Tumor eradication using methylene blue induced photodynamic therapy in mouse model: an experimental study

ABSTRACT

Thirty nude female Swiss Albino mice of weight 18-22gm were used in this study. The mice were subdivided into three subgroups of 10 mice each. Group A was to be maintained without any tumor cell implantation to provide normal cell readings on the spectrometer and to supply a reference for normal histopathological specimens. Groups B and C were to be injected with tumor cells and methylene blue dye intralesionally. Group C mice were to receive PDT treatment while Group B mice were intended to act as a control for such treatment modality by receiving MB+ injection only without any irradiation. Measurements performed included the change in tumor volume as compared to the value recorded at the start of the experiment, after a one week incubation period. Ceased mice were dissected to remove the tumor mass and thereby measuring its volume. Survival rates for each group were calculated throughout the following weeks. The tumor investigated in this study was a solid Ehrlich carcinoma which is a transplantable, poorly differentiated malignant tumor which appeared originally as a spontaneous breast carcinoma in mice. A line of Ehrlich ascites carcinoma [EAC] was supplied from the breading unit of the National Cancer Institute Cairo University. A line of 1X10 [EAC] cells was transplanted by subcutaneous inoculation in the submandibular area. The tumor developed seven days after injection at which time its size was considered appropriate for PDT treatment. Thereafter, Methylene blue 0.1 ML concentration 2% was injected into the lesion. The statistical significance of the therapeutic effect of PDT as a treatment modality was analyzed. Methylene blue intratumoral injection alone without irradiation did not result in any macroscopic cytotoxicity or inhibition of tumor growth. PDT as a treatment ' modality proved to be highly effective. Irradiation of the incubated tumors with 550 J/cm[2] led to almost complete macroscopic regression of the tumors towards the end of the third week. Histological examination of the scar proved complete destruction of the lesion. Overall comparison of tumor volume regardless of the time of death or sacrifaction showed a highly statistical significant difference in favor of PDT treated group. [p=0.001] Median overall survival was 4 weeks in PDT treated group compared to one week in the group injected with methylene blue only, [p = 0.02]. The variation in the structural integrity of the cells as studied by Raman spectroscopy indicated a] shifting of bands which indicate chemical reaction and structural changes, b] difference in relative intensities of the bands of untreated and treated specimens. Readings were recorded for Group A [normal cells] mice, Group B [methylene blue injected only] mice and Group C [PDT] treated mice. The results of the Raman spectroscopy of the test specimens were recorded. The spectra of transformed malignant tissues differed markedly from those of normal and treated tissues .In most regions of spectra, the difference was evident as increase in the intensity of the absorbance from malignant tissues in comparison to normal and treated tissues. It thus seems that Raman spectroscopy could be used for the study and characterization of cells at various stages of their life cycles. Our results support the feasibility of developing spectroscopy as an easy yet sophisticated method for detecting malignant cells and possibly also for discriminating among cells at various stages in their life cycles