Centrifugation of mammalian cells on gradients: a new rotor.

A short-arm rotor increases separation of viable mammalian cells, from mixtures, by low-speed centrifugation; continuous Ficoll density gradients in tissue-culture media are used. We describe the theory and experimental demonstration of the superior separation achieved with this new rotor.

Development of micrometastases: earliest events detected with bacterial lacZ gene-tagged tumor cells.

For the study of micrometastases at their earliest stages, we transfected the lacZ gene, which codes for beta-D-galactosidase in Escherichia coli, into BALB/c 3T3 cells transformed by the Ha-ras oncogene (also known as HRAS1) of a human EJ bladder carcinoma. These cells were subsequently injected into 6-week-old, female athymic NCR-NU nude mice by several routes. With chromogenic detection of the product of the lacZ gene (a heterologous gene not observed in animal cells) by use of 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside, we easily identified tumor cells implanted in the lungs minutes after intravenous injection by the intensely blue staining of the cells harboring the lacZ gene. The number of lung-associated tumor cells remained constant for several hours after intravenous injection but then decreased to a stable level by 24 hours. At most sites of lung invasion, multiple tumor cells, rather than single cells, were identified; this finding suggests that cooperation among multiple cells may be important in the early stages of micrometastasis development. Within several days, a few foci of micrometastases were expanding by proliferation and/or migration of individual tumor cells among host lung cells. These results confirm that the lacZ gene is an ultrasensitive histochemical marker for analyzing both qualitatively and quantitatively the earliest stages of micrometastasis development in the lung and in other organs where micrometastases may ensue.

Cytologic appearance of cells dissociated from rat colon and their separation by isokinetic and isopyknic sedimentation in gradients of Ficol.

Several methods for obtaining colon epithelial cells in suspension were compared. The largest number of epithelial cells per gram of colon was obtained in suspension when the colon was dissociated with 0.1% Pronase. Fivefold more cells per gram of tissue were obtained from the proximal 5 cm of colon than from the terminal 5 cm of colon. Several well-defined types of cells were observed in low frequency. They have not been described previously in normal colon. Suspensions of cells from colons of F344 rats always contained copious mucoid gel that was partially eliminated by washing the cells three times in culture medium with 10% fetal calf serum. Velocity sedimentation in a previously described isokinetic gradient of Ficoll in tissue culture medium permitted the separation of epithelial cells from lymphocytes and red blood cells. Type 2 cells, believed to be a subpopulation of epithelial cells, were obtained in a maximum purity of 95.7 +/- 1.2%. The frequency of 8 other cell types and their purification, when possible, are presented. Epithelial cells were separated from most bacteria and could be cultured with antibiotics after separation in the density gradient.

Purification and transplantation of hepatocytes from livers of carcinogen-treated rats.

Young adult male F344 rats were given 90 ppm diethylnitrosamine in drinking water for 5 weeks. Cells were obtained in suspension from the livers of these animals by in situ perfusion of collagenase. Cells were separated in a 2.7-16% (wt/wt) continuous gradient of Ficoll in tissue culture medium in the Sorvall TZ-28 reorienting zonal rotor for 10 minutes at 4 degrees C with a centrifugal force of 12 X g at the sample-gradient interface (26 X g at the gradient-cushion interface). Up to half a billion liver cells were separated without exceeding the band capacity. In all experiments the purest fractions contained more than 90% hepatocytes. After centrifugation, 72.4 +/- 6.6% of the purified hepatocytes had histochemically demonstrable gamma-glutamyl transpeptidase (GGT). When purified hepatocytes were injected into the mesenteric veins of rats given a diet containing 0.02% (wt/wt) N-2-fluorenylacetamide for 7 days and subjected to partial hepatectomy, all rats that received these cells developed foci that exhibited histochemically demonstrable GGT. Hepatocytes with histochemically demonstrable GGT make up all or part of what previously has been referred to as "liver colony-forming units." With the TZ-28 reorienting zonal rotor, these cells can be purified in biochemically preparative quantities.

Separation of cells exhibiting acid phosphatase activity from disaggregated hamster prostate cells in an isokinetic gradient of Ficoll in tissue culture medium.

Cells exhibiting histochemically demonstrable acid phosphatase were separated from suspensions of hamster prostate cells with velocity and isopyknic sedimentation. Unseparated suspensions of hamster prostate cells contained 57.2 plus or minus 11.3% cells with histochemically apparent acid phosphatase. After the cells were separated by velocity sedimentation in a previously described isokinetic gradient, the purest fractions from the gradient contained 97.2 plus or minus 0.8% cells with histochemically evident acid phosphatase. More than 99% of these separated cells excluded trypan blue. These cells were thought to be the acinar cells of the prostate. Isopyknic sedimentation was not as effective as velocity sedimentation for the purification of these cells.

Separation of two populations of cells with gamma-glutamyl transpeptidase from carcinogen-treated rat liver.

Noninbred Sprague-Dawley rats were maintained on a choline-deficient diet containing 0.05% ethionine. After 10-13 weeks, livers were dispersed with collagenase, lysozyme, collagenase and hyaluronidase. Pronase, or a selected batch of trypsin. The highest yield of cells with histochemically demonstrable gamma-glutamyl transpeptidase (GGT) was obtained with trypsin. After velocity sedimentation in an isokinetic gradient of Ficoll in tissue culture medium, two modal populations of cells with histochemically demonstrable GGT were observed. The first mode contained cells that were morphologically different from hepatocytes and that may be oval cells. The second, more rapidly sedimenting modal population of cells with GGT was morphologically similar to hepatocytes as assessed with Wright's stain; the location of this population in the gradient was the same as the location of cells with the appearance of hepatocytes that lacked iron and that had decreased glucose 6-phosphatase. In multiple experiments, the purest fractions contained 71.7 +/- 3.5% cells (mean +/- SD) with the appearance of hepatocytes with histochemically demonstrable GGT.

CWR22 xenograft as an ex vivo human tumor model for prostate cancer gene therapy.

BACKGROUND: Lack of well-defined relevant in vivo or in vitro tumor models is one of the major limitations in assessing candidate therapeutic regimens, especially gene therapy, for prostate cancer. Since gene therapy is emerging as a potentially powerful therapeutic modality, it is desirable to evaluate this approach for the treatment of human prostate cancer. PURPOSE: We sought to establish a relevant ex vivo tumor model for gene therapy studies of human prostate cancer. METHODS: We constructed and established a transgenic human tumor model consisting of three major components: 1) human primary prostate cancer cells, CWR22, reactivated for growth after storage in liquid nitrogen; 2) a collagen gel ex vivo tissue culture system useful for short-term maintenance and manipulation of CWR22 cells under in vitro experimental conditions; and 3) a high-velocity, particle-mediated gene transfer system that is highly efficient in the ex vivo transfection of target cells. Prostate-specific antigen (PSA) levels in the cell culture media were monitored after transfecting CWR22 cells with candidate therapeutic genes, including the cytokines human interleukin 2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), both as complementary DNAs [cDNAs]). CWR22 cells, transfected with firefly luciferase cDNA as a reporter gene, served as control cells for cytokine gene expression. CWR22 cells, transfected with the bacterial beta-galactosidase cDNA as a reporter gene, were used to assess the efficiency of gene transfer. Transcription of each of the cDNAs was driven by the cytomegalovirus (CMV) early gene promoter. RESULTS: The three-dimensional organization of tumor cells and functional characteristics of human prostate cancers were maintained in this ex vivo model of prostate cancer. Candidate therapeutic genes, CMV-IL-2 and CMV-GM-CSF, were expressed at peak levels of up to 38 ng of protein per 10(6) cells every 24 hours. IL-2 and GM-CSF secretion was sustained at approximately 40%-50% of peak levels during the entire experimental period (9-10 days in culture). At 7 days after gene delivery, a more than twofold reduction in the secretion of PSA was detected in the IL-2 (3.8 +/- 1.3 ng/10(4) cells every 24 hours [mean +/- standard deviation]) or GM-CSF (4.0 +/- 1.7 ng/10(4) cells every 24 hours) cDNA transfected cells as compared with the control cells transfected with luciferase cDNA (9.3 +/- 1.0 ng/10(4) cells every 24 hours). Up to 10% of the cells transfected with beta-galactosidase cDNA expressed measurable beta-galactosidase activity. CONCLUSION: This study demonstrated an efficient, rapid, and reliable system for gene transfer and expression in primary human prostatic carcinoma cells maintained in a collagen gel culture system. IMPLICATIONS: Our findings suggest a broad application of this CWR22 xenograft primary culture system as an ex vivo tumor model for the evaluation and characterization of various candidate therapeutic genes for human prostate cancer gene therapy, including a cytokine gene-modified tumor vaccine strategy.

K-ras mutations in putative preneoplastic lesions in human colon.

BACKGROUND: A mutation in c-K-ras (KRAS2) has long been implicated as one of the important early events in the development of a large proportion of human colon cancers. Aberrant crypt foci, putative preneoplastic lesions identified microscopically in wholemounts of colons, have been shown to occur with high frequency in the colons of animals treated with colon carcinogens and in the grossly normal mucosas of patients with colon cancer. PURPOSE: In this study, we asked whether the mutational activation of K-ras occurs in the aberrant crypt foci of human colon. METHODS: Grossly normal colonic mucosas were obtained from seven patients during surgery and were provided to us by the Western Division of the Cooperative Human Tissue Network located at Case Western Reserve University. A total of 42 samples, consisting of aberrant crypt foci and similarly sized normal crypt areas, were microdissected from the grossly normal colonic mucosas. The DNA region containing codon 12 of K-ras was amplified by polymerase chain reaction and analyzed for mutations by dot-blot hybridization with specific oligonucleotide probes complementary to normal or mutant sequences. RESULTS: Mutations in codon 12 of K-ras were found in 11 (73%) of 15 aberrant crypt foci but not in any of 27 morphologically normal crypt areas from the same patients. CONCLUSIONS: The observed high frequency of K-ras mutations in these microscopically identifiable lesions makes mutation in K-ras the earliest identified gene-mutational event in human colon tumorigenesis, establishes that it often occurs prior to the development of polyps, and is consistent with the hypothesis that aberrant crypt foci are the earliest identified precursors of human colon cancer. IMPLICATIONS: Further analysis of aberrant crypt foci may identify yet unknown early genetic events that precede human colon cancer.

Altered expression of RET proto-oncogene product in prostatic intraepithelial neoplasia and prostate cancer.

BACKGROUND: The RET proto-oncogene encodes a protein that belongs to the tyrosine kinase growth factor receptor family. Germline point mutations in RET are found in individuals with multiple endocrine neoplasia (MEN) syndromes, and gene rearrangements have been reported in papillary thyroid cancers. We recently identified transcripts of the RET proto-oncogene in human prostate cancer xenografts and prostate cancer cell lines by means of reverse transcription-polymerase chain reaction analyses. The purpose of this study was to investigate Ret protein expression in human prostate tissue. METHODS: Ret protein expression was evaluated immunohistochemically in formalin-fixed, paraffin-embedded whole-prostate sections. The prostate specimens were obtained from 30 patients with prostate cancer after radical prostatectomies. Ret protein expression was compared in tumor foci and benign prostatic tissue. Medullary thyroid carcinoma tissue associated with an MEN syndrome and papillary thyroid cancer tissue served as positive controls. RESULTS: Ret appeared to be overexpressed in high-grade (histopathologically advanced) prostatic intraepithelial neoplasia (PIN) and prostate cancer when compared with its expression level in benign prostatic secretory epithelium. In addition, there was an apparent increase in Ret protein expression with decreased cellular differentiation, i.e., increasing Gleason pattern. CONCLUSION: Expression of the RET proto-oncogene in benign prostatic epithelium, high-grade PIN, and histopathologically advanced prostate cancer suggests that RET may play a role in the growth of both benign and neoplastic prostate epithelial cells.

Xenografts of primary human prostatic carcinoma.

BACKGROUND: Prostatic carcinoma is both the most common invasive cancer and the second most common cause of cancer deaths in men in the United States. Before 1991, attempts to propagate prostatic carcinoma from primary tumors for periods longer than 3 months were unsuccessful in vivo and in vitro with rare exceptions. In 1991, we reported establishment of slowly growing tumors for six of 10 human primary prostatic carcinomas approximately 2-6 months after transplantation. However, none of the tumors were larger than 5 mm or serially transplantable. PURPOSE: Our purpose in this study was to determine whether human primary prostatic carcinoma could be grown as serially transplantable xenografts. METHODS: Cells from primary prostatic carcinomas obtained from transurethral prostatic resections or total prostatectomies in 20 patients were injected subcutaneously into male nude mice on the day of surgery. Sustained-release testosterone pellets were placed subcutaneously in the mice 2-24 days before transplantation of tumors and at intervals of 10-12 weeks. Serial transplantations in subsequent generations of mice were carried out by similar methods. Chromosome analysis was performed on six tumors. RESULTS: Six of 20 primary prostatic carcinomas have grown sufficiently to permit serial transplantation into second mice; four have been documented histopathologically in the second mouse and serially transplanted into three or more successive mice. When a single primary tumor was injected into several mice by the same procedure, tumors failed to grow in some recipients but became serially transplantable in others. Growth of these tumors is slow and irregular, with frequent regressions. Short-term cultures of 10 tumors, eight of which were injected into mice in parallel, were initiated on the day of surgery; CWR31, which was successfully transplanted serially, exhibited only aberrant metaphases and showed clonal, chromosomal changes in culture. Including CWR31, three of the six tumors for which chromosomal analysis was successful contained clonal aberrations. Preliminary studies of SCID (severe combined immunodeficient) mice suggest that they are not superior to nude mice for establishment of serially transplantable prostatic carcinoma xenografts. CONCLUSIONS: A proportion of human primary prostatic carcinomas can be grown as xenografts. Four new serially transplantable xenografts (CWR21, CWR31, CWR91, and CWR22) are currently propagated in our laboratory, a resource that was not previously available. IMPLICATIONS: Our experience suggests that the most important factor in serial transplantation is the collaboration of urologists and pathologists in expediting placement of the tumor in cold saline, examination of the frozen section, and transplantation.

Evidence of independent origin of multiple tumors from patients with prostate cancer.

BACKGROUND: In men with prostate cancer, the gland usually contains two or more widely separate tumors. A critical issue of prostatic carcinogenesis is whether these multiple tumors are independent in origin. Molecular analysis of microsatellite (i.e., highly repeated, short nucleotide sequences) alterations in the DNA from separate tumors in the same prostate can be used to determine whether or not these separate tumors arise independently. METHODS: Four microsatellite polymorphic markers (D8S133, D8S136, and D8S137, for a putative tumor suppressor gene on chromosome 8p, and D17S855, for the BRCA1 gene on chromosome 17q) were used to examine the pattern of allelic loss in prostate cancer from 19 patients who had two or more distantly separate tumors (i.e., located on contralateral sides or separated by at least half the anterior-posterior diameter of the prostate). Forty distantly separate tumors were microdissected, DNA samples were prepared from formalin-fixed, paraffin-embedded wholemount prostate tissue section, and the overall frequencies of loss of heterozygosity at the four loci were determined. RESULTS: The pattern of allelic loss was compatible with independent tumor origin in 15 of 18 informative cases. A random discordant pattern of allelic deletion was observed in distantly separate tumors, whereas the same allele was consistently lost in cells from different regions of the same tumor. For three patients, the results were compatible with either intraglandular dissemination or independent origin of prostate cancer. CONCLUSIONS: Our data suggest that multiple tumors in some patients with prostate cancer have independent origin.

Hormone therapy failure in human prostate cancer: analysis by complementary DNA and tissue microarrays.

BACKGROUND: The molecular mechanisms underlying the progression of prostate cancer during hormonal therapy have remained poorly understood. In this study, we developed a new strategy for the identification of differentially expressed genes in hormone-refractory human prostate cancer by use of a combination of complementary DNA (cDNA) and tissue microarray technologies. METHODS: Differences in gene expression between hormone-refractory CWR22R prostate cancer xenografts (human prostate cancer transplanted into nude mice) and a xenograft of the parental, hormone-sensitive CWR22 strain were analyzed by use of cDNA microarray technology. To validate the data from cDNA microarrays on clinical prostate cancer specimens, a tissue microarray of specimens from 26 prostates with benign prostatic hyperplasia, 208 primary prostate cancers, and 30 hormone-refractory local recurrences was constructed and used for immunohistochemical detection of protein expression. RESULTS: Among 5184 genes surveyed with cDNA microarray technology, expression of 37 (0.7%) was increased more than twofold in the hormone-refractory CWR22R xenografts compared with the CWR22 xenograft; expression of 135 (2.6%) genes was reduced by more than 50%. The genes encoding insulin-like growth factor-binding protein 2 (IGFBP2) and 27-kd heat-shock protein (HSP27) were among the most consistently overexpressed genes in the CWR22R tumors. Immunohistochemical analysis of tissue microarrays demonstrated high expression of IGFBP2 protein in 100% of the hormone-refractory clinical tumors, in 36% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P =.0001). Overexpression of HSP27 protein was demonstrated in 31% of the hormone-refractory tumors, in 5% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P =.0001). CONCLUSIONS: The combination of cDNA and tissue microarray technologies enables rapid identification of genes associated with progression of prostate cancer to the hormone-refractory state and may facilitate analysis of the role of the encoded gene products in the pathogenesis of human prostate cancer.

Separation of lymphocytes and mast cells from the Furth transplantable mast cell tumor in an isokinetic gradient of Ficoll in tissue culture medium.

Cell suspensions of the transplantable Furth murine mast cell tumor were separated both by velocity sedimentation in an isokinetic gradient and by isopyknic sedimentation. Prior to separation, the suspension of tumor cells contained 60.3+/-13.1% (S.D.) malignant mast cells, 9.8+/-10.4% lymphocytes, 4.3+/-2.1% granulocytes, 1.7+/-1.9% macrophages, 0.6+/-0.4% unidentified cells, and 22.8+/-8.5% red blood cells. After either isokinetic or isopyknic sedimentation, more than 97% of the nucleated cells in the purest modal fraction were malignant mast cells. Velocity sedimentation in the isokinetic gradient offered several advantages over isopyknic separation of this tumor; namely, in isokinetic sedimentation, the cells are exposed to a lower centrifugal force for a shorter period of time; a much larger proportion of mast cells were in the highly purified zone of the gradient following velocity sedimentation; and lymphocytes were more highly purified (88.9+/-10.1% of the nucleated cells) following velocity sedimentation. Granulocytes and macrophages were purified more than 8-fold over the nucleated cells in the starting sample suspension. The purified cells from this tumor offer the opportunity to study the interactions between highly purified, easily identified, malignant cells and cells that may participate in the defense of the host against cancer.

Bacterial lacZ gene as a highly sensitive marker to detect micrometastasis formation during tumor progression.

During tumor progression, micrometastases at their earliest stages have been difficult to analyze qualitatively or quantitatively because of a lack of suitably sensitive markers to discriminate small numbers of tumor cells from normal tissue cell populations. To overcome this problem, the Escherichia coli beta-galactosidase (lacZ) gene was introduced into human EJ Ha-ras oncogene-transfected BALB/c 3T3 cells with subsequent injection of transfected cells into athymic nude mice. Using a chromogenic substrate (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside), the lacZ-bearing tumor cells at primary tumor sites as well as at secondary organs stain intensely blue and can be easily distinguished from the host tissue cells hours, days, or weeks postinjection. Staining of lacZ-bearing tumor cells is specific and extremely sensitive in detecting micrometastatic foci in lungs and other organs, including brain and kidney for the first time. Stable integration of the lacZ and ras genes into cultured cells and subsequent tumor cells was verified by Southern blot analyses. The lacZ gene appears to be a stable marker during tumor progression in vivo based both on phenotypic (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside staining) and on genotypic (Southern blot analysis) evidence. Furthermore, 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside staining of tumor cells can also be used together with alkaline phosphatase staining relatively specific for endothelial cells to relate the topographies of metastatic cells and host blood vessels in embedded sections. By using the lacZ gene as a sensitive quantitative marker, analyses of micrometastasis development in the lung indicate that the ras oncogene contributes to the metastatic phenotype in this EJ Ha-ras model system, although further genetic and/or phenotypic alterations appear to be necessary for long-term growth and development into overt metastases. These findings demonstrate the effectiveness and sensitivity of the bacterial lacZ gene as a phenotypic marker in tumor progression studies, providing both a qualitative and a quantitative tool in virtually any tumor system for examining micrometastasis formation in target organs and the relationship of tumor cells to host organ microenvironments.

Inducible nitric oxide synthase (iNOS) is expressed similarly in multiple aberrant crypt foci and colorectal tumors from the same patients.

Aberrant crypt foci (ACF) are the earliest identified neoplastic lesions in the colon. Aberrant expression of inducible nitric oxide synthase (iNOS or NOS2) has been documented in colorectal tumors, but expression of iNOS has not been reported in human ACF or multiple neoplastic lesions from the same patient. Immunohistochemical expression of iNOS was evaluated in 42 ACF, 14 adenomas, and 25 carcinomas and their adjacent normal mucosa. iNOS was strongly expressed in the normal colonic epithelial cells of all patients; it was markedly reduced in 21 of 42 (50%) ACF and in 14 of 25 (56%) carcinomas. The expression of iNOS was remarkably similar in multiple lesions from the same patient (P < 0.0001). These results suggest that the reduced expression of iNOS is a very early event in the development of some human colorectal tumors, and that host factors control the expression of iNOS similarly in premalignant and malignant colonic epithelial cells.

Beta-catenin expression is altered in human colonic aberrant crypt foci.

The aberrant expression of beta-catenin in colon tumors and the discovery of beta-catenin mutations in small adenomas suggest that alterations of beta-catenin are early events in human colorectal carcinogenesis. Here, we describe the expression of beta-catenin in human aberrant crypt foci (ACF), the earliest identified neoplastic lesions in the colon. Paraffin-embedded sections of 94 ACF, 12 adenomas, and 10 carcinomas were evaluated for beta-catenin expression by immunohistochemistry. Normal colonic epithelial cells adjacent to these lesions showed strong membranous expression of beta-catenin and lacked cytoplasmic and nuclear expression. Cytoplasmic expression of beta-catenin was seen in 25 of 46 ACF with dysplasia and in 2 of 48 ACF with atypia. In ACF with dysplasia, reduced membranous expression of beta-catenin was associated with increased nuclear (P = 0.0013) and cytoplasmic (P = 0.0247) expression. The membranous (P = 0.0003) expression of beta-catenin was reduced, and the cytoplasmic (P = 0.0016) and nuclear (P = 0.0266) expressions increased in ACF according to their degree of dysplasia. Likewise, membranous (P = 0.0007) expression of beta-catenin was reduced, and the cytoplasmic (P = 0.0050) and nuclear (P = 0.0001) expressions increased from ACF to adenoma to carcinoma. These data suggest that ACF and their aberrant expression of beta-catenin play a role in colon tumorigenesis.

Transplantation of human prostatic carcinoma into nude mice in Matrigel.

Previous successful transplantation of human primary prostatic carcinomas into nude mice has been described as "close to zero." When injected in Matrigel instead of culture medium, 25,000-fold fewer cells of the PC-3 human prostatic carcinoma cell line were required for the growth of tumors in nude mice during a 3-month period of observation; similar enhancement was observed with two other human prostatic carcinoma cell lines. Six of ten primary human prostatic carcinomas were transplanted successfully into nude mice when Matrigel was used as the vehicle.

Arginase activity in prostatic tissue of patients with benign prostatic hyperplasia and prostatic carcinoma.

We studied arginase activity in human prostatic tissue in 15 patients with benign hyperplasia and 27 patients with prostatic carcinoma. Arginase specific activity is greater (p less than 0.0001) in prostatic carcinomas than in hyperplastic prostates. Arginase specific activity is correlated inversely (p less than 0.0001) with the histological grade of the tumor.

Tissue disaggregation of human renal cell carcinoma with further isopyknic and isokinetic gradient purification.

Five different human renal cell carcinomas were disaggregated with three combinations of enzymes. Significant tumor heterogeneity in response to the enzyme disaggregation was observed. A combination of collagenase (0.5 mg/ml) and trypsin (0.25%) was then used for routine disaggregation of 11 additional tumors. The viability of cells in suspension ranged between 63 and 98% with a mean viability of 83.2 +/- 10.7% (S.D.). The mean yield of total viable cells per g of tissue was 17.4 +/- 14.2 x 10(6). Tumor cells were further fractionated in isopyknic and isokinetic gradients. After isokinetic sedimentation, significant heterogeneity among tumors was seen, but lymphocytes were consistently located in Fraction 7 +/- 2, whereas tumor cells were predominantly in Fraction 22 +/- 1. Malignant epithelial cells were enriched to a 85.8 +/- 9.4% (range, 69.5 to 92.5%) purity by isokinetic gradient centrifugation. Lymphocytes could be successfully separated from tumor cells using an isopyknic gradient. Controlled rate freezing of cells provided material for repeated experiments while short-term tissue culture prior to cell separation increased the proportion of viable cells in the suspension. Disaggregation of human renal cell carcinoma and separation of malignant cells from tumor lymphocytes provides the foundation for characterizing these tumors biochemically and for analyzing hormonal responsiveness and the immunological characteristics of these tumors in vitro.

Large-capacity separation of malignant cells and lymphocytes from the Furth mast cell tumor in a reorienting zonal rotor.

The Furth murine mastocytoma was adapted to the ascitic form and separated into fractions enriched with respect to lymphocytes and malignant cells by velocity sedimentation in the SZ-14 reorienting zonal rotor or in the isokinetic gradient. Lymphocytes were more highly purified (p less than 0.01) in the isokinetic gradient than in the zonal rotor, i.e., lymphocytes comprised 99.1% of the nucleated cells in the purest fraction from the isokinetic gradient and 80.1% of the nucleated cells in the purest fraction from the zonal rotor. Neoplastic mast cells were similarly purified by the two methods; they comprised 67.7 and 78.5% of the nucleated cells in the purest fractions from the isokinetic gradient and zonal rotor, respectively. Up to 160 million tumor cells can be purified in a single step with the reorienting zonal rotor, whereas 30 to 40 million cells per gradient approach the limit of the isokinetic gradient. After centrifugation in the zonal rotor, recovery was 85.6 +/- 12% (S.D.) of the cells layered over the gradient; and the separated tumor cells retained their ability to form tumors when transplanted into mice. The separation of large numbers of lymphocytes and malignant cells from the same tumor in the SZ-14 rotor should aid in the biochemical and immunological characterization of cancer.