DYNAMICS OF LEUKOCYTE MIGRATION INTO THE MOUSE ASCITES TUMOR

There is a marked increase in the number of peritoneal leukocytes (lymphocytes, monocytes and granulocytes) during the growth of Ehrlich ascites tumor in mice. No local proliferation (as indicated by a labeling at 1 hr following a single ³H-TdR injection) was observed in the normal peritoneal leukocytes or those in the ascites tumor, except for a very minor labeling of some tumor macrophages. Kinetics of peritoneal leukocytes was studied with a series of twelve injections of ³H-thymidine (20 μCi every 8 hr) in normal mice as well as mice injected with 10⁶ tumor cells i.p. 2 hr after the last ³H-TdR injection. Animals were sacrificed at intervals up to 6 days. Granulocyte labeling in the blood as well as peritoneal space was near 100% in both groups of animals at all the intervals. Temporal changes in the labeling of lymphocytes (from 10% at 0 day to 22% at day 6), and monocytes (from 20% at 0 day to 57% at day 6) were identical in the blood and peritoneal space of normal animals, indicating a free exchange of cells between these compartments. Higher labeling indices than those in the controls were attained in the blood of tumor-bearing hosts (viz 40% for lymphocytes and 80% for monocytes at 6 days) suggesting an increased turnover of these cells in the circulation. In addition, peritoneal mononuclear cells of tumor-bearing mice showed even a higher labeling than those in the blood (viz 65% for lymphocytes and 92% for monocytes at 6 days) indicating a selective migration and/or retention of newly formed cells within the tumor, in contrast to a random migration into the normal peritoneal cavity. Furthermore, an identical labeling of macrophages to that of monocytes within the tumor indicated a short monocyte-macrophage transition. The preferential accumulation of young mononuclear cells into the tumor may be of functional importance.     

Rejection of tumor metastases in Fischer 344 rats following the administration of killed Corynebacterium parvum

Summary Fischer 344 rats received subcutaneous grafts of syngeneic 13762 mammary adenocarcinoma cells. Thereafter, at 20 days, each animal of a group received either daily (×2) permeating intratumor injections of killed Corynebacterium parvum (1.0 mg) saline, and weekly (×4) intravenous (i.v.) or intraperitoneal (i.p.) C. parvum or saline. In addition, a group each (4 groups) of rats were treated with surgical extirpation of the growing tumor nodule and i.v. and i.p. C. parvum or saline administered at weekly (×4) intervals. The results revealed that following intratumor injection of C. parvum there was rejection of tumor by all animals which exhibited long-term survival for 29 months (intratumor saline: 0% survival). The groups of rats treated with surgical extirpation of the tumor and parenteral administration of C. parvum exhibited 70–75% survival for 20 months (saline treated: 20–35% survival). The surviving animals exhibited tumor-specific protection to subsequent tumor cell challenges. Macrophages separated from the lung, peritoneum, and spleen of rats from the intratumor C. parvum group exhibited respectively, 46.2%, 62.9%, and 29.4% cytotoxicity towards target 13762 tumor cells as measured by ⁵¹ Cr release. Similar studies using macrophages from the group treated with surgery and parenteral C. parvum revealed similar tumor cytotoxicity (pulmonary: 49.2%; peritoneal: 65.7%; spleen: 34.4%). The splenic lymphocytes from the intratumor and parenteral C. parvum groups exhibited, respectively, 16.9% and 14.7% ⁵¹ Cr release following incubation with target tumor cells.This study was supported in part by Grant No. CA18582-01 from the National Cancer Institute     

Cell Proliferation In Emt6 Tumors Treated With Single Doses of X-Rays Or Hydroxyurea. I. Experimental Results

EMT6 mouse mammary tumors were treated in vivo with 5 mg/mouse of hydroxyurea (HU) or 300 rads of X-rays. the proliferation of the tumor cells was followed for 28 hr after treatment. Changes in the ³H-TdR labeling index, the mitotic index, the specific activity of the ³H-TdR-labeled DNA, and the proportion of suspended, clonogenic cells in the S phase of the cell cycle were examined and compared. Evidence was found for reassortment of the surviving cells in treated tumors into partially synchronous cohorts. the partial synchrony in the proliferation of the surviving cells was not accurately predicted by the changes in the labeling index and the mitotic index. the changes in DNA specific activity proved unacceptable as an indicator of cell proliferation in solid EMT6 tumors treated with low doses of radiation or HU.     

Kinetic Models of C3h Mouse Mammary Tumor Growth: Implications Regarding Tumor Cell Loss

Three models of tumor cell loss are described. the effects of cell loss on other cellular kinetic parameters are evaluated, and experiments which may distinguish among the models are discussed. Each model is based on a different cell-loss mechanism, and equations for the cell-cycle, cell-frequency distribution, the growth of both the proliferating and non-proliferating cell population, the growth fraction (GF), and the relative rate of volumetric growth, (dV/dt)/V, are derived. The following types of data are simulated for each model: the pulse labelling index, the mitotic index, and the labeling index as a function of time after a single or a series of ³H-TdR injections. the relative volumetric growth rate has the same mathematical form for each model. the PLM curves predicted by each model for the tumor lines studied (S102F and Slow) are not appreciably different. the predicted initial labeling index and mitotic index may differ significantly among the models depending upon the tumor line. the most striking difference among the models lies in the predictions regarding the labeling index as a function of time after a single or after a series of ³H-TdR injections. These types of labeling experiments should be valuable for distinguishing the different cell-loss mechanisms in solid tumors.     

Splenic lymphocytopoiesis and migration pattern of splenic lymphocytes

The spleens of young pigs were selectively labeled with tritiated thymidine ([³H]-TdR) and the relative and absolute numbers of labeled lymphocytes found 24 hr later in different lymphoid and nonlymphoid organs were determined autoradiographically. It was deduced that about 4.6 × 10⁹ lymphocytes (that is, about 15% of all splenic lymphocytes) are produced by the spleen per day and about 17% of the newly formed lymphocytes leave the spleen within the first day of labeling. Spleen-derived lymphocytes could be found in relatively high numbers in the lymph nodes, blood, gut-associated lymphoid tissues, and, surprisingly, in the bone marrow, whereas the concentration in the thymus was very low. In a second series, pigs were labeled with [³H]TdR and only the spleen was excluded from labeling. The labeling index of splenic small lymphocytes was about 10% 1 day later, indicating a high rate of influx of newly formed lymphocytes into the pig spleen. The spleen of the young pig is an important lymphocytopoietic organ and exports and imports newly formed lymphocytes at high rates.     

Rates of incorporation of radioactive molecules during the cell cycle

We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine (³H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) ³H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) ³H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing ³H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of ³H-TdR and ³⁵S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, ³HH-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside ³H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of ³⁵S-methionine increased continuously with cell size and DNA content. Incorporation of ³H-TdR in CHO cells was proportional to DNA synthesis.     

TUMOR ANGIOGENESIS : Rapid Induction of Endothelial Mitoses Demonstrated by Autoradiography

Walker ascites tumor cells and an extract derived from such cells (tumor angiogenesis factor, TAF) were injected into the subcutaneous tissue of rats by using a dorsal air sac technique. At intervals thereafter, thymidine-³H was injected into the air sac and the tissues were examined by autoradiography and electron microscopy. Autoradiographs of 1µ thick Epon sections showed thymidine-³H labeling in endothelial cells of small vessels 1–3 mm from the site of implantation, as early as 6–8 hr after exposure to live tumor cells At this time interval endothelial cells appeared histologically normal. DNA synthesis by endothelium subsequently increased and within 48 hr new blood vessel formation was detected. The presence of thymidine-³H-labeled endothelial nuclei, endothelial mitoses, and regenerating-type endothelium was confirmed by electron microscopy. TAF also induced neovascularization and endothelial cell DNA synthesis after 48 hr. A similar response was not evoked in saline controls. Formic acid, which elicited a more intense inflammatory response, was associated with less endothelial labeling and neovascularization at the times studied. Pericytes and other connective tissue cells were also stimulated by live tumor cells and TAF. The mechanism of new blood vessel formation induced by tumors is still unknown but our findings argue against cytoplasmic contact or nonspecific inflammation as prerequisites for tumor angiogenesis.     

Naïve rat umbilical cord matrix stem cells significantly attenuate mammary tumor growth through modulation of endogenous immune responses

Background aimsUn-engineered human and rat umbilical cord matrix stem cells (UCMSCs) attenuate growth of several types of tumors in mice and rats. However, the mechanism by which UCMSCs attenuate tumor growth has not been studied rigorously.MethodsThe possible mechanisms of tumor growth attenuation by rat UCMSCs were studied using orthotopic Mat B III rat mammary tumor grafts in female F344 rats. Tumor-infiltrating leukocytes were identified and quantified by immunohistochemistry analysis. Potential cytokines involved in lymphocyte infiltration in the tumors were determined by microarray and Western blot analysis. The Boyden chamber migration assay was performed for the functional analysis of identified cytokines.ResultsRat UCMSCs markedly attenuated tumor growth; this attenuation was accompanied by considerable lymphocyte infiltration. Immunohistochemistry analysis revealed that most infiltrating lymphocytes in the rat UCMSC-treated tumors were CD3⁺ T cells. In addition, treatment with rat UCMSCs significantly increased infiltration of CD8⁺ and CD4⁺ T cells and natural killer (NK) cells throughout tumor tissue. CD68⁺ monocytes/macrophages and Foxp3⁺ regulatory T cells were scarcely observed, only in the tumors of the phosphate-buffered saline control group. Microarray analysis of rat UCMSCs demonstrated that monocyte chemotactic protein-1 is involved in rat UCMSC-induced lymphocyte infiltration in the tumor tissues.ConclusionsThese results suggest that naïve rat UCMSCs attenuated mammary tumor growth at least in part by enhancing host anti-tumor immune responses. Naïve UCMSCs can be used as powerful therapeutic cells for breast cancer treatment, and monocyte chemotactic protein-1 may be a key molecule to enhance the effect of UCMSCs at the tumor site.     

CLEARANCE RATE OF EXOGENOUS 3H-THYMIDINE FROM THE PLASMA OF PREGNANT RHESUS MONKEYS

The plasma clearance rate of ³H-TdR was determined by thin-layer chromatography of samples from pregnant rhesus monkeys who had received a single intravenous injection of ³H-TdR. At 10 min after the injection the circulating levels of ³H-TdR had fallen to less than 0.2% of the initial theoretical distribution of the label. In general the plasma clearance rate can be described by a ‘double exponential’curve with half-lives of 1 min and 20 min. Thus, ³H-TdR disappears from the circulation of the pregnant monkey more rapidly than it does from the bloodstream of rodents, which correlates with previously detected interspecies differences in autoradiographic labeling of neurons. This means (1) that higher doses of ³H-TdR must be used for autoradiographic labeling of monkey tissues and (2) that the monkey may be useful for studying cell kinetics because it has a short pulse labeling of the DNA.     

Variability of the time at which PHA-stimulated lymphocytes initiate DNA synthesis

A method is presented for the study of the entrance of in vitro stimulated cells into their first poststimulation S phase. PHA-stimulated lymphocytes were incubated continuously with ¹⁴C-TdR. This isotope was then removed at different intervals and the cells were incubated for 8 h in medium containing ³H-TdR. Cells which had incorporated ³H-TdR but not ¹⁴C-TdR were considered to have entered their first post-stimulation S phase during the 8 h incubation with ³H-TdR. These cells were identified by double-layer autoradiography.The majority of PHA-stimulated lymphocytes entered their first period of DNA synthesis between 48 and 72 h after the addition of PHA. However, the variability was pronounced, some cells entering their first S phase at about 24 h and others some 100 h later. Cells entering their first S phase accounted for a considerable part of the population of cells in DNA synthesis still as late as 72 h after the addition of PHA.Calculation of the total number of cells that entered their first S phase during the 6 day culture period showed that DNA synthesis was initiated in some 40 % of the cells of the initial population.     

Dynamics of monocytes/macrophages and T lymphocytes in acutely rejecting rat renal allografts

We examined the infiltration of acutely rejecting renal allografts (DA→LEW) by ED1⁺ and ED2⁺ macrophages and T lymphocytes at intervals of 24 h after transplantation. Donor and recipient macrophages were differentiated by MHC class II antigen expression in double-staining experiments with ED1. Proliferation was assayed after pulse-labelling with BrdU. We subdivided allograft infiltration into three consecutive phases: 1) During phase I on days 1 to 2 after allogeneic kidney transplantation, perivascular infiltrates developed that contained numerous donor and recipient macrophages. Allograft rejection could already be diagnosed 24?h after transplantation by perivascular infiltration of T lymphocytes, whereas T cells were rarely found in isografts. 2) Phase II of allograft rejection from day 3 to 4 was characterized by massive propagation of the infiltrate. About equal numbers of interstitial donor and recipient macrophages were counted. Both macrophages and T lymphocytes proliferated in situ and macrophages outnumbered T cells until complete rejection. 3) During phase III the allograft was destroyed. Large intravascular monocytes surprisingly expressed the ED2 antigen. In the interstitium of viable graft regions, the population of recipient macrophages grew, whereas the population of donor macrophages and of T lymphocytes decreased.     

Nucleolar morphology and cell proliferation kinetics of thymic lymphocytes

Proliferation kinetics of cells of the lymphocytic series were studied in mouse thymuses using ³H-methyl-thymidine (³H-TdR) as a tracer of DNA synthesis and employing autoradiographic technique. Cells were allocated arbitrarily to several compartments according to their degree of maturity and nucleolar morphology. Serial sampling after continuous ³H-TdR injection showed that thymic cells of the lymphocytic series constitute a small highly proliferating pool that feeds into a large nonproliferating pool. Lymphoblasts with dense and trabeculate nucleoli and prolymphocytes with trabeculate nucleoli represent multiplicative compartments and belong to the proliferating pool. A fraction of multiplicative precursors enters a ‘dormant’ state and these immature lymphocytes are morphologically characterized by ring-shaped nucleoli. Multiplicative compartments and non-dividing compartments of immature lymphocytes differ significantly in labeling indices, kinetics of labeling in serial samples and in other kinetic parameters, namely in the efflux from the unlabeled pool, labeling increment and efflux from the labeled pool. Serially connected compartments of lymphoblasts with dense and trabeculate nucleoli, prolymphocytes with trabeculate nucleoli and mature lymphocytes, represent the main stream of cell differentiation and maturation. At least a portion of mature lymphocytes proceeds during maturation from the compartment of cells with trabeculate nucleoli to the compartment of cells with ring-shaped nucleoli. The presence of proliferating and ‘dormant’ precursors suggests that lymphopoiesis in thymuses may correspond to the advantaged logarithmic system of multiplication.     

Circulating T and B lymphocytes of the mouse. II. Lifespan

The average lifespan of circulating lymphocytes was investigated by determining the percentage of labeling of thoracic duct lymphocytes (TDL⁵) from mice injected with tritiated thymidine (³HT) for various periods. Percentage of labeling of TDL from normal CBA mice, which consist of approximately 85% T cells and 15% B cells, was found to be directly proportional to the time of ³HT administration. This technique thus failed to demonstrate the presence of more than one population of lymphocytes. Less than 50% of TDL were labeled after ³HT injection for 8 weeks.Percentage of labeling of TDL from nude mice (which consist solely of B cells) was likewise found to be directly proportional to the duration of ³HT injection but occurred at a rate three to four times faster than in non-T cell-depleted CBA mice. Further experiments, in which a marker for B cells was used, allowed the rate of ³HT labeling of B cells to be studied in normal CBA mice. These data corroborated the findings in nude mice and indicated that, with regard to lifespan, thoracic duct B cells consisted of a single population with an average lifespan of 5–7 weeks. Similarly it was calculated that the average lifespan of thoracic duct T cells was in the order of 4–6 months.Studies on the rate of formation of TDL during prolonged thoracic duct drainage of normal CBA mice indicated that the percentage of newly formed cells increased rapidly after 24-hr drainage. The total numbers of newly formed cells, however, were found to remain relatively constant throughout the period of drainage investigated (up to 9 days) except for a transient increase during the second and third day. Newly formed small lymphocytes were found to consist of approximately equal proportions of T cells, B cells, and other “mononuclear” cells which lacked surface markers for either T or B cells. The great majority of large lymphocytes, in contrast, were found to be neither T cells nor B cells and probably belonged to the plasma cell line. In nude mice, production of newly formed lymphocytes during prolonged thoracic duct drainage was found to be very low in comparison with normal CBA mice.     

Announcements

Human peripheral blood monocytes were reproducibly shown to lyse a variety of tumor cells in a 3- to 4-hr ⁵¹Cr release assay. Ficoll-Hypaque-purified mononuclear cells were suspended in medium supplemented with either 10% autologous serum or fetal calf serum (PCS). With either serum, highly purified (97–99%) and viable (>99%) monocyte suspensions were obtained by EDTA-reversible adherence to plastic surfaces which had been precoated with autologous serum. When used as effectors in cytotoxicity assays, the monocytes recovered from mononuclear cells suspended in FCS-supplemented medium exhibited higher cytolytic activity and were therefore used for further studies. Using FCS for both coating the plates and supplementing the suspension medium resulted in monocytes with low cytolytic activity. Tumor cell lysis measured by ⁵¹Cr release was detected within 2 hr of incubation and increased gradually with time. The level of lysis was dependent on the effector/target ratio and the tumor target cell employed. The involvement of natural killer lymphocytes in the observed tumoricidal activity was excluded. Detection of cytotoxic activity in a short-term assay will be very helpful in further studies of the mechanism of tumor cell killing by human monocytes since potential complicating effects of long-term in vitro cultivation will be minimized.     

A TECHNIQUE FOR DETERMINING THE PROPORTION OF THE CLONOGENIC CELLS IN S PHASE IN EMT6 CELL CULTURES AND TUMORS

The survival of cultured EMT6 cells was examined after treatment with hydroxyurea (HU) or high specific activity tritiated thymidine (³H-TdR). The concentrations of the agents, duration of exposure to the agents, and post-exposure treatment of the cultures were found to influence the cell survival; the effects of these factors are reported. Conditions were defined under which the proportions of cells killed by HU and by ³H-TdR were the same and were also the same as the proportion of labeled cells seen on autoradiographs of cultures labeled with small doses of ³H-TdR. Under these conditions, either ³H-TdR or HU could be used to determine the proportion of the clonogenic cells in S phase. Single cell suspensions prepared from solid EMT6 tumors were treated in vitro with HU or ³H-TdR, using the conditions found optimal for each agent with cultured cells. The proportion of the tumor cells killed by treatment with HU in vitro was the same as the proportion killed by HU in vivo and as the proportion labeled by ³H-TdR in vivo, and incubation of tumor cell suspensions with HU in vitro appeared to provide a valid measurement of the proportion of clonogenic tumor cells in S phase. Incubation of tumor cell suspensions with ³H-TdR in vitro proved difficult to perform and the results were relatively unreliable because of severe problems with reutilization of ³H-TdR during the incubation for colony formation.     

Human Y-chromatin : II. DNA replication

The DNA synthesis of the human Y-chromatin in its various morphological configurations was studied by labeling with tritiated thymidine (³H-TdR) and autoradiography. Continuous terminal and pulse labeling studies revealed that while condensed, the Y-body lagged in DNA synthesis behind the rest of the nucleus. The highest rate of incorporation of ³H-TdR by the Y-body occurred when it was dispersed. These observations are consistent with the replication characteristics of the Y chromosome as determined by conventional late labeling studies and strongly suggest that the Y-body uncoils at the time it replicates its DNA.     

A granulocytosis-inducing tumor inhibits the production of B lymphocytes in murine bone marrow

Mice bearing a transplantable CE mammary carcinoma have been shown to have greatly augmented rates of neutrophil production coupled with a marked diminution of bone marrow lymphocytes. The objective of the present study was to test whether the loss of lymphocytes, and especially of B cells, from the bone marrow and spleen of tumor-bearing animals was due to a reduced rate of cell production and if so, at what level this response was regulated. A modified 3H-TdR pulse and chase analysis was used to assess the rates of production of small lymphocytes and B cells (stained for c mu and s mu) at weekly intervals after CE tumor transplantation. 3H-TdR was infused continuously for 24 hr, and radioautographs were prepared of bone marrow and spleen cells 0, 24, and 48 hr after termination of the infusion. Pre-B cells (c mu+s mu-) essentially disappeared from the femoral bone marrow by the end of 1 wk of tumor growth, followed by a great reduction in the number of c mu+s mu+ cells in the marrow and s mu + cells in the spleen. Although pre-B cells appeared in the peripheral marrow (caudal vertebrae, metatarsal bones) and spleen of tumor-bearing mice, these cells could not compensate for the continued decrease in the numbers of more mature B cells. In normal mice, during the 48-hr chase period, newly formed, 3H-TdR-labeled, small lymphocytes and s mu+ cells continued to emerge from the prelabeled precursor compartment at a steady rate, but after 1 wk of tumor growth, the number of small lymphocytes and s mu+ cells emerging from the precursor compartment fell steadily during the 48-hr chase period. During the second and third weeks of tumor growth, a steady state appears to have been reached in B cell production, which was at a level approximately 10 times below that of normal. Because pre-B cells are normally maintained by a less mature precursor population (2), the initial disappearance of c mu+s mu- cells suggests that the CE mammary carcinoma exerts its modulatory influence on primary B cell production by inhibiting or eliminating the cells that eventually feed into the pre-B compartment. The nature of the regulatory factors apparently secreted by the tumor and the more precise identity of the target cells are under investigation.     

Induction of HLA-unrestricted and HLA-class-II-restricted cytotoxic T lymphocytes against MUC-1 from patients with colorectal carcinomas using recombinant MUC-1 vaccinia virus

We recently reported that immunization with a recombinant MUC-1 vaccinia virus (rVMUC-1) protected C57BL/6 mice from challenge with DF3/MUC-1-positive syngeneic tumors. To elucidate whether anti-MUC-1 tumor immunity, especially MUC-1-specific cytotoxic T lymphocytes (CTI), can be induced in cancer patients by rVMUC-1, we stimulated the peripheral blood lymphocytes from patients with DF3/MUC-1⁺ or DF3/MUC-1 colon carcinomas using the autologous monocytes infected with rVMUC-1 (rVAMN). The stimulated T lymphocytes from two patients with DF3/MUC-1-positive colorectal carcinomas (rVPY+T and rVPW+T) demonstrated HLA-unrestricted cytotoxicity against MUC-1, whereas those from the patient with DF3/MUC-1-negative colon carcinoma (rVPA-T) did not. The HLA-unrestricted cytotoxicity was demonstrated by the CD8⁺ T cells possibly recognizing an epitope present on the tandem repeats. Adoptive immunotherapy who performed three times with patient PY, at 4-week intervals. The adoptive transfer of the first stimulated lymphocytes, demonstrating a high level of HLA-unrestricted cytotoxicity against MUC-1, resulted in the significant reduction of the liver metastasis of patient PY. However, HLA-unrestricted cytotoxicity against MUC-1 was extremely reduced at the second transfer and finally eliminated at the third, whereas the CD4⁺ T cells demonstrating HLA-class-II-restricted cytotoxicity against MUC-1 predominantly proliferated at the third adoptive immunotherapy treatment. The liver metastasis and the serum levels of tumor markers (carcinoembryonic antigen CA19-9) demonstrated a rapid and marked increment after the second transfer and especially after the third. These results suggest that the HLA-unrestricted cytotoxic CD8⁺ T cells against MUC-1, induced in patients with DF3/MUC-1⁺ colorectal carcinomas using rVMUC-1, correlate with the antitumor activity in vivo. Received: 22 October 1997 / Accepted: 24 April 1998     

Differential Contribution of Acute and Chronic Inflammation to the Development of Murine Mammary 4T1 Tumors

Based on the notion that inflammation favors tumorigenesis, our experiments comparatively assessed the influence of acute and chronic inflammation on the development of a murine mammary tumor (4T1). In addition, we characterized angiogenic and inflammatory markers in the tumor tissue and systemically. Subcutaneous implantation of polyether-polyurethane sponge discs in Balb/c mice was used to host 4T1 tumor cells (1x10⁶), which were inoculated intraimplant 24h or 10 days post implantation. Flow cytometric analysis of enzyme-digested implants revealed that, after 24 hours, the population of leukocytes was primarily characterized by neutrophils (42.53% +/- 8.45) and monocytes (37.53% +/- 7.48), with some lymphocytes (16.27% +/- 4.0) and a few dendritic cells (1.82% +/- 0.36). At 10 days, macrophages were predominant (37.10% +/- 4.54), followed by lymphocytes (28.1% +/- 4.77), and monocytes (22.33% +/- 3.05), with some dendritic cells (13.60% +/- 0.55) and neutrophils (11.07% +/- 2.27). A mammary tumor grown in a chronic inflammatory environment was 2-fold when compared with one grown in acute inflammation and 5-fold when compared with tumor alone. The levels of pro-angiogenic cytokine (VEGF-Vascular Endothelial Growth Factor) were higher in implant-bearing tumor when 4T1 cells were grown in 10-day old implants as compared to the VEGF levels of the two other groups. Overall, the levels of the inflammatory markers evaluated (NAG -N-acetylglucosaminidase, TNF-α –Tumor Necrosis Factor- α) were higher in both groups of implant-bearing tumors and in serum from those animals when compared with the tumor alone levels. This inflammation-related difference in tumor growth may provide new insights into the contribution of different inflammatory cell populations to cancer progression.     

Monocyte chemoattractant protein-1 (MCP-1) gene transduction: an effective tumor vaccine strategy for non-intracranial tumors

Recently, there has been renewed interest in the concept of tumor vaccines using genetically engineered tumor cells expressing a variety of cytokines to increase their immunogenicity. Human MCP-1 (JE) is a potent chemoattractant and activator of monocytes and T lymphocytes and thus a good candidate gene for a tumor vaccine. We therefore evaluated the efficacy of vaccines consisting of irradiated tumor cells transduced with the murine MCP-1 gene in the syngeneic 9L gliosarcoma brain tumor model. 9L cell lines stably expressing murine MCP-1 (9L-JE) and control cell lines expressing neomycin 3 phosphotransferase (9L-Neo) were generated by infection with a Moloney murine leukemia retroviral vector. Fisher 344 rats were immunized with intradermal injections of 5×10⁵ or 2×10⁶ irradiated (5000 cGy) 9L-JE, 9L-Neo, and wild-type 9L (9L-WT) cells. Two weeks later immunized an non-immunized animals were challenged with varyious doses of intradermal (5×10⁶–5×10⁷) or intracerebral (2×10⁴–5×10⁵) 9L-WT cells. Intradermal tumors grew in all non-immunized animals. No tumors grew in animals immunized with irradiated 9L-JE or 9L-Neo cells and challenged with inocula of fewer than 5×10⁵ 9L-WT cells. With higher inocula up to 10⁷ cells, tumors appeared in all the animals. Tumors in animals immunized with 9L-JE were always smaller than tumors in the other groups. In addition, only the 9L-JE vaccine protected against tumor inocula of 5×10⁷ cells. Thus vaccination with MCP-1-expressing cells was able to protect animals against at least a 100-fold larger number of challenge tumor cells than vaccination with control cells. In contrast to studies with intradermal tumors, immunization with 9L-JE and 9L-Neo produced only minimal protection against intracerebral tumors. There was no significant difference between the 9L-JE and 9L-Neo vaccines in intracerebral challenge. This study suggests that tumor vaccines expressing cytokine genes such as MCP-1 can increase the antitumor response. However, the protective effect of these vaccines appears to be largely limited to intradermal tumors rather than intracerebral tumors.