Abstract
Background/Aim: Foxp3 is a transcription factor responsible for the formation of T regulatory lymphocytes. Foxp3 expression can be associated with either neoplastic progression or regression. The aim of the study was to evaluate Foxp3 expression in soft tissue tumours (fibromas and fibrosarcomas) of skin and subcutaneous tissue in dogs and to describe its relationship with tumour malignancy grade. Materials and Methods: The study was conducted on 71 skin and subcutaneous tumours including 31 fibromas and 40 fibrosarcomas. The samples underwent histological and immunohistochemical evaluation using anti-Foxp3, anti-Ki, and vimentin antibodies. Results: Cytoplasmic expression of Foxp3 protein in the cutaneous and subcutaneous fibrosarcomas in dogs was confirmed. Moreover, a positive relationship between the expression of Foxp3 and tumour malignancy grade and between Foxp3 and Ki-67 expression was noted. Conclusion: A positive correlation between the Foxp3 expression intensity and malignancy grade suggests a significant role of Foxp3 in the carcinogenesis of skin and subcutaneous fibrosarcomas in dogs. Increased expression of Foxp3 may have a positive effect on the progression of cancer.
Skin and subcutaneous tissue fibrosarcomas are malignant tumours of mesenchymal origin that belong to the soft tissue sarcomas (STS) group (1). STS constitute over 15% of all skin and subcutaneous tissue tumours in dogs (2). The pathogenesis of those tumours in dogs is not clear; there are reports suggesting a relationship between fibrosarcomas and changes in chromosomes 11 and 30 but also between fibrosarcomas and injections, inflammation, or trauma (3, 4). The tumours are usually locally invasive, infiltrating the surrounding tissue, although they can be accompanied by remote metastases in around 17% of cases. The metastatic foci are most often encountered in lungs, liver, bones, and brain (5, 6).
Despite the development of veterinary medicine, skin, and subcutaneous tissue fibrosarcomas are still diagnostically and therapeutically challenging. Early recognition and surgical resection provide the best therapeutic outcomes (7). In recent years immunohistochemical examination is performed more frequently; it is helpful in histological evaluation, especially in cases of low-differentiated tumours.
Fibromas derive from fibrocytes originating from mesenchymal tissue. In contrast to fibrosarcomas, they are benign tumours. They appear most often on the head and limbs of the animal. The surgical resection leads to complete cure in the majority of cases (8). Fibromas can be soft or hard; usually oval, often can be pedunculated. Microscopically they are usually uniform and mitotic figures appear rarely (9).
Foxp3 is a transcription factor localised in the nucleus and is responsible for gene expression (10). In physiological conditions it controls the formation of regulatory T cells (Treg) (11).
The influence of Foxp3 expression on neoplasms has been not clearly explained. Papers discussing that topic show inconsistent results. It has been shown that Foxp3 expression can be related to either progression or regression of the neoplasm (11-13).
Hitherto no research on Foxp3 expression in fibromas and fibrosarcomas in dogs has been conducted to date. Ki-67 expression is used as a prognostic marker in human neoplasms. In canine fibrosarcomas it is not a routinely used marker, mainly due to its cost. Ki-67 is a protein present only in the active phases of cell cycle; it is therefore considered a proliferative marker. Latest research presents the possibility of evaluating the presence of Ki-67 not only in neoplastic tissues but the canine serum as well. This provides the potential of assessing the risk of neoplastic disease occurrence in animals (14). Such research reflects on the continuous development of veterinary medicine and the necessity to implement advanced solutions in daily practice. Therefore, the study on the presence of Foxp3 in the neoplastic cells of canine fibrosarcomas can improve the understanding of tumour biology of these tumours. The analysis of animal material is also often useful in explaining the processes that occur in human tissues (15).
The aim of the study was to evaluate expression levels of Foxp3 in fibroma and fibrosarcoma cells to assess a correlation between the Foxp3 expression and degree of tumour histological malignancy and Ki-67 expression.
Materials and Methods
Samples. Experiments were conducted on tumours collected from dogs; the tumours were archived in the Division of Pathomorphology and Veterinary Forensics Wroclaw University of Environmental and Life Sciences. A total number of 71 tumours were studied including 40 fibrosarcomas and 31 fibromas. The research material was collected from animals aged 4-15 years, in 56% from females and 44% from males. The most frequent breed were crossbreeds (37%), followed by Labrador retrievers (10%), Bernese Mountain Dog, German Shepherds, Boxers (6% each), golden retrievers, Siberian husky (4% each), beagles, Yorkshire Terriers (3% each), Bavarian Mountain Hounds, English cocker spaniels, medium poodles, French bulldogs, American Staffordshire terriers, Doberman pinchers, Weimaraner, Polish Hunting Dogs, miniature schnauzers, English springer spaniels, pugs, Maltese dogs, English bulldogs and Shar Pei (1.5% each).
Histological evaluation. The obtained samples were fixed in 7% buffered formalin for 24h, embedded in paraffin blocks, cut in 4 μm thick slices and stained with haematoxylin-eosin (Figure 1). The histological sections were evaluated according to WHO guidelines (16). The evaluation included three parameters, each scored using 3-grade scale: 1) histological differentiation of tumour cells (1: cells well-differentiated; 2: cells poorly-differentiated; 3: cells without features of differentiation); 2) number of mitoses counted within the area of 2,37 mm2 (1: 0-9 mitoses; 2: 10-19 mitoses; 3: over 19 mitoses); 3) the extent of necrosis (0: no necrosis in the tumour; 1: necrosis covering ≤50% of the tumour area; 2: necrosis covering >50% of the tumour area). The score of each parameter was added leading to final grading: G1 (2-3 points), G2 (4-5 points) or G3 (6-8 points) (17).
Histological analysis of examined tumours. The figure shows a fibrosarcoma (malignancy grade: G3). Tumour shows sparse intracellular matrix with dense lying cells. Apparent anisocytosis, anisokaryosis and multiple mitotic figures are visible. Haematoxylin-eosin stain. Magnification: 400×.
The paraffin sections were placed on salinized microscope slides and underwent a standardised immunohistochemical staining procedure. First, deparaffinization, rehydration and antigen retrieval were performed using EnVision FLEX Target Retrieval Solution Low-pH (Dako, Glostrup, Denmark; 97°C, 20 min). Activity of endogenous peroxidase was blocked by 10 min incubation with EnVision FLEX Peroxidase-Blocking Reagent (DakoCytomation, Glostrup, Denmark). The sections were overlayed with primary antibodies, including a mouse monoclonal anti-Ki-67, clone MIB-1 (dilution 1:100; Dako); a mouse monoclonal anti-vimentin, clone V9 (dilution 1:100; Dako) and rabbit polyclonal anti-FOXP3 (dilution 1:200; Novus Biologicals, Warsaw, Poland) for 20 min. No secondary antibodies were used. Then slides were washed with EnVision™ FLEX WashBuffer and incubated with EnVision FLEX/HR SM802 (20 min). 3,3′-diaminobenzidine (DAB, Dako) was utilized as the peroxidase substrate and the sections were incubated for 10 min. Finally, all sections were counterstained with EnVision FLEX Hematoxylin (Dako) for 5 min. After dehydration in graded ethanol concentrations (70%, 96%, 99,8%) and in xylene, slides were closed with coverslips in Dako Mounting Medium (DakoCytomation). Sections immunostained in the absence of a primary antibody were used as negative controls.
Immunohistochemistry. The expression of examined proteins was evaluated in 400× magnification using Olympus BX53 light microscope equipped with SC50 CMOS digital camera (Olympus, Tokyo, Japan). The analysis was performed using cell^A and cell^B software (Olympus Soft Imaging Solution GmbH, Münster, Germany). The evaluation was performed independently by three pathologists.
Due to complete lack of nuclear reaction of Foxp3 in examined material (including fibromas and fibrosarcomas), only cytoplasmic reaction of the protein was evaluated. Levels of cytoplasmic Foxp3 expression were assessed using a modified semi-quantitative IRS scale according to Remmele (18). The method takes into account both the percentage of positive cells and the intensity of the reaction, and the final score presents as a product of both parameters ranging from 0 to 12 points (no reaction (−): 0 points; weak reaction (+): 1-2 points; moderate reaction (++): 3-5 points; strong reaction (+++): 6-12 points). The mitotic activity was visualized using anti-Ki-67 antigen (19). The expression of Ki-67 was evaluated quantitatively by assessing the percentage of positive nuclear reaction (no reaction (−): 0=5%; weak reaction (+): 6-25%; moderate reaction (++): 26-50%; strong reaction (+++): >50%). The mesenchymal origin of examined tumours was confirmed by a strong reaction of vimentin according to Vascelari et al. and Madej (20, 21).
Statistical analysis. Data were evaluated using Statistica 13.3 (TIBCO Software Inc, Kraków, Poland) and appropriate statistical tests. Data normality was tested using Shapiro-Wilk analysis. The relationship between the data was tested using Spearman correlation analysis. The differences between sexes were tested using Mann-Whitney U analysis. The tests were considered statistically significant if p<0.05.
Results
No expression of Ki-67 and Foxp3 was found in the examined fibromas although they presented intense vimentin reaction. The lack of Ki-67 expression in fibromas is probably related to a low mitotic activity of these benign tumours. We cannot explain the concomitant lack of Foxp3 expression in fibromas as data in the literature are scarce. We hypothesize that it can be related to the benign character of tumours.
In fibrosarcomas, in addition to vimentin expression, both Foxp3 and Ki-67 expression was observed (Figure 2, Figure 3, Figure 4). Almost 30% of fibrosarcomas showed moderate or strong Foxp3 expression. Statistical analysis revealed a positive correlation between histological malignancy grading and the intensity of Foxp3 (p<0.05; r=0.54). Moreover, a positive correlation between the intensity of Foxp3 expression and Ki-67 expression was noted (p<0.05; r=0.44). The results are shown in Table I.
Expression of vimentin in the cytoplasm of fibrosarcoma cells showing intense reaction. The histological malignancy grade of the tumour is G2. Magnification: 400×.
Expression of Ki-67 in the nuclei of fibrosarcoma cells showing a moderate reaction. The histological malignancy grade of the tumour is G3. Magnification: 400×.
Expression of Foxp3 in the cytoplasm of fibrosarcoma cells showing a moderate reaction. The histological malignancy grade of the tumour is G2. Magnification: 400×.
Comparison of histological malignancy grading, Foxp3 expression and Ki-67 expression in fibrosarcoma tumours.
Discussion
The expression of various novel proteins and their relationship with the tumour histological appearance and its biological behaviour is the topic of recent research (22). The use of immunohistochemistry and molecular biology allows to update the classification of benign and malignant tumours (22). It also allows a better understanding of tumour biology, opening possibilities of new treatment strategies (22).
The presence of Foxp3 in neoplastic lesions has been previously evaluated by various researchers, although these studies mainly focused on its role as a transcription factor. Foxp3 plays a key role in the differentiation of Treg cells (10, 11). Nonetheless, the results presented in the literature do not allow for unambiguous conclusions to be drawn: they are inconsistent or even conflicting. It was noted that overexpression of Foxp3 can be related to either positive or negative prognosis and seems to be dependent on the type of the neoplasm. Moreover, interactions between the neoplastic cells and lymphocytes in the tumour microenvironment may play a role (10).
Foxp3 expression has been noted in cells of various types of neoplasms, including lung cancer, colon cancer, breast cancer and melanoma, where it was related to a neoplastic regression (10, 11). Moreover, study on human ovarian cancer showed a relation between protein expression and limitation of tumour invasiveness and tumour cell migration (12).
Opposite conclusions were drawn by Zhang et al. in a study of Foxp3 expression in human stomach cancer. Their study showed presence of protein in both cellular nucleus and cytoplasm. Higher expression was positively correlated with a higher number of recurrences and higher malignancy grade. Expression of Foxp3 was not stated in normal stomach tissue (23).
Similar results were obtained in a study on human cervical carcinoma. The research conducted by Luo et al. showed that the presence of Foxp3 expression results in neoplastic progression (13). A higher expression of the protein was noted in neoplastic cells of tumours with higher malignancy grade. The expression of Foxp3 was stated in both cellular nucleus and cytoplasm. It was not observed in normal cervical cells.
Our study was focused on the presence of Foxp3 in neoplastic cells. There is no previous relevant investigation of Foxp3 expression in canine fibroma and fibrosarcoma, therefore our results will contribute to expanding the diagnostic possibilities and understanding the biology of those tumours.
Foxp3 protein as a transcription factor is present in the cell nucleus (24), although our study did not detect any nuclear expression; only cytoplasmic expression was noted. Similar results were noted in studies on human pancreatic cancer (25, 26). Some studies suggest that cytoplasmic expression of Foxp3 can have a clinical relevance. Nonetheless, as the expression of Foxp3 can be related to both neoplastic progression and regression, this hypothesis requires further examination (27).
Hitherto the role of Foxp3 present outside the cell nucleus in neoplastic tissue has not been clarified. In our study, we tried to determine whether there is a relationship between the protein presence in cytoplasm and malignancy grading and the expression of Ki-67 – a protein related to tumour proliferative potential.
Zhang et al. (2017) noted that overexpression of Foxp3 in cells (in both nucleus and cytoplasm) of human stomach cancer favour tumour proliferation, migration, and invasiveness, therefore is connected with poor prognosis. Simultaneously, a weak or no expression entailed with lower aggressiveness of the neoplasm (23). Our research showed similar results in case of fibrosarcoma: a positive correlation between the intensity of cytoplasmic expression of Foxp3 and histological malignancy grading of the tumour. Comparable results were noted by Hinz et al. (2007), who showed that Foxp3 present in the human pancreatic cancer cells enabled the tumour to avoid the immunological response of the organism and therefore enhanced tumour’s proliferation (28).
Our study showed a positive correlation between the expression of Ki-67 and Foxp3 together with a relevant up-regulation of Ki-67 expression with the malignancy grading of the tumour. Ki-67 protein is a well-known marker of cell proliferation; it was noted several times in fibrosarcomas in dogs. Frequently it was related to poor prognosis (29).
Conclusion
The results of our study are promising and showing that the presence of Foxp3 in fibrosarcoma in dogs can be associated with the progression of the neoplastic process. A positive correlation between the Foxp3 and Ki-67 expression suggests a significant role of Foxp3 in the carcinogenesis of skin and subcutaneous fibrosarcomas in dogs. A similar conclusion can be drawn from a positive correlation between Foxp3 expression intensity and malignancy grade. A future utility of Foxp3 examination in the evaluation of soft tissue sarcomas, including fibrosarcomas is herein proposed.
Acknowledgements
The Authors would like to express their appreciation for the technical staff of the Department of Pathology at Wroclaw University of Environmental and Life Sciences for their support.
Footnotes
Authors’ Contributions
Design of the study and conduction of the experiment was accomplished by MMO, MKG, MN. Histological and IHC evaluation was performed by MMO, MKG, MN. The statistical analysis was performed by IJ. Writing and editing the paper – MMO, MKG, IJ, JB, MN. All Authors have approved the final manuscript.
Conflicts of Interest
The Authors declare that they have no competing interests in regard to this study.
- Received December 11, 2022.
- Revision received December 23, 2022.
- Accepted January 17, 2023.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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