Murine tumor cells transduced with the gene for tumor necrosis factor-alpha. Evidence for paracrine immune effects of tumor necrosis factor against tumors

Studies of the anti-tumor activity of TNF-alpha in vivo have been hampered by the need to administer systemically toxic doses of the cytokine to obtain a curative response. To facilitate studies of the effect of high local concentrations of TNF-alpha on tumor growth and host immunity, a newly induced murine sarcoma was transduced with the gene for human TNF-alpha and the biologic characteristics of these cells were examined. We identified high and low TNF-producing tumor clones which exhibited stable TNF secretion over time. Significant amounts of membrane associated TNF were found in a high-TNF producing clone as well. No difference in the in vitro growth rates between TNF-producing and nonproducing cell lines was observed. In contrast, in vivo studies demonstrate that although unmodified parental tumor cells grew progressively when implanted s.c. in animals, tumor cells transduced with the TNF gene were found to regress in a significant number of animals after an initial phase of growth. This effect correlated with the amount of TNF produced and could be blocked with a specific anti-TNF antibody. Regressions of TNF-producing cells occurred in the absence of any demonstrable toxicity in the animals bearing these tumors. TNF-producing tumor cells could function in a paracrine fashion by inhibiting the growth of unmodified, parental tumor cells implanted at the same site. The ability of tumor cells to regress was abrogated by in vivo depletion of CD4+ or CD8+ T cell subsets and animals that had experienced regression of TNF-producing tumors rejected subsequent challenges of parental tumor. Our studies thus show that tumor cells elaborating high local concentrations of TNF regress in the absence of toxicity in the host and that this process requires the existence of intact host immunity. Studies of the lymphocytes infiltrating the gene modified tumors and attempts to use TNF gene modified tumor infiltrating lymphocytes to deliver high local concentrations of TNF to the tumor site without inducing systemic toxicity are underway.     

Preclinical Evaluation of Engineered Oncolytic Herpes Simplex Virus for the Treatment of Pediatric Solid Tumors

Recently, investigators showed that mice with syngeneic murine gliomas that were treated with a neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ₁34.5 gene, enabling replication in tumor cells but precluding infection of normal cells. Previous studies have shown antitumor effects of other oHSV against a number of adult tumors including hepatocellular carcinoma and renal cell carcinoma. The purpose of the current study was to investigate the oncolytic potential of M002 against difficult to treat pediatric liver and kidney tumors. We showed that the oHSV, M002, infected, replicated, and decreased cell survival in hepatoblastoma, malignant rhabdoid kidney tumor, and renal sarcoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly increased survival and decreased tumor growth. Finally, these studies showed that the primary entry protein for oHSV, CD111 (nectin-1) was present in human hepatoblastoma and malignant rhabdoid kidney tumor specimens. We concluded that M002 effectively targeted these rare aggressive tumor types and that M002 may have potential for use in children with unresponsive or relapsed pediatric solid tumors.     

Stimulation of glycosaminoglycan production in murine tumors

Three types of murine tumors, B-16 melanoma, A-10 carcinoma, and S-180 sarcoma, were shown to contain elevated glycosaminoglycan (GAG) concentrations in vivo as compared to normal muscle or subcutaneous tissue. Hyaluronate was especially concentrated in the A-10 carcinoma, which contained approximately six times more hyaluronate than subcutaneous tissue and 18 times more than muscle. In all three tumors, chondroitin sulfates, especially chondroitin-4-sulfate, were present in higher concentrations than in the normal tissues. In culture, however, all three tumor cell lines produced less than 5% as much GAG as mouse fibroblasts, when measured by incorporation of [3H] acetate or by chemical analysis. Varying the culture passage number or the medium composition, ie, glucose, serum, and insulin concentrations, had little effect on GAG synthesis by the tumor cells. The low GAG levels in the tumor cell cultures were not due to hyaluronidase activity in their media. In an attempt to mimic possible host-tumor cell interactions that could account for the elevated GAG levels in vivo, tumor cells were cocultured with fibroblasts, but no stimulation above the amount made by the tumor cells alone plus that by the fibroblasts alone was observed. Conditioned media from the tumor cells, either dialyzed or not against fresh complete medium, had no effect on fibroblast GAG synthesis. Tumor extracts, however, were found to stimulate synthesis of hyaluronate by fibroblasts. Stimulation by extracts of A-10 carcinoma was greater than and additive to that of serum. The above results strongly suggest that GAG production in these tumors is in part regulated by host-tumor interactions.     

Transforming growth factors (TGFs): Properties and possible mechanisms of action

Transforming growth factors (TGFs) are growth-promoting polypeptides that cause phenotypic transformation and anchorage-independent growth of normal cells. They have been isolated from several human and animal carcinoma and sarcoma cells. One TGF is sarcoma growth factor (SGF) which is released hy murine sarcoma virus-transformed cells. The TGFs interact with epidermal growth factor (EGF) cell membrane receptors. TGFs are not detectable in culture fluids from cells which contain high numbers of free EGF cell membrane receptors. SGF acts as a tumor promoter in cell culture systems and its effect on the transformed phenotype is blocked by retinoids (vitamin A and synthetic analogs). The production of TGFs by transformed cells and the responses of normal cells to the addition of TGFs to the culture medium raise the possibility that cells “autostimulate” their own growth by releasing factors that rebind at the cell surface. The term “autocrine secretion” has been proposed for this type of situation where a cell secretes a hormone-like substance for which it has external cell membrane receptors. The autocrine concept may provide a partial explanation for some aspects of tumor cell progression.     

Nuclear transport in 3T3 fibroblasts: effects of growth factors, transformation, and cell shape

Nucleocytoplasmic transport of fluorescent-labeled macromolecules was investigated in transformed and nontransformed 3T3 fibroblasts. Insulin and epidermal growth factor enhanced transport three-fold after 1-2-h incubation with nontransformed adhering fibroblasts; no enhancement of transport was observed for spherical unattached fibroblasts. The concentration of growth factor for maximal enhancement was 3-10 nM. Nuclear transport for Kirsten murine sarcoma virus-transformed BALB/c 3T3 fibroblasts, however, was maximally enhanced before addition of growth factors; addition of insulin or epidermal growth factor causes no additional transport enhancement. Transformation also minimizes cell shape effects on macromolecular nuclear transport. These results provide evidence that protein growth factors and oncogenic transformation may use a similar mechanism for activation of nuclear transport.     

Systemic endocrine instigation of indolent tumor growth requires osteopontin

The effects of primary tumors on the host systemic environment and resulting contributions of the host to tumor growth are poorly understood. Here, we find that human breast carcinomas instigate the growth of otherwise-indolent tumor cells, micrometastases, and human tumor surgical specimens located at distant anatomical sites. This systemic instigation is accompanied by incorporation of bone-marrow cells (BMCs) into the stroma of the distant, once-indolent tumors. We find that BMCs of hosts bearing instigating tumors are functionally activated prior to their mobilization; hence, when coinjected with indolent cells, these activated BMCs mimic the systemic effects imparted by instigating tumors. Secretion of osteopontin by instigating tumors is necessary for BMC activation and the subsequent outgrowth of the distant otherwise-indolent tumors. These results reveal that outgrowth of indolent tumors can be governed on a systemic level by endocrine factors released by certain instigating tumors, and hold important experimental and therapeutic implications.     

Selective host range restriction of goat cells for recombinant murine leukemia virus and feline leukemia virus type A

We isolated a strain of normal goat fibroblasts which was uniquely selective in that it allowed the replication of xenotropic murine leukemia virus but not polytropic recombinant murine leukemia virus. In addition, feline leukemia virus type A replication was severely diminished in these goat cells, whereas feline leukemia virus type B and feline endogenous RD114-CCC viruses replicated efficiently. No other known cells exhibit this pattern of virus growth restriction. These goat cells allow the study of xenotropic murine leukemia virus in mixtures which also contain recombinant murine leukemia virus and may be helpful in eliminating feline leukemia virus type which often coexists in feline sarcoma or leukemia virus mixtures with other feline leukemia virus types.     

Cellular responsiveness to growth factors correlates with a cell's ability to express the transformed phenotype

Five random subclones of the rat fibroblast line F2408 vary in their frequency of transformation by the unrelated Kirsten murine sarcoma virus and Abelson murine leukemia virus. The same pattern of sensitivity is displayed when the cells are induced to anchorage-independent growth (transformed) by epidermal, platelet-derived, and sarcoma growth factors, or by whole serum. Our results demonstrate that a growth factor's ability to render cells anchorage independent is not unique to transforming growth factors, but common to many growth factors; anchorage-independent growth is a function of the total growth factor concentration in the medium; cells vary in their inherent responsiveness to growth-factor-induced anchorage-independent growth; and cells resistant to growth-factor-induced anchorage-independent growth are also resistant to transformation by a variety of tumor viruses. We conclude that the way a cell responds to growth factors plays a central role in the expression of the transformed phenotype.     

Comparison and modulation of angiogenic responses by FGFs,VEGF and SCF in murine and human fibrosarcomas

The effects of angiogenic growth factors on the growth, vascular architecture and the downstream cytokine signaling of sarcomas are unknown. These are of potential great importance since sarcoma, like endothelium, is of mesodermal origin and therefore could grow in response to these factors. Three human sarcomas (leiomyosarcoma SK-LMS-1, liposarcoma SW872 and fibrosarcoma SW684) and one murine fibrosarcoma (KHT) were grown in nude and C3H/He mice, respectively. Tumor structural vessels, perfused vessels and hypoxia were quantified immunohistochemically. Fast-growing murine KHT tumors had a markedly higher number of structural vessels compared with the human sarcomas. In both murine and human sarcomas, approximately half of the total structural vessels were perfused, and the numbers of perfused vessels decreased with increasing tumor volume. In vitro, basal mRNA expression of several angiogenic growth factors and their receptors differed between two of the human sarcoma cell lines, SK-LMS-1 and SW872. Compared with SK-LMS-1, untreated SW872 cells had higher levels of mRNA expression for FGF11, FGF14, angiopoietin, CD105 and VEGFR1. Two sarcoma cell lines were also treated with 10 ng/ml of six angiogenic growth factors (FGF1, FGF2, FGF7, FGF10, VEGF and SCF) for 24 h, and mRNA expression of endogenous FGF family members (FGF1, FGF2, FGF10, FGF11, FGF13 and FGF14) were quantitatively measured using RNase protection at various times following treatments. Again, SW872 cells were more responsive to exogenous growth factor treatment compared with SK-LMS-1 cells in terms of the elevation of endogenous FGF mRNA expression. In the SW872 cells, all of the exogenous angiogenic growth factor treatments, except for VEGF, upregulated endogenous FGF1, FGF2 and FGF14 mRNA expression. The SK-LMS-1 cells, in contrast, only responded to exogenous FGF1, FGF7 and FGF10, but did not respond to VEGF.     

Tumor-stroma interactions

The importance of stromal cells and the factors that they express during cancer initiation and progression has been highlighted by recent literature. The cellular components of the stroma of epithelial tissues are well-recognized as having a supportive role in carcinogenesis, where the initiating mutations of a tumor originate in the epithelial cells. The use of mouse models and xenografts suggests that mutations in the stromal fibroblasts can also initiate epithelial tumors. Many of these tumors result from the alteration of paracrine growth factor pathways that act on the epithelia. However, the tissue specificity of the responses to the growth factors is a mystery not yet solved.     

Suppression of in vivo tumor growth by the transfection of the interleukin-5 gene into colon tumor cells

To investigate the influence of tumor producing interleukin-5 (IL-5) on growth kinetics of tumors, we transduced the murine IL-5 gene into murine colon C26 tumor cells. Two IL-5-secreting clones, low-level IL-5 producer C26-8B and high-level IL-5 producer C26-6F, were established. Both tumors, C26-6F and C26-8B, grew more slowly than the mock C26 tumor, although the in vitro growth rate of these IL-5 transfectants was much the same as that of the mock C26 cells. There was a significantly decreased number of colonies in the lung of mice given C26-6F or C26-8B tumors i.v. than in mice given mock C26 tumors i.v. Moreover, in mice given C26-6F cells i.v., a smaller number of tumor colonies in the lung was observed, as compared to the case with C26-6B cells. While the growth rate of C26-8B tumors in mice treated with anti-IL-5 mAb was more rapid than that seen in control mAb-treated mice, growth of C26-6F tumors in anti-IL-5-mAb-treated mice was slightly more rapid compared to findings in control mAb-treated mice. The isotypematched mAb did not alter the in vitro growth of mock-C26 cells or of the IL-5-gene-modified C26 cells. Growth of IL-5-secreting C26 tumors transplanted in nude mice was also inhibited. These results suggest that tumor-producing IL-5 inhibits growth of colon tumors mediated through T-cell-independent protective mechanisms of the host.     

Suppression of in vivo tumor growth by the transfection of the interleukin-5 gene into colon tumor cells

To investigate the influence of tumor producing interleukin-5 (IL-5) on growth kinetics of tumors, we transduced the murine IL-5 gene into murine colon C26 tumor cells. Two IL-5-secreting clones, low-level IL-5 producer C26-8B and high-level IL-5 producer C26-6F, were established. Both tumors, C26-6F and C26-8B, grew more slowly than the mock C26 tumor, although the in vitro growth rate of these IL-5 transfectants was much the same as that of the mock C26 cells. There was a significantly decreased number of colonies in the lung of mice given C26-6F or C26-8B tumors i.v. than in mice given mock C26 tumors i.v. Moreover, in mice given C26-6F cells i.v., a smaller number of tumor colonies in the lung was observed, as compared to the case with C26-6B cells. While the growth rate of C26-8B tumors in mice treated with anti-IL-5 mAb was more rapid than that seen in control mAb-treated mice, growth of C26-6F tumors in anti-IL-5-mAb-treated mice was slightly more rapid compared to findings in control mAb-treated mice. The isotypematched mAb did not alter the in vitro growth of mock-C26 cells or of the IL-5-gene-modified C26 cells. Growth of IL-5-secreting C26 tumors transplanted in nude mice was also inhibited. These results suggest that tumor-producing IL-5 inhibits growth of colon tumors mediated through T-cell-independent protective mechanisms of the host.     

Expression of developmentally regulated endothelial cell locus 1 was induced by tumor-derived factors including VEGF

Developmentally regulated endothelial cell locus 1 (Del1) is a new angiogenic molecules expressed specifically in early embryonic endothelial cells. We investigated the relationship between Del1 and tumor cell-derived vascular endothelial growth factor (VEGF). Dunn osteosarcoma cells and high- and low-metastatic murine sarcoma cells did not express Del1. However, the expression of Del1 was observed in these primary tumor tissues and the pulmonary metastatic tissues after subcutaneous inoculation in vivo. Every tumor cell-conditioned medium containing VEGF induced the expression of Del1 in murine lung microvascular endothelial (MLE) cells, although control MLE cells did not express Del1. The anti-mouse VEGF monoclonal antibody inhibited the induction of the Del1 expression. In addition, mouse recombinant interleukin-1alpha and tumor necrosis factor-alpha also induced Del1 in MLE cells. Del1 may play an important role in tumor angiogenesis through the effects of tumor-derived factors including VEGF.     

A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo

We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) formation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of embryos surviving to assessment time with visible tumors on the CAM; v) maintainability of the cell, both in the original host and the embryonic chick, or 'shuttle maintainability'; vi) detection by the naked eye, and vii) cost/benefit relation. The murine melanoma cell lineage, B16F10, which efficiently forms distinct, pigmented tumor masses and metastases on the CAM, performed better in this model than the murine B61 cell line. In vitro transduction of B16F10 cells with a recombinant adenovirus carrying a construct of the E. coli LacZ gene followed by inoculation onto the CAM resulted in beta-galactosidase expression in the tumor mass growing on the CAM. This model is potentially applicable to preclinical evaluation of gene vectors, especially for gene therapy of cancer.     

Thermal sensitivity of endothelial cells

Experimental work indicates that one of the mechanisms of tumor control by hyperthermia may be damage to blood vessels, resulting in decreased blood flow to the neoplasms. Among the various elements of the microvasculature, endothelial cells are the most important possible targets of thermal injury. Furthermore, neoplasms have a significantly higher proportion of proliferating endothelial cells than do normal tissues. Thus it is necessary to establish the thermal sensitivity of endothelial cells and to explore possible differences in response between resting and proliferating endothelium. We studied the in vitro thermal sensitivity of murine and human capillary endothelial cells compared to human fibroblasts by following cell survival and growth recovery. Nonstimulated endothelial cells are more sensitive than fibroblasts. Their sensitivity is dose dependent within the range of 42 to 45 degrees C/30 min. Stimulation to proliferate by endothelial cell growth factor (ECGF) renders these cells even more sensitive. Morphologic studies confirm these thermal effects in endothelial cells and fibroblasts. These findings support a direct effect of hyperthermia on endothelial cells, which appears to be more severe in proliferating cells. This may explain the reduced blood flow in heated tumors and may indicate a valuable therapeutic gain for hyperthermia.     

Chemical and viral transformation of cultured skin fibroblasts from patients with familial polyposis coli

Treatment of skin fibroblasts from an FPC patient with 4NQO or MNNG followed by sequential passaging caused morphological changes of the cells, which showed characteristics of transformed cells such as a high frequency of colony formation in agarose, increased growth ability, and chromosomal abnormalities. This and other fibroblast lines from 5 of 12 FPC patients had an increased susceptibility to 4NQO cytotoxicity, which was caused by enhanced 4NQO-reductase activity rather than by reduced DNA repair. However, the susceptibility to cytotoxicity of MNNG and repair of MNNG-damaged DNA were normal in FPC cells. The tumor promoters TPA and DHTB enhanced the frequency of chemical transformation of the FPC fibroblasts, and protease inhibitors suppressed the promoter-enhanced transformation. The skin fibroblasts from many FPC patients exhibited increased susceptibility to transformation by murine sarcoma viruses. Analysis of the viral DNA and RNA after infection revealed that the increased susceptibility is determined at an early stage of transformation. Two out of 5 MNNG-transformed clones of FPC fibroblasts, isolated from agarose, had increased expression of c-Ki-ras or c-Ha-ras, and 4 of 4 MSV-transformed clones showed high expression of viral Ki-ras. These clones grew further after isolation from agarose, but were mortal and did not form tumors in nude mice. The present results suggest that additional changes in morphologically transformed FPC fibroblasts are required for malignant transformation.     

A novel function of p38-regulated/activated kinase in endothelial cell migration and tumor angiogenesis

The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in both suppression and promotion of tumorigenesis. It remains unclear how these 2 opposite functions of p38 operate in vivo to impact cancer development. We previously reported that a p38 downstream kinase, p38-regulated/activated kinase (PRAK), suppresses tumor initiation and promotion by mediating oncogene-induced senescence in a murine skin carcinogenesis model. Here, using the same model, we show that once the tumors are formed, PRAK promotes the growth and progression of skin tumors. Further studies identify PRAK as a novel host factor essential for tumor angiogenesis. In response to tumor-secreted proangiogenic factors, PRAK is activated by p38 via a vascular endothelial growth factor receptor 2 (VEGFR2)-dependent mechanism in host endothelial cells, where it mediates cell migration toward tumors and incorporation of these cells into tumor vasculature, at least partly by regulating the phosphorylation and activation of focal adhesion kinase (FAK) and cytoskeletal reorganization. These findings have uncovered a novel signaling circuit essential for endothelial cell motility and tumor angiogenesis. Moreover, we demonstrate that the tumor-suppressing and tumor-promoting functions of the p38-PRAK pathway are temporally and spatially separated during cancer development in vivo, relying on the stimulus, and the tissue type and the stage of cancer development in which it is activated.