Bovine lactoferrin and lactoferricin, a peptide derived from bovine lactoferrin, inhibit tumor metastasis in mice.

We investigated the effect of a bovine milk protein, lactoferrin (LF-B), and a pepsin-generated peptide of LF-B, lactoferricin (Lfcin-B), on inhibition of tumor metastasis produced by highly metastatic murine tumor cells, B16-BL6 melanoma and L5178Y-ML25 lymphoma cells, using experimental and spontaneous metastasis models in syngeneic mice. The subcutaneous (s.c.) administration of bovine apo-lactoferrin (apo-LF-B, 1 mg/mouse) and Lfcin-B (0.5 mg/mouse) 1 day after tumor inoculation significantly inhibited liver and lung metastasis of L5178Y-ML25 cells. However, human apolactoferrin (apo-LF-H) and bovine holo-lactoferrin (holo-LF-B) at the dose of 1 mg/mouse failed to inhibit tumor metastasis of L5178Y-ML25 cells. Similarly, the s.c. administration of apo-LF-B as well as Lfcin-B, but not apo-LF-H and holo-LF-B, 1 day after tumor inoculation resulted in significant inhibition of lung metastasis of B16-BL6 cells in an experimental metastasis model. Furthermore, in in vivo analysis for tumor-induced angiogenesis, both apo-LF-B and Lfcin-B inhibited the number of tumor-induced blood vessels and suppressed tumor growth on day 8 after tumor inoculation. However, in a long-term analysis of tumor growth for up to 21 days after tumor inoculation, single administration of apo-LF-B significantly suppressed the growth of B16-BL6 cells throughout the examination period, whereas Lfcin-B showed inhibitory activity only during the early period (8 days). In spontaneous metastasis of B16-BL6 melanoma cells, multiple administration of both apo-LF-B and Lfcin-B into tumor-bearing mice significantly inhibited lung metastasis produced by B16-BL6 cells, though only apo-LF-B exhibited an inhibitory effect on tumor growth at the time of primary tumor amputation (on day 21) after tumor inoculation. These results suggest that apo-LF-B and Lfcin-B inhibit tumor metastasis through different mechanisms, and that the inhibitory activity of LF-B on tumor metastasis may be related to iron (Fe3+)-saturation.

Persistence of dormant tumor cells in the bone marrow of tumor cell-vaccinated mice correlates with long-term immunological protection.

Live proliferation-competent and irradiated proliferation-incompetent L5178 murine lymphoma cells (Eb cell line) were compared for their potency to induce systemic anti-tumor immunity in syngeneic DBA/2 mice. The tumorigenic potential in vivo of live Eb cells was suppressed through local secretion of interleukin 4 (IL4) or alternatively by injection of parental cells at a site refractory to tumor growth. Inoculation of nontumorigenic doses of live Eb or Eb-IL4 cells led to long-lasting specific and systemic T-cell-mediated antitumor response requiring both CD4+ and CD8+ T lymphocytes. Irradiated cells offered only limited short-term protection, which could be marginally improved by IL4. The more effective protection offered by vaccination with live tumor cells correlated with rapid migration and persistence of tumor cells in the bone marrow of host animals after tumor cell inoculation. In contrast, irradiated Eb-lacZ cells had a short persistence. Tumor cells recovered from the bone marrow of host animals injected with live Eb-IL4 cells still expressed IL4. These observations indicate that in the course of vaccination with live Eb or Eb-IL4 cells, a fraction of these cells escaped destruction by host mechanisms and persisted in a dormant state in the bone marrow for long periods of time. Persistence of dormant tumor in the bone marrow correlated with the duration of anti-tumor immunity.

B30-MDP, a synthetic muramyl dipeptide derivative for tumour vaccination to enhance antitumour immunity and antimetastatic effect in mice.

The effect of a muramyl dipeptide derivative (B30-MDP) on the augmentation of antitumour immunity against highly metastatic L5178Y-ML25 mouse lymphoma cells was examined in CDF1 (Balb/c x DBA/2) mice. Mice immunized with a mixture of X-irradiated tumour cells (10(3)) and B30-MDP (100 micrograms) on 7 days prior to challenge by viable tumour cells displayed a significant decrease in metastasis towards the target organs, liver and spleen, compared with that of untreated mice. Immunization of mice with the mixture on day 5 or 7 after tumour challenge, when the level of glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) in sera of mice inoculated with viable tumour cells was observed to be normal, caused less metastasis than immunization with X-irradiated tumour cells alone. Sensitization with X-irradiated tumour cells admixed with B30-MDP induced almost two times higher cytotoxicity of spleen cells against L5178Y-ML25 lymphoma cells than sensitization with X-irradiated tumour cells without B30-MDP. In contrast, cytotoxic activity of spleen cells against another target, L1210 lymphoma cells derived from BDF1 mice, was not observed by immunization with X-irradiated L5178Y-ML25 cells with or without B30-MDP. Specific lysis by splenic cells of the immunized mice against L5178Y-ML25 cells decreased to the normal level when T cells were deleted from the immunized spleen cells by the treatment of rabbit anti-mouse Thy1.2 antibody and rabbit complement. These results indicate that B30-MDP is able to augment a specific tumour immunity due to the enhancement of cytotoxicity mediated by T lymphocytes, and is useful as an immunopotentiating agent for active immunization of inactivated tumour cells.