Intralesional delivery of dendritic cells engineered to express T-bet promotes protective type 1 immunity and the normalization of the tumor microenvironment.

T-bet (Tbx21), a T-box transcription factor, has been previously identified as a master regulator of type 1 T cell polarization. We have also recently shown that the genetic engineering of human dendritic cells (DCs) to express human T-bet cDNA yields type 1-polarizing APCs in vitro (1). In the present study, murine CD11c(+) DCs were transduced with a recombinant adenovirus encoding full-length murine T-bets (DC.mTbets) and analyzed for their immunomodulatory functions in vitro and in vivo. Within the range of markers analyzed, DC.mTbets exhibited a control DC phenotype and were indistinguishable from control DCs in their ability to promote allogenic T cell proliferation in MLR in vitro. However, DC.mTbets were superior to control DCs in promoting Th1 and Tc1 responses in vitro via a mechanism requiring DC-T cell interaction or the close proximity of these two cell types and that can only partially be explained by the action of DC-elaborated IL-12p70. When injected into day 7 s.c. CMS4 sarcoma lesions growing in syngenic BALB/c mice, DC.mTbets dramatically slowed tumor progression (versus control DCs) and extended overall survival via a mechanism dependent on both CD4(+) and CD8(+) T cells and, to a lesser extent, asialoGM1(+) NK cells. DC.mTbet-based therapy also promoted superior tumor-specific Tc1 responses in the spleens and tumor-draining lymph nodes of treated animals, and within the tumor microenvironment it inhibited the accumulation of CD11b(+)Gr1(+) myeloid-derived suppressor cells and normalized CD31(+) vascular structures. These findings support the potential translational utility of DC.Tbets as a therapeutic modality in the cancer setting.

[Evaluation of the toxicity of tirapazamine plus cisplatin in a mouse tumor model]. / Untersuchungen zur Toxizität von Tirapazamine plus Cisplatin in einem Maus-Tumormodell.

BACKGROUND AND PURPOSE: Tirapazamine (TPZ) is an anticancer drug that is selectively activated by the low oxygen environment in solid tumors. Furthermore, TPZ also enhances the tumor cell-killing effect of cisplatin. So far, detailed information on the toxicity of combined treatment is rare. The authors evaluated the toxicity of TPZ in combination with cisplatin in a mouse tumor model. For this purpose, general toxicity was monitored and all inner organs were examined histologically. MATERIAL AND METHODS: RIF-1 fibrosarcomas of murine origin growing in the right hindfoot dorsum of C3H mice were used. The animals were treated with 10 x 2 Gy irradiation plus six i.p. injections of 4 mg/kg cisplatin (total dose 24 mg/kg) together with varying doses of TPZ (0-28 mg/kg per injection; total dose 0, 43.2, 86.4, 129.6, 151.2, 172.8 mg/kg). Treatment was applied within 2 weeks (Figure 1). Total observation period was up to 35 days. RESULTS: Combined treatment with TPZ led to a dose-dependent, significant decrease in motor activity (Table 1) and body weight and an increase in mortality (Figures 2 and 3, Tables 2 and 3). Histological analyses showed areas of necrosis in the heart, liver and kidney and gastric ulcers (Table 4). Cisplatin alone produced no severe toxicity. Tumor doubling times were TPZ dose-dependent and comparable with data from the literature (Figures 4 and 5, Table 3). CONCLUSION: Unlike most data from the literature a dose-dependent increase in toxicity was seen when adding TPZ to a standard treatment of cisplatin plus irradiation. To the authors' knowledge this is the first study histologically examining in detail the organ toxicity of TPZ in a mouse model. Furthermore, they expand the rare data on long-term toxicity after TPZ plus cisplatin in a fractionated therapy regimen. The results question the usefulness of frequently performed therapeutic studies where only short-term treatment and observation endpoints are used, since essential toxicities are likely to be overlooked.

A new strategy for treatment of malignant tumor: intra-bone marrow-bone marrow transplantation plus CD4- donor lymphocyte infusion.

Donor lymphocyte infusion (DLI) is clinically used for the treatment of malignant tumors. We have found recently that intra-bone marrow-bone marrow transplantation (IBM-BMT) can be used to treat various autoimmune diseases, even when radiation doses are reduced. In addition, recently we have found that IBM-BMT can prevent not only graft failure but also graft-versus-host disease (GvHD). Based on these findings, we attempted to prevent and treat the progression of a tumor (Meth-A cell line: BALB/c-derived fibrosarcoma) by DLI plus IBM-BMT. When the tumors had grown to approximately 10 x 10 mm, the tumor-bearing BALB/c (H-2(d)) mice were irradiated with 5 Gy, and whole spleen cells from C57BL/6J (B6) (H-2(b)) mice (as DLI) were then intravenously injected into the BALB/c mice. Simultaneously, bone marrow cells (BMCs) from B6 mice were injected directly into the bone marrow cavity of the BALB/c mice (IBM-BMT). The tumors decreased in size, but the mice died of GvHD. However, when CD4(+) T-cell-depleted spleen cells were used for DLI, the recipients showed only mild GvHD and survived longer, due to the slow growth of the tumor. In contrast, when CD8(+) T-cell-depleted spleen cells were used for DLI, the recipients showed more severe GvHD than those injected with whole spleen cells. These results suggest that IBM-BMT plus DLI (the depletion or reduction of a certain cell population like CD4(+) T cells) could be helpful to suppress both GvHD and tumor growth.

CTLA-4 blockade enhances the therapeutic effect of an attenuated poxvirus vaccine targeting p53 in an established murine tumor model.

p53 is overexpressed by half of all cancers, and is an attractive target for a vaccine approach to immunotherapy. p53 overexpression is frequently the result of point mutations, which leaves the majority of the protein in its wild-type form. Therefore, the majority of p53 sequence is wild type, making it a self-protein for which tolerance plays a role in limiting immune responses. To overcome tolerance to p53, we have expressed wild-type murine p53 in the nonpathogenic attenuated poxvirus, modified vaccinia virus Ankara (recombinant modified vaccinia virus Ankara expressing wild-type murine p53 (rMVAp53)). Mice immunized with rMVAp53 vaccine developed vigorous p53-specific CTL responses. rMVAp53 vaccine was evaluated for its ability to inhibit the outgrowth of the syngeneic murine sarcoma Meth A, which overexpresses mutant p53. Mice were inoculated with a lethal dose (5 x 10(5) cells injected s.c.) of Meth A tumor cells and vaccinated by i.p. injection 3 days later with 5 x 10(7) PFU of rMVAp53. The majority of mice remained tumor free and resistant to rechallenge with Meth A tumor cells. We wished to determine whether rMVAp53 immunization could effect the rejection of an established, palpable Meth A tumor. In subsequent experiments, mice were injected with 10(6) Meth A tumor cells, and treated 6 days later with anti-CTLA-4 Ab (9H10) and rMVAp53. The majority of treated mice had complete tumor regression along with lasting tumor immunity. In vivo Ab depletion confirmed that the antitumor effect was primarily CD8 and to a lesser extent CD4 dependent. These experiments demonstrate the potential of a novel cell-free vaccine targeting p53 in malignancy.

Esters of chlorambucil with 2-substituted 1,4-dihydroxy-9,10-anthraquinones as multifunctional anticancer agents.

Novel twelve esters of chlorambucil with 2-(1-hydroxyalkyl)-1,4-dihydroxy-9,10-anthraquinone were synthesized and tested for their antitumor activity in mice bearing S-180 ascitic cells as well as cytotoxic activity against L1210 cells. Eight of them were highly cytotoxic on L1210 cells (ED(50), <6 microg mL(-1)) and derivatives 1 and 12 (T/C, 200 and 205%) appeared more active in vivo than chlorambucil (T/C, 168%).

Mouse SaI sarcoma tumor model.

SaI is an A/J-derived (H-2K(k)D(d)) transplantable mouse fibrosarcoma that produces a solid tumor when inoculated subcutaneously, intradermally, or intramuscularly, and an ascites tumor when inoculated intraperitoneally into syngeneic mice. This unit describes the establishment of primary solid SaI/N tumor, the establishment of SaI ascites tumor, and the removal for sampling of in vivo-passaged SaI ascites tumor cells in mice.

Effect of estrogen and antiestrogen therapy in rats bearing mesenchymal tumors.

We applied both hormonal and antiestrogen treatment in female Wistar rats to analyze the estrogen dependence of the growth of sarcomas induced with 9,10-dimethyl-1,2-benzanthracene. Animals bearing tumors of 10 mm in diameter were divided at random into five groups and submitted to different treatments during 24 weeks. The treatment with ovariectomy and tamoxifen in tumor-bearing animals resulted in tumor growth suppression and prolonged survival by a protection against the lethal tumor. On the other hand, the estrogen treatment exerted an adverse effect showing a faster growth of the tumors and a great decrease in survival. In summary, the antiestrogen treatment can have an antitumor effect in mesenchymal tumors, possibly by modifying the immunological status of the host.

Lack of carcinogenesis by the baked mushroom Agaricus bisporus in mice: different feeding regimen [corrected].

Agaricus bisporus, the cultivated mushroom of the western hemisphere, was baked at 220-230 degrees C for 10 minutes and subsequently fed to mice for 12 hours each day, five days each week throughout their life. After each feeding cycle, the animals received a well-balanced semisynthetic diet for 12 hours each day for five days plus the remaining two full days each week. The estimated average daily mushroom consumption per animal was 4.8 g for a female and 4.2 g for a male. Randomly bred Swiss mice, six weeks old at the start of the experiment, were used. In the baked mushroom-fed group, the incidences of tumors in the lungs, blood vessels, cecum, and colon increased when compared to the untreated controls. These increases were not, however, statistically significant. In another previous experiment, both the raw and the baked mushrooms, when used in different feeding regimens, induced statistically significant incidences of cancers in several organs of the mice. It is possible that the negative finding in the current study was due to insufficient mushroom consumption.

Role of endogenous interferon gamma in murine tumor growth and tumor necrosis factor alpha antitumor efficacy.

BACKGROUND: The anticancer role of tumor necrosis factor-alpha (TNF-alpha) has been limited by toxicity. These experiments evaluate blocking endogenous interferon-gamma (IFN-gamma) activity to abrogate TNF-alpha toxicity. METHODS: C57B1/6 mice bearing MCA 105 tumor were treated with TNF-alpha and anti-IFN-gamma antibody (Ab) to evaluate the effect on the acute lethality of TNF-alpha and their efficacy as evaluated by tumor growth rate, tumor histology, and survival. RESULTS: Anti-IFN-gamma Ab decreased TNF-alpha lethality. Anti-IFN-gamma Ab alone increased tumor growth significantly more than did nonimmune IgG (p2 < 0.0001). Tumor-bearing mice that received nonimmune IgG and TNF-alpha had slower tumor growth (p2 < 0.02) and a trend toward improved survival (p = 0.07) compared with saline-treated controls. Anti-IFN-gamma Ab abrogated the antitumor effect of TNF-alpha, prevented acute tumor necrosis histologically, and resulted in tumor growth rate and host survival similar to that of controls. The findings in mice that received anti-IFN-gamma Ab and high-dose TNF-alpha were comparable with those in mice that received a lower, equitoxic dose of TNF-alpha alone. CONCLUSIONS: Blocking endogenous IFN-gamma accelerates tumor growth in this model and partially abrogates the toxic and antitumor activity of exogenous TNF-alpha equally. This suggests that blocking endogenous IFN-gamma activity is not a useful strategy for limiting TNF-alpha treatment toxicity.

Influence of an anti-angiogenic treatment on 9L gliosarcoma: oxygenation and response to cytotoxic therapy.

Tissue oxygen tensions were measured in subcutaneously growing rat 9L gliosarcoma under normal air and carbogen breathing conditions prior to and after i.v. administration of a perflubron emulsion. When these animals were treated with the anti-angiogenic agents TNP-470 and minocycline for 5 days prior to oxygen measurement, tumor hypoxia was decreased compared with untreated tumors. Hypoxia, defined as the percent of pO2 readings < or = 5 mm Hg, was decreased from 71% in untreated air-breathing controls to 34% in animals treated with the anti-angiogenic agents, the perflubron emulsion and carbogen breathing. These effects were manifest in the increased response of the tumor to single-dose (10, 20 and 30 Gy) radiation therapy. Twenty-four hours after treatment with BCNU oxygenation of the tumors was not altered; however, 24 hr after administration of adriamycin oxygenation of the tumors was increased such that hypoxia in adriamycin-treated tumors in animals receiving the perflubron emulsion and carbogen was reduced to 21%. Tumor growth delay in the s.c. tumors was increased by the addition of treatment with the anti-angiogenic agents from day 4 through day 18 post-tumor cell implantation along with BCNU or adriamycin on days 7-11. Administration of the perflubron emulsion and carbogen breathing resulted in increased tumor growth delay with the chemotherapeutic agents alone and in combination with the anti-angiogenic agents. Life span in animals bearing intracranially implanted 9L gliosarcoma progressively increased with administration of the anti-angiogenic agents and then the anti-angiogenic agents and perflubron emulsion/carbogen compared to treatment with BCNU or adriamycin.

Bone sarcoma characteristics and distribution in beagles fed strontium-90.

A total of 66 primary bone sarcomas were diagnosed in 47 beagles; 43 of these dogs were part of the 403 beagles fed 90Sr and 4 were part of the 162 controls. Multiple primary bone sarcomas were found in 15 of the 47 beagles (32%). The incidence of multiple primary bone sarcoma was restricted to the two highest dose groups, except for a single control dog which developed two bone sarcomas. A threshold-like radiation dose response was observed; no sarcomas were observed in the lowest three dose groups, but the number of primary bone sarcomas increased rapidly in the higher dose groups. Of the 66 primary sarcomas, 49 were osteosarcomas (74%). As the dose increased, the proportion of osteosarcomas increased sharply, 4/10 (40%), 26/29 (90%), and 16/18 (89%), in the three highest dose groups. Thirteen of the bone sarcomas of other types occurred in males, and 4 in females, whereas 21 osteosarcomas occurred in males, and 28 in females. The ratio of bone sarcomas of the appendicular skeleton to those in the axial skeleton was 40:26, with osteosarcomas occurring more often in the appendicular than the axial skeleton (32:17), whereas nonosteogenic tumors showed no predilection (8:9). A statistical study of the distribution of bone sarcomas among 16 separate bone groups showed a correlation only with the distribution of cancellous bone volume-to-surface ratio and not with either skeletal mass distribution or dose distribution. The highest occurrence of sarcomas was in the humeri, femora, and mandible, and no occurrence in the coccygeal vertebrae, paws, or sternum. It is postulated that the distribution of bone sarcomas reflects a critical combination of the osteosarcoma precursor cell population, their cell division rate, and the radiation dose absorbed by these cells.

[Incidence of the development of 90Sr-induced osteosarcomas depending on the age of the animals]. / Chastota razvitiia osteosarkom, indutsirovannykh 90Sr, v zavisimosti ot vozrasta zhivotnykh.

In experiments with albino male rats, a study was made of the influence of age on the incidence of osteosarcomas induced by 90Sr. It was shown that the incidence of tumors decreases exponentially with age.

Cholera toxin pretreatment protects against tumor necrosis factor lethality without compromising tumor response to therapy.

Antitumor therapy with tumor necrosis factor is limited by systemic toxic effects. We studied whether cholera toxin, a bacterial exotoxin that adenosine diphosphate-ribosylates the alpha-subunit of Gs proteins, could separate the lethal from the antitumor effects of tumor necrosis factor. A single dose of intravenous cholera toxin protected non-tumor-bearing mice from a lethal dose of Escherichia coli endotoxin administered 6 or 24 hours later. On the basis of these results, tumor-bearing mice were randomized to receive either cholera toxin or saline, followed 6 hours later by either human tumor necrosis factor (400 micrograms/kg) or saline. Tumor-bearing mice pretreated with cholera toxin had (1) reduced treatment-related mortality (0/11 vs 5/11 for saline controls) and (2) tumor regression similar to that of controls. In a separate experiment in tumor-bearing mice, intravenous human tumor necrosis factor treatment induced an increase in serum levels of murine tumor necrosis factor to a peak of 500 pg/mL at 1 hour in saline-pretreated controls, while a similar increase could not be detected in those mice pretreated with cholera toxin. These results suggest that pretreatment with cholera toxin can reduce the endogenous tumor necrosis factor response to administered tumor necrosis factor and separate the lethal from the antitumor effects. Cholera toxin may prove to be a useful tool for investigating the mechanisms underlying the varied effects of tumor necrosis factor.

Effective regional therapy of experimental cancer with paralesional administration of tumour necrosis factor-alpha + interferon-gamma.

Systemically administered tumour necrosis factor (TNF) has anti-tumour effects in animal tumour models but its clinical application is limited by severe toxicity. Interferon-gamma(IFN-gamma) has been shown to augment the anti-tumour effect of TNF. We evaluated the effect of paralesional (p.I.) injections of TNF plus IFN-gamma in a murine tumour model and compared the toxicity and anti-tumour effect with that seen with systemic administration. C57BL6 mice with 10-day subcutaneous MCA sarcomas were treated with daily p.I. injections of recombinant huTNF +/- IFN-gamma for 5 days. Optimal mean survival and 30-day cure rate was seen with doses of 5 micrograms TNF-alpha + 5000 U IFN-gamma (P < 0.05 vs. control or IFN-gamma alone). Tumour response after a single i.v. injection of 0-15 micrograms TNF + 5000 U IFN-gamma was then compared with five daily p.I. injections of the same dose of TNF-alpha and IFN-gamma. All animals with p.I. injections of > 5 micrograms TNF had initial complete necrosis of tumour with a variable degree of surrounding tissue necrosis, with rapid regrowth of tumour seen in some animals. Although treatment-related mortality was similar between i.v. and p.I. therapy, there was a higher percentage of animals cured with p.I. injections with overall cure rates in treated animals at 30 days of 17% vs. 72% (i.v. vs. p.I., P < 0.01) and 13% vs. 67% (P < 0.04) in a repeat study. 2+ clinical applications.

Long-term effects of flavone acetic acid on the growth of a rat tumour.

A rat tumour (MC7 sarcoma), growing subcutaneously, has been shown to be sensitive to a single application of flavone acetic acid. Thirteen rats were still alive after 50 days and 8 of these were tumour free, as compared with control rats which survived for 15.7 +/- 2.53 days. The 8 tumour free animals were rechallenged with MC7 sarcoma 40 weeks later, without further FAA treatment. The tumour grew initially but in all cases the animals became tumour free within 24 days. After a further 30 days they were rechallenged with D23 hepatoma which grew as effectively as in the controls.

The role of bone fracture at the site of carcinogen exposure on nickel carcinogenesis in the soft tissue.

The influence upon nickel subsulfide (alpha-Ni3S2; Ni-SS)-induced carcinogenesis in the soft tissue of bone fracture at the site of Ni-SS exposure was studied using female Fischer rats. During the one year of the experiment, the group subjected to bone fracture exhibited the shortest tumor induction time and survival time, and a significantly higher metastatic rate. The present study may suggest a model for the metastasis of human soft tissue tumors.

[Effects of organic fluorosilicone compounds on the development of tumors in mice]. / Vliianie ftorkremniiorganicheskikh soedinenii na razvitie nekotorykh opukholei u myshei.

The influence of new synthesized fluoro-silicium-organic complexes on the virus-induced Rauscher leukosis and cell-transferred MX-11 mouse sarcoma was studied. We also studied the cytotoxic effects of these complexes in vitro in the human CaOv cells. Two complexes from seven studied were cytotoxic for CaOv cells. Five complexes from seven studied diminished the mortality of animals with MX-11 tumors on the 27-th day of observation, but the total life duration of the animals in the experimental group was the same as in controls. One complex from seven studied increased the life duration of mice with MX-11 tumors. No effects were noted in relation to mice virus-induced Rauscher leukosis.

[The inhibitory effect of ascorbic acid on the estrogen-stimulated promotion of uterine sarcoma development in mice]. / Tormoziashchii éffekt askorbinovoi kisloty na stimulirovannuiu éstrogenom promotsiiu sarkomogeneza matki u myshei.

The administration of estradiol propionate after the discontinuation of 1,2-dimethylhydrazine (DMH) treatment increased the incidence of uterine sarcoma in CBA mice from 32.5 (DMH alone) to 62.5%. The addition of the ascorbic acid (0.3% solution in drinking water) to estradiol propionate was followed by a decrease in the tumor incidence to 35%, i. e. the acid levelled the promoting effect of estradiol propionate almost completely. Sodium ascorbate did not share that effect. Mechanisms underlying the antiestrogenic effect of the ascorbic acid are discussed.

Effect of combined intrapleural administration of Lactobacillus casei (LC9018) and adriamycin on experimental malignant pleurisy in mice.

The combined effect of Lactobacillus casei YIT9018 (LC9018) and adriamycin (ADR) on malignant pleurisy was investigated using an experimental model in BALB/c mice in which Meth A fibrosarcoma cells were intrapleurally implanted. The control mice died from dyspnea due to pleural effusion, before significant growth of tumor nodules could be achieved in the thoracic cavity. Intrapleural (ipl) administration of LC9018 (20-200 micrograms/head) on days 1 and 5 reduced the effusion volume and induced pleural adhesions in a dose-related manner. A statistically significant and reproducible prolongation of survival was observed at a dose of LC9018 200 micrograms/head: increase of lifespan (ILS) values of 15-39% were obtained. An ipl administration of ADR (2-4 mg/kg) on day 1 was also effective in prolonging survival without severe toxicity (ILS values of 100-122%). The combined use of ADR and LC9018 induced a high incidence of pleural adhesions, a delay in effusion accumulation, and an additive prolongation of lifespan (ILS values of 133-178%), compared with ADR monotherapy. In the combination therapy group, a marked and continuous ipl exudation of neutrophils, macrophages, and lymphocytes was observed with a significant decrease in pleural tumor cells. These findings suggest that ipl administration of LC9018 enhances the effect of ADR, probably through both host-mediated tumoricidal activity and sclerosing effects on the pleura.

The cause of death in non-metastasizing sarcoma-bearing mice. A study with relevance for tumor treatment experiments in mice.

Adult sarcoma-bearing mice were used to demonstrate whether hypoglycemia was the immediate cause of death in experimental animals with rapidly growing tumors without metastases. This kind of tumor model is representative of the majority of animal models used in experimental cancer research. Tumor-bearing animals died with severe hypoglycemia under all experimental conditions, while pair-killed controls were normoglycemic. Anorexia prevented tumor-bearing animals from attenuating the hypoglycemia by drinking glucose-containing water while completely starved control animals survived more than 14 days with glucose-containing water as the only energy source. Adrenalectomy shortened survival in tumor-bearing animals, but survival of adrenalectomized tumor-bearing animals could be normalized by daily injections of pharmacologic doses of hydrocortisone (25 mg/25 g body wt/day) but not by physiologic replacement (20 micrograms/25 g body wt/day). Injections of pharmacologic doses of hydrocortisone did not influence on survival or body composition in tumor-bearing animals with intact adrenals. Glucagon was without effect on either survival, tumor growth or body composition. Based on the results in this study and in our previous reports we conclude that hypoglycemia is the cause of death in the majority of murine tumor models. This hypoglycemic theory is important, since any treatment modality in animal experiments that influences glucose metabolism in the host may indirectly change tumor growth and may thus be misinterpreted as a direct tumor effect.