Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos

Abstract

The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.

Figure 1. Inoculation site of P1.HTR cells on chick embryo chorioallantoic membrane (CAM) (A). Note the blood vessel-free area between large CAM vessels (white arrow). Thirty minutes after inoculation, a small bubble persists and the surrounding vessels become more evident (B, black arrow), with a pronounced hyperemia (blue arrow) and spotted hemorrhagic area (yellow arrow) 6 h later (C). “Cobblestone-like” pattern of blood vessel distribution 24 h post-inoculation (D).

Figure 2. Assessment of chick embryo chorioallantoic membrane 24 h post-inoculation of P1.HTR cells. Mast cells were grouped around the main vessels (red circle) and a high density of small blood vessels convergent to mast cell clusters was observed (A, white arrows, Alcian blue/safran method on uncut membrane). Histology of the chorioallantoic membrane 24 h post-inoculation (B). Note a high density of split small perfused and unperfused blood vessels in treated specimens compared to normal chick embryo chorioallantoic membrane (C). Immunostaining with FVIII-related antigen highlighted a heterogenous distribution of a positive reaction in endothelial cells from newly formed blood vessels in treated specimens. Note the homogeneous intense positive reaction in the capillary plexuses beneath the chorionic epithelium (red arrow). Weak or moderate expression of FVIII-related antigen in the newly formed perfused and unperfused blood vessels from chick chorioallantoic membrane after mast cell inoculation (green arrows) (D).

Figure 3. In ovo image of the mast cell tumor (mastocytoma) 7 days post-inoculation (A). Note the disorganized vascular network and the macroscopic appearance of tumor blood vessels. “Wheel spoke-like” arrangement of blood vessels around the tumor (red circle); peritumoral blood vessels showed higher density compared to the adjacent chorioallantoic membrane vascular network (B). Whole mount Alcian blue/safran staining of the chick embryo chorioallantoic membrane with a mastocytoma, before cutting (C). A mixture of both safran- and Alcian blue-positive mast cells composed the mast cell tumor, surrounded by a rich network of newly formed blood vessels. By immunohistochemistry, mastocytoma tumor cells were shown to be positive for both chymase (D left, yellow arrows) and tryptase (D right, red arrows) with a granular cytoplasmic pattern.