International Journal of Molecular Sciences Article Canine Mammary Neoplasia Induces Variations in the Peripheral Blood Levels of CD20, CD45RA, and CD99 Makchit Galadima 1, Iuliia Kotova 2, Ronny Schmidt 2, Josep Pastor 1, Christoph Schröder 2 , Joan Enric Rodríguez-Gil 1 and Maria Montserrat Rivera del Alamo 1, * 1 Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; makchitgaladima@gmail.com (M.G.); josep.pastor@uab.cat (J.P.); juanenrique.rodriguez@uab.cat (J.E.R.-G.) 2 Sciomics GmbH, Karl-Landsteines-Straβe 6, 69151 Neckargemünd, Germany; iuliia90kotova@gmail.com (I.K.); ronny.schmidt@sciomics.de (R.S.); schroeder@sciomics.de (C.S.) * Correspondence: mariamontserrat.rivera@uab.cat; Tel.: +34-935811045 Abstract: The idea of using tumour biomarkers as diagnostic tools is progressively increasing. Of these, serum biomarkers are of particular interest, as they can provide rapid results. In the present study, serum samples from 26 bitches diagnosed with mammary tumours, plus 4 healthy bitches, were obtained. The samples were analysed using CD antibody microarrays targeting 90 CD surface markers and 56 cytokines/chemokines. A total of five CD proteins, namely CD20, CD45RA, CD53, CD59, and CD99, were selected and further analysed, utilizing immunoblotting techniques to validate the microarray results. CD45RA showed a significantly lower abundance in the serum samples from the bitches carrying mammary neoplasia in comparison to the healthy animals. Regarding CD99, the serum samples from the neoplastic bitches showed it in a significantly higher abundance than those from the healthy patients. Finally, CD20 showed a significantly higher abundance in bitches carrying a malignant mammary tumour in comparison to healthy patients, but no differential expression between malignant and benign tumours was observed. According to these results, both CD99 and CD45RA are indicators of mammary tumour presence, but without distinguishing between malignant and benign. Citation: Galadima, M.; Kotova, I.; Schmidt, R.; Pastor, J.; Schröder, C.; Rodríguez-Gil, J.E.; del Alamo, Keywords: canine mammary tumours; serum biomarkers; CD99; CD45RA; CD20 M.M.R. Canine Mammary Neoplasia Induces Variations in the Peripheral Blood Levels of CD20, CD45RA, and CD99. Int. J. Mol. Sci. 2023, 24, 9222. 1. Introduction https://doi.org/10.3390/ijms24119222 Mammary tumour is a life-threatening condition in both humans and bitches [1–3], al- Academic Editors: Galliera Emanuela though dogs display the highest incidence of mammary tumours among all the mammalian Rita and Elena Vianello species [1,4,5]. Canine mammary tumour (CMT) is the second most common tumour after skin cancer in dogs [1–3,6], especially in intact bitches [6–10]. About 50% of CMTs Received: 26 April 2023 are diagnosed as malignant and can metastasize to other organs through the vascular Revised: 17 May 2023 or lymphatics systems [8,11,12]. CMTs are prevalent globally, with a reported incidence Accepted: 19 May 2023 rate of 200/100,000 dogs/year, showing lower incidences in countries that practice early Published: 25 May 2023 spaying [8]. Mammary tumour development, like other neoplasia, is associated with inflammation [13,14], which is a normal quick response to acute tissue damage result- Copyright: © 2023 by the authors. ing from physical injury, ischemic injury, toxins, or other types of injury [13,14]. Whatever Licensee MDPI, Basel, Switzerland. its origin, inflammation in the tumour microenvironment (TME), mainly chronic, has many This article is an open access article cancer-promoting effects on and aids in the proliferation and survival of malignant cells, distributed under the terms and as well as promotes angiogenesis and metastasis [15]. The TME is composed of innate conditions of the Creative Commons immune cells (including macrophages, neutrophils, mast cells, myeloid-derived suppressor Attribution (CC BY) license (https:// cells, dendritic cells, and natural killer cells), adaptive immune cells (T and B lymphocytes), creativecommons.org/licenses/by/ tumour cells, and their surrounding stroma, which consist of fibroblasts, endothelial cells, 4.0/). pericytes, and mesenchymal cells [13–15]. Immune cells provide diverse molecules to this Int. J. Mol. Sci. 2023, 24, 9222. https://doi.org/10.3390/ijms24119222 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 9222 2 of 14 TME, such as growth factors, proangiogenic factors, and extracellular-matrix-modifying enzymes, among others [16–18]. Thus, the link between inflammation, mainly chronic, and immune cells in tumorigenesis is evident. To date, canine mammary diagnoses are usually performed by means of histopathol- ogy techniques. However, using tumour biomarkers as diagnostic tools has gained interest [11]. The promising mammary tumour biomarkers in both humans and canines are cancer-associated stroma (CAS) or TMEs, circulating tumour cells and tumour DNA (ctDNA), exosomes and miRNAs, and metabolites [19]. Variations in the expression of these markers may be helpful for achieving an early diagnosis, providing a prognosis, following the course of anti-tumoral therapy, predicting the response or resistance to specific therapies and the surveillant after primary surgery [11,20]. Nevertheless, CMT biomarkers are poorly studied and choosing the most appropriate biomarker remains the biggest challenge in optimizing tumour diagnoses [11,21]. Since early detection is crucial for the evolution of patients with mammary tumours, the determi- nation of these biomarkers is key to evaluating the disease progression and response to treatments [22]. However, tissue biomarkers, although useful, do not provide an improve- ment in CMT in terms of immediacy, whereas serum biomarkers open up an interesting possibility of improving the celerity of the diagnosis. The literature focused on biomarkers in the peripheral blood is abundant in human research, but it is nearly absent when focusing on CMT. Against this background, this study aimed at two different goals. The first objective aimed to determine the putative variations in the expressions of both the inflammatory and immune molecules in peripheral blood. On the other hand, it aimed to determine the potential of these molecules as serum biomarkers in canine mammary tumours. For these purposes, serum samples from 30 bitches were analysed through CD antibody microarrays targeting 90 CD surface markers and 56 cytokines/chemokines (Sciomics GmnH, Neckargemünd, Germany). Furthermore, the CD molecules showing differential expressions were validated using immunoblotting techniques. 2. Results 2.1. Animals and Histopathology Twenty-six patients carrying mammary neoplasia were submitted to the teaching veterinary hospital at the Universitat Autònoma de Barcelona (Bellaterra, Spain) for surgical removal. After performing the surgery, tissue samples were submitted to the laboratory for a histopathology evaluation. The histology analyses yielded a total of 12 bitches with benign mammary tumours, 10 carrying mammary carcinoma, and 4 carrying both mammary carcinoma and adenoma. On the other hand, the 4 healthy bitches were proven, using histopathology, to have normal mammary tissue. 2.2. Microarrays When comparing the samples from the healthy animals and the samples from the bitches carrying benign mammary tumours, 20 molecules recorded differential protein abundances in terms of either their log FC or p values (Table 1). However, only two proteins showed differential abundances in terms of both the log FC and p-value simultaneously, namely ITAX and CD177. The proteins with positive log FC values showed a higher abundance in the samples from the bitches with benign mammary tumours, whereas those with negative values showed a higher abundance in the samples from the healthy bitches. |log FC| > 1 and > −1 indicated a differential abundance between the analysed groups. p < 0.05 indicated statistically significant differences. Int. J. Mol. Sci. 2023, 24, 9222 3 of 14 Table 1. Proteins with differential abundance, benign tumours, cancer (n = 12) vs. healthy (n = 4) animal. Protein Antibody ID log FC AveExp Adj. p-Val Uniport-Link CD20 S0288 1.51 13.45 0.270 P11836 ICAM1 S0333 1.25 11.50 0.920 P05362 CD53 S0332 1.23 13.91 0.650 P19397 ICAM1 S0334 1.22 12.28 0.920 P05352 DAF S0335 1.08 9.49 0.370 P08174 MCP S0325 1.05 12.60 0.960 P15529 CD20 S0286 −0.96 11.50 0.039 P11836 CD139 S0438 −1.01 14.31 0.950 PDCD1 S0003 −1.01 12.57 0.960 Q15116 IL1β S0387 −1.04 14.53 0.950 P01584 IL6 S0530 −1.06 10.01 0.490 P05231 CCL3 S0393 −1.07 14.54 0.920 P10147 IL16 S0529 −1.09 11.88 0.720 Q14005 CD86 S0356 −1.10 10.98 0.960 P42081 TGM2 S0405 −1.14 9.07 0.650 P21980 CD79A S0354 −1.15 11.66 0.920 P11912 ITAX S0268 −1.22 11.04 0.036 P20702 KIT S0001 −1.24 13.51 0.960 P10721 CD24 S0294 −1.33 10.84 0.960 P25063 CD177 S0368 −1.34 10.62 0.039 Q8N6Q3 MPRI S0369 −1.42 11.61 0.090 P11717 IFNA1 S0402 −2.10 13.94 0.720 P01562 When comparing the samples from the healthy animals and samples from the bitches carrying malignant mammary tumours, 36 molecules recorded differential protein abun- dances in terms of either their log FC or p values (Table 2). When considering both the log FC and p values, six proteins showed differential abundances in terms of both the log FC and p value simultaneously, namely CD20, CD53, ITAX, FCG3A, CD177, and MPRI. The proteins with positive log FC values showed a higher abundance in the samples from the bitches with malignant mammary tumours, whereas those with negative values showed a higher abundance in the samples from the healthy bitches. |log FC| > 1 and >−1 indicated differential abundances between the analysed groups. p < 0.05 indicated statistically significant differences. Finally, when comparing the samples from the bitches carrying malignant mammary tumours or benign mammary tumours, 17 molecules recorded differential protein abun- dances in terms of either their log FC or p values (Table 3). When considering both the log FC and p values, seven proteins showed differential abundances in terms of both the log FC and p value simultaneously, namely PDCD1, IFNA1, CD44, CD20, CD15, IL37, and TNR8. The proteins with positive log FC values showed a higher abundance in the samples from the bitches with malignant mammary tumours, whereas those with negative values showed a higher abundance in the samples from patients carrying benign mammary tumours. |log FC| > 1 and >−1 indicated differential abundances between the analysed groups. p < 0.05 indicated statistically significant differences. 2.3. Immunoblotting The microarrays analysis yielded an elevated number of differential proteins between the groups of both the CD surface markers and the cytokines/chemokines. Of these, the five most relevant CD molecules were selected for further analyses, utilizing Western blotting for validation, namely CD20, CD45RA, CD53, CD59, and CD99 (Figure 1). The data obtained from the Western blotting were analysed using two different ap- proaches. On the one hand, the data were analysed by distributing the samples in the healthy and neoplasia groups. On the other hand, the data were distributed in the healthy, benign mammary neoplasia, and malignant mammary neoplasia groups. Int. J. Mol. Sci. 2023, 24, 9222 4 of 14 Table 2. Proteins with differential abundance, malignant tumours, cancer (n = 14) vs. healthy animal (n = 4). Protein Antibody ID log FC AveExp Adj. p-Val Uniport-Link CD20 S0288 2.57 13.45 0.002 P11836 MCP S0325 2.05 12.60 0.820 P15529 CD3 S0407 2.05 12.86 0.290 P07766 PD1L1 S002 1.96 12.88 0.820 Q9NZQ7 CD53 S0332 1.92 13.91 0.040 P19397 AMPN S0378 1.54 13.32 0.710 P15144 ICAM1 S0.334 1.47 12.28 0.480 P05362 CD44 S0424 1.43 14.10 0.190 P16070 CD22 S0292 1.40 11.35 0.420 P20273 CD99 S0360 1.31 11.75 0.430 P14209 CD8A S0254 1.15 12.13 0.480 P01732 CD44 S0315 1.12 12.40 0.520 P16070 EGLN S0363 1.02 11.72 0.820 P17813 CD20 S0286 −0.87 11.50 0.049 P11836 CEAM6 S0503 −1.00 12.45 0.630 P40199 IL7 S0388 −1.01 13.42 0.910 P13232 IL16 S0529 −1.02 11.88 0.470 Q14005 CEAM1 S050 −1.02 12.76 0.480 P13688 IL8 S0389 −1.08 11.41 0.190 P10145 LFA3 S0340 −1.10 12.29 0.910 P19256 CEAM5 S0348 −1.12 12.35 0.500 P06731 IL8 S0475 −1.15 10.33 0.420 P10145 ITAX S0268 −1.16 11.04 0.019 020702 CD24 S0294 −1.16 10.84 0.820 P25063 NTF4 S0397 −1.22 13.88 0490 P34130 CCL7 S0394 −1.22 13.59 0.710 P80098 FCG3A S0277 −1.23 10.50 0.011 P08637 CD177 S0368 −1.24 10.62 0.044 Q8N6Q3 CD53 S0331 −1.35 12.03 0.250 P19397 TGM2 S0405 −1.36 9.07 0.230 P21980 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEWCCL11 S0382 −1.37 14.23 0.370 5 of 14 P51671 IL15 S0391 −1.39 14.48 0.290 P40933 CD139 S0438 −1.42 14.31 0.480 IL1b S0387 −1.43 14.53 0.480 P01584 2.3. Immunoblotting BDNF S0381 −1.50 14.15 0.400 P23560 TheIL18 microarrays analysis −1.52 number of14.34 S0392yielded an elevated 0.290be- differential proteins Q14116 MPRI tween the S069 groups of both the −1.65and the cytokines/chemokines. CD surface markers 11.61 Of0.017 these, P11717 the fiveCCL3 most relevant CD S0393 −1.75 for further analyses, molecules were selected 14.54 utilizing Western 0.230 P10147 CD86 S0356 −2.12 10.98 0.390 P42081 blotting for validation, namely CD20, CD45RA, CD53, CD59, and CD99 (Figure 1). Figure 1. Western blotting images. H: healthy bitches; B: bitches carrying benign mammary tu- Figure 1. Western blotting images. H: healthy bitches; B: bitches carrying benign mammary tumours; mours; M: bitches carrying malignant mammary tumours; and MK: molecular weight marker. M: bitches carrying malignant mammary tumours; and MK: molecular weight marker. The data obtained from the Western blotting were analysed using two different ap- proaches. On the one hand, the data were analysed by distributing the samples in the healthy and neoplasia groups. On the other hand, the data were distributed in the healthy, benign mammary neoplasia, and malignant mammary neoplasia groups. When comparing the healthy and neoplastic samples, CD99 showed a significant over- expression in the neoplastic samples (Figure 2D), whereas CD45RA showed significantly Int. J. Mol. Sci. 2023, 24, 9222 5 of 14 Table 3. Proteins with differential abundance, malignant (n = 14) vs. benign tumours (n = 12). Protein Antibody ID log FC AveExp Adj. p-Val Uniport-Link PDCD1 S0003 1.82 12.57 0.012 Q15116 IFNA1 S0402 1.72 13.94 0.008 P01562 CD44 S0315 1.31 12.40 0.007 P16070 KIT S0001 1.24 13.51 0.370 P10721 CD3 S0407 1.11 12.86 0.170 P07766 PD1L1 S0002 1.08 12.88 0.550 Q9NZQ7 CD20 S0288 1.07 13.45 0.011 P11836 MCP S0325 1.00 12.60 0.640 P15529 IL37 S0473 −0.42 10.27 0.012 Q9NHZ6 PTPRC S0323 −068 11.29 0.012 P08575 CD38 S0308 −0.78 10.15 0.011 P28907 IL8 S0475 −0.80 10.33 0.021 P10145 NCAM1 S0337 −0.94 11.60 0.013 P13591 CD86 S0356 −1.03 10.98 0.200 P42081 CD15 S0273 −1.04 11.41 0.012 LFA3 S0340 −1.04 12.29 0.550 P19256 IL37 S0474 −1.11 11.36 0.008 Q9NZH6 TNR8 S0302 −1.13 12.34 0.021 P28908 When comparing the healthy and neoplastic samples, CD99 showed a significant over- Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW expression 6 of 14 in the neoplastic samples (Figure 2D), whereas CD45RA showed significantly lower values in the serum from the bitches with mammary neoplasia (Figure 2D). Comparative expression Figure 2. Comparative expression of of peripheral peripheralblood bloodlevels levelsfor for CD53 CD53 (A), (A), CD59 CD59 (B), (B), CD99 CD99 (C), (C), CD45RA (D), and CD20 CD20 (E) (E) between between healthy healthy (n == 4) 4) and and mammary mammary neoplasia (n = 26) bitches. Results are expressed inin terms terms of of mean mean±±SDSDfrom fromtwo twoimmunoblotting immunoblotting replicates. Statistically replicates. significant Statistically (p significant < 0.05) (p differences < 0.05) areare differences marked markedwith asterisks. with asterisks. When comparing the healthy, benign, and malignant samples, CD99 showed a sig- nificant overexpression in the neoplastic samples, regardless of if they were benign or malignant, in comparison to the healthy samples (Figure 3D). In contrast, only the carci- noma samples showed a significant overexpression of CD20 (Figure 3E) in comparison to Int. J. Mol. Sci. 2023, 24, 9222 6 of 14 Figure 2. Comparative expression of peripheral blood levels for CD53 (A), CD59 (B), CD99 (C), CD45RA (D), and CD20 (E) between healthy (n = 4) and mammary neoplasia (n = 26) bitches. Results theexpressed are healthy bitches. However, in terms of nofrom mean ± SD significant difference between two immunoblotting theStatistically replicates. benign and malignant significant (p 1 and a p value < 0.05. The differences in the protein abundances between the different samples or sample groups were presented as log-fold changes (log FC) calculated for the basis 2. In a study comparing samples versus controls, a log FC = 1 means that the sample group has, on average, a 21 = 2-fold higher signal than the control group. log FC = −1 stands for 2−1 = 1/2 of the signal in the sample, as compared to the control group. The raw data obtained from the immunoblotting technique were analysed by applying a Shapiro–Wilk test, which was used to study the normality distribution. When a param- eter did not follow a normal distribution, a non-parametric test was used to assess the differences between the groups. The Kruskal–Wallis test was used when more than two variables were compared with multiple comparison tests and the Mann–Whitney test when two variables were compared. The correlations between the parameters were studied using Pearson or Spearman correlation coefficients, according to their distribution. The statistical analysis was performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, CA, USA, (URL: www.graphpad.com). 5. Conclusions Canine mammary tumours induce variations in the peripheral concentrations of CD20, CD45RA, and CD99. However, these differences are only significant between animals carrying mammary tumours and healthy bitches, thus not allowing for a differentiation between malignant and benign CMT. Further research is warranted to determine the novel serum biomarkers that allow for more accurate and celeritous diagnoses. Author Contributions: Conceptualization, R.S., C.S., J.E.R.-G. and M.M.R.d.A.; methodology, M.G. and I.K.; data curation, I.K., J.P., R.S. and M.M.R.d.A.; writing—original draft preparation, M.G., I.K. and M.M.R.d.A.; writing—review and editing, J.P., J.E.R.-G. and M.M.R.d.A. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The animal study protocol was approved by the Ethics Committee of the UNIVERSITAT AUTÒNOMA DE BARCELONA (protocol code CEEAH 1127, 20 March 2012). Int. J. Mol. Sci. 2023, 24, 9222 11 of 14 Informed Consent Statement: Not applicable. Data Availability Statement: Data are available at the authors’ request. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Table A1. List of evaluated proteins by means of microarrays. ANCA CD45RO CYTL BDNF CD46 Eotaxin-1 CD1a CD47 GDC CD2 CD48 GM-CSF CD3 CD49d HCD137 CD4 CD50 HLA CD5 CD52 HLA-ABC CD6 CD53 HLA-DP CD7 CD54 HLA-DR CD8 CD55 HLA-I CD9 CD56 IFN CD10 CD57 IFN alpha CD11a CD58 IFNg CD11b CD59 IFNy CD11c CD61 IgE CD13 CD62L IL-1 CD14 CD62p IL-10 CD15 CD63 IL-12B CD16 CD66e IL-12p70 CD17 CD69 IL-13 CD18 CD71 IL-15 CD19 CD72 IL-16 CD20 CD79a IL-18 CD21 CD80 IL-1a CD22 CD86 IL-1b CD23 CD95 IL-37 CD24 CD97 IL-4 CD25 CD98 IL-6 CD27 CD99 IL-7 CD28 CD99R IL-8 CD29 CD105 MICP-2 CD30 CD106 MCP-E CD31 CD116 MIP-1a CD33 CD117 MIP-4 CD34 CD123 MPO CD35 CD139 NT-4 CD36 CD147 NTAL CD37 CD162 P53 CD38 CD177 P72Syk CD40 CD222 Pan CD41 CD235a PD1 CD41a CD235ab PDL1 CD41b CDw131 RANTES CD42b CEACAM1 TNFa CD43 CEACAM3 TRAIL CD44 CEACAM5 TSLP CD45 CEACAM6 TSLPR CD45RA CEACAM8 tTG CD45RB COX1 CYTL Int. J. Mol. Sci. 2023, 24, 9222 12 of 14 References 1. 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