Macrophage-mediated inhibition of melanoma cell growth in nude mice

The effects of macrophages or sera from tumor-transplanted or control syngeneic and allogeneic mice on the latency and growth rate of P51 murine melanoma cells were determined after transplantation into congenitally athymic (nude) mice (tumor neutralization test). Syngeneic macrophages from tumor-bearing mice (TBM) inhibited melanoma growth in the nude mouse more than control macrophages, additionally macrophages from sensitized allogeneic mice inhibited melanoma growth to a greater degree than did allogeneic control macrophages. Sera from TBM inhibited melanoma growth as compared to control cells alone. Macrophages obtained after 14 days were also cytolytic towards the melanoma target in vitro. Despite the growth of large local masses, no evidence of distant metastases was found. The nude mouse thus provides an appropriate model for this tumor to portray in vivo immunotherapy.     

Eradication of melanoma in vitro and in vivo via targeting with a Killer-Red-containing telomerase-dependent adenovirus

Melanoma is a highly recalcitrant cancer and transformative therapy is necessary for the cure of this disease. We recently developed a telomerase-dependent adenovirus containing the fluorescent protein Killer-Red. In the present report, we first determined the efficacy of Killer-Red adenovirus combined with laser irradiation on human melanoma cell lines in vitro. Cell viability of human melanoma cells was reduced in a dose-dependent and irradiation-time-dependent manner. We used an intradermal xenografted melanoma model in nude mice to determine efficacy of the Killer-Red adenovirus. Intratumoral injection of Killer-Red adenovirus, combined with laser irradiation, eradicated the melanoma indicating the potential of a new paradigm of cancer therapy.     

A novel 10B-enriched carboranyl-containing phthalocyanine as a radio- and photo-sensitising agent for boron neutron capture therapy and photodynamic therapy of tumours: in vitro and in vivo studies.

The synthesis of a Zn(ii)-phthalocyanine derivative bearing four 10B-enriched o-carboranyl units (10B-ZnB4Pc) and its natural isotopic abundance analogue (ZnB4Pc) in the peripheral positions of the tetraazaisoindole macrocycle is presented. The photophysical properties of ZnB4Pc, as tested against model biological systems, were found to be similar with those typical of other photodynamically active porphyrin-type photosensitisers, including a singlet oxygen quantum yield of 0.67. The carboranyl-carrying phthalocyanine was efficiently accumulated by B16F1 melanotic melanoma cells in vitro, appeared to be partitioned in at least some subcellular organelles and, upon red light irradiation, induced extensive cell mortality. Moreover, ZnB4Pc, once i.v.-injected to C57BL/6 mice bearing a subcutaneously transplanted pigmented melanoma, photosensitised an important tumour response, provided that the irradiation at 600-700 nm was performed 3 h after the phthalocyanine administration, when appreciable concentrations of ZnB4Pc were still present in the serum. Analogously, irradiation of the 10B-ZnB4Pc-loaded pigmented melanoma with thermal neutrons 24 h after injection led to a 4 day delay in tumour growth as compared with control untreated mice. These results open the possibility to use one chemical compound as both a photosensitising and a radiosensitising agent for the treatment of tumours by the combined application of photodynamic therapy and boron neutron capture therapy.     

Morphological changes in human melanoma cells following irradiation with thermal neutrons

Morphological changes in two human melanoma cell lines, MM96 and MM418, following irradiation with thermal neutrons, were studied using light and electron microscopy. The results show that the response of human malignant melanoma cells to neutron irradiation is both cell line dependent and dose dependent, and that in any given cell line, some cells are more resistant to irradiation than others, thus demonstrating heterogeneity in respect to radiosensitivity. Cells repopulating MM96 flasks after irradiation were morphologically similar to the cells of origin whereas in MM418 flasks cells differentiated into five morphologically distinct subgroups and showed increased melanization. The results also show that radiation causes distinctive morphological patterns of damage although ultrastructural changes unique to the high LET particles released from boron 10 neutron capture are yet to be identified.     

The survival of mice and the metallothionein content of their livers and kidneys as criteria for assessing exposure to pulsed neutron radiation

The effect of pulsed neutron radiation was studied in comparison with continuous neutron radiation and continuous gamma-radiation. Animal survival and induction of metallothionein (MT) synthesis in liver and kidney of mice exposed to equivalent doses were chosen as criteria for evaluation of radiation effects. It was found that the level of MT in liver and kidney of mice exposed to neutron radiation decreased 24 hours after irradiation and then continued decreasing in kidney for 48 hours after irradiation. This is evidence of more intensive free-radical processes initiated by pulsed neutron radiation. At the same time, RBE values of pulsed neutrons did not differ significantly from that of continuous neutron radiation.     

Experimental Boron Neutron Capture Therapy for Melanoma: Systemic Delivery of Boron to Melanotic and Amelanotic Melanoma

The boron-containing melanin precursor analogue p-boronophenylalanine (BPA) has previously been shown to selectively deliver boron to pigmented murine melanomas when administered in a single intragastric dose. If boron neutron capture therapy is to become a clinically useful method of radiation therapy for human malignant melanoma, the boron carrier must be capable of delivering useful amounts of boron to remote tumor sites (metastases) and to poorly pigmented melanomas. We have now determined the ability of BPA to accumulate in several nonpigmented melanoma models including human melanoma xenografts in nude mice. The absolute amount of boron in the nonpigmented melanomas was about 50% of that observed in the pigmented counterparts but was still selectively concentrated in the tumor relative to normal tissues in amounts sufficient for effective neutron capture therapy. Single intragastric doses of BPA resulted in selective localization of boron in the amelanotic Greene melanoma carried in the anterior chamber of the rabbit eye and in a pigmented murine melanoma growing in the lungs. The ratio of the boron concentration in these tumors to the boron concentration in the immediately adjacent normal tissue was in the range of 3:1 to 4:1. These distribution studies support the proposal that boron neutron capture therapy may be useful as a regional therapy for malignant melanoma.     

人黑色素瘤细胞裸鼠肺转移模型的建立

目的建立整合素αvβ3高表达的黑色素瘤细胞肺转移模型。方法通过将不同数目的M21细胞经尾静脉接种裸鼠,适时处死后计数肺表面癌结节。通过实时定量PCR和明胶酶谱的方法比较肺转移灶细胞与亲代M21细胞差异。结果M21细胞1×10^6、2×10^6、5×10^6尾静脉接种裸鼠,均能形成肺转移灶,癌结节数目均值分别为：84±8、70±6、88±12,三组之间没有明显差异,阴性对照组未形成转移灶。M21肺转移灶细胞与亲代细胞相比,增殖增快,MMP-2活性增高,整合素αv和β3mRNA表达水平明显增高。结论M21细胞1×10^6经尾静脉接种裸鼠50d内即可100%成瘤。M21肺转移灶细胞具有更快的增殖能力,整合素αvβ3和MMP-2表达水平明显增高。本实验建立了稳定的肺转移模型,为黑色素瘤和整合素αvβ3的研究提供重要的动物模型。     

Quantitative evaluation of anticancer agents against human melanoma cells implanted in nude mice

BRO human melanoma cells, which are exceptionally tumorigenic and lethal for nude mice, were inoculated intraperitoneally or intracerebrally in varying numbers. An inverse linear correlation was observed between the logarithm of the number of cells inoculated and host survival. In mice bearing 10(7) cells intraperitoneally, 2.4-2.8 log10 units of cell kill were obtained after a single intraperitoneal injection of vincristine, and some mice inoculated with 10(5) cells were cured by this treatment. Fewer cells were killed by L-phenylalanine mustard. Vincristine did not prolong survival of nude mice with intracerebral BRO tumors. Cell kill after administration of anticancer agents can be quantitated for BRO cells inoculated intraperitoneally or intracerebrally.     

The effect of graded doses of fission neutrons or X rays on the stromal compartment of the thymus in mice

The effect of irradiation on the supportive role of the thymic stroma in T cell differentiation was investigated in a transplantation model using athymic nude mice and transplanted irradiated thymuses. In this model, neonatal CBA/H mice were exposed to graded doses of whole-body irradiation with fast fission neutrons of 1 MeV mean energy or 300 kVp X rays. The doses used varied from 2.75 up to 6.88 Gy fission neutrons and from 6.00 up to 15.00 Gy X rays at center-line dose rates of 0.10 and 0.30 Gy/min, respectively. Subsequently, the thymus was excised and a thymus lobe was transplanted under the kidney capsule of H-2 compatible nude mice. One and two months after transplantation, the T cell composition of the thymic transplant was investigated using immunohistology with monoclonal antibodies directed to the cell surface differentiation antigens Thy-1, Lyt-1, Lyt-2, MT-4, and T-200. Furthermore, the stromal cell composition of the thymic transplant was investigated with monoclonal antibodies directed to MHC antigens and with monoclonal antibodies defining different subsets of thymic stromal cells. To investigate the reconstitution capacity of the thymic transplant, the peripheral T cell number was measured using flow cytofluorometric analysis of nude spleen cells with the monoclonal antibodies anti-Thy-1, anti-Lyt-2, and anti-MT-4. The results of this investigation show that a neonatal thymus grafted in a nude mouse has a similar stromal and T cell composition as that of a normal thymus in situ. In addition, grafting of such a thymus results in a significant increase of the peripheral T cell number. Irradiation of the graft prior to transplantation has no effects on the stromal and T cell composition but the graft size decreases. This reduction of size shows a linear dose-response curve after neutron irradiation. The X-ray curve is linear for doses in excess of 6.00 Gy. The RBE for fission neutrons for the reduction of the relative thymic graft size to 10% was equal to 2.1. Furthermore, the peripheral T cell number decreases with increasing doses of irradiation given to the graft prior to transplantation. The present data indicate that the regenerative potential of thymic stromal cells is radiosensitive and is characterized by D0 values equal to 2.45 and 3.68 Gy for neutrons and X rays, respectively. In contrast, the ability of the thymic stromal cells to support T cell maturation is highly radioresistant.     

Tumor induction in BALB/c female mice after fission neutron or gamma irradiation

This study was designed to examine the dose-response relationships for tumor induction after neutron irradiation in female BALB/c mice, with emphasis on the response in the dose range 0 to 50 rad. Tumors induced after radiation exposure included ovarian tumors, lung adenocarcinomas, and mammary adenocarcinomas. For comparison the dose responses for induction of these tumors after 137Cs gamma irradiation were also examined. As previously described for the female RFM mouse, the data for ovarian tumor induction after neutron and gamma irradiation were consistent with a threshold model. For lung and mammary tumors the dose-response curve after neutron irradiation appeared to "bend over" in the dose range 10 to 20 rad. The factors responsible for this bend-over and their relative contributions to the overall form of the dose-response relationship are not presently known. However, these data strongly indicate that extrapolation from data above 50 rad could result in a significant underestimate of risks. Further, it is clear that current models of neutron carcinogenesis are inadequate, since such a bend-over is not predicted at these low dose levels.     

Adeno-associated virus sensitizes HeLa cell tumors to gamma rays.

Infection with the helper virus-dependent human parvovirus adeno-associated virus (AAV) is known to interfere with cellular transformation in vitro and oncogenesis in vivo. Here we report on sensitization to gamma irradiation by AAV infection of cells in culture and of tumors established from HeLa cells grafted into immunodeficient (nude) mice: infection of HeLa cells with AAV type 2 enhanced cell killing and reduced plating efficiency after irradiation compared with uninfected cells. Similarly, HeLa cell tumors in nude mice displayed a reduced growth rate and were more sensitive to gamma irradiation when the animals were infected with AAV type 2 prior to or after tumor cell inoculation. Since no pathogenicity is known for AAV, the ability of this virus to render radiotherapy of human tumor cells more efficient may up open novel approaches in cancer treatment.     

Effects of murine tumor necrosis factor on heterotransplanted human tumors.

A partially purified glycoprotein fraction (the G-200 II fraction) obtained from sera of CD-1 mice sensitized with Corynebacterium parvum and treated with endotoxin was designated as tumor necrosis factor (TNF). Human melanoma cells exposed to this factor in vitro had decreased tumorigenicity when injected into nude mice. Human melanoma, embryonal adenocarcinoma of the testis and colon carcinoma heterotransplanted in nude mice exhibited regressions in size following intraperitoneal injections of TNF. The responses were related to dose and duration of exposure.     

Cell binding and tumor inhibiting functions of a new antihuman melanoma murine monoclonal antibody.

Murine, antihuman melanoma cell monoclonal antibody (mAb) 16.C8 was generated by fusing the murine myeloma cell line P3X63/Ag8.653 with splenocytes from a nude mouse bearing a human melanoma xenograft, after reconstitution with splenocytes from syngeneic immunocompetent BALB/c mice. The antibody reacted strongly with fresh human melanoma cells and exhibited preferential reactivity with established human melanoma and neuroectodermal tumor cell lines. Electrophoresis and Western blotting experiments indicated that 16.C8 is directed against a sialoglycoprotein antigen with a molecular weight of 110-120 kDa. mAb 16.C8 mediated lysis of melanoma cells in vitro in antibody-dependent cellular cytotoxicity assays using human mononuclear effector cells isolated from normal volunteers or malignant melanoma patients. In addition, the administration of mAb 16.C8 to nude mice bearing established human melanoma lung and liver metastases resulted in significant inhibition of tumor growth as shown by gross and histologic examination. In contrast, animals treated with Hanks' balanced salt solution or nonspecific immunoglobulin exhibited a large tumor burden. These results suggest that mAb 16.C8 may be of value in treatment of metastatic melanoma in humans.     

Induction of external abnormalities in offspring of male mice irradiated with 252Cf neutron.

To assess the genetic effects of fission neutron, the induction of external malformations was studied in F1 fetuses after F0 male mice were irradiated. Male mice of the ICR:MCH strain were irradiated with 252Cf neutron at doses of 0.238, 0.475, 0.95 and 1.9 Gy. They were mated with non-irradiated female mice at 71-120 days after the irradiation. Pregnant females were autopsied on day 18 of gestation and their fetuses were examined for deaths and external abnormalities. No increases of pre- and post-implantation losses were noted at any dose. External abnormalities were observed at rates of 1.40% in the 0.238 Gy, 2.23% in the 0.475 Gy, 3.36% in the 0.95 Gy and 3.26% in the 1.9 Gy groups; the rate in the control group was 1.65%. The dose-response curve was linear up to 0.95 Gy, and then flattened out; the induction rate of external abnormalities was 2.7 x 10(-4)/gamete/cGy based on the linear regression. These results indicated that fission neutron effectively induces external abnormalities in F1 fetuses after spermatogonial irradiation.     

Response of human squamous cell carcinoma xenografts of different sizes to irradiation: relationship of clonogenic cells, cellular radiation sensitivity in vivo, and tumor rescuing units

The relationship of clonogenic cells, cellular radiation sensitivity at tumor control does in vivo, and tumor rescuing units at different tumor sizes was investigated in the human squamous cell carcinoma FaDu growing in NCr/Sed nude mice. The composition of the tumors was determined in single cell suspensions and compared to tumor control data after single-dose irradiation. To avoid the influence of varying oxygen concentrations in the tumors, all irradiations were performed under clamp hypoxia. Nude mice and animals further immunosuppressed by 6-Gy whole-body irradiation were used to assess the immunological effects. The numbers of total cells, cells excluding trypan blue, host cells, and colony-forming cells increased linearly with the weight of FaDu tumors. Comparable results were obtained for cell suspensions prepared from tumors growing in nude of pretreated nude mice. The radiation dose required to control 50% of tumors (TCD50) of different sizes between 36 and 470 mm3 increased from 52.1 to 60.1 Gy when the tumors were maintained in normal nude mice and from 50.8 to 61.3 Gy in whole-body-irradiated mice. The D0 of FaDu cells in vivo was calculated by regression analysis of TCD50 vs the logarithm of the clonogenic cell number, assuming an oxygen enhancement ratio of 3.0. The resultant D0S of 1.1 and 1.2 Gy in vivo correspond well to the radiosensitivity of FaDu cells in vitro determined previously. Assuming the single-hit multitarget model of cell killing and extrapolation numbers between 2 and 20, the mean number of tumor rescuing units would be 10(5) to 10(6) for a 100-mm3 tumor growing in whole-body-irradiated nude mice. Comparison of the number of tumor rescuing units to the estimated number of clonogenic cells does not conflict with the assumption that every surviving clonogenic cell is able to repopulate FaDu tumors after irradiation; however, it seems more likely that more than one clonogenic cells is necessary. The proportion of tumor rescuing units in the clonogenic cell population is independent of tumor size.     

Radioprotective properties of ATP and modification of acid phosphatase response after a lethal dose of whole body p(66MeV)/Be neutron radiation to BALB/c mice.

The intraperitoneal administration of exogenous ATP prior to a lethal dose (7 Gy) of whole body neutron irradiation increased the radioresistance of BALB/c mice. This radiation used the beam from a neutron therapy facility produced by the reaction p(66 MeV)/Be. Survival of the mice, determined 7 days post-irradiation as the endpoint, was increased from 26% to 86% by the action of the exogenous ATP. Furthermore, the response of acid phosphatase activity as an indicator of the acute radiation effects showed a marked augmentation in both tissues studied, testes and small intestine. The activity of the enzyme after neutron irradiation with prior administration of ATP showed smaller increases when compared with the increases observed after neutron irradiation alone. This implies that exogenous ATP reduces the effect of the lytic enzyme and, hence, damage. Finally, changes were observed in the activity of acid phosphatase in the testes and intestine with different concentrations of exogenous ATP. In both tissues there was a monotonic decrease in the activity of the enzyme with increase of the concentration of exogenous ATP administrated before radiation. These results reflect the protective ability of exogenous ATP as an adaptive defence mechanism to reduce radiation damage in normal tissues after a lethal dose of neutron radiation.     

Anti-cachectic effect of ghrelin in nude mice bearing human melanoma cells

Ghrelin is a novel brain-gut peptide that stimulates food intake and body weight gain. We studied the anabolic effect of ghrelin in a cancer cachexia mouse model. SEKI, a human melanoma cell line, was inoculated into nude mice to examine the effects of ghrelin on food intake and body weight. The intraperitoneal administration of ghrelin twice a day (6 nmol/mice/day) for 6 days suppressed weight loss in SEKI-inoculated mice and increased the rate of weight gain in vehicle-treated nude mice. Ghrelin administration also increased food intake in both SEKI- and vehicle-treated mice. Both the weight of white adipose tissue and the plasma leptin concentration were reduced in tumor-inoculated mice compared with vehicle-treated mice; these factors increased following ghrelin administration. The levels of both ghrelin peptide and mRNA in the stomach were upregulated in tumor-inoculated mice. The anabolic effect of ghrelin efficiently reverses the cachexia in mice bearing SEKI human melanoma. Ghrelin therefore may have a therapeutic ability to ameliorate cancer cachexia.     

Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and ⁷Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.     

Metastatic behavior of human melanoma cell lines in nude mice correlates with urokinase-type plasminogen activator, its type-1 inhibitor, and urokinase-mediated matrix degradation

Five out of six human melanoma cell lines tested were able to degrade in vitro a smooth muscle cell extracellular matrix in a plasmin-dependent way. In three of these five cell lines, this process was mediated by tissue-type plasminogen activator (t-PA) and in the other two cell lines by urokinase-type plasminogen activator (u-PA). All melanoma cell lines produced t-PA mRNA and protein, whereas only the two cell lines showing u-PA-mediated matrix degradation produced u-PA mRNA and protein. These latter cell lines also produced plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) mRNA and protein. u-PA receptor (u-PA-R) mRNA and binding of radiolabeled u-PA was found in all melanoma cell lines. The metastatic capacity of these cell lines was studied in nude mice. All cell lines were able to develop primary tumors at the subcutaneous inoculation site. The production of plasminogen activators, their inhibitors and urokinase receptor by subcutaneous tumors corresponded with the production by the parental cell lines in vitro. The two u-PA and PAI-1 producing cell lines showed the highest frequency to form spontaneous lung metastases after subcutaneous inoculation, whereas five of the six cell lines formed lung colonies after intravenous inoculation. In conclusion, u-PA mediated matrix degradation in vitro and production of u-PA and PAI-1 by human melanoma cell lines correlated with their ability to form spontaneous lung metastasis in nude mice. No correlation was found with the ability to form lung colonies after intravenous injection. These findings suggest a role for u-PA and PAI-1 in a relatively early stage of melanoma metastasis.     

Effect of chemotherapy on human tumor heterografts and lymphocytes in athymic mice

Sensitivity of human serially transplanted tumours (melanoma, hepatoma, lung, stomach and bladder cancer) to monochemotherapy was studied in nude mice. The following groups of nude mice were examined: intact and those with human xenografts (control and mice treated with alkylating agents). Changes in peripheral blood morphology have been revealed and functional activity of the energetic enzymes (SDH and alpha-GPDH) has been cytochemically discovered.