Abstract
Background: Cyclo-oxygenase-2 (COX-2) and cancer associated fibroblasts (CAFs) play an important role in the development and progression of tumor malignancy in humans and animals, showing that both can influence the tumor microenvironment. However, the impact of these two markers in feline mammary carcinogenesis has not yet been addressed. Materials and Methods: In the present study, the clinicopathological significance of COX-2 immunoexpression and alpha-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs) was determined and correlated with disease-free and overall survival of 50 felines with malignant mammary tumors. Results: COX-2 overexpression was positively associated with mitotic index (p=0.031), degree of malignancy (p≤0.001), lymph node metastasis (p≤0.001), vascular invasion (p=0.002), disease recurrence (p=0.019) and distant metastasis (p=0.036). α-SMA-positive CAFs were associated with mitotic index (p=0.004), lymph node metastasis (p=0.027), vascular invasion (p=0.05), disease recurrence (p≤0.001) and distant metastasis (p≤0.001). Additionally, both markers were correlated with disease-free and overall survival, emerging as predictors of poor prognosis. Conclusion: Our results indicate for the first time that the presence of two markers, COX-2 and α-SMA, is associated with carcinogenesis and worse prognosis in feline mammary cancer and that α-SMA-positive CAFs have a role in feline mammary tumorigenesis, cancer development, and clinical outcome.
Cyclo-oxygenase-2 (COX-2) is an inducible enzyme that belongs to the prostaglandin H-synthase family. It catalyzes the rate-limiting step in prostanoid biosynthesis, a process known for its involvement in tumor progression and dissemination (1). During oncogenesis, the tumor microenvironment alters the essential functions of COX-2, inducing its overexpression. Encoded by a fast-response gene, this enzyme acts early to promote increased cell survival, enhanced tumor cell invasiveness, stimulation of neovascularization, and evasion from the host’s immune system. This facilitates angiogenesis and proliferation, allowing for malignant transformation and rapid somatic evolution (1-3). Through biomolecular studies, it is currently known that in breast cancer, this molecule is responsible for increasing the survival of tumor cells by stimulating growth, invasion, angiogenesis, and down-regulating tumor apoptosis (4-9).
Cancer-associated fibroblasts (CAFs) undergo transformation in the tumor microenvironment, induced by interactions with cancer cells. These interactions lead to CAFs acquiring a modified phenotype, similar to fibroblasts associated with wound healing (10-14). CAFs have been described as promoting tumor progression in breast cancer in in vitro and in vivo studies (14, 15). Activated CAFs secrete a series of growth factors that lead to the survival and support of surrounding malignant cells (10, 15, 16), functioning as key mediators for proliferation, angiogenesis, and metastasis, as well as poor patient prognosis (11, 16, 17). The expression of alpha-smooth muscle actin (α-SMA) by CAFs has been used to identify them in the tumor microenvironment (18).
COX-2 and α-SMA-positive CAFs play an important role in the development and progression of tumor malignancy, showing that both can influence the tumor microenvironment. Moreover, these components also showed an effective role in the participation and stimulation of the metastatic cascade, a fundamental phase for neoplasms to invade the primary tissue or organ and later acquire the ability to invade tissues or organs elsewhere (1-3, 11, 15, 18, 19, 20).
Malignant feline mammary tumors are among the most aggressive, leading to low survival rates after diagnosis (21-24). The identification of a subset of cases, through the identification of new prognostic biomarkers, that might benefit from stronger chemotherapeutic regimes is useful and needed (25). To date, no published studies have investigated the role of COX-2 and α-SMA-positive CAFs in feline mammary tumors (FMT). Therefore, this study aimed to investigate their expression and prognostic value in cases of malignant FMT, examining their value as prognostic indicators.
Materials and Methods
Patient selection and tissue sample collection. In this study, 50 cases of malignant FMT were included. The sample collection comprised tumors excised by surgery from cats with malignant FMT received for treatment at Onevet, Veterinary Hospital of Porto, Porto, Portugal.
Histopathological examination. Tumor samples were fixed in 10% neutral buffered formaldehyde, placed in synthetic Histoplast® Shandon® paraffin and cut into 3-μm sections according to routine methodology. The histopathological diagnosis was made based on hematoxylin-eosin-stained sections according to the World Health Organization classification of FMT (26) and tumors were then classified based on their predominant histological features. The clinicopathological variables evaluated in this study were tumor size (T1 ≤2 cm; T2 ≥2 cm and <3 cm; T3 ≥3 cm), animal breed, age at the time of diagnosis, reproductive status, prior use of contraceptives, presence/absence of skin ulceration, histological type of the tumor, presence/absence of histological necrosis, mitotic Index, nuclear pleomorphism/nuclear grade, degree of tumor differentiation/tubular formation, histological grade of malignancy, and presence/absence of lymph node metastasis. The Nottingham system was used to assess the histological grade of malignancy, in which the variables tubular formation, nuclear pleomorphism and mitotic index are considered (27).
Immunohistochemistry. Paraffin-embedded sections were deparaffinized, rehydrated in graded concentrations of alcohol and the methodology used to detect the proteins under study was performed the same for both COX-2 and α-SMA. The detection system used was the commercial kit Novolink™ Polymer Detection Systems (Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK) according to the manufacturer’s instructions. Antigen retrieval was performed by microwave treatment for 20 min for COX-2 and 5 min for α-SMA at 750 W in 0.01 M citrate buffer, pH 6.0, followed by cooling for 20 min at room temperature. All sections were incubated with the primary specific COX-2 antibody (1:100; Richard Allan Scientific Co., Kalamazoo, MI, USA) and α-SMA antibody (1:40, Clone 1A4; Richard Allan Scientific Co.) for 24 h at 4°C. The antibody reaction products were observed using the chromogen 3,3′-diaminobenzidine tetrachloride at 0.05% with 0.01% hydrogen peroxide (30%). After a final wash in distilled water, the sections were counterstained with hematoxylin, dehydrated, cleared, mounted, and viewed under a light microscope. For negative controls, phosphate-buffered saline was used instead of the primary antibody. In each procedure, positive controls suitable for each case were used: As a positive control for α-SMA, normal feline mammary gland with attached skin was used; for COX-2, sections of normal kidney (macula densa) from a young cat were used.
Evaluation of COX-2 and α-SMA-positive CAFs staining. The immunohistochemical evaluation was performed by two observers (FQ and JC) in a process blinded to the clinical status of the feline patients. The evaluation of COX-2 immunoreactivity was performed on tumor cells using a semiquantitative method previously described (28). The percentage of COX-2–positive tumor cells was graded as 0 when 0% positive cells were present, 1 when there were <10% positive cells, 2 for 10-50% positive cells), 3 for 51-80% positive cells and 4 for >80% positive cells; and the intensity of COX-2 immunoreactivity was classified as 0 for no staining, 1 for weak, 2 for moderate and 3 for strong staining. Each COX-2 score was the product of the scores for the percentage of positive tumor cells and the staining intensity, ranging from 0 to 12. Final scores were defined as low (≤6) or high (>6) COX-2 immunoreactivity.
To evaluate α-SMA-positive CAFs in tumor stroma, a semiquantitative methodology was used according to the method previously described (18). In brief, the percentage of α-SMA-positive CAFs was graded as 0 for 0% positive cells, 1 for 1-10% positive cells, 2 for 11-50% positive cells, 3 for 51-80% positive cells and 4 for 81-100% positive cells. The immunolabelling intensity was classified as 0 for no staining, 1 for weak, 2 for moderate and 3 for Intense and strong staining. The final score was obtained by the product of percentage of positive cells and staining intensity, ranging from 0 to 12. Final scores were defined as low (<6) or high (≥6) immunoreactivity for α-SMA-positive CAFs.
Follow-up data. After the surgical excision of tumors, clinical follow-up was carried out for a mean period of 393 days (minimum 53, maximum 973 days). Disease-free survival (DFS) was defined as the period between surgery and local recurrence or development of distant metastasis. Overall survival (OS) was defined as the period between surgery and natural death due to the tumor or euthanasia in advanced stages of the disease (confirmed at necropsy).
Statistical analyses. The Statistical Package for the Social Sciences, version 26.0 (IBM, Armonk, NY, USA) was used for all statistical analyses. The chi-square test with Fisher’s exact test (when appropriate) was used to study the categorical variables. Survival curves were generated by the Kaplan-Meier method and survival rates were compared using the log-rank test. Values of p<0.05 were accepted as denoting significant differences.
Results
Clinicopathological data. The 50 FMT samples included in this study were histologically classified according to the World Health Organization criteria as: tubulopapillary carcinoma in 28 (56%), tubular carcinoma in 11 (22%), solid carcinoma in 7 (14%) and cribriform carcinoma in 4 (8%). The tumor size was T1 in 21 (42%), T2 in 13 (26%) and T3 in 16 (32%). The histological grade of malignancy was classified as I in 9 (18%), II in 5 (10%) or III in 36 (72%) and 27 cases (54%) had lymph node metastases.
Immunoreactivity scores for COX-2 and α-SMA-positive CAFs in malignant FMT. Representative examples of immunostaining are shown in Figure 1. Regarding COX-2 immunoreactivity, 15 (30%) of the cases presented low immunoreactivity, while 35 (70%) cases presented high immunoreactivity. For α-SMA-positive CAFs, 16 (32%) of the cases presented low immunoreactivity, while 34 (68%) cases presented high immunoreactivity.
Immunoreactivity for cyclo-oxygenase-2 (COX-2) (A) and alpha-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs) (B) expression. A: Low COX-2 expression in tubulopapillary carcinoma with a low degree of malignancy (left panel). High expression of COX-2 in solid carcinoma with a high degree of malignancy (right panel). B: Low immunoreactivity for α-SMA-positive CAFs in tubulopapillary carcinoma with a low degree of malignancy (left panel). High immunoreactivity for α-SMA-positive CAFs in tubulopapillary carcinoma with a high degree of malignancy (right panel). Bars=100 μm.
Associations of clinicopathological features with COX-2 and α-SMA-positive CAFs in malignant FMT. Our analysis identified an association between the presence of aggressive disease and high immunoexpression of these two markers. As summarized in Table I, high COX-2 expression was statistically significantly associated with characteristics, such as a high mitotic index (p=0.031), high histological degree of malignancy (p≤0.001), presence of lymph node metastasis (p≤0.001), presence of vascular invasion (p=0.002), disease recurrence (p=0.019) and development of distant metastasis (p=0.036) (Table I). For α-SMA-positive CAFs, high immunoreactivity scores were statistically significantly associated with high mitotic index (p=0.004), presence of lymph node metastasis (p=0.027), presence of vascular Invasion (p=0.05), disease recurrence (p≤0.001) and development of distant metastasis (p≤0.001).
Relationship between cyclo-oxygenase-2 (COX-2) and α-smooth muscle actin (α-SMA) expression and clinicopathological parameters in feline mammary tumors.
Follow-up study. The present data showed that DFS statistically significantly differed between animals whose malignant tumors had high COX-2 expression compared to those that had low expression. The group that had low COX-2 expression had an average DFS of 607 days (range=402-811 days), while those with a high COX-2 expression had an average of 338 days (range=227-448 days) (p=0.048) (Figure 2A, left panel). The right panel of Figure 2A demonstrates that there was also a significant difference in OS between the animals whose tumors had a higher COX-2 expression [326 days (range=207-445 days)], compared to those with low COX-2 immunoreactivity [489 days (range=339-639 days)] (p=0.034).
Kaplan-Meier curves for disease-free (DFS) (left) and overall (OS) (right) survival according to immunoreactivity scores for cyclo-oxygenase-2 (COX-2) (right (A) and alpha-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs) (B). A COX-2 immunoreactivity score of >6 was associated with significantly worse DFS (p=0.048) and OS (p=0.034). An immunoreactivity score of ≥6 for α-SMA-positive CAFs was associated with significantly worse DFS (p=0.004) and OS (p=0.043).
Looking at Figure 2B (left panel), it can be seen that there is a significant difference between animals whose malignant tumors had a high α-SMA-positive CAFs score compared to those that had a low expression regarding the duration of DFS. Animals whose tumors had low immunoreactivity score for α-SMA-positive CAFs had an average DFS of 611 days (range=469-754 days), while those with a high score had an average DFS of 305 days (range=204-405 days) (p=0.004). In the right panel of Figure 2B, it can be seen that animals with high score for α-SMA-positive CAFs had a mean OS of 348 days (range=241-455 days), while those with low immunoexpression had a mean OS of 558 days (range=404-711 days) (p=0.043).
Discussion
Cancer has increasingly become a significant aspect of global life expectancy over the years, surpassing other diseases in both prevalence and prominence. The rising incidence and mortality rates can be attributed, in part, to more aggressive screening and detection efforts aimed at identifying and combating this disease (29).
Breast cancer is a chronic and multifactorial disease that is classified by the uncontrolled growth of tumor cells in the breast tissue and is the second -leading cause of cancer-related death in women worldwide (30-33).
In the context of human breast cancer, rodent models have played a crucial role in advancing both basic and translational research (34, 35). However, in recent decades, dog and cat with mammary cancers have also been demonstrated to be good models to study this human neoplastic disease (19). What makes companion animals a particularly suitable model for human cancer research is the spontaneous development of cancer in pets, in the same environment shared with humans (22, 36, 37).
Feline malignant mammary tumors, aside from being spontaneous and hormonal-dependent, exhibit notable similarities to their human counterparts at clinical, histological, and molecular levels (38). These similarities hold the potential to make substantial contributions to the progression of dependable translational studies and the development of cancer therapies applicable to humans (39).
COX-2, an enzyme within the pro-inflammatory family, has been implicated in several steps of breast oncogenesis leading to dissemination (40-42). Elevated COX-2 expression has been associated with cancer progression, the metastatic process, and dysregulation of apoptotic and inflammatory processes, ultimately contributing to a poor prognosis (1, 43, 44).
In this study, COX-2 demonstrated an association with several clinicopathological characteristics of histological and clinical tumor aggressiveness. The results we obtained are not in full agreement with the study carried out by Millanta et al. (45), where the authors did not find significant correlations between COX-2 overexpression, high histological grade, and presence of lymph node metastasis. However, our data are similar to several reports on human breast cancer and canine mammary tumors (4, 28, 44-47). A possible explanation for this may be related to the use of distinct primary antibodies; however, we cannot rule out the possibility of differences in material conservation prior to immunohistochemical detection.
Fibroblasts are cells usually programmed to participate in the healing process, however, when this program is disrupted, normal fibroblasts turn into CAFs (11). CAFs are important cells in the tumor environment, where they are responsible for synthesizing and secreting several types of growth factors such as transforming growth factor β and platelet-derived growth factor that, with other components of the tumor microenvironment, maximize neoplastic progression (11,13, 48, 49). Numerous studies have highlighted the significance of α-SMA-positive CAFs in the development and progression of human breast cancer. These investigations have revealed that stromal expression of α-SMA is associated with a high number of lymph node metastases (14, 15) and a poorer clinical outcome in patients with breast cancer (16). Additionally, α-SMA-positive CAFs have been shown to enhance angiogenesis, affect tumor growth in vivo, and be correlated with a higher frequency of cancer stem cells (17).
To the best of our knowledge, this is the first study to investigate the role of α-SMA-positive CAFs in feline mammary tumorigenesis, cancer development, and clinical outcome. In the present study, α-SMA-positive CAFs were positively associated with high mitotic index and presence of lymph node metastasis, revealing their link to a more aggressive biological behavior in accordance with that described in human breast cancer (50-52). Furthermore, there was also a statistically significant association of high immunoreactivity for α-SMA-positive CAFs with a shorter DFS as well as with reduced OS. Despite the absence of previous similar studies in FMT, the prognostic value of α-SMA-positive CAFs has been described in human breast cancer (53, 54).
The overexpression of COX-2 in tumoral cells and the high scores for α-SMA-positive CAFs show these play a pivotal role in breast cancer progression (54, 55). In this investigation, the evaluation of COX-2 and α-SMA-positive CAFs through immunohistochemistry aimed to explore their influence on tumor aggressiveness and prognosis. The present results are both important and exciting. While the impact of COX-2 on feline mammary tumorigenesis, cancer development, and clinical outcomes has been previously studied, this research represents the first attempt, to our knowledge, to address these aspects specifically for α-SMA-positive CAFs.
Conclusion
This study evaluated the role of COX-2 and α-SMA in feline mammary carcinomas. Both markers were raised as indicators of poor prognosis in the cases analyzed, indicating that they may play a role in tumor progression and poorer prognosis. Future and more in-depth research, namely investigations on tumor genetic signatures, will be essential to understand the role of COX-2 and α-SMA positive CAFs in FMT more clearly.
Acknowledgements
This work was financed by National Funds (FCT/MCTES, Fundação para a Ciência e a Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) under the project UIDB/00772/2020. The Authors also want to thank the support received by projects UIDB/00211/2020and LA/P/0059/2020, from FCT/MCTES.
Footnotes
Authors’ Contributions
FLQ and IP conceived and designed the study; GP performed data collection; JP performed diagnosis; IP and JCMG performed the experimental analysis; FLQ and JCMG analyzed the results; JCMG and FLQ wrote the article; JCMG, GP, JP, IP, FLQ reviewed and approved the submitted version.
Conflict of Interest
None of the Authors has a financial or personal conflict of interest to declare.
- Received November 8, 2023.
- Revision received December 21, 2023.
- Accepted December 29, 2023.
- Copyright © 2024 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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