We have previously reported that pre-treatment of mice with gelatin or its derivatives can induce resistance to transplantation of Ehrlich carcinoma 1-5). The tested gelatin products were derived non-mouse species such as cows, pigs, and whales and were thus xenogeneic to mice. In this study, we carried out the same experiment using allogeneic gelatin.

Materials and methods

Crude gelatin was obtained from mouse (ddY) skin according to the procedure described in the Japanese Pharmacopoeia. The crude gelatin was re-lysed, filtered, centrifuged, concentrated, and purified to obtain samples with a protein content of 55.4% (albumin equivalent).

Sample 1: mouse (ddY) gelatin
Sample 2: mouse (ddY) gelatin with amino groups methylene-polymerized with formaldehyde
Sample 3: gelatin (Merck)
Sample 4: untreated control

Aliquots (0.3 ml) of 1% aqueous solutions of gelatin samples 1, 2, and 3 were injected subcutaneously into the inguinal region of mice (ddY) once a week for a total of 5 times, and this was followed by subcutaneous transplantation of 5 x106 Ehrlich carcinoma cells, which had been subcultured in mice of the same strain (ddY), in the gluteal region 7 days after the last gelatin injection.
The results were as follows: the number of mice without engraftment of carcinoma cells out of the 10 mice injected with gelatin samples 1, 2, 3, and 4 was 0, 2, 4 and 0, respectively.

Method:

Although administration of polymerized gelatin reduced the occurrence of DMBA-induced mammary carcinoma, this observation is not adequate to draw a conclusion. Regardless of whether or not the altered immune status following the administration of polymerized gelatin affected the carcinogenic mechanism of DMBA, emerging carcinoma cells were rejected by the aforementioned anti-transplant immunity at a very early stage of tumor-bearing state. The fact that polymerized gelatin was more effective when given before, rather than after, the administration of DMBA suggests that its effect is not simply mediated by its pharmacologic activity.

1) Aliquots (0.3 ml) of 1% aqueous solution of the aforementioned ddY mouse (white fur)-derived gelatin was subcutaneously injected into the inguinal region of CRJ:C3H/Hecr mice (black fur) once a week, for a total of 7 times, and this was followed by subcutaneous transplantation of 5 x106 Ehrlich carcinoma cells, which had been subcultured in ddY mice, in the gluteal region 7 days after the last gelatin injection. Carcinoma cells were engrafted, and increased mass was observed in all 6 mice, resulting in death of all mice due to carcinoma.

2) In the control group, Ehrlich carcinoma cells, which had been subcultured in ddY mice, were transplanted into untreated CRJ:C3H/Hecr mice in the same manner. Red bean-sized masses were palpable in all 6 mice but subsequently reduced in size and eventually disappeared in 4 mice. These 4 mice survived while the remaining 2 died due to carcinoma within 1 month.

Conclusion and discussion

Mice injected with xenogeneic gelatin products, such as bovine/porcine gelatin, polymerized gelatin, collagen, and crude collage, developed resistance to Ehrlich carcinoma transplants 1). In ddY mice injected with allogeneic gelatin derived from mice of the same strain, no resistance to Ehrlich carcinoma cell transplants was observed. Methylene-polymerization of amino groups not only enhanced the anti-transplant immunity-inducing activity of xenogeneic gelatin, but also provided allogeneic gelatin with this activity. While some of the C3H mice transplanted directly with Ehrlich carcinoma cells, which had been subcultured in ddY mice, rejected the transplants, all C3H mice pre-treated with ddY gelatin accepted the ddY-subcultured carcinoma cells. Gelatin is generally considered a low-immunogenic material. However, in carcinoma transplantation studies, xenogeneic gelatin induced resistance to carcinoma transplants, allogeneic gelatin derived from the same strain of mice had no effect, and allogeneic gelatin derived from a different strain of mice facilitated engraftment of carcinoma transplants, as described above.

Mouse (ddY) gelatin was kindly produced and provided by Mr. Koichi Takahashi of Maruho Co., Ltd. This study was conducted at the Department of Microbiology, Osaka Medical College.