Immunobiology of heterotransplanted human tumors in nude mice

The immunobiology of heterotransplanted human tumors was investigated following transplantation into nude mice of human bronchogenic, colon, rectal, ovarian, gastric, endometrial, vaginal, bladder, renal, esophageal, embryonic cell, pancreatic, and breast carcinoma, as well as fibrosarcoma, rhabdomyosarcoma, malignant melanoma, astrocytoma, Wilm's tumor, endometrial hyperplasia, and hydatidiform mole. Several of these tumors were passaged up to 15 generations. During these passages no changes in latency period for tumor development or in histology were noted. There were significant differences between several tumors in the minimum number of cells required for successful transplantation; such differences were independent of the basic biologic aggressiveness of the individual tumors. Nude mice that received transplants of fibrosarcoma and endometrial carcinoma had increased serum IgM and numbers of spleen cells and complement receptor lymphocytes. No such changes were noted for mice that received transplants of malignant melanoma, In contrast, there were no apparent differences in the responses of nude mice, who were given transplants of human tumors, to be T-cell mitogens concanavalin A or phytohemagglutinin or in the number of theta-bearing spleen cells. The success rate for transplantation was significantly improved when explants, rather than single-cell suspensions, were performed. Tumors transplanted to nude mice derived from strictly homozygous matings behaved like tumors transplanted to mice born of heterozygous mothers. Finally, despite the dramatic size of subcutaneous tumor nodules, there were no examples of invasion or distant metastases.     

Rehabilitation approach to children with sequelae of hypoxic encephalopathy

OBJECTIVES: The dependence of human prostate carcinoma growth on hormone was studied in xenotransplants in nude mice. The objective was to determine differences in cell kinetic parameters and volume growth of tumors growing with alpha-dehydrotestosterone (alphaDHT) and without alphaDHT. These differences could be used as arguments pro and contra the adaptation versus the clonal selection hypothesis. METHODS: Human prostate carcinomas were xenotransplanted into nude mice. Growth of tumors was observed in castrated male mice without and with implanted osmotic pumps secreting alphaDHT. In a further series of experiments the alphaDHT tubes were removed when the tumors had reached a volume of 0.3 cm3. Tumor volume was measured to determine tumor doubling time with and without alphaDHT. Detailed cell kinetics were analyzed using the bromodeoxyuridine (BrdUrd) method with flow cytometry. Applying the relative movement (RM) and a simulation analysis to parallel single and multiple BrdUrd labelling experimental data we determined transit times through the phases of cell cycle, potential doubling time Tpot, growth fraction (GF) and cell loss. RESULTS: Five human prostate carcinomas were xenotransplanted into nude mice. Tumor take was only achieved when androgen hormone was present. However, when alphaDHT was removed when the tumors had grown to a volume of 0.3 cm3, they continued to grow at nearly the same Td as those tumors with continued alphaDHT application. The BrdUrd experiments, on the other hand, showed considerable increase of Tc and Tpot upon withdrawal of alphaDHT in 4 out of 5 tumors. The GF and labelling index (LI) were maintained at about the same level as alphaDHT consuming tumors. CONCLUSION: While small transplanted tumor pieces do not grow without alphaDHT, larger tumors grow with the same Td after removal of alphaDHT. The slower proliferation shown by the increased Tc and Tpot is balanced by less cell loss. Since GF and LI were maintained at about the same level, we conclude that in our tumors the majority of cells adapted to hormone independence. There was no evidence for the selection model since the tumors continued to grow at about the same speed after hormone depletion. All cell kinetic parameters showed a considerable inter- and intratumoral heterogeneity. A clinical implication may be that hormone ablation therapy should always be supplemented by some other therapy.     

The invasion-MTT assay as a predictor of liver metastasis using human gastrointestinal carcinomas transplanted in nude mice

The invasion-3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, which evaluates invasive potential into the reconstituted basement membrane, Matrigel, was performed on 49 human gastrointestinal carcinomas transplanted in nude mice. There were 19 colorectal carcinomas, 10 pancreatic carcinomas, 10 gastric carcinomas, 8 esophageal carcinomas, and 2 bile duct carcinomas. The percent invasion (PI) value of each tumor by the invasion-MTT assay expresses the invasive rate of tumor cells into the Matrigel as a percentage. There were no significant differences in correlations between the PI values and primary tumor site, clinicopathological findings, tumor doubling time, or DNA index; however, the PI values of primary tumors and lymph nodes with liver metastases were significantly higher than those of primary tumors without liver metastasis (P < 0.05). Furthermore, the primary tumors with synchronous (P < 0.05) or asynchronous (P < 0.01) liver metastases showed significantly higher PI values compared with the primary tumors without liver metastases. These results suggest that PI is not only an independent factor to predict liver metastasis, but it also correlates closely with liver metastasis. Thus, the invasion-MTT assay for primary tumors might be clinically useful to predict liver metastasis in patients following surgery for gastrointestinal carcinomas.     

A comparison of three fluid replacement strategies for maintaining euhydration during prolonged exercise

SSIN mice are very sensitive to tumor promoters in two-stage skin carcinogenesis protocols. It was recently reported that SSIN mice have fewer CD8+ T-cells than other strains of mice and develop a weaker cytotoxic T-cell response upon challenge with an allogeneic tumor transplant. The significance of this muted immune response to processes involved in two-stage carcinogenesis depends on the immunogenicity of the tumors generated in such protocols. Although they have low CD8+ T-cell contents, SSIN rejected a variety of subcutaneously transplanted allogeneic murine tumors. Analyses of the growth of primary papillomas derived from 7,12-dimethylbenz[a]anthracene-initiated/12-O-tetradecanoylphorbol-13-ac etate-promoted SSIN mice and then subcutaneously transplanted into triple-deficient (bg-nu-xid), athymic nude and immune-competent and immunosuppressed SSIN mice revealed that few tumors took and tumor takes were not markedly influenced by the immumological status of the transplant recipient. Two tumor cell lines (RS1 and RS2) were derived from the transplantation studies and could be passaged in normal SSIN mice (H-2q haplotype). Both tumors were squamous cell carcinomas (SCCs) by the second in vivo passage and were rejected in allogeneic mice (BALB/c) but grew in FVB/N mice, a strain having the H-2q haplotype. Transplantation studies revealed that prior exposure to RS1 and RS2 did not prime SSIN mice to reject a subsequent tumor challenge. Three primary SCC tumors derived from SSIN mice in a two-stage carcinogenesis protocol also grew when subcutaneously transplanted in SSIN mice and could be serially passaged. Consequently, the epidermal SCCs that develop in two-stage carcinogenesis protocols appear to be nonimmunogenic.     

Reversible tumorigenesis in mice by conditional expression of the HER2/c-erbB2 receptor tyrosine kinase

In the present study we describe the reversible transformation of NIH3T3 fibroblasts by overexpression of the HER2/c-erbB2 receptor tyrosine kinase. Cell lines expressing HER2 under control of a tetracycline-responsive promoter were isolated. Induction of HER2 expression resulted in cellular transformation in vitro as depicted by growth in soft agar and focus formation in tissue culture. Subsequent treatment of these cells with the effector anhydrotetracyline switched-off HER2 expression and induced morphological and functional changes characteristic for non-transformed cells. Subcutaneous transplantation of cells in nude mice resulted in the formation of solid tumors. Interestingly tumor formation was completely suppressed by treatment of the animals with anhydrotetracyline. Our findings indicate that overexpression of HER2 induces the transformed phenotype of NIH3T3 cells and is required for tumor formation and progression in nude mice. By linking the expression of the marker gene secreted placental alkaline phosphatase to the expression of HER2, a sensitive monitoring of tumor development in nude mice was feasible.     

Multifocal or chaotic atrial rhythm: report of nine infants, delineation of clinical course and management, and review of the literature

Although the transplantation of human neoplasms in immunodeficient mice is now a well-established procedure, the majority of primary malignancies cannot be successfully maintained for long periods of time in adult athymic (nude) and asplenic-athymic (lasat) mice. Various lipids such as cholesterol, cholesterol oleate, stearic and palmitic acid esters markedly depress the RES phagocytic activity and immunocompetence of mammals. In view of the immunosuppressive properties of certain lipids and in order to graft and grow as many tumors as possible, further studies into the effects of lipids on the growth of heterotransplanted human tumors is warranted. Lipids may enhance local growth and facilitate the development of metastases rarely seen in nude and lasat mice bearing xenogeneic cancer cells. Lipids may accelerate human malignant cell proliferation in mice by both depressing further the defense of host and modifying the cancer cell membrane. The relationship of lipids to the onset and progression of 'spontaneous' tumors in humans is not known.     

Human vestibular schwannoma growth in the nude mouse: evaluation of a modified subcutaneous implantation model

HYPOTHESIS: Based on the hypothesis that vestibular schwannomas can be successfully implanted and grown in the nude mouse model, an in vivo experiment was designed for subcutaneous implantation of solid vestibular schwannoma tissue. BACKGROUND: Vestibular schwannomas are benign tumors arising from Schwann cells of cranial nerve VIII. Little in vivo research has been carried out with these tumors, due in part to the difficulty to grow cells in culture or maintain tumor in an animal model. Recently, vestibular schwannomas have been implanted in nude mice with moderate success. The current study evaluates a modification of prior techniques in an effort to establish a dependable research model. METHODS: Thirty-six nude mice were implanted with variable-sized vestibular schwannoma tissue from three human subjects. Volumes implanted ranged from 14-170 mm3. Mice were observed for 28 days and individual volumes recalculated. Eleven of the mice were observed for a total of 56 days with volumes re-evaluated, and tumors subsequently were removed for assessment of viability and vascularity. RESULTS: At 28 days, 36 tumors (100%) showed take with 34 tumors (94%) showing macroscopic growth. The 11 tumors observed for 56 days showed a trend of stable or decreased size at 56 days compared with that of the 28-day measurement. Overall growth from time of implantation to measurements at 56 days was noted in 8 (73%) of 11 tumors when measured at the skin and in 10 (91%) of 11 tumors when direct tumor volume was measured. One hundred percent of tumors evaluated microscopically at 56 days was viable. All tumors at the time of removal had significant vascularity with a mean of 70.68% (SD = 23.42) of surface covered with vessels. There were no significant differences in take and growth for the larger tumor specimens compared with those of smaller sizes. CONCLUSION: Human vestibular schwannomas successfully can be implanted and maintained in the subcutaneous pocket of the nude mouse. This in vivo tumor model provides a reliable, accessible base for further research with vestibular schwannomas.     

Comparison of the pattern of integrin expression between primary tumors and liver metastasis of gastric and colorectal cancers

To investigate the interrelationship between integrin VLA3 overexpression and liver metastasis, immunohistochemical studies of VLA3 were made in 73 cases of gastric cancer (66 cases without liver metastasis, 7 cases with liver metastasis) and 15 cases of colorectal cancer (3 cases without liver metastasis, 12 cases with liver metastasis). The rate of integrin VLA3 expression in 69 primary gastric and colorectal cancers without liver metastasis was 41%, that was higher than that (0%) in the 19 primary tumors of gastric and colorectal cancers with liver metastasis. In contrast, the positive rate for integrin VLA3 staining in 19 cases involving liver metastasis of gastric and colorectal cancers was 58% (11/19), which was higher than that (0%) in primary tumors. These findings suggest that VLA3 may play an important role in the process of liver metastasis of gastric and colorectal cancers.     

Metastatic retinoblastoma and L-phenylalanine mustard-dianhydrogalactitol

Retinoblastomas heterotransplanted to nude mice were shown to be sensitive to a combination of phenylalanine mustard and dianhydrogalactitol. A patient with metastatic retinoblastoma was treated with this combination and had a significant response with complete clinical clearance of her bone marrow and metastatic tumors. However, the patient's response was similar to that observed in nude mice; recurrences of tumors developed after complete clinical remission.     

Human pancreatic beta-cell deoxyribonucleic acid-synthesis in islet grafts decreases with increasing organ donor age but increases in response to glucose stimulation in vitro

Human pancreatic beta-cell proliferation may be crucial for the success of islet transplantation. The aim of this study was to test the hypothesis that adult human beta-cells proliferate in vitro and in vivo and respond with increased rates of replication to factors known to promote rodent islet-cell proliferation, i.e. glucose, human recombinant GH, and FCS. For this purpose, human islets were prepared from a total of 19 adult heart-beating organ donors and cultured for 48 h with or without the additives described above. 3H-thymidine was added to the medium during the last 60 min of culture. After immunohistochemical staining for insulin and autoradiography, the labeling index (LI; i.e. % of labeled beta-cells over total number of beta-cells) was estimated by light microscopy. Islets also were transplanted under the kidney capsule of normal or alloxan-diabetic nude mice. After 2 weeks, 3H-thymidine was injected and the islet grafts prepared for determination of LI, as described above. Islets cultured at 5.6 mM glucose showed an increased beta-cell proliferation compared with islets cultured at 2.8 mM glucose (P < 0.05). However, culture at 11 mM glucose failed to further increase beta-cell proliferation. Addition of GH (1 microg/ml) to the medium, in the presence of 1% FCS and 5.6 mM glucose, did not influence the rate of beta-cell proliferation. In islets transplanted to hyperglycemic nude mice, beta-cell proliferation was similar to that observed in islets grafted into normoglycemic nude mice. Proliferation, however, decreased with increasing organ donor age. This study shows that pancreatic beta-cells from adult man are able to proliferate both in vitro and in vivo. Moreover, beta-cells from adult human donors respond with increased proliferation to glucose in vitro and show a decreased proliferation in vivo with increasing donor age.     

Methylcholanthrene-induced sarcomas in nude mice have short induction times and relatively low levels of surface MHC class I expression

In order to study the role of the T-cell-mediated immune defense in tumor development, a total of 93 sarcomas were induced using different doses (8 micrograms (0.1%), 40 micrograms (0.5%) and 400 micrograms (5%)) of 3-methylcholanthrene in athymic nude Balb/c mice and phenotypically normal immunocompetent Balb/c mice. A shorter tumor induction time and a higher tumor incidence after treatment with low doses of methylcholanthrene were seen in nude mice than in immunocompetent mice, indicating that they have a lower resistance to the carcinogen. Contrary to expectations we found that the MHC class I expression of tumors from nude mice was lower than that of tumors from normal mice. Higher surface expression of MHC class I was demonstrated on high dose tumors from normal mice than on low dose tumors from normal mice. The cellular composition of the individual tumors raised in nude mice was more heterogeneous with respect to MHC class I expression. Since the mice differ genetically only with respect to the nu gene, these results indicate that a lack of T-cell-mediated defense mechanisms may confer upon the bearer a lower resistance to 3-methylcholanthrene and a different MHC profile of the ensuing tumor.     

Establishment and characterization of six new human endometrial adenocarcinoma cell lines

The endometrial carcinoma cell lines EC-MZ-1, 2, 3, 5, 9, and 11 were established between 1986 and 1990. Four cell cultures were started from endometrial tissue, one from ascites, and one from a lymph node metastasis. Lines have to date been subcultured up to 180 times and the doubling time varies between 26 hr and 3 weeks. Immunocytochemically the coexpression of cytokeratin (predominantly simple-epithelial cytokeratin polypeptides) and vimentin intermediate filaments was detectable in all cell lines, but three lines (EC-MZ-5, 9, 11) expressed vimentin only at low level. By transmission electron microscopy the tumor cells exhibited features of epithelial differentiation. After subcutaneous transplantation into nude mice three lines (EC-MZ-1, 2, 5) produced slow-growing tumors. The histological classification of these tumors ranged from moderately differentiated adenocarcinoma to undifferentiated carcinoma and closely corresponded to the original tumor. Even after long-term in vitro culture, without any addition of estrogens to the culture medium, the moderately differentiated receptor-positive cell line (EC-MZ-2) retained its morphological differentiation. The cells were propagated without estrogens in the culture medium. The estrogen and progesterone receptor levels of cultured cells were determined. Three lines (EC-MZ-1, 2, 3) were positive for the progesterone receptor in low passage number only, the other cell lines were negative for both receptors. The transplantable lines were investigated for hormonal receptor expression in ovariectomized nude mice. In the moderately differentiated cell line (EC-MZ-2) we observed an enhanced expression of the estrogen receptor under optimal stimulation of the nude mouse with estradiol benzoate. There was no effect on the expression of the progesterone receptor.     

Invasion of fibrosarcoma cells transfected with PAI-2 gene

The effect of PAI-2 on the invasion of fibrosarcoma cells in vitro and in vivo was investigated. The control cells (C+, C+ pem) and PAI-2 transfectants (C+ exp) were used in the assay of the degradation of isotopically labelld 3H-ECM and were injected sc into athymic/nude mice. Recombinant PAI-2 could inhibit efficiently the degradation of 3H-ECM by tumor cells (86.5%). The PAI-2 transfectants remained tumorigenic in nude mice, but tumors originating from the PAI-2 transfectants showed histologically the presence of a thick capsule which was absent in tumors from control cells.     

Expression of antisense CD44 variant 6 inhibits colorectal tumor metastasis and tumor growth in a wound environment

Up-regulation of CD44 variant isoforms has been linked to the progression of epithelial tumors and the metastatic phenotype. Here we report a functional role for CD44 variant isoforms in colorectal cancer metastasis. An antisense mRNA approach was used to down-regulate CD44 variant isoforms containing CD44 variant 6 (v6) in the metastatic colorectal tumor cell line HT29. Cell lines stably expressing antisense CD44 exon 10 (v6) showed reduced expression of alternatively spliced CD44 variant isoforms but no significant change in expression of CD44 core protein, as judged by immunohistochemical analysis using CD44 domain-specific monoclonal antibodies. Expression of antisense exon 10 (v6) had no effect on HT29 tumor cell proliferation in vitro or the ability of the cells to bind immobilized hyaluronan, but it resulted in a reduced capacity to form liver metastases in nude mice following intrasplenic injection. Metastases were not detected in nude mice inoculated with antisense CD44 exon 10 (v6)-expressing cell lines after 4 months, against a background of a 30% metastasis rate in the control HT29 parental and vector alone transfected lines. Furthermore, whereas 82% of mice intrasplenically injected with control HT29 parental and vector alone cell lines developed tumors in incisional wound sites, none of the mice injected with antisense exon 10 expressing HT29 cells developed similar tumors. This is the first demonstration that antisense RNA can be used to selectively inhibit expression of specific domains of a molecule generated through alternative mRNA splicing while allowing expression of core domains to remain unaffected. Furthermore, these results provide direct evidence for a functional role of CD44 variant isoforms in the metastasis of human colorectal tumor cells and may suggest a critical role for CD44 variants in promoting cell growth specifically in the cytokine/growth factor-enriched environment of a wound site.     

Prognostic factors in colorectal cancer. Literature review for clinical application

PURPOSE: Identification of prognostic factors is a primary basis for planning the treatment and predicting the outcome of patients with colorectal cancer. Reviewing studies from the literature performed using univariate and multivariate analyses and their own study, the authors critically discuss the prognostic value of the clinicopathologic parameters of the tumor. METHODS: Among 853 patients with colorectal tumors seen at the Department of Clinical Surgery of the Catholic University of Rome, Italy, 690 cases that were curatively resected the study. Overall survival rate, related to the clinicopathologic variables, was calculated, and univariate and multivariate analyses were performed. RESULTS: Five-year and ten-year overall survival rates were 70 and 55 percent, respectively. Univariate and multivariate analyses showed that node involvement, distant metastases, bowel obstruction, and patient gender are factors independently related to outcome. CONCLUSIONS: Data from the literature and the present study suggest that only a few clinical parameters, particularly bowel obstruction, and some pathologic factors (tumor stage, vessels invasion, and tumor ploidy) are related to patient survival rate and are the most reliable prognostic criteria. In prospective clinical studies, any other new pathologic or molecular factors should be matched with these parameters to confirm their value in outcome prediction.     

Meloxicam inhibits the growth of colorectal cancer cells

Cyclooxygenase-2 has been reported to play an important role in colorectal carcinogenesis. The effects of meloxicam (a COX-2 inhibitor) on the growth of two colon cancer cell lines that express COX-2 (HCA-7 and Moser-S) and a COX-2 negative cell line (HCT-116) were evaluated. The growth rate of these cells was measured following treatment with meloxicam. HCA-7 and Moser-S colony size were significantly reduced following treatment with meloxicam; however, there was no significant change in HCT-116 colony size with treatment. In vivo studies were performed to evaluate the effect of meloxicam on the growth of HCA-7 cells when xenografted into nude mice. We observed a 51% reduction in tumor size after 4 weeks of treatment. Analysis of COX-1 and COX-2 protein levels in HCA-7 tumor lysates revealed a slight decrease in COX-2 expression levels in tumors taken from mice treated with meloxicam and no detectable COX-1 expression. Here we report that meloxicam significantly inhibited HCA-7 colony and tumor growth but had no effect on the growth of the COX-2 negative HCT-116 cells.     

Absence or reduction of Fhit expression in most clear cell renal carcinomas

The FHIT gene at human chromosome region 3p14.2 straddles the common fragile site, FRA3B, and numerous homozygous deletions in cancer cell lines and primary tumors. Also, the 3p14.2 chromosome breakpoint of the familial clear cell kidney carcinoma-associated translocation, t(3;8)(p14.2;q24), disrupts one FHIT allele between exons 3 and 4, fulfilling one criterion for a familial tumor suppressor gene: that one allele is constitutionally inactivated. Because the FHIT gene sustains biallelic intragenic deletions rather than mutations, there has not been evidence that the FHIT gene frequently plays a role in kidney cancer, although replacement of Fhit expression in a Fhit-negative renal carcinoma cell line suppressed tumor growth in nude mice. We have now assessed 41 clear cell renal carcinomas for expression of Fhit by immunohistochemistry. Normal renal tubule epithelial cells express Fhit uniformly and strongly, whereas 51% of the tumors are completely negative, 34% of tumors show a mixture of positive and negative cells, and 14% are uniformly positive, although usually less strongly positive than the normal epithelial cells. Most interestingly, there was a correlation between complete absence of Fhit and the G1 morphological grade and early clinical stage. Morphological grades G2 and G3 exhibited a mixture of positive and negative cells with a tendency for a higher fraction of negative cells in G3. Fhit inactivation is likely to be an early event in G1 tumors and may be associated with progression in G2 and G3 tumors.     

Networking rural health resources. The woman who cried wolf

OBJECTIVE: To study the genetic stability and to establish a method for detection of micrometastasis using microsatellite DNA in human breast cancer xenograft in nude mice. METHODS: Fresh tissue of human breast cancer was xenotransplanted in nude mice or thotopically. Genomic DNA extracted from tissues of human breast cancer, xenotransplanted tumors and metastatic foci in nude mice were PCR amplified at three microsatellite loci (D14S68, D18S69, D20S199) and were analysed by electrophoresis and silver stain on PAGE. RESULTS Microsatellite DNA in genome of the xenotransplanted tumors and metastatic foci in nude mice were identical with that of the human breast cancer. CONCLUSION: This study has demonstrated in nude mice the xenotransplanted tumors and metastatic foci that originated from human breast cancer. The genetic stability in human breast cancer is evident in the processes of xenotransplantation, serial passages in nude mice, metastasis and in vitro culture. This method is sensitive and specific for the discrimination of metastatic tumor cells.     

Establishment of new epithelial carcinoma cell lines by blocking monolayer formation

Human endometrial carcinoma cell lines were established by using a new cell culture technique. The malignant endometrial tumors were grossly disaggregated by mechanical means and cultivated in suspension culture. Adhesion to the bottom of the culture flasks was prevented by first coating the flasks with a thin agarose layer. Four cell lines were derived from 17 samples by this new technique. The cell lines obtained in this way were fully characterized, including karyotyping, intermediate filament staining and transplantation to nude mice. This new technique of initial suspension culture may also be applicable to other human tumors that are equally difficult to cultivate in vitro.     

Pharmacokinetic characteristics and tolerability of a novel intravenous immunoglobulin preparation

Cathepsin D content and activity were determined in matched paired sets of colorectal tumor tissue and normal mucosa and correlated with a number of biological and clinical parameters. Significantly higher cathepsin D activity was measured in tumor cytosol compared to paired normal mucosa (p < 0.02), in Dukes' stage A tumors compared to Dukes' B and C (p < 0.05), in tumors < 5 cm compared to those > 5 cm, or in tumors with a low proliferation rate compared to those with a high proliferation rate (p < 0.05). Moreover, significant differences in enzyme activity between tumor tissue and paired normal mucosa were observed in node-positive and G2 tumors (p < 0.05). No significant correlation between cathepsin D activity and other biological parameters was found. Further, no differences in cathepsin D content between tumor tissue and paired normal mucosa were observed except in Dukes' stage A tumors (p < 0.02). A significantly increased cathepsin D content was also observed in tumors > 5 cm compared to tumors < 5 cm (p < 0.01). No relationship between tumor cathepsin D content and clinical stage was detected. However, a significant correlation (p < 0.05) was observed between the tumor-specific content of this enzyme and tumor grade. Finally, there was no relationship between tumor-specific cathepsin D activity and content (r = -0.27, p = 0.23). These data suggest that cathepsin D activity rather than content correlates with the malignant progression of colorectal cancer. This phenomenon should be taken into consideration when clinical studies are undertaken to assess the potential prognostic value of proteolytic enzymes involved in tumor progression.