A serum-mediated mechanism for concomitant resistance shared by immunogenic and non-immunogenic murine tumours.

Resistance of tumour-bearing mice to a second tumour challenge, that is concomitant resistance, was evaluated in euthymic and nude mice using nine tumours with widely different degrees of immunogenicity. Two temporally separate peaks of concomitant resistance were detected during tumour development. The first one was exhibited only by small immunogenic tumours; it was tumour specific and mediated by classical immunological T-cell-dependent mechanisms. The second peak was shared by both immunogenic and non-immunogenic large tumours; it was non-specific, thymus independent and correlated with the activity of a serum factor (neither antibody nor complement) that inhibited the in vitro proliferation of tumour cells. This factor was eluted from a Sephadex G-15 column at fractions corresponding to a molecular weight of approximately 1000 Da and it was recovered from a high-performance liquid chromatography column in one peak presenting maximum absorption at 215 and 266 nm. The data presented in this paper suggest for the first time, to our knowledge, that in spite of the differences between immunogenic and non-immunogenic tumours, a common serum-mediated mechanism seems to underlie the concomitant resistance induced by both types of tumours at late stages of tumour development.     

Humoral mediated macrophage response during tumour growth.

Reticuloendothelial (RE) phagocytic and circulating plasma opsonic activity was evaluated in rats transplanted with the Walker 256 carcinoma tumour in an attempt to evaluate the role of opsonic protein in governing the functional state of the macrophage system. Animals transplanted intramuscularly with 2 X 10(4) viable tumour cells manifested 2 peaks of RE stimulation at 6 and 14 days post-transplantation with a subsequent decline in the phagocytic activity over the 14-30 day period. Increased phagocytic activity as determined by colloid clearance was primarily a reflection of hepatic Küpffer cell hyperphagocytosis while the decline in phagocytic activity was related to a decrease in Küpffer cell function. The initial peak of RE stimulation was associated with an elevation in the blood opsonin level and no significant enlargement of the liver and spleen. In contrast, the second peak of RE stimulation at 14 days was associated with both an elevation in opsonin levels and an associated hepatic and splenic enlargement. The decline in phagocytic activity over the 14-30 day interval was associated with a progressive decline in the plasma opsonic activity, a return of the spleen to its normal size in relationship to the body weight, and a persistent hepatomegaly. These findings suggest that the alterations in macrophage function during tumour growth may be mediated in part by changes in the opsonic or phagocytosis promoting capacity of plasma. Since opsonic protein contributes to the discriminatory capacity of macrophages, it is suggested that changes in the blood opsonin level may condition the anti-tumour capacity of the macrophage system with respect to host defence aginst malignant disease.     

Inhibitation of pro-inflammatory cytokine gene expression and papilloma growth during murine multistage carcinogenesis by pentoxifylline

Topical application of 12-O-tetradecanoylphorbol-13-acetate(TPA) to the dorsal epidermis of Sencar mice induces synthesisof pro-inflammatory cytokines, including interleukin-1     

Modification of immunogenic tumor growth by adrenalectomy in a syngeneic murine system

The immunosuppressive action of adrenal glucocorticosteroids is well-known, and depressed cell-mediated immunity and adrenal cortical hyperplasia have been described in tumor-bearing animals. This study was designed to evaluate the effect of removing the source of lympholytic steroids by adrenalectomy upon tumor growth rate, thymus weight, and thymocyte incorporation of iodine 125 (125I) deoxyuridine into DNA. Newly derived methylcholanthrene-induced immunogenic fibrosarcomas were used in male syngeneic mice. Log dosages of 10(4), 10(5), and 10(6) viable tumor cells as single cell suspension were injected subcutaneously into the popliteal space of adrenalectomized and control mice. Tumor size was followed serially with caliper measurements, and the animals were killed 4 weeks after inoculation. Adrenalectomized mice inoculated with 10(4) cells had smaller tumors (P less than 0.02), heavier thymi (P less than 0.01), and more thymic DNA synthesis (P less than 0.05) than their tumor-bearing controls. No differences were seen between populations receiving 10(5) or 10(6) tumor cell inoculations. A second experiment was carried out in which intact controls, adrenalectomized animals, and sham adrenalectomy animals were inoculated with 10(4) tumor cells and killed 28 days later. Tumor growth rate and volume were significantly decreased for the adrenalectomized mice, which had higher thymus weights and DNA synthesis. These findings suggest that pretreatment adrenalectomy slows the growth of antigenic tumor cells and prevents thymic involution after tumor growth in a syngeneic murine system.     

Further studies of the relationship between lymphatic dissemination and lymphnodal metastasis in non-immunogenic murine tumours.

In all 6 different murine tumours of spontaneous origin, a high proportion (22-95%) of the regional lympgh nodes draining small intradermal tumours gave rise to tumours after their isogeneic transplantation as whole nodes. In separate experiments with 4 of these tumours, equivalent tumour-bearing mice had their tumours surgically excised and were observed for the development of regional nodal corresponding frequency of tumour formation by transplanted nodes. After high-dose radiotherapy of intradermal carcinomas, there was a progressive fall in the incidence of positive regional node transplants from 48 to 96 h after irradiation. It is concluded that continual lymphatic dissemination of viable cancer cells is characteristic of malignant tumours, but that there is a relatively small chance of such cells giving rise to nodal metastatic growth. Related studies showed that the ability of a small number of cancer cells to give rise to tumours was very much greater if they were incorporated in a lymph node at transplantation than if they were transplanted directly as a suspension.     

Lysis and growth stimulation of a murine melanoma determined by density of macrophage populations

Activated macrophages were found to induce opposite effects on the growth of the F10 variant of B16 melanoma in both in vivo and in vitro systems. Low macrophage numbers had lytic effects and high numbers stimulated the growth of the melanoma. In fact, the number of macrophages per area (namely, their density) was found to determine whether lysis or growth stimulation would occur. Low density macrophages were found to be structurally and functionally different from high density macrophages. Macrophages in sparse cultures were round, separate, and lysed tumor cells, while crowded macrophages appeared spread, with pseudopodia, and caused enhancement of tumor growth.     

Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors

We have created a novel cellular vehicle for gene therapy of malignant gliomas by transfection of murine bone marrow stroma cells (MSCs) with a cDNA encoding epidermal growth factor receptor (EGFR). These cells (EGFR-MSCs) demonstrate enhanced migratory responses toward glioma-conditioned media in comparison to primary MSCs in vitro. Enhanced migration of EGFR-MSC was at least partially dependent on EGF-EGFR, PI3-, MAP kinase kinase, and MAP kinases, protein kinase C, and actin polymerization. Unlike primary MSCs, EGFR-MSCs were resistant to FasL-mediated cytotoxicity and were capable of stimulating allogeneic mixed lymphocyte reaction, suggesting EGFR-MSCs possess suitable characteristics as vehicles for brain tumor immuno-gene therapy. Following injection at various sites, including the contralateral hemisphere in the brain of syngeneic mice, EGFR-MSCs were able to migrate toward GL261 gliomas or B16 melanoma in vivo. Finally, intratumoral injection with EGFR-MSC adenovirally engineered to secrete interferon-alpha to intracranial GL261 resulted in significantly prolonged survival in comparison to controls. These data indicate that EGFR-MSCs may serve as attractive vehicles for infiltrating brain malignancies such as malignant gliomas.     

Captopril, an angiotensin-converting enzyme inhibitor, promotes growth of immunogenic tumors in mice.

PURPOSE: Antitumor potential of angiotensin-converting enzyme inhibitors has been shown in different preclinical settings, which always involved immunocompromised organisms or nonimmunogenic tumor models. In our study, we wanted to evaluate the effect of captopril on growth of immunogenic tumors in immunocompetent animals. EXPERIMENTAL DESIGN: We used different murine tumor models to evaluate the effect of captopril on tumor take and survival of tumor-bearing immunocompetent and immunocompromised mice. We used an orthotopic renal cell cancer model and highly immunogenic tumor model, which were based on kidney subcapsular injection of RenCa cells or s.c. injection of MethA cells, respectively. To show the influence of captopril on antigen-specific immune responses, we have used two model antigens (green fluorescent protein and beta-galactosidase). RESULTS: Captopril decreased survival of RenCa-bearing, immunocompetent mice in a dose-dependent manner and in adjuvant setting. In nephrectomized mice, captopril shortened their survival. Captopril promoted formation of immunogenic MethA sarcoma tumors but had no effect on nonimmunogenic melanoma cells (B78-H1). Treatment of immunocompromised mice bearing MethA tumors or RenCa kidney tumors with captopril did not affect tumor formation nor survival, respectively. Captopril-treated mice immunized with AdLacZ or AdGFP vectors did not generate or generated decreased numbers of antigen-specific CD8+ T cells, respectively. However, they showed B-cell responses represented by infiltration of MethA tumors with activated B cells and dramatically increased serum level of beta-galactosidase-specific antibodies. CONCLUSIONS: Our results show a novel role of captopril in tumor biology and the tumor-promoting properties of captopril seem to be associated with its immunomodulatory potential.     

Effects of hyperoxia on growth characteristics of metastatic murine tumors in the lung

Suspensions of an oxygen-sensitive (MT-7) and of an oxygen-insensitive(M109) tumor cell line were injected i.v. into BALB/c mice. Exposure to 100% O2 after injection of the cells did not modify the initial arrest of either cell line in the lung. Exposure of animals given injections of MT-7 cells for 60 h to 100% oxygen decreased the number of lung colonies formed even when onset of oxygen exposure was delayed up to 10 days after injection of the cell suspension. Cell cycle time and growth fraction in lung colonies growing in vivo were estimated from an analysis of the percentage of mitoses labeled. In lung colonies formed by MT-7 cells, hyperoxia produced a mitotic delay and a 30 to 40% reduction in the growth fraction. In M109-derived colonies, oxygen did not change cell cycle times or reduce growth fraction. In earlier experiments done in vitro and reported by others it had been found that, in tumor cell lines other than the ones used in the present study, a prolongation of the early prophase was the most oxygen-sensitive event. The present data show that in vivo oxygen inhibits lung colony formation in MT-7 cells by a similar mechanism.     

Cellular immunity to murine sarcoma virus-induced tumors as measured by macrophage migration inhibition assays.

C57BL/6N mice immunized with regressor murine sarcoma virus (MuSV) were studied at different times after inoculation for their cellular immune responses as measured by migration inhibition assays. We used both the direct capillary and indirect agarose dropiet methods and, as the source of antigens, viable tumor cells and 3 M KCl-solubilized extracts. Migration inhibitory factor (MIF) production coulctivity becoming undetectable after 21 days. However, the level of activity and the kinetics of production of this lymphokine were strongly influenced by the source of the antigen and the form in which it was presented to the immune spleen cells (ISC). Using RBL-5 ct 9-10 days, a decrease to low levels at 14 days, and a second peak of activity between 17 and 21 days. However, uith MBL-2 cells or with 3 M KCl-solubilized antigen from fresh RBL-5 ascites cells, MIF production was observed as early as 9-10 days after tumor induction, peaked at 14 days, and decreased substantially by 21 days. T-cells appeared to be required for migration inhibition reactivity, since ISC depleted of T-lymphocytes by treatment with antibody plus complement were unable to produce MIF after antigen stimulation. The results obtained from specificity studies on the response of ISC in migration inhibition to 11 different tumor lines agreed with the results previously obtained from cytotoxicity studies. With the use of RBL-5 cells as the antigen, there appeared to be an inverse relationship between the development of specific cytotoxic effector cells in 51Cr-release assay and the development of specific effector cells needed for MIF production. However, after removal of adherent cells, the level of cytotoxicity observed correlated with MIF release by immune lymphocytes.     

Divergent effects of macrophage toxins on growth of primary tumors and lung metastases in mice

The effects of silica and carrageenan on primary tumor growth and metastases were evaluated in c57bl/6 and BALB/c mice transplanted with the poorly immunogenic Lewis lung (3ll) carcinoma, mFS6 sarcoma and Madison 109 carcinoma spontaneously metastasizing to the lungs. Silica and carrageenan significantly enhanced lung metastases and decreased primary tumor weight in all three experimental models. A similar augmentation of lung secondaries was found after i.v. inoculation of 3LL tumor cells. The effects of carrageenan on primary 3LL tumor growth and metastases were observed also in thymus-deprived animals. The effect of macrophage toxins was studied also in C57BL/6 mice transplanted with the M5076/73A ovarian carcinoma. This tumor spontaneously metastasizes to various abdominal organs, but not to the lung. After treatment with carrageenan, lung metastases were observed, but no effect on secondaries at other anatomical sites or on the primary tumor was detectable. It is suggested that host defense mechanisms impaired by silica and carrageenan may have divergent effects in the regulation of growth of some primary tumors and spontaneous lung metastases.     

Distribution of cardiac output during development of two metastasizing murine tumors

The study of cardiac output (CO) distribution to tumors and metastases is of interest for a better understanding of tumor biology and for pharmacological approaches. A radioactive microsphere method was developed to assess CO distribution in C57BL/6J mice bearing syngeneic 3LL or BALB/c mice with JWS. At the initial stages of cancer growth, the CO relative fractions per g of tissue (%CO/g) to 3LL and JWS were similar to those in surrounding tissues. In both tumors a positive, significant correlation was found between tumor weight and tumor CO fraction, but %CO/g was lower in 3LL 2 and 3 weeks after transplantation, whereas it did not change in JWS. Indeed, much larger necrotic areas developed in 3LL than in JWS. The %CO/g to the lungs increased in both models when metastases were not yet visible; subsequently, the appearance of lung nodules was accompanied by a decrease of %CO/g in JWS and a further increase in 3LL. This corresponded to the much higher ratio of metastatic to intact lung tissue in JWS than in 3LL. In fact, isolated lung metastases had a significantly lower blood supply than the surrounding tissue. This might be due to a different vascularization pattern and/or smaller amounts of vasodilator substances being produced by metastatic nodules; the latter is suggested by lower generation of prostacyclin activity in isolated lung metastases than in intact pulmonary tissue.     

Dose-dependent effects of hydralazine on microcirculatory function and hyperthermic response of murine FSall tumors

The effects of the vasodilator hydralazine (HYD) on microcirculatory function and hyperthermic response were studied in early generation isotransplants of a spontaneous C3Hf/Sed mouse fibrosarcoma (FSall). Red blood cell flux (RBC flux) in superficial tumor regions was assessed using laser Doppler flowmetry. A differential microcirculatory response was seen between tumor and normal skin after 0.25 micrograms/g i.p. HYD, the tumor showing a transient increase in flow and the skin remaining almost stable. At 1.0 micrograms/g i.p., the differential response continued, this time with a transient fall in tumor blood flow but again no change in skin flow. High dose hydralazine (10.0 micrograms/g i.p.) was associated with a dramatic and prolonged decrease in tumor blood flow but a lesser and only transient decline in skin flow. Identical doses of hydralazine were given 30 min prior to heat treatment (43.5 degrees C for 15, 30, or 60 min). Tumor growth was measured daily and compared to controls (HT without hydralazine). Hydralazine at 0.25 micrograms/g i.p. did not affect heat induced growth delay. At 1.0 micrograms/g i.p., it significantly increased growth delay upon heat exposures of 15 min, but not after 30 or 60 min HT. Hydralazine at 10 micrograms/g i.p. increased growth delay for all heat doses (P less than 0.05). Hydralazine alone had no influence on growth delay of sham-heated tumors. The results obtained clearly indicate that tumor and normal tissues have microcirculatory differences in the time-course, degree and/or direction of response after hydralazine, and that hydralazine has potential for increasing the response of tumor to HT.     

Impairment of fibrinolysis during the growth of two murine mammary adenocarcinomas

The fibrinolytic activity present in the euglobulin (EU) fraction of BALB/c mice before and during the growth of M3 and MM3 murine mammary adenocarcinomas was characterized. The main plasminogen activator (PA) form contained in EUs from control mice was defined as murine urokinase-type PA (uPA). Overall fibrinolytic activity decreased significantly during tumor development. Zymographies showed that this fall was associated with a reduction in the free uPA band (47 kDa) and to the detection of a tissue-type PA (tPA) complexed band (117 kDa). Western blotting showed free tPA protein (68 kDa) in control mice, that disappeared in M3 tumor-bearing mice. In this model, high subcutaneous tumor burden induces a severe impairment in the circulating fibrinolytic system.     

Liposomal quercetin efficiently suppresses growth of solid tumors in murine models.

PURPOSE: Quercetin is a potent chemotherapeutic drug. Clinical trials exploring different schedules of administration of quercetin have been hampered by its extreme water insolubility. To overcome this limitation, this study is aimed to develop liposomal quercetin and investigate its distribution in vivo and antitumor efficacy in vivo and in vitro. EXPERIMENTAL DESIGN: Quercetin was encapsulated in polyethylene glycol 4000 liposomes. Biodistribution of liposomal quercetin i.v. at 50 mg/kg in tumor-bearing mice was detected by high-performance liquid chromatography. Induction of apoptosis by liposomal quercetin in vitro was tested. The antitumor activity of liposomal quercetin was evaluated in the immunocompetent C57BL/6N mice bearing LL/2 Lewis lung cancer and in BALB/c mice bearing CT26 colon adenocarcinoma and H22 hepatoma. Tumor volume and survival time were observed. The mechanisms underlying the antitumor effect of quercetin in vivo was investigated by detecting the microvessel density, apoptosis, and heat shock protein 70 expression in tumor tissues. RESULTS: Liposomal quercetin could be dissolved in i.v. injection and effectively accumulate in tumor tissues. The half-time of liposomal quercetin was 2 hours in plasma. The liposomal quercetin induced apoptosis in vitro and significantly inhibited tumor growth in vivo in a dose-dependent manner. The optimal dose of liposomal quercetin resulted in a 40-day survival rate of 40%. Quantitative real-time PCR showed that liposomal quercetin down-regulated the expression of heat shock protein 70 in tumor tissues. Immunohistochemistry analysis showed that liposomal quercetin inhibited tumor angiogenesis as assessed by CD31 and induced tumor cell apoptosis. CONCLUSIONS: Our data indicated that pegylated liposomal quercetin can significantly improve the solubility and bioavailability of quercetin and can be a potential application in the treatment of tumor.     

Reconstituted basement membrane (Matrigel) promotes the survival and influences the growth of murine tumors.

The effects of reconstituted basement membrane (Matrigel) on in vivo survival and growth of several murine tumors were studied. Survival of tumor cells was enhanced in all experiments which resulted in increased incidence and/or in increased tumor mass. While basement membrane enhanced the in vivo growth of B16F6 melanoma cells, survival of these mice was prolonged. Basement membrane increased the incidence but reduced the growth of Ehrlich ascites tumor. Walker-256 hypercalcemic breast carcinosarcoma growth was enhanced and glandular-like structures were observed when grown on Matrigel. The results indicate that the enhanced survival of tumor cells in the presence of basement membrane is not unequivocally linked with increased malignancy.     

Selenium and the genesis of murine mammary tumors

One hundred forty-seven female Sprague-Dawley rats were dividedinto 5 groups and at 60 days of age were treated i.g. with 5mg of 7,12-dimethylbenzanthracene (DMBA) suspended in 1.0 mlof sesame oil. Selenium (Se), as selenium dioxide (SeO₂), wasadministered in the drinking water to 4 of the 5 groups (30rats/group) at 2 doses (2 and 4 mg/1) from 30–90 daysof age (series 1) and from 90–150 days of age (series2) prior to the onset of palpable mammary tumors. One groupof rats (27 rats) served as controls. All rats were palpatedweekly for mammary tumors and sacrificed 28 weeks after DMBAtreatment. Total number of palpable mammary carcinomas whichdeveloped in each group were: controls, 60; series 1, 2 mg Sedose, 27, 4 mg Se dose, 29; series 2, 2 mg Se dose, 24, 4 mgSe dose, 32. Each dose level of Se in each series significantly(P <0.05) reduced the incidence of mammary carcinomas. Theseresults provided evidence that Se can inhibit the early promotingphases of polycyclic hydrocarbon induced mammary carcinogenesisin female rats. Two hundred twenty-six nulliparous and 99 multiparousGR mice were treated daily with estrogen and progesterone for13–16 weeks. Se (SeO₂) was administered in the drinkingwater (2 mg/1) to one-half of these mice. Total number of mammarycarcinomas in control nulliparous and multiparous mice were119 and 90, respectively; in Se treated nulliparous and multiparousmice, 113 and 81, respectively. Se did not significantly effectmammary carcinoma incidence in hormone treated nulliparous andmultiparous GR mice.