Activation and expansion of tumor-derived activated cells for therapeutic use.

We obtained tumor specimens from patients with cancer in an effort to activate and expand the tumor-infiltrating lymphocytes for therapeutic use. With the use of finely minced tumor preparations from eight different tumor types, recombinant interleukin-2, and lymphokine-activated killer cell-conditioned medium, lymphocytes were expanded in vitro. After 4 weeks, the tumor cells were virtually absent from the cultures. At this point, the lymphocytes were termed "tumor-derived activated cells" (TDACs). Over 90% of the TDACs from each of the different tumor types were T lymphocytes, and the percentage of cells expressing either CD4 or CD8 varied considerably from population to population. The lymphocytes showed specific cytolytic activity in melanoma and colon and renal cell carcinomas. Continued expansion and long-term growth of the TDACs, as well as maintenance of the cytolytic activity, were achieved by periodic stimulation of the TDACs with irradiated autologous tumor cells. In a clinical study of 28 patients with cancer, we generated a mean number of 1.2 X 10(11) TDACs in an average time in culture of 69 days. These TDACs were subsequently infused into the patients with cancer. TDACs appear to represent an important resource for biotherapy of patients with cancer.

Characterization of rat mammary tumor cell populations.

Nodules of tumor cells have been isolated from 7, 12-dimethylbenz (alpha)anthracene-induced rat mammary tumors. These nodules consist of at least two cell populations which can be subfractionated by selective attachment in calcium-free medium. One cell population which attaches in the absence of calcium (basal cells) stains intensively with antibodies against type IV collagen and also with antibodies against keratin. The nonattaching population, the epithelial cells, stains much more weakly with either antibody. Biochemical analyses indicate that 27% of the protein labeled in basal cell culture is type IV collagen, while 8% is keratin. With epithelial cells, only 0.35% of the protein made is collagen, and only 2.4% is keratin. Basal cells contain about 2 times as much calmodulin as epithelial cells but only about one-tenth as many estrogen receptors. In culture, the basal cells stimulate the attachment and/or division of the epithelial cell population. The epithelial cells have little effect on the division or attachment of the basal cells. Interaction between the two cell populations may be important for tumor growth in vivo.

Sensitivity of N-nitrosomethylurea-induced rat mammary tumors to cis-hydroxyproline, an inhibitor of collagen production.

The growth of primary N-nitrosomethylurea-induced rat mammary tumors was depressed by cis-hydroxyproline (CHP). This growth arrest appeared to be related to the ability of CHP to inhibit the deposition of basement membrane collagen as based on the following observations: (a) in vitro and in vivo, tumor cells synthesized type IV collagen, the collagen uniquely localized in basement membranes; (b) in vitro, the inhibition of tumor cell growth was preceded by a specific decrease in collagen accumulation with no effect on non-collagen protein synthesis; (c) a transplantable N-nitrosomethylurea-induced rat mammary tumor accumulated no type IV collagen as determined by polyacrylamide gel electrophoresis and indirect immunofluorescence. The growth of this tumor was not influenced by CHP; (d) an established human mammary tumor cell line, MCF-7, did not accumulate type IV collagen and was not inhibited by CHP. At the doses which effectively blocked the growth of primary N-nitrosomethylurea-induced mammary tumors, CHP and no toxic effects, and serum prolactin levels were not altered. The inhibitory effect was thus apparently due to the direct action of CHP upon the accumulation of collagen in cells which required type IV collagen production for continued growth.

Blocking basement membrane collagen deposition inhibits the growth of 7,12-dimethylbenzanthracene-induced rat mammary tumors.

7,12-Dimethylbenzanthracene-induced rat mammary tumors contain basement membrane collagen in vivo and in vitro in primary culture, as determined by immunofluorescence. The proline analogue dis-hydroxyproline, which selectively blocks collagen accumulation, inhibits tumor cell growth in vitro. In vivo cis-hydroxyproline blocks tumor growth and produces epithelial cell degeneration and tumor necrosis at a non-toxic dose. This compound has no effect on the growth of the mammary carcinoma cell line 64/24, which makes no basement membrane collagen. As we have previously described for the normal proliferating rat mammary gland, basement membrane collagen deposition may be necessary for the growth of some rat mammary tumors.

Glutathione levels in cultured heart cells. Influence of buthionine sulfoximine, an inhibitor of glutathione synthesis.

In primary cultures of heart cells in mid-growth phase, levels of acid-soluble glutathione were 99 nmoles/mg protein and increased to 178 nmoles/mg protein at confluent growth. Glutathione disulfide accounted for less than 9% of the total. Levels of protein-bound mixed disulfide in mid-growth phase cells were 58 nmoles/mg protein and decreased to 36 nmoles/mg protein at confluent growth. Buthionine sulfoximine (BSO, 10(-4) M) depleted the levels of both glutathione and glutathione disulfide with no influence on the levels of protein-bound mixed disulfide. BSO had no influence on the multiplication rate of heart cells in primary culture. In secondary passage cultures, the levels of glutathione were less than half those of the primary cultures. BSO depressed cell growth and soluble glutathione, whereas the levels of protein-bound mixed disulfide were increased. These results showed that BSO depletes heart cells of soluble glutathione, whereas protein-bound thiol remains unchanged or increases.

Estrogen receptors and growth response in cultured human periodontal ligament cells.

The periodontal ligament (PDL) is a connective tissue involved in the remodeling process associated with tooth development and positioning. PDL cells grown in culture were analyzed for the capacity to specifically bind steroid hormones and for growth response to estradiol-17 beta. Using [3H]estradiol-17 beta as the ligand, PDL cells in first passage cultures exhibited a specific estrogen binding capacity of 881 fmol/mg cell protein. With [3H]dexamethasone as a ligand, the binding capacity of the glucocorticoid receptor was 143 fmol/mg protein. With [3H]R5020 as a ligand, the progestin receptor exhibited a binding capacity of 5 pmol/mg protein. Scatchard analysis of estradiol binding at 37 degrees revealed a dissociation constant of 2.7 X 10(-9) M, representative of the estrogen receptor. The addition of estradiol-17 beta at concentrations of 10(-9) and 10(-8) M to culture media induced a dose-dependent decrease in growth (DNA content) to 62% and 38% control values, respectively. The addition of the antiestrogens tamoxifen and 4-hydroxytamoxifen at concentrations of 10(-7) and 10(-6) M similarly depressed cell growth. These results show that PDL cells contain high affinity receptors for several steroid hormones and further that these cells are targets for the action of estrogens.

Growth of tumor-derived activated T cells for the treatment of advanced cancer.

In 1994, we reported on a series of patients treated with T-cell therapy (Study #1). This paper (Study #2) is an update of our experience through 1999 in the production of tumor-derived activated cells (TDAC), also called tumor-infiltrating lymphocytes (TIL), from tumor biopsies. TDAC were successfully grown in medium containing Interleukin-2 from 75% of the 366 tumor biopsies tested. There was no significant difference in success (growth to 1 x 10(9) cells) comparing primary and metastatic tumors. Many of the tumors were shipped to the laboratory by overnight delivery from distant sites. Success rate did decrease with the length of time for tumor transport in excess of 24 hours. Certain additional cytokines were tested when cultures did not grow. Interleukin-4 was beneficial in the development of 1 of 4 TDAC cultures which did not grow with IL-2 alone. In order to produce TDAC to treat patients, cells were grown in gas permeable plastic bags or in artificial capillary bioreactor cultures. Approximately 1 x 10(9) were seeded from an initially successful "feasibility study" to bulk produce cells for treatment. Harvest was carried out after about 3 weeks. Sixty-three patients were treated at least once with a minimum of 1 x 10(10) TDAC given by intravenous infusion. On the average, the number of cells per treatment was 3 x 10(10) with a viability of 87%. TDAC cultures contained T cells with variable ratios of CD4 to CD8 cells. Secreted granulocyte-monocyte colony stimulating factor, interferon gamma and tumor necrosis factor alpha were measured in TDAC conditioned medium. Only 34 patients received the full course of 4 TDAC treatments. The cells were well tolerated with mild fever and dyspnea. Partial responses were observed in 8 patients, including the dramatic regression of scalp nodules in a patient with renal cancer. These results showed that therapeutic amounts of TDAC can be produced in cell culture in a reasonable and cost-effective manner. The cells were well tolerated and responses were seen in renal and melanoma patients resistant to IL-2 with bulky, advanced cancer.

Cancer biotherapy with interferon, interleukin-2 and tumor-derived activated cells (TDAC).

Interleukin-2 (Il-2) allows for the therapeutic augmentation of immunity in vivo and/or in vitro. In extensive studies in nearly 1000 patients with advanced cancer, we have demonstrated that Il-2 by continuous infusion, with cellular therapy (LAK or TDAC), is feasible, cost effective and useful in selected patients with melanoma and kidney cancer. Studies in patients with other tumor types are underway.

Influence of selenium on the growth of N-nitrosomethylurea-induced mammary tumor cells in culture.

Selenium is an essential dietary trace element which has anticancer properties. Among its effects in rats, selenium has been shown to inhibit the development of carcinogen-induced mammary tumors by interfering with the postinitiation, promotion phase of carcinogenesis. We studied the effects of selenium on the growth of rat mammary tumor cells in primary culture. Our objective was to determine whether selenium had any direct influence on cell growth which might explain its influence on tumor development. Rat mammary tumors were induced by N-nitrosomethylurea. Tumor epithelium was prepared by collagenase dispersion and the cells were separated by Ficoll gradient centrifugation. The tumor epithelium was grown in primary culture using a defined serum-free medium. The addition of low concentrations of sodium selenite, less than 1.0 micrograms/ml, stimulated tumor cell proliferation. Protein synthesis and the production of type IV collagen increased within the first hour of exposure, prior to any measurable increase in DNA synthesis. Concentrations of selenite greater than 1.0 micrograms/ml inhibited cell proliferation, the synthesis of protein, and the replication of DNA in a dose-related manner. These studies demonstrated that selenium has the potential to influence the postinitiation phase of rat mammary tumorigenesis by directly altering the growth of tumor cells, possibly through the regulation of protein synthesis.

Cell culture of human metastatic breast carcinomas: a review.

The literature concerned with culture of primary and metastatic breast carcinomas is reviewed historically and summarized in the tables. The development of cell culture methodology for these tissues and its usefulness is discussed in conjunction with the problems encountered in culture of breast carcinomas.

Steroid-hormone-binding proteins in normal and neoplastic mammary tissues from C3H mice fed diethylstilbestrol.

Mammary tumors develop earlier and in greater numbers in C3H mice fed diets containing up to 1000 ppb diethylstilbestrol or 5000 ppb estradiol-17 beta. We have analyzed the steroid-hormone-binding proteins in cytosols of normal target tissues and in neoplastic mammary tissues of control and estrogen-fed mice to determine whether the capacity for hormone response was altered by dietary estrogens. Using estradiol-17 beta as a ligand, estrogen-binding proteins were measured with Kd = 10(-11)-10(-10) M and sedimentation constants of 8 and 4 S. The binding capacity (per mg cytosol protein) of uterus exceeded that of normal mammary gland 10-20-fold, while mammary tumors only contained half the binding capacity of virgin mammary gland. Binding proteins for the glucocorticoid, triamcinolone acetonide, exhibited a sedimentation constant of 7-8 S and Kd = 10(-9)-10(-8) M. The glucocorticoid binding capacity of mammary tumors exceeded that of virgin mammary gland. Binding proteins for progestins were measured using the synthetic ligand R5020. Mammary gland contained progestin-binding proteins with 4 S sedimentation constants and Kd = 10(-9)-10(-8) M. Not all mammary tumors contained progestin-binding proteins, and the binding capacities varied considerably among those that did. The molecular characteristics of the steroid receptors in tumors were the same as those found in normal target tissues. There was nothing to suggest that dietary estrogens altered the characteristics of the three steroid-binding proteins.

Steroid hormone receptors in MCCLX, a transplantable lactogen-dependent mammary tumor of the rat.

MCCLX is a transplantable rat mammary tumor which, for sustained growth, requires the elevated levels of circulating lactogen provided by pregnancy or the implantation of an estrogen pellet. High affinity receptors for estradiol, as well as for the glucocorticoids, dexamethasone and triamcinolone acetonide and the progestin R5020 were measured in the cytosols of these tumors. Estrogen binding capacities were significantly lower in the cytosols of tumors from estrogen pellet treated animals compared with tumors from pregnant animals. Ligand exchange assays demonstrated that nuclei of tumors from estrogen-treated rats contained 3-4 times the estrogen receptors but that there was a definite decrease in total estrogen binding capacity compared with tumors from pregnant rats. It was concluded that this lactogen-dependent tumor contains steroid receptors with molecular properties similar to those of normal target tissues, including estrogen receptors capable of nuclear translocation, the levels of which are modulated by the specific growth conditions.

Structural alterations within N-nitrosomethylurea-induced mammary tumors after in vivo treatment with cis-hydroxyproline.

N-Nitrosomethylurea-induced mammary tumors in Buffalo rats were examined ultrastructurally and compared with tumors from rats treated in vivo for 9 days with cis-hydroxyproline (CHP). The tumors from untreated animals were heterogeneous histologically but often had their cells arranged in clusters called pseudolobules. At the periphery of the pseudolobules in control tumors, a few myoepithelial cells showed evidence of basal lamina secretion. Outlining the pseudolobules were many mast cells. Treatment with CHP inhibited the growth of the tumors. It increased the extent of atrophy and regression and also induced cyst-like areas to form within the pseudolobules. These "pseudocysts" were actually part of the stromal compartment and they sometimes contained multilayered basal lamina and mast cells. The extensive multilayering of the basal lamina observed after CHP-treatment was thought to arise from extensive infoldings as the pseudolobules "shrank" due to atrophy and loss of cells. The multilayering and cell debris disappeared from the pseudocysts, leaving many spaces bounded by myoepithelial cells. The remaining myoepithelial cells appeared atrophied, but often had an expanded rough endoplasmic reticulum. We did not observe evidence of basal lamina secretion by cells in treated tumors, which correlates with CHP's ability to block type IV collagen production.

Influence of phomopsin and ivalin on steroid-hormone binding and growth of MCF-7 human breast cancer cells.

Ivalin is a plant alkaloid that inhibits the induction of tumors in animals. Phomopsin is a mycotoxin known to be carcinogenic. To determine if these compounds influence endocrine responsiveness, their effect on steroid receptors was measured. Neither of these toxins had a direct effect on either the binding capacity or the rate of steroid association of [3H]estradiol-17 beta, [3H]R5020, or [3H]dexamethasone to their respective receptors in cytosol of human breast cancer and rat liver. However, steroid receptor levels of MCF-7 cells, grown in tissue culture, were altered by ivalin and phomopsin. Ivalin at 10(-6) M depressed estrogen receptor levels, while glucocorticoid receptor levels were increased. At 10(-6) M, phomopsin was inhibitory of both progestin and glucocorticoid binding capacities. Data obtained from the proliferation of MCF-7 cells indicated that ivalin and phomopsin at 10(-6) M decreased the number of cells grown in tissue culture. Phomopsin exhibited an inhibitory effect on both [3H]thymidine and [3H]glycine incorporation, while ivalin stimulated [3H]glycine incorporation.

Growth of tumor derived activated T-cells for the treatment of cancer.

This report describes the production of Tumor Derived Activated Cell cultures (TDAC, also called tumor infiltrating lymphocytes) from patient tumor biopsies and our preliminary experience growing these cells to therapeutic levels using artificial capillary bioreactor cultures. TDAC were successfully grown in medium containing Interleukin 2 from 80% of the 113 tumor biopsies tested. There was no significant difference in success (growth to 1 x 10(9) cells) comparing primary and metastatic tumors. Many of the tumors were shipped to the laboratory from distant sites. Success rate did decrease with the length of time for tumor transport. Interleukin 4 was beneficial in the development of 1 of 4 TDAC cultures which did not grow with IL-2 only. Seventy-seven bioreactor cultures were initiated for 31 patients. On the average, 1.9 x 10(9) TDAC were inoculated per bioreactor; 3.3 x 10(10) were harvested in 22 days. Twelve liters of medium were required per 1 x 10(10) TDAC produced. TDAC cultures contained T cells with variable ratios of CD4 to CD8 cells. Secreted granulocyte monocyte colony stimulating factor, interferon gamma and tumor necrosis factor were measured in the bioreactor cartridge conditioned medium. Twenty three patients were evaluated. Partial responses were observed in 4 patients including a dramatic remission of scalp nodules in a patient with renal cancer. Results showed that therapeutic amounts of TDAC cells may be produced in a reasonable and cost effective manner using artificial capillary bioreactor cultures.

Chemosensitivity testing in a tumor acquisition, propagation, and preservation program.

Chemosensitivity assays were carried out as part of a tumor acquisition, propagation, and preservation program for cancer biotherapy. In addition to biopsy specimens, tumor cells propagated in culture or tumor xenografts grown in nude mice were submitted for chemosensitivity assay when sufficient biopsy material was unavailable. Chemosensitivity was tested utilizing the adhesive tumor cell culture system. A total of 154 specimens was submitted for testing; 96 specimens were assayed. Success rates were 55% for primary cancer biopsies, 67% for metastases, 69% for xenografts, and 70% for cell lines. There were no significant differences evident when the sensitivity to drugs of tumor cells originating from biopsies, xenografts, or tissue culture were compared. Sufficient data were available for 18 patients to compare clinical results of drug treatment with predictive results from the chemo-sensitivity assay. Assay results indicating insensitivity appeared to predict resistance; however, assays indicating sensitivity were not predictive. These results suggest that propagated tumor material, such as xenografts and cultured cell lines, may be useful when biopsy tissue is not available.

Tumor-derived activated cells: preliminary laboratory and clinical results.

It is well known that T lymphocytes can mediate significant anti-tumor responses. A limiting factor has always been the ability to expand T cells, whether from the peripheral blood, spleen, or tumor. The recent availability of recombinant interleukin-2 (r-IL2) has demonstrated the feasibility of expanding T cells and the clinical efficacy of these cells as anti-tumor effectors in murine models. Concomitantly, researchers discovered that lymphokine-activated killer cells--peripheral blood cells functionally distinct from T cells--could be cultured, expanded, and re-infused in patients, with significant clinical effects. For many years, the infiltrating lymphocytes have been recognized in tumor biopsies and known to be cytolytically active. Major limiting factors were the ability to culture large numbers of these infiltrating cells and the limited understanding of the tumor antigens involved for T-cell stimulation. Restimulation by antigen (tumor cells) appears to provide the ongoing antigen stimulation needed to maintain selective killing of tumor cells. By defining various factors in the medium that support and enhance T-cell growth and activation, the components are becoming available to develop a broad attack on advanced cancer by using this laboratory-based technology of stimulation and expansion of tumor-derived activated cells.

Interleukin-15 and the growth of tumor derived activated T-cells.

Interleukin-15 was tested to determine whether this recently discovered cytokine was capable of stimulating the growth of tumor derived activated T cells in culture (TDAC, also referred to as tumor infiltrating lymphocytes). When established cultures of IL-2 induced, IL-2 dependent TDAC were tested, IL-15 stimulated growth in a dose dependent manner, alone or in the presence of IL-2. One established TDAC was cultured with IL-15 alone for 18 passages over a 10 week period. Comparing IL-2 and IL-15 treated cultures, growth rate with IL-15 was slower. IL-15 doubled the secreted interferon alpha and granulocyte-macrophage colony stimulating factor. IL-15 and IL-2 were compared in primary TDAC cultures. IL-15 induced TDAC outgrowth in 3 of 6 cultures. IL-2 induced outgrowth in all 6. Tumor cells were eliminated as TDAC grew out in both IL-2 and IL-15 treated cultures. These results suggested that IL-15 like IL-2, is capable of stimulating the growth of TDAC with antitumor activity, but with certain distinct effects which may be of interest therapeutically.

Cultured breast cystosarcoma phylloides cells and applications to patient therapy.

Malignant cystosarcoma phylloides (CP) is a relatively rare cancer of the breast. A CP tumor was processed as part of a tumor acquisition, propagation, and preservation program in patient biotherapy. Two tissue culture cell lines were developed from this tumor, one directly from the biopsy, another from a xenograft tumor grown in athymic mice. The two cell lines were similar in character. There was strong immunochemical reactivity with antibodies to vimentin, type I collagen, and type III collagen. There was no reactivity with antibodies to cytokeratin and epithelial membrane antigen. Both cell lines were aneuploid, clonogenic in soft agar, and tumorigenic in nude mice. 5 alpha-dihydrotestosterone and thyroxine added to the culture medium stimulated growth, while testosterone, 17 beta-estradiol, and 4-hydroxytamoxifen were without effect. Dexamethasone and cortisol were inhibitory at high doses (10(-6) M). Dibutyryl cyclic AMP, theophylline, and vitamin C were all inhibitory. The biopsy contained tumor-infiltrating lymphocytes which proliferated in cultures containing interleukin 2. The expanded lymphocytes were activated T cells which had the capacity to lyse tumor cells. These results suggest possibilities in the therapy of cystosarcoma phylloides involving vitamin C, certain hormones, and tumor-infiltrating lymphocytes.

Tumor acquisition, propagation, and preservation. The culture of human colorectal cancer.

Fourteen new colorectal cancer cell lines were developed as part of a tumor acquisition, propagation, and preservation program for biotherapy. Fifty-six specimens were received. Nine cell lines were generated from biopsies; seven of these cell lines were from metastatic lesions. Five additional cell lines were developed from xenografts grown in nude mice. Biopsies that produced three of these xenografts gave rise to parallel culture cell lines. Biopsy-derived and xenograft-derived cell lines from the same tumor behaved similarly in culture and exhibited similar markers when assessed immunohistochemically. Collagen substrate was beneficial in the primary culture of 50% of the specimens tested. Collagen was required for the successful propagation of two cell lines.