Dr. João António Ferreira

Ana Catarina Urbano

The Landscape of Tumor-Infiltrating Immune Cells in Feline Mammary Carcinoma: Pathological and Clinical Implications

Tumor microenvironment has gained great relevance due to its ability to regulate distinct checkpoints mediators, orchestrating tumor progression. Serum programmed cell death protein-1 (PD-1) and programmed death ligand-1 (PD-L1) levels were compared with healthy controls and with serum cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and tumor necrosis factor-alpha (TNF-α) levels in order to understand the role of PD-1/PD-L1 axis in cats with mammary carcinoma. PD-1 and PD-L1 expression was evaluated in tumor-infiltrating lymphocytes (TILs) and cancer cells, as the presence of somatic mutations. Results showed that serum PD-1 and PD-L1 levels were significantly higher in cats with HER2-positive (p = 0.017; p = 0.032) and triple negative (TN) normal-like mammary carcinomas (p = 0.004; p = 0.015), showing a strong positive correlation between serum CTLA-4 and TNF-α levels. In tumors, PD-L1 expression in cancer cells was significantly higher in HER2-positive samples than in TN normal-like tumors (p = 0.010), as the percentage of PD-L1-positive TILs (p = 0.037). PD-L1 gene sequencing identified two heterozygous mutations in exon 4 (A245T; V252M) and one in exon 5 (T267S). In summary, results support the use of spontaneous feline mammary carcinoma as a model for human breast cancer and suggest that the development of monoclonal antibodies may be a therapeutic strategy.

18 August 2022 in Cells

Journal Article

Diagnostic Value of VEGF-A, VEGFR-1 and VEGFR-2 in Feline Mammary Carcinoma

Feline mammary carcinoma (FMC) shares key molecular and clinicopathological features with human breast cancer. We have herein studied the inflammatory infiltrate of FMC in order to uncover potential therapeutic targets and prognostic markers. To this end, the expression of different markers (CD3, CD4, CD8, CD20, CD56, FoxP3, CD68 and CD163) was analyzed in total, stromal (s) and intratumoral (i) tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs), in 73 feline mammary carcinomas. The results revealed that higher percentages of sCD8⁺ TILs were associated with longer disease-free survival (p = 0.05) and overall survival (p = 0.021). Additionally, higher percentages of iCD4⁺ TILs correlated with positive lymph node status (p = 0.003), whereas CD163⁺ TAMs were associated with undifferentiated tumors (p = 0.013). In addition, sCD3⁺ (p = 0.033), sCD8⁺ (p = 0.044) and sCD68⁺ (p = 0.023) immune cells were enriched in triple negative normal-like carcinomas compared to other subtypes. Altogether, our results suggest that specific subsets of immune cells may play a major role in clinical outcome of cats with mammary carcinoma, resembling what has been reported in human breast cancer. These data further support the relevance of the feline model in breast cancer studies.

1 January 2021 in Cancers

Journal Article

PD-1 and PD-L1 are diagnostic biomarkers and PD-L1 a promising therapeutic target in HER2-positive and triple negative normal-like feline mammary carcinoma subtypes

Vascular endothelial growth factor (VEGF-A) plays an essential role in tumor-associated angiogenesis, exerting its biological activity by binding and activating membrane receptors, as vascular endothelial growth factor receptor 1 and 2 (VEGFR-1, VEGFR-2). In this study, serum VEGF-A, VEGFR-1, and VEGFR-2 levels were quantified in 50 cats with mammary carcinoma and 14 healthy controls. The expression of these molecules in tumor-infiltrating lymphocytes (TILs) and in cancer cells was evaluated and compared with its serum levels. Results obtained showed that serum VEGF-A levels were significantly higher in cats with HER2-positive and Triple Negative (TN) Normal-Like subtypes, when compared to control group (p = 0.001, p = 0.020). Additionally, serum VEGFR-1 levels were significantly elevated in cats presenting luminal A, HER2-positive and TN Normal-Like tumors (p = 0.011, p = 0.048, p = 0.006), as serum VEGFR-2 levels (p = 0.010, p = 0.046, p = 0.005). Moreover, a positive interaction was found between the expression of VEGF-A, VEGFR-1, and VEGFR-2 in TILs and their serum levels (p = 0.002, p = 0.003, p = 0.003). In summary, these findings point to the usefulness of VEGF-A and its serum receptors assessment in clinical evaluation of cats with HER2-positive and TN Normal-Like tumors, suggesting that targeted therapies against these molecules may be effective for the treatment of these animals, as described in human breast cancer.

27 April 2020 in bioRxiv

Preprint

Ana Catarina Urbano

Clinical Validation of a Size-Based Microfluidic Device for Circulating Tumor Cell Isolation and Analysis in Renal Cell Carcinoma

Tumor microenvironment has gained great relevance due to its ability to regulate distinct checkpoints mediators, orchestrating tumor progression. In order to understand the role of the PD- 1/PD-L1 axis in cats with mammary carcinoma, serum PD-1 and PD-L1 levels were compared with healthy controls and with serum CTLA-4 and TNF- α levels. PD-1 and PD-L1 expression was evaluated in TILs and cancer cells, as the presence of somatic mutations. Results showed that serum PD-1 and PD-L1 levels were significantly higher in cats with HER2-positive (p=0.017; p=0.032) and triple negative (TN) normal-like mammary carcinomas (p=0.004; p=0.015). Besides, cats presenting these tumor subtypes showed a strong positive correlation between serum PD-1, PD-L1, CTLA-4 and TNF- α levels. In tumors, PD-L1 expression in cancer cells was significantly higher in HER2-positive samples than in TN normal-like tumors (p=0.010), as the percentage of PD-L1-positive TILs (p=0.038). Results from the PD-L1 gene sequencing identified two heterozygous mutations in exon 4 (A245T, 3.8%; V252M, 42.3%) and one in exon 5 (T267S, 3.8%). In summary, results support that serum PD-1 and PD-L1 levels can be used as diagnostic biomarkers of HER2-positive and TN normal-like feline mammary carcinomas and suggest that the development of monoclonal antibodies may be a good therapeutic strategy.

7 May 2023 in International Journal of Molecular Sciences

Journal Article

Renal cell carcinoma (RCC) presents as metastatic disease in one third of cases. Research on circulating tumor cells (CTCs) and liquid biopsies is improving the understanding of RCC biology and metastases formation. However, a standardized, sensitive, specific, and cost-effective CTC detection technique is lacking. The use of platforms solely relying on epithelial markers is inappropriate in RCC due to the frequent epithelial-mesenchymal transition that CTCs undergo. This study aimed to test and clinically validate RUBYchip™, a microfluidic label-free CTC detection platform, in RCC patients. The average CTC capture efficiency of the device was 74.9% in spiking experiments using three different RCC cell lines. Clinical validation was performed in a cohort of 18 patients, eight non-metastatic (M0), five metastatic treatment-naïve (M1TN), and five metastatic progressing-under-treatment (M1TP). An average CTC detection rate of 77.8% was found and the average (range) total CTC count was 6.4 (0–27), 101.8 (0–255), and 3.2 (0–10), and the average mesenchymal CTC count (both single and clustered cells) was zero, 97.6 (0–255), and 0.2 (0–1) for M0, M1TN, and M1TP, respectively. CTC clusters were detected in 25% and 60% of M0 and M1TN patients, respectively. These results show that RUBYchip™ is an effective CTC detection platform in RCC.

6 January 2023 in Cancers

Journal Article

TIM-3 Is a Potential Immune Checkpoint Target in Cats with Mammary Carcinoma

Sofia Valente