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

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.


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The results obtained showed that cats presenting mammary carcinoma subtypes associated with

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Results from the immunostaining analysis revealed that in HER2-positive mammary carcinoma, presenting HER2-positive carcinomas, indicating that sPD-1 can bind to PD-L1 attached to the cell 197 membrane of dendritic cells, inhibiting T cell function and proliferation, as previously described [27].

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Lastly, three non-redundant heterozygous mutations were found in the feline PD-L1 gene, with 199 none of them located at the PD-L1 extracellular domain, which is recognized by the recent and

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Serum was separated from clotted blood by centrifugation (1500 g, 10 min, 4°C) and stored at -80°C 212 until further use. All samples that showed hemolysis were discarded, as recommended for humans 213 [28].

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Regarding the molecular-based subtyping of FMC [15,29], cats enrolled in this study were

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All the animals enrolled in this study were anesthetized before surgical procedures, with serum 227 and tumor samples being collected during anesthesia. Therefore, as there was no interference with animal well-being and all the procedures involving the manipulation of animals were consented by the owners, the Commission on Ethics and Animal Wellbeing considered that there was no reason 230 for additional advice.

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The plates were thereafter sealed and incubated for 2 hours at RT. After another washing step (3x400 255 μl/well PBS-Tween 0.05%), 100 μl/well of detection antibodies were added and incubated for 2 hours 256 at RT. Later and after three washes with 400 μl/well of PBS-Tween 0.05%, 100 μl/well of streptavidin-

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HRP were added and incubated for 20 min at RT, avoiding placing the microplate in direct light.

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(1:1 mixture of H2O2 and tetramethylbenzidine) were added and incubated for 20 min at RT in the 260 dark. Finally, the reaction was stopped by adding 50 μl/well of 2 N H2SO4 and gently shaking the 261 plate. The absorbance was measured by a spectrophotometer (Fluostar Optima Microplate Reader,

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BMG, Ortenberg, Germany), following the manufacturer's recommendations. negative controls and feline lymph nodes as internal controls. All slides were independently subjected to blind scoring by two independent pathologists.
Genomic DNA extraction was performed in 26 primary mammary tumors using a QIAamp 294 FFPE kit (Qiagen, Dusseldorf, Germany) following the manufacturer's recommendations. Initially,

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tumor samples were homogenized with Tissue Lyser II (Qiagen) and digested with protease K in a 296 concentration of 20 mg/ml (Qiagen). After several washing steps, the genomic DNA was eluted from 297 the extraction columns and its quantity and quality were measured by NanoDrop ND-100

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Spectrophotometer (Thermo Fischer Scientific). Then, the exons of interest in PD-L1 gene were 299 amplified by using specific primers (

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Statistical analysis was conducted in SPSS software version 25.0 (IBM, Armonk, USA) and the two-sided p-value <0.05 was considered statistically significant. Results were presented as median