Figure 1.
Tumor morphology changes with tumor progression.
(A) Control –The characteristic organization and thickness of epidermis and dermis are observed in a section of normal skin from a control mouse. (B) Phase I –This phase is characterized by epidermal proliferation, a pronounced thickening of the dermis and the formation of a papilloma. (C) Phase II - The dermis is thicker and the papilloma is more prominent. (D) Phase III - Keratin cysts (*) are observed, as well as a thickening of the stratum corneum.
Figure 2.
Mature and immature mast cell numbers increase in the dermis during tumor progression.
(A) Mature metachromatic mast cells (arrow) are observed in the dermis of a control mouse. (B) At phase I a pronounced increase in mature (arrows) and immature lightly metachromatic mast cells (arrowhead) are observed in the dermis. In phases II (C) and III (D) the number of mature (arrows) and immature mast cells (arrowheads) is higher than in phase I. Inset in phase I show an immature mast cell (Toluidine blue). (E) Flow cytometry analysis of mast cells during tumor progression using AA4-FITC. (F) The percentage of immature mast cells is increased at phase I of tumor progression and continues to increase during the phases II and III. (G) Histamine content also increased during tumor progression. (n = 3) *P<0.05.
Figure 3.
Expression of chymase, tryptase and carboxypeptidase A were altered during tumor progression.
Western blots of cell lysate from control, phases I, II and III of tumor progression. The levels of mMCP-4 are constant in the 3 phases. mMCP-5 is only expressed during tumorigenesis. The levels of mMCP-6 increase progressively during the three phases. mMCP-7 was not detected in control animals, begging to expression in phase I and remained unchanged in phases II and III. The expression of mMC-CPA is almost not detectable in controls, but increases in phases I and II, and remains unchanged in phase III. Alpha-actin was used as a loading control. Mean optical density of blots presenting the data of the mean values ± SEM of 3 independent experiments. *P<0.05.
Figure 4.
Enzymatic activity of chymase and tryptase increase with tumor progression.
The activities of both chymase and tryptase increase mainly in phases II and III. The data represent the mean values ± SEM. (n = 3) *P<0.05.
Figure 5.
Blood vessel number and diameter increase during tumor progression.
(A) Representative sections of control skin and tumors during Phases I, II and III demonstrating the increase in the number and diameter of blood vessels (arrows) with tumor progression. Blood vessels were immunostained for von Willebrand factor. (B) The number of blood vessels, (C) the percentage of area covered by blood vessels and (D) the diameter of the vessels increases considerably in phase I and II and remains constant in phase III. (n = 3) *P<0.05.
Figure 6.
Mast cell proteases induce cell spreading and tube formation in SVEC4-10 cells.
(A) When cells were incubated in absence of proteases, only a few cells were spread on the surface, beginning to form tubes (arrow) and most of the cells remained unspread. (B) mMCP-6 augments cell spreading and tube formation in comparison to the control cultures (arrows). (C) mMCP-7 is more efficient in inducing cell spreading and tube formation than mMCP-6. Most of cells are spread (arrowhead) or in the form of tubes (arrows). Phase contrast microscopy. (D) The graphics show the percent of cells that were spread on the surface (Geltrex™) after 5 h incubation. *P<0.05.