids under the same culturing conditions. High levels of ALDH1 seem to keep tumor cells in an undifferentiated, stem cell-like state indicated by the low expression of beta-III-tubulin. In contrast, ALDH1 inhibition induces premature cellular differentiation and reduces clonogenic capacity. Primary cell cultures obtained from fresh tumor samples approve the established GBM cell line results.     

Characterization of lymphoma-derived cell lines: comparison of cell lines positive and negative for Epstein-Barr virus nuclear antigen. I. Physical, cytogenetic, and growth characteristics

Sixteen lymphoid cell lines were derived from patients with undifferentiated lymphoma of Burkitt's or non-Burkitt's type. They were obtained directly from tumor biopsies, from serous effusions, or from bone marrow. In 10 of the cell lines, the Epstein-Barr virus (EBV) nuclear antigen (EBNA) was undetectable; the remaining 6 lines were EBNA-positive (EB-pos). Of the 16 lines, 15 were aneuploid, with detectable chromosome "14q+ markers (11 had +8;14 translocations). These 15 lines, which included the EBNA-negative (EB-neg) lines, were believed to be of tumor cell origin. The remaining line consisted predominantly of diploid cells derived from normal lymphocytes, but some cells of tumor origin were present. Four EB-pos cell lines derived from EB-neg tumors had an aneuploid karyotype consistent with an origin from tumor cells (including no.8;14 translocation in two), which suggested that either tumor cells were infected with EBV in vitro or a tiny fraction of EB-pos tumor cells (or potential tumor cells) present in vivo gave rise to the predominant cell of the line. EB-neg B-cell lines and EB-pos cell lines established from undifferentiated lymphomas differed greatly. EB-neg lines had consistently smaller electronic mean cell volumes and narrow-angle light scatter than did EB-pos lines. This finding correlated with a lower nuclear:cytoplasmic ratio in EB-pos lines. EB-neg lines also had higher saturation cell densities than did EB-pos lines under standard culture conditions. The data indicate either that EBV influences the morphologic and physiologic characteristics of lymphoid cell lines or that EB-neg B-cell lines and EB-pos cell lines are derived ultimately from different lymphocyte subpopulations or that both may apply.