Cell therapy of a highly invasive human breast carcinoma implanted in immunodeficient (SCID) mice

Although enormous progress has been made in the detection and treatment of localized (nonmetastatic) breast cancer, there has been relatively moderate progress toward the effective treatment of advanced disease. This study investigates the antitumor efficacy of a potent MHC nonrestricted cytotoxic human T cell line (TALL-104) upon transfer into a clinically relevant mouse model of metastatic breast cancer. Fragments from a surgical specimen of a patient with infiltrating ductal carcinoma were implanted s.c. in the flank region of severe combined immunodeficient (SCID) mice. One hundred % of the animals developed a local tumor mass that metastasized to subaxillary and inguinal lymph nodes, bones, lungs, liver, kidneys, ovaries, and brain, very closely mimicking the human disease. Multiple i.p. transfers of gamma-irradiated (nonproliferating) TALL-104 cells into mice bearing low tumor burden (the primary tumor mass weighed only 150 mg) completely arrested local tumor growth and prevented systemic spread into local lymph nodes and distant organs. Remarkably, cell therapy administered in an advanced disease stage (when the tumor weighed 2 g) induced a significant or total regression of established metastasis with no obvious effects on the primary tumor mass. Profound antitumor effects against both local and systemic disease were instead seen in mice that received cell therapy after surgical excision of the primary tumor. The implications of these data in adjuvant breast cancer therapy are discussed.     

Role of CD38 and its ligand in the regulation of MHC-nonrestricted cytotoxic T cells

Human CD38 is a type II transmembrane glycoprotein that regulates lymphocyte adhesion, proliferation, and cytokine production. The mAb Moon-1 recognizes a ligand for CD38 (CD38L) and specifically inhibits CD38-mediated cell adhesion. To analyze the role of CD38 and its ligand in MHC-nonrestricted T cell activation, we examined the effects of Moon-1 and the anti-CD38 mAb IB4 on the effector functions of the IL-2-dependent T cell line TALL-104 (CD3/TCR-alphabeta+, CD8+, CD56+) and of LAK cells (90% CD3+). TALL-104 cells were almost 100% reactive with both mAbs, whereas the reactivity of LAK cells for IB4 and Moon-1 ranged from 10 to 60% among different donors. From 78 to 94% of the cytotoxic CD8+/CD56+ LAK subset was CD38L+. Like mAb OKT3 (anti-CD3), and at variance with IB4, Moon-1 drastically enhanced the cytotoxicity of TALL-104 and CD8+ LAK cells against a resistant tumor target. Granule exocytosis did not appear to play a role in Moon-1-induced cytotoxicity. Moreover, neither IB4 nor Moon-1 induced [Ca2+]i mobilization in LAK and TALL-104 cells. Whereas stimulation of CD3 and CD38 resulted in a dramatic induction of cytokine (granulocyte-macrophage-CSF, IFN-gamma, TNF-alpha, and TNF-beta) release by both TALL-104 and LAK cells, ligation of CD38L was not followed by cytokine production in TALL-104 cells. Thus, cytotoxicity and cytokine release are independently regulated, at least in this system. These data demonstrate that CD38 and its ligand can regulate some T cell functions using signaling pathways distinct from those of CD3.     

Phase I clinical trial with a human major histocompatibility complex nonrestricted cytotoxic T-cell line (TALL-104) in dogs with advanced tumors

The human TALL-104 cell line is endowed with a uniquely potent MHC nonrestricted tumoricidal activity across several species. In view of the potential applicability of TALL-104 cells as an anticancer agent, this study was conducted to evaluate the possible toxicity and efficacy of this new cell therapy in a superior animal model with spontaneous tumors. Nineteen canine cases with advanced, refractory malignancies of various histological types were entered in the study. All dogs had failed all other available treatments and had very limited life expectancy. Cyclosporin A was administered p.o. (10 mg/kg/day) starting from the day before TALL-104 cell administration throughout the treatment to prevent rejection of the xenogeneic effectors. Lethally irradiated (40 Gy) TALL-104 cells (10(8)/kg) were administered systemically following two treatment schedules. In the first schedule, the cells were given every other day for 2 weeks in a row and then once a week for 3 additional weeks; in the second schedule, TALL-104 cells were administered daily for a total of 5 days. None of the 19 cases showed significant clinical or laboratory toxicity; in addition, none of the dogs had to be withdrawn from the study because of immediate adverse reactions to the infusions. The severe side effects usually associated with classical lymphokine-activated killer therapy in association with high doses of interleukin 2, such as "capillary leak syndrome," were absent in this study. Remarkably, TALL-104 therapy induced various degrees of antitumor effects in 7 of the 19 dogs, including 1 complete response (continuing at +13 months), three partial responses (duration of 2 months, 3 months, and continuing at +2 months), and three transient responses. Clinical responses and immunological parameters correlated well in each case. Taken together, these data indicate that systemic administration of lethally irradiated TALL-104 cells in the absence of exogenous interleukin 2 may be regarded as a safe and promising adjuvant type of treatment for advanced cancer patients.     

Preclinical evaluation of 111In-labeled B3 monoclonal antibody: biodistribution and imaging studies in nude mice bearing human epidermoid carcinoma xenografts

Biodistribution and imaging characteristics of monoclonal antibody B3 were evaluated in nude mice bearing A431 human epidermoid carcinoma xenografts. B3 is a murine IgG1k, recently isolated, reacting with a carbohydrate epitope abundantly and uniformly expressed by most carcinomas. B3 was conjugated to a new backbone-substituted derivative of diethylenetriaminepentaacetic acid, 2-(p-isothiocyanato benzyl)-cyclohexyl-diethylenetriaminepentaacetic acid, and labeled with 111In. Tumor-bearing mice were given i.v. injections of approximately 5 microCi of either 111In-B3 or 111In-MOPC-21, an isotype-matched control, and sacrificed in groups of five at 6 h and daily up to 168 h. Imaging was performed at 24, 72, and 144 h. Significant differences were observed in tumor uptake at all time points with peak values at 48 h (25 +/- 5.2% versus 6.3 +/- 0.4% of the injected dose/g tissue) (mean +/- SD) for 111In-B3 and 111In-MOPC-21, respectively (P < 0.001). All tumor to organ ratios increased with time for 111In-B3. In particular, tumor:liver ratios rose from 3.2 +/- 0.6 at 24 h to 6.3 +/- 1.2 at 168 h. Imaging results showed selective and progressive accumulation of 111In-B3 at the tumor site, whereas 111In-MOPC-21 did not show specific localization. In summary, 111In-labeled B3 demonstrated good and specific tumor targeting, which warrants its future clinical evaluation.     

Immunotherapy for medullary thyroid carcinoma by a replication-defective adenovirus transducing murine interleukin-2

We have evaluated the feasibility of gene transduction using replication-defective adenovirus vector as a novel therapy for medullary thyroid carcinoma (MTC), a thyroid C cell neoplasm. Replication-defective adenoviruses were constructed to express murine interleukin-2 (mIL-2) gene and Escherichia coli beta-galactosidase (beta-gal; lacZ) gene under the control of the human cytomegalovirus (CMV) promoter (AdCMVmIL2, AdCMVbeta-gal) by homologous recombination. The efficiency of transduction was evaluated using AdCMVbeta-gal at different conditions. The gene transduction efficiency was dependent on multiplicity of infection, duration of exposure to the virus, and viral concentration. The expression of functional mIL-2 in transduced tumor cells was verified both in vitro and in vivo. Two cell lines (rat MTC and mMTC) secreted large amounts of functional mIL-2 after transduction, as tested in cytotoxic T lymphocyte (CTL) L-2 cells. When AdCMVmIL2-infected mMTC cells were injected s.c. into their host animals, tumors developed in 2 of 10 animals, in contrast to 9 of 10 animals injected with AdCMVbeta-gal-infected mMTC cells and all 10 animals injected with parental mMTC cells. Moreover protected animals developed a long lasting immunity against mMTC tumor cells and their splenocytes, showing cytotoxicity to parental tumor cells, and active natural killer (NK) cell activity. BALB/c-SCID (severe combined immune deficiency) mice were also used to evaluate the function of NK cells in antitumor activities. No tumor developed in SCID mice injected with AdCMVmIL2-infected cells, whereas all animals injected with either AdCMVbeta-gal-infected or parental mMTC cells developed tumors. Our data indicate that IL-2 production by MTC cells leads to rejection in syngeneic animals and suggest that both cytotoxic T cells and NK cells may play an important role. In addition, transduction of adenoviral vectors into tumor cells produces some nonspecific antitumor effects.     

Drug distribution. The forgotten relative in clinical pharmacokinetics

The purpose of this study was to test the three-step targeting of tumors in mice using biotinylated antibody, streptavidin and radiolabeled biotin. Nude mice bearing subcutaneous LS180 human colon cancer xenografts were intravenously administered with 200 microg of the biotinylated anti-Tn monoclonal antibody MLS128, and 2 days later they got intravenous injection of 50 microg of streptavidin. They were intravenously injected 1, 4 or 7 days later with 0.5 microg of 111In-diethylenetriamine pentaacetic acid (DTPA)-biotin. The tumor uptake, determined 2 h later, was 1.4, 0.5 and 0.6% injected dose/gram of tissue (ID/g), respectively, and the blood radioactivity was 1.0, 0.2 and 0.2% ID/g, respectively. When the interval between the streptavidin and radiolabeled biotin injections was prolonged from 1 day to 7 days, the tumor-to-blood ratio 2 h after injection of 111In-labeled biotin increased from 1.5 to 4.0. Clear tumor images were obtained as early as 2 h after injection of radiolabeled biotin. In conclusion, these preliminary data suggested that the three-step method using the streptavidin-biotin system would be applicable in an experimental mouse tumor model and provides images of tumors rapidly and clearly after injection of radiolabeled biotin.     

High cytokine production and effective antitumor activity of a recombinant vaccinia virus encoding murine interleukin 12

We have constructed a recombinant vaccinia virus (recVV), vKT0334 mIL-12, containing the genes encoding the p35 and p40 subunits of murine interleukin-12 (mIL-12). In vitro experiments demonstrated that vKT0334 mIL-12 efficiently infected a variety of murine and human tumor cell lines and produced very high amounts (1.5 micrograms/10(6) cells/24 h) of biologically active mIL-12. Mice injected s.c. with 10(6) MCA 105 sarcoma cells, followed by injection at the same site with saline or a control recVV, vKT033, containing no mIL-12 genes, all developed progressively growing tumor, whereas 60% of animals injected with vKT0334 mIL-12 remained tumor free (P < 0.0005). Furthermore, tumor growth was significantly reduced in the remaining mice treated with vKT0334 mIL-12 that did develop tumor compared with mice treated with vKT033 (P < 0.03) or saline (P < 0.0001). We conclude that recVV expressing high levels of mIL-12 offers an effective in vivo method of cytokine gene delivery and expression in tumors with subsequent antitumor effect.     

Intravital videomicroscopic evidence for regulation of metastasis by the hepatic microvasculature: effects of interleukin-1alpha on metastasis and the location of B16F1 melanoma cell arrest

There have been few reported visual observations of metastatic cancer cell arrest in vivo. To seek evidence that inducible vascular adhesive properties can regulate hepatic metastasis, groups of 9-14 c57bl/6 mice were given 1.5 microg of interleukin-1alpha (IL-1alpha) 4 h before the injection of 3 x 10(5) B16F1 melanoma cells into a mesenteric vein. After 7 days, these mice had an 11-22-fold greater hepatic tumor burden than controls given i.p. saline. In both groups, small metastases were seen in the portal tract region. Twice as many 125I-labeled UdR-labeled B16F1 cells were detected in the livers of IL-1alpha-treated animals 5 min after injection, and 7 times as many were found after 24 h. Intravital videomicroscopy showed marked differences in the arrest pattern of the B16F1 cells between controls and IL-1alpha-treated mice. In controls, arrest occurred at a median distance of 32 microm beyond the sinusoidal inlet, where the median sinusoidal diameter was 16 microm. However, in IL-1alpha-treated mice, arrest occurred in the presinusoidal portal vein branches, which had a median diameter of 34 microm. Maximum observed tumor cell velocities were 2-fold less in the IL-1alpha-treated mice, although there was no significant difference in the flow rate of RBCs. To look for effects on the adhesive properties of the hepatic microvasculature, 5 x 10(4) B16F1 cells were incubated for 15 min on 5-microm sections of liver from control and IL-1alpha-treated mice. Three-fold more cells adhered to sections of liver from IL-1alpha-treated mice. This phenomenon was blocked by GRGDS peptides and by antibodies to E-selectin, ICAM-1, VCAM-1, and the alpha v integrin subunit. We postulate that pretreatment of mice with IL-1alpha alters a number of adhesive interactions between B16F1 cells and the hepatic microvasculature, contributing to the site of arrest and to the subsequent fate of the arrested cells.     

Role of spontaneous and interleukin-2-induced natural killer cell activity in the cytotoxicity and rejection of Fas+ and Fas- tumor cells

In the current study, we investigated whether the naive, poly I:C or interleukin-2 (IL-2)-induced natural killer (NK)/lymphokine-activated killer (LAK) cells use perforin and/or Fas ligand (FasL) to mediated cytotoxicity. We correlated these findings with the ability of mice to reject syngeneic Fas+ and Fas- tumor cells either spontaneously or after IL-2 treatment. The spontaneous NK-cell-mediated cytotoxicity was primarily perforin based, whereas the poly I:C and IL-2-induced NK/LAK activity was both FasL and perforin dependent. L1210 Fas+ tumor targets were more sensitive than L1210 Fas- targets to poly I:C and IL-2-induced cytotoxicity in wild-type, gld/gld, and perforin knockout mice. When L1210 Fas+ and Fas- tumor cells were injected subcutaneously (sc) or intraperitoneally into syngeneic mice, Fas- tumor cells caused mortality earlier than Fas+ tumor cells. Also, approximately 20% of the mice injected sc with L1210 Fas+ tumor cells survived the challenge(>60 days), whereas all mice injected similarly with L1210 Fas- tumor cells died. When immunotherapy using IL-2 (10,000 U, three times/d for a week, followed by once/d for an additional week) was attempted in mice injected sc with tumor cells, IL-2 treatment was very effective against mice bearing L1210 Fas+ (40% survival) but not L1210 Fas- (0% survival) tumors. These data correlated with the finding that the LAK cells from IL-2-injected mice caused increased cytotoxicity against L1210 Fas+ when compared with L1210 Fas- targets. Also, L1210 Fas+ tumor-bearing mice showed increased tumor-specific cytotoxic T lymphocyte (CTL) activity when compared with those bearing L1210 Fas- tumor cells. Together our studies show for the first time that expression of Fas on tumor targets makes them more immunogenic as well as susceptible to CTL- and IL-2-induced LAK activity. The Fas+ tumor cells are also more responsive to immunotherapy with IL-2.     

Interleukin 1 increases thymidine labeling index of normal tissues of mice but not the tumor

PURPOSE: This study was conducted to investigate the action of human recombinant interleukin 1 as a radioprotector for different mouse normal cells other than bone marrow cells. METHODS AND MATERIALS: Semi-continuous injections of tritiated thymidine were administered every 6 h, over 24 h to determine thymidine labeling index. Mice were injected with recombinant human interleukin 1 24 h prior to tritiated thymidine and were compared to control animals that did not receive interleukin 1. Mice were killed 1 h after the last thymidine injection. The 24 h thymidine labeling index for normal tissues and RIF-1 tumor was determined. Labeling indices were also determined 1-14 days after a series of fractionated irradiations with or without pretreatment with a single dose of interleukin 1 administered 24 h prior to the first radiation. RESULTS: The thymidine labeling index of normal tissues was higher following the injection of recombinant human interleukin 1 24 h before radiolabeling. This was found in all normal tissues tested, including the lip and tongue mucosal basal cell layers, crypt cells of the duodenum, alveolar cells of the lung, hepatocytes, and basal skin cells. The thymidine labeling index of RIF-1 fibrosarcoma was not affected by interleukin 1 injection. A single interleukin 1 injection 24 h before the first radiation fraction also increased the thymidine labeling indices of normal tissues after localized fractionated irradiation. The thymidine labeling index of RIF-1 tumor was not increased by interleukin 1 administration except after relatively high radiation doses (20 Gy in five fractions). The ability of interleukin 1 to enhance the thymidine labeling index declined after the first day following the completion of fractionated irradiation. CONCLUSION: Recombinant human interleukin 1 increased the 24 h thymidine labeling index in normal tissues in mice, but not in RIF-1 tumor. Fractionated irradiation could maintain the effect of a single dose of interleukin 1, administered 24 h prior to the first fraction, up to 24 h after the end of radiation.     

P-selectin deficiency attenuates tumor growth and metastasis

Selectins are adhesion receptors that normally recognize certain vascular mucin-type glycoproteins bearing the carbohydrate structure sialyl-Lewisx. The clinical prognosis and metastatic progression of many epithelial carcinomas has been correlated independently with production of tumor mucins and with enhanced expression of sialyl-Lewisx. Metastasis is thought to involve the formation of tumor-platelet-leukocyte emboli and their interactions with the endothelium of distant organs. We provide a link between these observations by showing that P-selectin, which normally binds leukocyte ligands, can promote tumor growth and facilitate the metastatic seeding of a mucin-producing carcinoma. P-selectin-deficient mice showed significantly slower growth of subcutaneously implanted human colon carcinoma cells and generated fewer lung metastases from intravenously injected cells. Three potential pathophysiological mechanisms are demonstrated: first, intravenously injected tumor cells home to the lungs of P-selectin deficient mice at a lower rate; second, P-selectin-deficient mouse platelets fail to adhere to tumor cell-surface mucins; and third, tumor cells lodged in lung vasculature after intravenous injection often are decorated with platelet clumps, and these are markedly diminished in P-selectin-deficient animals.     

Model of implantation of tumor cells simulating recurrence in colonic anastomosis in mice

PURPOSE: Local recurrence after colorectal cancer surgery is usually perianastomotic. An experiment was designed to investigate whether free intraluminal cells can penetrate through a colonic anastomosis and thereby cause local recurrence. METHODS: BALB/c and C57/BL mice underwent ascending colotomy followed by watertight anastomosis. Thereafter, CT-26 murine colon carcinoma cells were injected into the cecal lumen 2 cm proximal to the anastomosis of syngeneic BALB/c mice, whereas B-16 murine melanoma cells were injected in the same fashion into C57/BL mice. Control animals without anastomosis received similar injections. Animals were killed 24 hours, 72 hours, and 30 days after surgery and were checked for tumorigenesis. RESULTS: Results of peritoneal fluid cytology were negative after 24 hours, whereas after 72 hours cancer cells were identified in the peritoneal fluid of 80 percent of mice with colotomy and anastomosis compared with 20 percent of control mice. Thirty days after surgery, 11.1 percent of the control BALB/c mice developed pericecal tumor growth, similar to the overall rate of murine melanoma in C57/BL. In mice with anastomoses, perianastomotic tumor growth was observed in 47.5 percent of BALB/c mice (P < 0.001) and was correlated with the number of injected cells. Tumor growth reached approximately 75 percent tumor take with high cell densities, whereas in C57/BL mice no difference was found between the experimental and control groups. CONCLUSIONS: The findings suggest that free intraluminal cancer cells of colonic origin may penetrate through watertight anastomoses and implant on the anastomotic or peritoneal surface and initiate tumor growth. This anastomotic penetration is cell-mass dependent. The reported experimental model is simple, reproducible, and advantageous for studies of colonic anastomosis.     

Gene structure of rat testicular cell adhesion molecule 1 (TCAM-1), and its physical linkage to genes coding for the growth hormone and BAF60b, a component of SWI/SNF complexes

The aim of this study was to develop as effective as possible autologous tumor vaccine which would be at the same time easy to produce, highly controllable, and without undesired side effects on normal tissue. Therefore, irradiated autologous - syngeneic B-16 tumor cells admixed with a non-specific immunomodulator MVE-2 (a polymer fraction of 1,2-co-polymer of divinyl ether and maleic anhydride) were used for subcutaneous (s.c.). or intraperitoneal (i.p.) prevaccination of experimental mice. Compared to the control mice, a statistically significant delay in tumor development of s.c. tumors was achieved in prevaccinated mice (p<0.05). An even better effect was observed in mice challenged i.p. with viable tumor cells. Using a single prevaccination complete protection was obtained in between 40-85% of the experimental mice. When the survivors from the groups injected once with the tumor vaccine were rechallenged with viable tumor cells (101 day after the first tumor challenge, no additional prevaccination), 15.7% remained free of tumor, while the survivors from the groups injected with the tumor vaccine twice and 101 day later rechallenged with viable tumor cells remained free of tumor in 60% of the cases. Based on these results we can postulate that our vaccine is effective for prevention of tumor development. The achieved protection can be augmented with serial vaccinations and can be maintained for a longer period of time.     

Regression of bladder tumors in mice treated with interleukin 2 gene-modified tumor cells

This study explored the use of interleukin 2 (IL-2) and interferon gamma (IFN-gamma) gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor used is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology, and responds to treatment in a manner similar to its human counterpart. Using retroviral vectors, the human IL-2 and mouse IFN-gamma genes were introduced and expressed in MBT-2 cells. The tumor-forming capacity of the cytokine gene-modified MBT-2 cells was significantly impaired, since no tumors formed in mice injected intradermally with either IL-2- or IFN-gamma-secreting cells, using cell doses far exceeding the minimal tumorigenic dose of parental MBT-2 cells. Furthermore, mice that rejected the IL-2- or IFN-gamma-secreting tumor cells became highly resistant to a subsequent challenge with parental MBT-2 cells, but not to 38C13 cells, a B cell lymphoma of the same genetic background. To approximate the conditions as closely as possible to the conditions prevailing in the cancer patient, inactivated cytokine-secreting cells were used to treat animals bearing tumors established by orthotopic implantation of MBT-2 cells into the bladder wall of the animal. Treatment of mice carrying a significant tumor burden with IL-2-secreting MBT-2 cells had a significant inhibitory effect on tumor progression with extended survival. Moreover, in 60% of the mice the tumor regressed completely and the animals remained alive and free of detectable tumor for the duration of the observation period. Treatment of tumor-bearing animals with IL-2-secreting MBT-2 cells was superior to the use of cisplatin, a chemotherapeutic agent used in the treatment of bladder cancer. The therapeutic effect of IFN-gamma-secreting cells was minimal and treatment with unmodified MBT-2 cells had no effect on tumor growth or survival, showing that the parental MBT-2 cells were nonimmunogenic in this experimental setting. Most importantly, mice that exhibited complete tumor regression after treatment with IL-2-secreting MBT-2 cells became resistant to a subsequent challenge with a highly tumorigenic dose of parental MBT-2 cells, indicating that long-term immunological memory was established in the "cured" mice.     

Vaccination with tumor cells engineered to secrete interleukin 2-immunoglobulin G fusion protein induces tumor rejection

Here we provide proof that the injection of tumor cells engineered to secrete interleukin 2 (IL-2)-IgG chimeric proteins locally induces potent antitumor responses, which are more effective than tumor transfection with IL-2 alone. Murine plasmacytoma cells (J558L) were stably transfected with DNA coding for a human IL-2-IgG1 or a murine IL-2-IgG2b fusion protein and were injected s.c. into syngeneic BALB/c mice. Evaluation of tumor growth and rejection patterns showed that IL-2-IgG secretion by transfected J558L tumor cells induced their rejection in all animals tested, similar to the rejection of J558L cells engineered to secrete IL-2 alone, whereas treatment with parental cells was lethal. However, mice treated with IL-2-IgG-secreting J558L cells (human IL-2-IgG1 and murine IL-2-IgG2b) exhibited a significantly stronger tumor immunity against a later challenge with parental J558L cells than mice treated with IL-2-secreting tumor cells.     

Diurnal uptake of circulating interleukin-1alpha by brain, spinal cord, testis and muscle

The effects, synthesis, and release of cytokines show diurnal patterns. We used recombinant human interleukin-1alpha radioactively labeled with 125I (I-IL) to determine whether its uptake by brain, spinal cord, testis and muscle showed a diurnal rhythm when tested every 4 h in mice. Each tissue showed statistically significant diurnal variation in their uptakes of I-IL ranging from a nearly 10-fold difference for the spinal cord to less than a 2-fold difference for muscle. All nadirs occurred at either 04.00 or 24.00 h and all peaks at 08.00 or 12.00 h. The pharmacokinetics of intravenously injected I-IL did not show any significant variations in blood. Recombinant human tumor necrosis factor-alpha, which does not cross the blood-brain barrier, did not show a diurnal rhythm in its uptake by any of these tissues. This diurnal variation in the rate of uptake of cytokines by tissues could underlie differences in potency when given at different times of the day.     

Keratinocyte growth factor (KGF/FGF-7) has a paracrine role in canine prostate: molecular cloning of mRNA encoding canine KGF

BACKGROUND: Recent studies have demonstrated the usefulness of gene-modified tumor cells for immunotherapy. Using the tumorigenic murine fibrosarcoma, MCA 106, we investigated the effects of localized interferon-gamma (IFNg) secretion on tumorigenicity and on long-term memory. METHODS: The murine IFNg (MuIFNg) gene was introduced into tumor cells. High and low IFNg-secreting clones were isolated. C57BL/6 mice were injected subcutaneously (s.c.) with either parental (P), high or low IFNg-secreting (H- or L-IFNg) cells, and tumor growth was assessed weekly. Spleens were harvested on different days postinjection (p.i.) to assess in vitro cytolytic activity. In parallel, tissues from injection sites were stained with macrophage-, CD4-, and CD8-detecting antibodies. Mice were injected s.c. with H-IFNg MCA106 tumor. After 150 days the animals were rechallenged s.c. with MCA106P in one leg and with irrelevant syngeneic tumor in the other. RESULTS: Both P- and L-IFNg cells had similar growth, whereas the H-IFNg cells never grew. Only splenocytes from the H-IFNg animals showed in vitro CTL activity persisting until day 30 p.i. Histological data revealed a macrophage and CD4+ infiltrate much earlier in the H-IFNg group compared with the P group. Only the irrelevant, syngeneic tumor grew in animals previously injected with H-IFNg cells, whereas both P and irrelevant syngeneic tumors grew in controls. CONCLUSIONS: Transduction of MCA106 cells with the MuIFNg gene diminished in vivo tumorigenicity in proportion to the amount of IFNg secreted. Immunization with H-IFNg cells elicited a host response characterized by macrophages and CD4+ cells. Long-term tumor-specific memory was seen after immunization with H-IFNg cells.     

Retinoic acid and interferon alpha act synergistically as antiangiogenic and antitumor agents against human head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy in which multiple independent lesions develop over time throughout the mucosa of the upper aerodigestive tract. Therefore, the comprehensive treatment of this neoplasm must include a chemopreventive arm to hold premalignant lesions in check, a role well-suited to antiangiogenic agents. Retinoic acid (RA) and interferon alpha (IFN-alpha), drugs with known biological activity against HNSCC when used individually, are also inhibitors of angiogenesis. Here we show that they are remarkably synergistic antiangiogenic agents able to inhibit both the growth and the neovascularization of HNSCC injected into the floor of the mouth of nude mice. The mechanism of action of these drugs as antiangiogenic agents was 2-fold. They decreased the angiogenic activity of the tumor cells, and they caused the endothelial cells to become refractory to inducers of angiogenesis. When tumor cells were treated in vitro with IFN-alpha A/D, there was a dramatic drop in their secretion of interleukin-8, the major angiogenic factor produced by these tumors. When combined with RA, which causes tumor cells to secrete an inhibitor of angiogenesis, there was a synergistic inhibition of both tumor cell growth and secreted angiogenic activity. The combination of RA and IFN-alpha also acted synergistically on endothelial cells by reducing their responsiveness to both interleukin-8 and tumor conditioned media. Doses of each drug could be reduced by two logs without loss of activity. When animals bearing human HNSCC tumor cells were treated systemically with a combination of RA and IFN-alpha A/D at doses that were ineffective when used alone, dramatic decreases in both tumor growth and tumor angiogenesis were seen. These data suggest that the use of antiangiogenic mixtures may be a particularly effective way to design future chemoprevention protocols against HNSCC.     

Divergent effects of TNF alpha in the adoptive immunotherapy of a murine sarcoma

We have previously described an in vitro sensitization (IVS) procedure which enabled the generation of therapeutic T cells from tumor-bearing mice for adoptive immunotherapy. The procedure involved culture of tumor-draining lymph node (TDLN) cells with irradiated tumor in the presence of interleukin-2 (IL-2). The availability of many recombinant cytokines affords an opportunity to examine their effects on the immune response to tumor. In this study, we investigated the effect of tumor necrosis factor-alpha (TNF alpha) on the generation and function of IVS cells utilized in adoptive immunotherapy of the murine MCA 106 sarcoma. TNF alpha administered iv at nontherapeutic doses was found to enhance the antitumor efficacy mediated by IVS cells plus IL-2 in the treatment of pulmonary metastases. In contrast, TNF alpha administration to mice bearing progressive footpad tumors had inhibitory effects on the sensitization of tumor-reactive cells in TDLN since IVS cells generated from these animals displayed a diminished antitumor effect. This effect appeared to be due to a reduced number of tumor-reactive lymphoid cells in the TDLN since TNF alpha added to IVS cultures did not alter the antitumor efficacy of the resultant IVS effector cells. These findings indicate the divergent effects of TNF alpha on the immune response to tumor and adoptive immunotherapy with IVS cells.