Cytostatic murine B16 cell-pulsed dendritic cells induce effective protection against melanoma in mice.

BACKGROUND: Peripheral blood mononuclear cells (PBMCs) present an antitumor activity in vitro on doxorubicin-resistant B16 melanoma (B16R) spheroids, but do not inhibit tumor growth in vivo. This study aimed to improve in vivo the antitumor immune response by using antigen presenting cells. MATERIALS AND METHODS: After injection of B16R cells, mice received either tumor cell lysate-pulsed PBMCs, or naive or cytostatic tumor cell-pulsed bone marrow-derived dendritic cells (DCs). Tumor development and mouse survival were followed and spleen cell cytotoxic activity against B16R was estimated in vitro by Lactate dehydrogenase activity measurement. RESULTS: The best results were obtained with peritumoral injections of cytostatic tumor cell-pulsed DCs which induced tumor regression, increased mouse survival and exhibited a higher splenocyte cytotoxic activity against B16R cells. When injected at a distant site, the efficacy was reduced. Furthermore, preventative injections of pulsed DCs induced protection of mice against B16R cells. CONCLUSION: These results show the important role of cytostatic tumor cell-pulsed DCs in a specific antitumor immunity establishment. Consequently, they could be used combined with other treatments to improve clinical outcomes.

Anti-tumoural activity of peripheral blood mononuclear cells against melanoma cells: discrepant in-vitro and in-vivo effects.

As tumour cells use multiple mechanisms to escape from chemotherapeutic drugs, the anti-tumoural activity of naive mouse peripheral blood mononuclear cells was examined in this study, using a mouse melanoma cell subline resistant to doxorubicin (B16R). Multicellular spheroids are known to be the most adapted in-vitro model to mimic solid tumours in vivo and are used to investigate many aspects of tumour biology. For in-vitro studies, murine peripheral blood mononuclear cells recovered by Ficoll gradient centrifugation after caudal puncture were co-cultured with multicellular tumour spheroids of B16R cells. Morphological investigations show that peripheral blood mononuclear cells were gathered and focused around the spheroids after 14 h of co-culture and contacts were established within 32 h. Between 38 and 62 h of co-culture, the size of the spheroids decreased significantly. The peripheral blood mononuclear cells exerted cytolytic effects that correlated with the induction of cell death in spheroids of B16R melanoma cells. Immunological investigations to localize and identify peripheral blood mononuclear cells that exerted anti-tumoural effects have shown that spheroids were deeply infiltrated by monocytes/macrophages at a stage in which a significant cytolytic activity and a strong cell death rate were observed. For in-vivo studies, intratumoural injections of syngeneic naive peripheral blood mononuclear cells were administered. A weak potential in-vivo anti-tumoural effect of these cells was observed (inhibition of B16R melanoma growth by 20-25%) but the median survival time of mice treated with peripheral blood mononuclear cells did not increase compared with untreated control mice. Thus, despite anti-tumoural activities of peripheral blood mononuclear cells against the poorly immunogenic and highly metastatic chemoresistant B16 melanoma cells in vitro, a potential anti-melanoma effect in vivo, if present, did not increase the life span of B16R melanoma-bearing mice.

In vitro and in vivo anti-tumoral effect of curcumin against melanoma cells.

Curcumin, the active ingredient from the spice turmeric (Curcuma longa Linn), is known to be an anti-oxidant and an anti-inflammatory agent. It has been demonstrated recently to possess anti-angiogenic effects and pro-apoptotic activities against Ehrlich ascites tumor cells. In the current study, curcumin was found to be cytotoxic in vitro for B16-R melanoma cells resistant to doxorubicin either cultivated as monolayers or grown in three-dimensional (3-D) cultures (spheroids). We have demonstrated that the cytotoxic effect observed in the 2 culture types can be related to the induction of programmed cell death. In our in vivo studies, we examined the effectiveness of a prophylactic immune preparation of soluble proteins from B16-R cells, or a treatment with curcumin as soon as tumoral appearance, alone or in combination, on the murine melanoma B16-R. The combination treatment resulted in substantial inhibition of growth of B16-R melanoma, whereas each treatment by itself showed little effect. Moreover, animals receiving the combination therapy exhibited an enhancement of their humoral anti-soluble B16-R protein immune response and a significant increase in their median survival time (> 82.8% vs. 48.6% and 45.7% respectively for the immunized group and the curcumin-treated group). Our study shows that curcumin may provide a valuable tool for the development of a therapeutic combination against the melanoma.

Three-dimensional culture and multidrug resistance: effects on immune reactivity of MCF-7 cells by monocytes.

BACKGROUND: Multicellular spheroids are known to be the most adapted model to keep the in vitro resistance properties of cells. This in vivo-like tissue-culture representation was applied to investigate the immune reactivity of MCF-7 cells by monocytes. MATERIALS AND METHODS: Human blood monocytes, obtained by elutriation, were co-cultured with multicellular tumor spheroids of drug-sensitive (MCF-7S) and doxorubicin-resistant (MCF-7DXR) MCF-7 breast cancer cells. RESULTS: Tumor cells, according to their phenotype, induced differential recruitment and behavior of the immune cells towards the two types of spheroids. The secretion of various cytokines and the expression of several adhesion molecules were analysed. The MCF-7DXR/monocytes co-culture supernatant showed higher levels of IL-6 and IL-8 than the MCF-7S/monocytes co-culture supernatant. Cells from the MCF-7DXR spheroids expressed some adhesion molecules, CD-44 and CD-54, leading to a strong cellular cohesion in comparison with the sensitive spheroids. CONCLUSION: The two spheroid phenotypes represented an excellent model system for determining the precise tumor microenvironment in which cells move, the crucial molecular requirements and the mechanisms by which immunotherapeutic strategies could be developed to eradicate chemo-resistant tumors.

Change in chromatin organization related to in vivo transcriptional activity and histone synthesis independent of DNA replication during differentiation (germination) of Physarum spherules.

During the germination of Physarum spherules, increases have been observed, at the same moment, in the level of in vivo transcriptional activity as measured by [3H] uridine incorporation, and the accessibility of DNA for ethidium bromide staining as shown by flow cytometric measurements. We suppose that the changes observed in these two processes are due to a difference in chromatin organization between the first and the second period of the premitotic germination stage. In the second period, the four nucleosome core histories are synthesized in the absence of DNA replication and may correspond to a replacement of spherulation histone variants by plasmodial histone types in nucleosomes. The synthesis of historic H4 clearly distinguishes the second period of the premitotic germination stage from a growing plasmodium G2 phase, though nuclei exhibit a G2 phase DNA content. The same pattern of histone synthesis has been found during the cell cycle following the first mitosis after germination and the growing plasmodium cell cycle, with a synthesis of two histories H2B and H2A and the high mobility group (HMG)-like protein AS3 during the G2 phase, i.e. in the absence of DNA synthesis.

In vitro control of blastema cell proliferation by extracts from epidermal cap and mesenchyme of regenerating limbs of axolotls.

The presence of a mitogenic activity in limb blastemas of axolotls was detected in crude extracts of blastemas at the mid-bud stage. The mitogenicity of the extracts was estimated from the mitotic index of blastema cells grown for 6 days in the presence of limb blastema extracts, with colchicine present for the last 2 days. All the extracts tested (whole blastema, blastemal mesenchyme, epidermal cap) significantly enhanced proliferation of blastema cells. The highest stimulation factors we observed were 7 × with 7 µg protein/ml whole blastema extracts, 5.2 × with 14 µg/ml blastemal mesenchyme extracts, and 11 x with 3.5 µg/ml epidermal cap extracts. Hence the epidermal cap extracts appeared to be the most mitogenic. Extracts from the blastemal mesenchyme, although less mitogenic than the epidermal cap extracts, were more potent than nerve extracts [Albert P, Boilly B (1986) Biol Cell 58:251-262]. These results are discussed with regard to the production of growth factors during limb regeneration.

Effect of transferrin on amphibian limb regeneration: a blastema cell culture study.

In order to study mitogenic control during axolotl limb regeneration, we have developed a primary blastema cell culture as a very sensitive bioassay for blastema mitogens. Transferrin, an iron-binding glycoprotein which has been shown to be the neurotrophic factor for muscle cells, is the mitogen which has been analysed in the present report. Addition of approximately 2 µg human transferrin/ ml of serum-free culture medium enhances blastema cell proliferation 11-fold over control levels and 2-fold over that produced by the addition of nerve extracts or purified growth factors extracted from nerve tissues (basic and acidic fetal growth factor, FGF). At a higher concentration (20 µg/ml), transferrin alone has no mitogenic effect unless the medium is also supplemented with FeCl3 (100 µM). The results are discussed with regard to the sensitivity of the blastema cell culture bioassay and in the context of the neurotrophic theory of urodele limb regeneration.