Immunohistochemical Detection of Autophagy‐Related Proteins in Canine Appendicular Osteosarcoma

Abstract

Dogs diagnosed with appendicular osteosarcoma commonly succumb to metastatic disease despite undergoing the standard of care treatment for curative intent: amputation of the affected limb, followed by adjuvant chemotherapy. Post‐surgical chemotherapy improves one‐ and two‐year survival rates, but prognosis is still poor. In response to adverse environmental conditions normal cells are capable of undergoing autophagy, a survival mechanism involving self‐degradation and recycling of cellular components. Neoplastic cells take advantage of this mechanism to enhance their survival during chemotherapy. Autophagy's role in osteosarcoma has been investigated in humans however similar studies in dogs are rare. Our objective was to examine the expression patterns of a select group of autophagy‐related proteins in canine osteosarcoma amputation samples and to identify potential correlations with survival. Immunohistochemistry for beclin‐1, LC3, phospho‐mTOR, phospho‐AKT1, phospho‐S6 ribosomal protein, and p62 was performed on a tissue microarray containing naïve canine appendicular osteosarcoma cores from dogs who began the standard of care treatment (≥1 dose of chemotherapy). For survival analyses, Kaplan‐Meier survival curves for different expression patterns were compared using the log‐rank test and interactions were considered significant if p < 0.05. The median survival time from the date of diagnosis was 250 days and survival time ranged from 67–897 days (n=20, 95% CI: 122–381). Intense phospho‐S6 ribosomal protein labeling was identified most consistently adjacent to osteoid matrix. Phospho‐mTOR labeling was most evident in the nuclear compartment, however cells undergoing mitosis displayed strong cytoplasmic labeling. Canine osteosarcoma cells exhibited a mixture of diffuse and punctate LC3 cytoplasmic labeling, potentially distinguishing between free cytoplasmic LC3‐I and autophagosome anchored LC3‐II, respectively. Western blot analysis of the beclin‐1 antibody produced a 58 kDa band as predicted. Three distinct intensities of cytoplasmic labeling were observed for beclin‐1 in canine osteosarcoma cells. Strong cytoplasmic beclin‐1 labeling in the majority of neoplastic cells was not observed in any cases with greater than one‐year survival (0/6); this pattern was observed only in cases with less than one‐year survival (5/14). Dogs with tumours having light (n=4), moderate (n=9), and strong (n=5) cytoplasmic labeling had median survival times of 294, 301, and 122 days, respectively. Strong cytoplasmic labeling was a significantly correlated with decreased survival time (Chisq=6.9, 2 df, p < 0.05). This preliminary study provides evidence that canine osteosarcoma cells undergo autophagy in vivo, with a similar LC3 labeling pattern to that described in other tumour types in various species. Additionally, it identifies beclin‐1 as a promising candidate biomarker of chemoresponsiveness in canine appendicular osteosarcoma.Support or Funding InformationFunded by Pet Trust