Detection of bimodal survivin expressions in canine cancer types by flow cytometry compared to immunohistochemistry

Abstract

Animal practice requires both convenience for the owner and risk management for the animal's health. Deterioration due to cancer may associate with poor prognosis under general anesthesia, which need to partial excision for pathological diagnosis. This study aimed to establish rapidly detecting the expression of survivin antigens for cancer vaccines or molecular targeted therapies via flow cytometry (FCM) using the intracellular staining method in tumor samples obtained via needle biopsy without anesthesia. Therefore, survivin expression patterns in each cell lines of canine melanomas, a murine mast cell tumor, a murine colon carcinoma, and a murine melanoma was analyzed by FCM and immunofluorescence microscopy, and compared with immunohistochemical analysis and western blot method. Interestingly, FCM results of the bimodal expression pattern of survivin were suggested to reflect the high fluorescence intensity of its nuclear-cytosol localization and the weak fluorescence intensity of its cytosol alone localization. In a case of canine cancer disease, it was confirmed that survivin expression patterns can be detected via FCM using needle biopsy samples in actual clinical settings. In this study, a novel method via FCM was proposed to quickly determine also survivin localization not only whether the survivin is expressed in cancer cells. The application of cancer vaccine or chemical therapy via this technology can be expected to contribute to improved animal care due to the "one-day first program," which has been proposed in convenience for owners.

Keywords: B16F10; CMM2; CMeC2; CT26; LMeC; flow cytometry; p815; survivin.

Figure 1

Analysis of survivin expression sites by IHC and WB. (A) Images of HE staining and survivin expression via IHC in canine melanoma cell lines (CMM2, CMeC, and LMeC) are shown. (B) Images of HE staining and survivin expression via IHC in murine cell lines (p815, CT26, and B16F10) are shown. (C) The cytosol and total survivin expression patterns via WB are indicated. (D) The expression levels of total survivin (blue bar) and cytosolic survivin (orange bar) corrected on the basis of β-actin expression are shown via density of plot analysis via ImageJ software.

Figure 2

Analysis of survivin expression patterns by FCM and IM and the tests in a clinical case. (A) The fluorescence intensity of APC-survivin in each cell line detected by FCM is shown. (B) Representative survivin expression patterns in each cell line by IM are shown. (C) The percentages of high fluorescence intensity of survivin expression in each cell line are indicated. *p < 0.05, one-way ANOVA. (D) Survivin expression after needle biopsy in a clinical case of canine oral melanoma (female, 9 years old) was analyzed via FCM. The percentage of cells with high fluorescence intensity was 57.46%. Similar findings were detected in four other cases of oral melanoma.

Figure 3

“One-day first program” for a cancer check and vaccination. At the university's veterinary hospital, after an interview and an informed consent form (ICF) with the owner in the morning, the patient dog is taken in, and a general CBC blood test is performed to check its health condition. Needle biopsy will be performed on the tumor, the cells will be stained via an APC-conjugated anti-survivin mAb after detergent treatment following cell membrane fixation, and the presence or absence of survivin expression will be examined by FCM. If the tumor expresses survivin, the vaccine after production will be administered intradermally, such as through the axilla. After confirming that there is no anaphylactic reaction, the patient's dog waits until the owner returns to pick up after 5 p.m.