The purpose of these studies was to determine whether the ability of tumor cells to grow in an organ parenchyma selects for cells with enhanced potential to metastasize to this organ. B16-F1 melanoma cells (with low metastatic potential) were cultured in vitro on fragments of mouse lung or kidney suspended in medium supplemented with only 1 per cent fetal bovine serum. Seven days later, the organs were enzymatically dissociated and tumor cells recovered and expanded in monolayer culture. Tumor cells were then harvested and seeded onto fresh organ fragments. This sequence was repeated six times. The cells designated as B16 Lung-6 and B16 Kidney-6 were then injected intravenously into C57BL/6 mice and the number of experimental metastases counted after 21 days. B16 Lung-6 and, to a lesser degree, B16 Kidney-6 cells produced significantly more lung tumor colonies than B16-F1 cells. Some factor(s) in the organ environment did appear, therefore, to select out cells with greater metastatic potential from the low metastatic B16-F1. Forty-five clones of the B16-F1 melanoma, isolated by limiting dilution, were screened for their ability to grow on explants of mouse lung in a low-serum medium. Four clones exhibiting least growth and four clones exhibiting most growth, as assessed by examining histological sections of the lung explants, were injected intravenously into syngeneic mice. The eight clonal populations differed in experimental metastatic potential, but this behavior did not correlate with the ability of cells to grow in lung explants in vitro. The data suggest that selecting for cells with enhanced ability to grow in an organ is a necessary but not sufficient condition for isolating cells with high metastatic potential.

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PubMedDate not supplied

Correlation between Ha-ras gene amplification and spontaneous metastasis in NIH 3T3 cells transfected with genomic DNA from human skin cancers.

Our previous studies have shown that DNA from some human skin cancers contained activated Ha-ras oncogenes capable of inducing tumorigenic transformation when introduced into NIH 3T3 cells by DNA-mediated gene transfer. In addition, we found that NIH 3T3 cells transfected with DNA from one of the human skin cancers not only induced s.c. tumors at the site of injection but also metastasized spontaneously to the lungs in 100 per cent of nude mice injected. In this present study we examined the relationship between Ha-ras oncogene amplification and metastatic potential in tumors induced by various human skin cancer DNA-transfectants. Total cellular RNA was extracted from nude mouse tumor cell lines and analyzed by northern blot hybridization to a 32P-labeled, nick-translated Ha-ras probe. The metastatic potential of nude mouse tumor cell lines was assessed by their ability to form lung colonies after i.v. or s.c. injection. It was found that only the tumors expressing high levels of Ha-ras gene transcripts induced spontaneous metastasis after s.c. injection. There appeared to be little correlation between the level of Ha-ras oncogene amplification and experimental metastasis. These results suggest that amplification and overexpression of Ha-ras oncogene may play a role in the escape of cells from the primary tumor rather than in the ability of cells to survive in the circulatory system and colonize secondary sites.

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Characterization of the invasive and metastatic phenotype in human renal cell carcinoma.

The purpose of these studies was to identify some characteristics of metastatic cells and deficiencies of non-metastatic cells in the heterogeneous SN12 human renal cell carcinoma. The SN12 parental line and several isolated variants with different metastatic potential were studied both in vivo and in vitro. We compared the ability of metastatic and non-metastatic cells to adhere to components of the extracellular matrix or to endothelial cells, to migrate and invade, to form multicell aggregates, to survive in the circulation, and to produce experimental and spontaneous lung metastases. In general, highly metastatic SN12 cells capable of producing spontaneous lung metastases demonstrated invasion through reconstituted basement membrane-coated filters; the cells also released diffusible collagenolytic activity into the culture medium that could enhance invasion by otherwise non-invasive and non-metastatic SN12 cells. In addition to enhanced invasion, metastatic cells produced more homotypic aggregation then non-metastatic cells and survived to produce experimental metastasis. Collectively, these data confirm that metastatic cells must complete all steps of the process; in this process, failure to produce metastasis is probably due to one or more deficiencies.

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PubMedDate not supplied

Organ distribution of experimental metastases of a human colorectal carcinoma injected in nude mice.

The metastatic behavior of the HT-29 human colorectal carcinoma cell line was studied following injection into nude mice by different routes. After intrasplenic injection, experimental metastases formed in the livers of most mice. Variant lines were established in culture from the liver lesions and from tumors growing at the site of injection, the spleen. Cells of the HT-29 LMM line exhibited slightly enhanced ability to form liver metastases compared with cells of the non-selected parent line. When injected i.v., the HT-29 cells produced only a few small experimental metastases in the lungs, but in most of the mice macroscopic tumors were found in various lymph nodes and the interscapular fat. Analyses of the distribution of IdUrd-labeled cells did not reveal a preferential localization of the HT-29 cells in sites where metastases subsequently formed. This suggested that the growth of the human colon carcinoma cells in those sites might be the result of a stimulatory interaction between the tumor and host cells as opposed to growth in sites such as the lungs, where numerous cells arrested after i.v. injection but only a few, small metastases were seen 60 days later.

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PubMedDate not supplied

Distinctive karyotypes and growth patterns in nude mice reveal cross-contamination in an established human cancer cell line.

A human cancer cell line was found to be heterogeneous for expression of the epidermal growth factor receptor (EGFR). Clones and variants of this cell line were separated on the basis of EGFR expression level, and those expressing high EGFR had different growth characteristics, in vitro and in vivo, than variants expressing low levels of EGFR. Karyotype analysis revealed that the heterogeneity was the result of mixing of two lines, the 2774 ovarian cancer cell line, and the SW620 colon cancer cell line. Our results reinforce the necessity for accurate identification of cell lines. Also, that measurement of gene expression on a single cell level, for example by flow cytometric analysis, can be more informative than measurements of cell lysates, since the initial indication of heterogeneity would not have been detected by northern or western blotting. The different cell types retained characteristic growth patterns when injected i.p. in nude mice, i.e. peritoneal carcinomatosis and ascites formation by the 2774 ovarian cancer cells, and liver metastasis and growth of discrete abdominal tumors by the SW620 colon cancer.

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PubMedDate not supplied

Epidermal growth factor receptor (EGFR) antisense transfection reduces the expression of EGFR and suppresses the malignant phenotype of a human ovarian cancer cell line.

An EGFR-expressing clone of the human ovarian cancer line 2774 was transfected with an antisense construct of EGFR to test how suppression of this gene modulates the malignant phenotype. Transfected clones were screened for EGFR expression by Western blot and FACS analysis. Anchorage-independent growth was used to assess the effect of reduced EGFR on the malignant behavior of the cells. Several transfected clones with decreased EGFR (40-50% reduction) were identified. A correlation was noted between reduced EGFR and decreased anchorage-independent growth, with the transfected clones losing the ability to grow in agarose and responsiveness to exogenous EGF. These results suggest that EGFR may be an important factor in the malignant behavior of this ovarian cancer cell line.

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Correlation of growth capacity of cells in hard agarose with successful transfection by the activated c-Ha-ras oncogene and in vivo proliferative capacity at metastatic sites.

The purpose of this study was to determine whether the degree of anchorage-independent growth of rodent or human cells in increasing concentrations of agarose correlated with successful transfection of the cells with an activated c-Ha-ras oncogene and tumorigenicity in nude mice. NIH 3T3 cells, C3H 10T1/2 fibroblasts, four clones of the murine K-1735 melanoma with different metastatic capacities and the TE85 human osteogenic sarcoma line were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, which confers resistance to neomycin (pSV2-neoEJ). Cells transfected with pSV2-neo, a plasmid containing the neo gene, served as controls. Cells from parental or transfected lines (selected by Geneticin) were plated into medium containing 0.3%, 0.6% 0.9%, or 1.2% agarose. These cells were also injected subcutaneously and intravenously into nude mice. The production of tumor cell colonies in dense agarose (greater than or equal to 0.6%) correlated with successful transfection with pSV2-neoEJ and production of experimental metastases in the lung of nude mice. We conclude that the degree of anchorage-independent growth of cells predicts successful transfection with activated c-Ha-ras oncogene and tumorigenic behavior in vivo. Thus this technique may be useful for the detection of cells transfected with transforming oncogenes.

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PubMedDate not supplied

Binding of 125I-labelled concanavalin A by cells from spontaneously arising murine mammary carcinomas and experimentally induced metastases.

Measurement of the quantity of 125I-labelled Concanavalin A bound by disaggregated cells from a series of 20 primary murine mammary carcinomas has shown that there is no relationship between the binding of this lectin and capability of the cells to colonise the lungs after intravenous injection. However, cells taken from pulmonary deposits formed by those tumors able to colonise the lungs consistently bound greater amounts of the radio-labelled lectin than cells from the corresponding primary tumour (paired t-test, p less than 0.05). Also cells obtained from tumours formed by transplantation of pulmonary deposits into the mammary fat pad bound still more of this lectin than cells from either corresponding pulmonary deposits or primary tumours. The findings indicate that the higher lectin binding characterising cells from metastatic deposits is not site-induced and may reflect either a selective advantage of cells with these properties for metastasis formation or phenotypic changes in the cells during the metastatic process.

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PubMedDate not supplied

Accelerated growth of human colon cancer cells in nude mice undergoing liver regeneration.

The purpose of this study was to determine whether liver regeneration induced by partial hepatectomy (PH) influences the growth of human colon cancer (HCC) cells implanted into athymic nude mice. HCC KM12C cells were injected subcutaneously into nude mice and then the mice were randomized to undergo PH, laparotomy, or no surgery. The latent period to development of measurable tumors was shorter and the growth rate of HCC tumors was significantly faster in hepatectomized mice. Accelerated tumor growth directly coincided with liver regeneration. Peak mitotic activity in both the regenerating liver and HCC occurred on the second day following PH. No enhancement in growth of tumors occurred in mice implanted with HCC cells 3 weeks after PH (i.e. 2 weeks after completion of liver regeneration). The accelerated tumor growth was specific to HCC. We base this conclusion on results of control experiments where cells from human melanoma, colon, breast, prostate, and renal cancer were injected into nude mice that were then randomized to undergo PH or laparotomy. Only HCC grew faster in hepatectomized mice. No significant differences in expression of epidermal growth factor receptor (EGF-R) and c-met were found between HCC tumors in mice with PH or laparotomy, suggesting that over-expression of EGF-R or c-met is not an essential component of this phenomenon. The accelerated growth of HCC cells at a site distant from surgical trauma suggests that circulating growth factors involved in liver regeneration can specifically stimulate the growth of HCC cells.

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PubMedDate not supplied

Host-tumor interactions in the progression of breast cancer metastasis.

The dissemination of cells from a primary tumor, resulting in the progressive growth of metastatic carcinoma in distant sites, including bone, lungs, liver and brain, is the most common cause of death of breast cancer patients. The results of studies using rodent tumors and human cancer cell lines, combined with clinical observations, suggest that metastasis is not a random event. It is the result of a sequence of selective events, many of which involve interactions with elements of the microenvironment of the primary and the metastatic tumors. A greater understanding of the metastatic phenotype from cellular and molecular analyses will provide a rationale approach for controlling this most lethal aspect of breast cancer.

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