Background: Neuroblastoma is the most common extracranial solid tumor in children. The survival rate in high-risk neuroblastoma is less than 50 % despite intensive multimodal therapy, and there is thus an immense need for new treatment options. In a previous preclinical study conducted at Uppsala University, treatment with sunitinib was found to inhibit tumor growth and angiogenesis in an orthotopic model of high-risk neuroblastoma.  Aim: The present study aimed to further explore the effect of sunitinib on tumor stroma, focusing on whether it was possible to detect and quantify tumor-associated neutrophils (TANs) in tumor sections from the above-mentioned study using immunohistochemistry (IHC). Methods: Tissue sections from formalin-fixated paraffin-embedded tumors were stained with anti-Ly-6G/Ly-6C, anti-ITGAM, or hematoxylin-eosin, and the number of granulocytes was quantified manually using a light microscope. An independent samples two-tailed t-test was used for statistical analysis. Results: The average number of granulocytes increased by 40 % in animals treated with sunitinib compared to control animals (p = 0.003) in hematoxylin-eosin stained tumor sections of orthotopic neuroblastomas. The results from the staining with anti-Ly-6G/Ly-6C or anti-ITGAM, on the other hand, were impossible to quantify due to the high background staining despite the concentration of antibody used. Conclusion: In conclusion, this report indicates that the density of TANs in an orthotopic murine neuroblastoma model is increased by treatment with sunitinib. However, to confirm this result, the study should be repeated once a reliable IHC method for the detection of TANs has been developed.

Neuroblastoma (NB), an extracranical solid tumor, is among the most prevalent cancers affecting children, particularly those under the age of five. High-risk NB presents a survival rate just below 50 %. Angiogenesis, a crucial process in NB, is induced by various pro-angiogenic factors. The compound SU11657 has demonstrated efficacy in inhibiting angiogenesis and tumor progression. Tumor-associated macrophages (TAMs) and Tumor-associated neutrophils (TANs) contribute to tumor progression, including angiogenesis, and their heightened levels within the tumor has been correlated with a poor clinical prognosis. This study aimed to quantify TANs and TAMs in NB tumors through manual assessment and the development of an automated digital image analysis. Unfortunately, due to time constraints, TAMs were not subjected to detailed analysis. Immunohistochemistry using antibody ab2557 and DAB staining was employed, and cell content analysis was performed through both manual assessment and digital analysis using QuPath. Successful differentiation of TANs was achieved with ab2557. The manual assessment observed a decrease of TANs between the control and treatment groups in UB7 and UB8, with UB7 being statistically significant (p<0.05), based on a two-tailed t-test. QuPath analysis noted increases in the percentages of TANs between the control and treatment groups, with the t-tests being non-significant (p>0.05). While digital image analysis is gaining importance in clinical applications, imperfections persist, underscoring the imperative for further research and development to accurately distinguish biomarkers.