ARTICLE |
Correspondence to: Lloyd A. Culp, Dept. of Molecular Biology and Microbiology, Case Western Reserve U., School of Medicine, Cleveland, OH 44106.
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Summary |
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Metastasis to organs other than lung is rarely observed in animal model systems of human prostate carcinoma (PCA), with the exception of already metastatic isolates of human PCA cultured for long periods of time. To analyze more directly the evolution of metastatic variants from primary PCA tumor isolates, the lacZ histochemical marker gene was transfected into the CWR22Rv1 cell line isolated from the CWR22R xenograft (primary tumor). Three clones of varying lacZ-expression stability were analyzed for tumorigenicity and progression in athymic nude mice. Clones B and D were highly tumorigenic in the subcutis; however, lacZ expression was highly unstable. In contrast, clone H demonstrated highly stable lacZ expression for >25 passages in culture or in animals. Clone H, injected sc in a PBS vehicle, gave a 1540% tumorigenic take. All primary tumor-bearing animals exhibited micrometastases in lung and other organs. Clone H injected in a Matrigel vehicle gave 100% tumorigenicity, with all animals displaying micrometastases in lung, liver, and/or bone (lower frequency in brain and kidney). Overall, the relative frequency of micrometastasis to multiple organs was lung>liver=bone>>brain>kidney. Overt metastases were never observed in the lung or bone but were occasionally found in liver. lacZ-transfected clone H CWR22Rv1 cells represent a much more accurate model of metastasis of PCA to the organs normally involved in progression of the human disease. Use of marker gene-tagged cells and other high-resolution molecular techniques will now permit analyses of the earliest events in PCA progression and micrometastasis. (J Histochem Cytochem 48:643651, 2000)
Key Words: histochemical marker gene, prostate carcinoma, micrometastasis, target organs
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Introduction |
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Studies of human prostate carcinoma (PCA) in animal model systems have been hampered by the paucity of human PCA cells available in tissue culture (
The xenograft approach for isolating human PCA tissue and its passage in nude mice offers an intermediate stage in the development of new cell systems (
CWR22Rv1 cells therefore offer an excellent opportunity to test possible metastatic spread to multiple organs from a cell population originally isolated from a human primary tumor and not from already metastatic subpopulations. If metastasis is observed with these cells to organs that are normally involved in the human disease, then we have a more relevant model system for evaluating mechanisms for PCA progression and metastasis.
Our laboratory has maximized sensitivity of progression and metastasis studies in animal models of tumor systems by transfecting a histochemical marker gene (e.g., bacterial lacZ or human placental alkaline phosphatase gene) into tumor cells, isolating relatively stable-expressing lacZ or PAP transfectants and testing for micrometastases in many target organs of athymic nude mice using ultrasensitive histochemical staining (
In the analyses reported here, the lacZ gene was transfected into cultured CWR22Rv1 cells, three clones were isolated, and the tumorigenic progression and metastasis of these clones were evaluated. These analyses identify one transfectant clone, referred to as LZ-CWR22R-H, that closely mimics metastatic progression normally observed in the human disease to lung, liver, and bone with a reasonable frequency. They also demonstrate remarkable stability of micrometastases in the lung and bone, as well as overt metastasis in the livers of this experimental animal system.
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Materials and Methods |
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Isolation of lacZ-transfected CWR22R Cells
Early-passage CWR22Rv1 cells (
Tumorigenicity Studies
Athymic nude mice (HSD nu/nu) were bred and housed in the Athymic Animal Facility of the CWRU/Ireland Cancer Center under AAALAC-I approval. All procedures were executed under supervision of the Animal Care and Use Committee of Case Western Reserve University. Nude mice (46-week-old males) received two sets of India ink tattoos on each side of their hind flanks, between which cells were injected into the subcutis (
Tissue Isolation and Histochemical Staining
Primary tumors were isolated from sacrificed animals when they were generally >4 mm in their shortest dimension and <10 mm in their longest. In most cases, the entire primary tumor and the surrounding normal mouse tissues were isolated as a unit for X-gal staining. In select cases, the primary tumor was isolated, bisected with a scalpel, and then the halves fixed and X-gal-stained. Lungs and other organs were isolated intact from sacrificed animals as described previously (
X-gal staining was executed on fixed tissues as described previously at pH 7.4 (overnight at room temperature) to minimize background tissue staining (
Materials
Matrigel was obtained from Collaborative Research (Bedford, MA), X-gal from Research Organics (Cleveland, OH), G418 and RPMI 1640 medium from Gibco (Grand Island, NY), potassium ferricyanide, potassium ferrocyanide, and formaldehyde from Sigma Biochemicals (St Louis, MO), tissue culture plastic ware from Becton-Dickinson Labware (Oxnard, CA), and fetal calf serum and RPMI 1640 medium from Irvine Scientific (Santa Ana, CA).
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Results |
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Primary Tumor Development
When LZ-CWR22R Clone B, D, or H was injected into the subcutis of nude mice in a PBS vehicle, there were differences in the tumorigenicity of the three clones. Clone B yielded tumors in all animals (Table 1). However, the vast majority of these tumors failed to give uniform X-gal stainability. For example, Fig 1A illustrates a tumor with only a minor fraction retaining expression of lacZ. In the case of Clone D, many animals yielded tumors when injected with >1 x 106 cells, but none of these tumors displayed any staining (not shown), consistent with the considerable instability of lacZ expression in cultured populations of Clone D. Because of this rapid loss of X-gal stainability by Clones B and D, they could not be studied any further to test for development of micrometastasis in various organs.
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Results for Clone H were very different. Only 1540% of the animals injected with either 1 x 106 or 5 x 106 cells in a PBS vehicle developed tumors, even as long as 6 months post injection (Table 1). However, all these primary tumors in the subcutis were uniformly stainable with X-gal (Fig 1B), demonstrating the persistent expression of lacZ in this particular clone. Fig 1B shows a sizeable and highly stained primary tumor surrounded by fascia that is not X-gal-stainable. When any of these Clone H primary tumors were first sliced into halves with a scalpel and then fixed for X-gal staining, the uniformity of the stainability was again evident ( Fig 1C). This shows that non-expressing variants of the tumor cells do not arise at later time points of primary tumor development (e.g., at the most interior regions of the primary tumor). The extensive vasculature of the primary tumor is also evident at these later times [red-staining blood vessels against the intense blue background (Fig 1C)].
In contrast to the relatively low tumorigenicity of Clone H when injected in PBS, the same cells injected in Matrigel were 100% tumorigenic when 15 x 106 cells were used (Table 1). These Matrigel-borne primary tumors developed identically to PBS-borne tumors when pathological parameters were considered (not shown).
Micrometastasis to Multiple Organs of PBS-injected Cells
With PBS-injected Clone H cells, there was little evidence of overt metastases developing in most organs (except for liver; see below). However, lacZ stainability enables us to detect the very smallest of micrometastases. Micrometastases were observed in the lung for most mice with large tumors when PBS was used as the injection vehicle ( Table 2). An example of one lung micrometastasis is shown in Fig 2A. With lower frequency, micrometastases were observed in other organs as well (Table 2). Fig 2B shows a micrometastasis that has established itself reasonably close to a blood vessel in the liver. Fig 2C shows micrometastases close to a blood vessel in the brain. The relative distribution of micrometastases in animals bearing primary tumors and using PBS-injected cells was lung (>90%) >liver=bone (50%) >>kidney=brain.
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Micrometastasis to Multiple Organs of Matrigel-injected Cells
When Matrigel was the vehicle, Clone H primary tumors yielded micrometastases in the lungs of all animals examined (Table 2). A lung micrometastasis is shown in Fig 3A, a liver micrometastasis in Fig 3B, and kidney micrometastases in Fig 3C. Micrometastasis frequency for all tumor-bearing animals was as follows: lung (100%) >bone (60%) >liver (20%) >>kidney or brain.
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Several precautions were taken in these studies to guarantee that blue-staining foci truly contain tumor cells. First, a clonal population of CWR22Rv1 cells, shown to be tumorigenic in nude mice, was used for transfection of the lacZ gene. This obviates any possibility for another cell type from the original xenograft to be transfected with this gene. Second, a chromosome-integrating plasmid was used in these studies to obviate any episomal plasmid being expressed and possibly being transmitted to neighboring host tissue cells (
Micrometastasis to Bone
Bone has been the most difficult and elusive target for PCA metastasis studies in animal model systems (
Micrometastasis Outgrowth into Overt Metastases
The question arises of whether micrometastases in this LZ-CWR22R-H cell system can give rise to overt metastases. In none of the animals examined in this study were overt metastases ever observed in the lungs (not shown). This is a remarkable finding in light of the several hundred micrometastases that were scored in animals with very large primary tumors and in light of all animals that yielded lung micrometastases when primary tumors had developed. The same applied to bone micrometastases.
In contrast, overt metastases were observed in the livers of some animals. An example from a PBS-injected population is shown in Fig 5. Because micrometastases were much less common in the liver than in the lung and because comparable long time periods were involved, it is unlikely that insufficient time is the basis for poor outgrowth of overt metastases in the lung or bones. Apparently, there are environmental cues in the liver that permit effective outgrowth in the liver of Clone H cells, and these cues are missing in lung and in bone.
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Discussion |
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These studies have identified a highly stable lacZ transfectant of a new human PCA cell line isolated from a primary tumor population, CWR22Rv1 (
Tumorigenesis of LZ-CWR22R-H cells was limited to 1540% of animals injected when cells were injected sc in a PBS vehicle. However, all tumor-bearing animals yielded micrometastases in their lungs and in some cases in liver and bone, with brain and kidney micrometastases the most infrequent. The relative distribution of micrometastases with PBS-injected cells was lung (>90%) >liver=bone (50%) >>kidney= brain. The reasonably high level of progression of these cells into liver and bone offers optimism that this is a more accurate model of the human disease from an unselected primary tumor population of cells (
When Matrigel, a now commonly used vehicle for PCA cells (
A notable surprise in our analyses was the divergence in competence for forming overt metastases. With PBS- or Matrigel-injected cells, overt metastases were never observed in the lung or bone but were occasionally observed in the liver. That the liver received fewer tumor cells than either the lung or bones suggests that time or cell dosage was not a critical element in determining overt metastasis. Perhaps outgrowth in the lung and the bones is retarded by organ-specific environmental cues that are not present in the liver (
This study is not the first to use a marker gene to track PCA cells in an animal model system.
The lacZ-tagged CWR22R tumor cells described here provide a valuable system for evaluating gene regulation at the level of single tumor cells. The ability to identify X-gal-stained single tumor cells in any tissue, combined with laser-capture microdissection (
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Acknowledgments |
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Some of these studies were supported in part by the Comprehensive Cancer Center of the Ireland Cancer Center at Case Western Reserve University (NCI-supported via P30-CA43703) for pilot studies on PCA metastasis. More extensive support was provided by research grant DAMD17-98-1-8587 from the US Army on PCA metastasis.
Athymic nude mouse experiments were conducted in the Athymic Animal Facility (AAALAC-I-approved) of the Case Western Reserve University/Ireland Cancer Center and were approved by the Animal Care and Use Committee of this University. The assistance of Pamela Steele and Kathy Pustai of this facility is greatly appreciated. Special gratitude is extended to Drs Thomas and Theresa Pretlow, as well as Joseph Giaconia of their laboratory, for conveying their extensive knowledge of PCA xenograft biology to the authors during the execution of these studies. Special gratitude is extended to Dr James Jacobberger of the Ireland Cancer Center for the donation of tissue culture-adapted CWR22Rv1 PCA cells.
Received for publication September 29, 1999; accepted January 9, 2000.
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