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. 2024 Aug 6:46:e001524.
doi: 10.29374/2527-2179.bjvm001524. eCollection 2024.

Analysis of VEGFR-2 and PDGFR-β expression in canine splenic hemangiosarcoma to identify drug repositioning candidates

Igor Simões Tiagua Vicente  1 , Fernanda Barthelson Carvalho de Moura  2 , Juliana Moreira Rozolen  3 , Denner Santos Dos Anjos  4 , Renata Afonso Sobral  5 , Carlos Eduardo Fonseca Alves  1   4   6
Affiliations

Analysis of VEGFR-2 and PDGFR-β expression in canine splenic hemangiosarcoma to identify drug repositioning candidates

Igor Simões Tiagua Vicente et al. Braz J Vet Med. .

Abstract
in English, Portuguese

Splenic tumors are very common in dogs, and canine hemangiosarcoma (HSA) is one of the most important malignant splenic tumors. Surgery followed by chemotherapy (anthracycline-based protocols) is recommended for treating canine HSA; however, patients still do not achieve long-term survival. Therefore, this research aimed to assess vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet-derived growth factor receptor-β (PDGFR-β) gene expression in formalin-fixed tissues, evaluate the quality of mRNA for quantitative polymerase chain reaction (qPCR) analysis and identify drug repositioning candidates based on VEGFR-2 and PDGFR-β. qPCR analysis identified the relative expression of heterogeneous VEGFR-2 and PDGFR-β, with samples showing no transcripts or very low expression and those with higher relative quantification for both genes. We then used immunohistochemistry to correlate the relative quantification of VEGFR-2 and PDGFR-β transcripts with respective higher protein expression to validate our results. In the next step, we evaluated drug repositioning candidates and identified small molecule inhibitors (i.e. sorafenib) and natural compounds (curcumin and resveratrol) with the ability to block VEGFR-2 and PDGFR-β genes. Overall, our results indicated that VEGFR-2 and PDGFR-β expression is highly variable among canine HSA samples and different drugs can block the expression of both genes. Therefore, a personalized approach could be useful for selecting anti-VEGFR-2 and PDGFR-β therapies and both genes are potential candidates for future oncological panels.

Os tumores esplênicos são muito comuns em cães, e o hemangiosarcoma (HSA) é um dos tumores esplênicos malignos mais importantes em cães. A cirurgia seguida de quimioterapia (protocolos baseados em antraciclinas) é a abordagem terapêutica mais recomendada para o tratamento da HSA canino; no entanto, os pacientes ainda não alcançam longa sobrevida após tratamento. Portanto, esta pesquisa teve como objetivo avaliar a expressão do receptor do fator de crescimento endotelial vascular-2 (VEGFR-2) e do receptor do fator de crescimento derivado de plaquetas-β (PDGFR-β) em tecidos fixados em formalina e identificar candidatos ao reposicionamento de medicamentos baseado na expressão desses genes. A análise qPCR identificou a expressão relativa heterogênea de VEGFR-2 e PDGFR-β, com amostras sem transcritos ou com expressão muito baixa ou amostras com alta quantificação relativa para ambos os genes. Em seguida, foi realizada o exame imuno-histoquímico para correlacionar a quantificação relativa dos transcritos de VEGFR-2 e PDGFR-β com a respectiva maior expressão proteica para validar nossos resultados. Na próxima etapa, avaliamos candidatos ao reposicionamento de medicamentos e identificamos inibidores de moléculas pequenas (ou seja, sorafenibe) e compostos naturais (curcumina e resveratrol) com capacidade de bloquear os genes VEGFR-2 e PDGFR-β. No geral, nossos resultados indicaram que a expressão de VEGFR-2 e PDGFR-β é altamente variável entre amostras caninas de HSA e diferentes drogas podem bloquear a expressão de ambos os genes. Portanto, uma abordagem personalizada poderia ser útil para selecionar terapias anti-VEGFR-2 e PDGFR-β e ambos os genes são potenciais candidatos para futuros painéis oncológicos.

Keywords: dogs; drug repurposing; endothelial cells; spleen.

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Conflict of interest statement

Conflict of interests: Two authors of this study are all current employees of VetPrecision Laboratory (I.S.T.V. and C.F.F-A) and Institute of Veterinary Oncology – IOVET (C.E.F-A), and hold vested or unvested equity in both companies. This does not alter the authors’ adherence to Cell Insight policies.

Figures

Figure 1
Figure 1. Representative immunohistochemistry scores for VEGFR-2 and PDGFR-β expression. (A) VEGFR-2 score of 4+ in a capillary histopathological subtype; (B) PDGFR-β expression in a capillary tumor with a score of 4+. Capillary HSA with a score of 3+ for VEGFR-2 (C) and PDGFR-β (D); (E) Solid HSA representing a score of 1+; (F) Solid HSA negative for PDGFR-β. Counterstain was performed with Harris Hematoxylin, 40x.
Figure 2
Figure 2. Statistical analysis of VEGFR-2 and PDGFR-β gene and protein expression in canine hemangiosarcoma (HSA). A positive correlation is observed between relative gene and protein expressions of VEGFR-2 (A) and PDGFR-β (B); (C) Absence of statistical difference when comparing VEGFR-2 transcripts with histopathological subtypes; (D) Strong association between histological subtypes and relative expression of PDGFR- β.
Figure 3
Figure 3. Multivariate analysis for VEGFR2 and PDGFR-β. When the expression of VEGFR2 and PDGFR-β receptors was assessed, there was a lower number of correlations with the patients´ clinical data. In the multivariate analysis, there was a moderate negative correlation between PDGFR-β expression and patient survival (R = -0.42). Younger patients showed moderate correlation with a higher probability of metastasis (R = -0.43).
Figure 4
Figure 4. Survival analysis of VEGFR-2 and PDGFR-β gene expression and patient survival time. Expression of both the genes was not associated with patient survival.
Figure 5
Figure 5. Representation of the different drug classes that can block VEGFR-2 and PDGFR-β expression.
See this image and copyright information in PMC

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