Introduction

The incremental evidence shows breast cancer has the highest rate of incidence in both dog (more than 180 cases per 100,000) and human (over 2 million new cases) and accounts for one of the main influential caus- es of death in women and intact female dogs (Pang et al., 2011; Winters et al., 2017; Bray et al., 2018). However ,along with progres- sive development in molecular pathology and different therapeutic strategies, there is increasing concern over recurrence, metasta- sis and high range of cancer-related deaths (Case et al., 2017). A small heterogeneous population of tumor mass with mostly sim- ilar properties of normal stem cells, includ- ing self-renewal capacity and differentiation to various cellular lineages has received meticulous attention as a main cause of tu- mor recurrence (Rogez et al., 2018). These so-called cancer stem cells (CSCs) are ade- quately equipped to overcome conventional cancer therapies in comparison to other dif- ferentiated cancer cells (Madjd et al., 2012; Rybicka et al., 2016). Different researches identified widely variable ranges of CSCs from 2% in breast cancers to 27% in mela- noma (Im et al., 2015; Parmiani, 2016). Dif- ferent approaches are applied to detect and characterize these infrequent cells, including immunohistochemical staining (IHC), flow cytometry, and functional assay (Rybicka et al., 2016).

The well-accepted stem  cell  biomarkers for human breast cancer (HBC) and canine mammary glands cancer (CMGC), including ALDH1, CD44 and CD24, are used to detect these tumor initiating cells as a well-charac- terized phenotype named ALDH1+/CD44+/ CD24-/Low (Lee et al., 2011; Gavhane et al., 2016; Rybicka et al., 2016). Besides being therapy resistant, CSCs are able to create dif-

ferent biologic behaviors in cases with ap- parently similar histopathology but different metastatic potentials and patient outcomes.  So, choosing an accurate therapeutic regimen based on specific molecular features of indi- vidual's cancer for both dog and human be- comes challenging (Case et al., 2017).

In managing CMGC, veterinary oncologists come across challenges closely comparable to HBC. These spontaneously occurring tumors, in addition to clinical and molecular biologic similarities to HBC provide an opportunity to achieve the true meaning of public health with regard to both human and animal patients, with two-directional way of exchanging the latest  therapeutic  strategies  from  medicine to veterinary oncology and transferring new molecular discoveries and  vice  versa  (Pang et al., 2011; Case et al., 2017; Nguyen et al., 2018; Marconato et al., 2019).  Furthermore, in an attempt to discover CSCs potential in- teractions, the role of comparative oncology becomes increasingly apparent, in that it is possible to investigate probable correlations between these relapse-inducing cells and clin- icopathologic parameters in the most relevant animal cases that have crucially significant priorities over experimental methods such as in vitro research (Queiroga et al., 2011; Ry- bicka et al., 2016; Nguyen et al., 2018).

Since the main issues addressed in oncol- ogy research are first finding prognostic fac- tors involved in metastasis cascades and then targeting them combined with conventional methods, this research intends to evaluate prognostic capability of CSCs in CMGC as in HBC via IHC staining based on CD44, CD24, ALDH1 and to determine the probable asso- ciations between CSCs and clinicopathologi- cal features. Based on similar features in this regard, HBC treatment protocols can be used

in dog patients and all advances gained from researches in animal mammary gland cancer can be utilized in new preclinical and clinical designs for further progression. To our knowl- edge, the present study is the first Iranian re- garding CSCs existence in CMGC.

Materials and Methods

This experiment was designated as a cross-sectional study which was extended  for one year. A total of 40 samples were tak- en from referred mammary gland masses to veterinary hospitals from canines that under- went surgery during 2018-2019 and were di- agnosed as CMGC by pathologist.

Tumor histotypes

Based on Goldschmit classification (2011) just epithelial malignancies were included.

Clinical data

All clinical data, including patient ages, tumor laterality and surgical procedures were obtained from patient records and pathology reports were extracted.

Tumor grade

All slides were rechecked in order to re- grade tumor samples, based on Nottingham grading score system (Cassali et al., 2017) which categorized all breast malignancies to three grades.

Lymphovascular invasion

To determine whether lympho-vascular in- vasion (LVI) is present or not, the H&E stained slides were examined to investigate presencee of tumors cells around or inside the vessels which were reported as positive or negative cases.

IHC Procedure

Paraffin embedded blocks were selected based on proper criteria  for  IHC  staining, as follows: the largest volume of tissue, the least necrosis and the  most  invasive  part  of tumor. After cutting to 3 μm, all slides

were deparaffinized by xylene, rehydrated through ethanol and antigen retrieval was done by putting immersed slides on Tris buffer in microwave. Following that, perox- idation process was performed. Then, slides were incubated by monoclonal primary anti- bodies to assess CSCs presence at room tem- perature for 30-60 min, ALDH1 with CAT number (Sc-166362) (Mouse monoclonal antibody (H-8), dilution: 1:50-1:500), CD44 with CAT number (NBP1-47386) (CD44 antibody (8E2F3), dilution: 1:200-1:1000) and CD24 with CAT number (Sc-19651) (Rat monoclonal antibody (M1/69), dilu- tion: 1:50-1:500) were  used. Then  washing 3 times in Tris buffer, each time for 5 min was done.

After completing the exposure to second- ary antibody and HRP polymer of MACH 1 Universal HRP-polymer, Biocare Medical Co. (Pacheco, California, USA) at room temperature for 1 hr, incubation at room temperature for 30-60 min, then washing 3 times, each time 5 min in Tris buffer were done. Amplification of antigen- antibody links with betazoid DAB chromogen was done. Then samples were counterstained by hematoxylin and immersed in alcohol for dehydration and xylene for clearing, finally they were mounted.

IHC evaluation

According to modified Alred Scoring Sys- tem (Qureshi et al., 2010), semiquantitative analysis of epithelial tumoral cells immuno- reactivity at invasive parts with cutoff ≥ 1% was made. Intensity and proportion rate of CD44 and CD24 as membranous markers and ALDH1 as a cytoplasmic antigen with this cut point were estimated. The main fo- cus of the experiments was to detect CSCs- like cells, so the widely accepted phenotype, ALDH+/CD44+/CD24-/Low, was used to

call cases as positive and the remaining phenotypes were called  negative.  Posi- tive control was done based on Santa Cruz (Dallas, USA) and Novus Biological (Col- orado, USA) Company’ protocols to vali- date antibodies specificities. According to each protocol, human gall bladder tissue, formalin-fixed, paraffin-embedded mouse blood smear and paraffin-embedded human lung cancer tissue are control positives for ALDH-1, CD24 and CD44, respectively, and normal mammary gland tissue accounts for negative control.

Statistical analysis

Since this study was a pilot one, exclu- sively descriptive data were generated for variables, including patient age, surgical methods, tumor laterality, tumor histolo- gy, and statistical analysis was performed only for tumor grade, CSCs and LVI sta- tus. SPSS software was used for data anal- ysis and correlation between quantitative and qualitative parameters was assessed by t-test and Chi Square test, respective- ly. It should be noted that P≤ 0.05 was considered significant.

Results

Clinicopathological results

In the present study, 40 cases of canine mammary gland cancers were examined with the mean age of 8.61 ± 2.41 years (ranging from 5 to 14 years). Age data was not available in four cases. Tumor laterality was divided into the left and right side with 60.7% and 39.2%, respectively. Surgical methods were operated in 28 cases includ- ing: simple regional mastectomy (53.5%), unilateral mastectomy (28.7%) and lumpec- tomy (17.8%). Cancerous mammary glands were diagnosed in 78.6% of cases as sin- gle, while only 21.4% of cases had cancer- ous mass in two or more mammary glands. Involvement of regional mammary glands was categorized as thoracic, abdominal or both with 21.4%, 75.0% and 3.6%, respec- tively. Tumor grades as I, II and III were observed in 42.5%, 47.5% and 10% of cas- es, respectively. More details with regard to clinicopathologic characteristics of samples were shown in Table 2 and Figure 1.

Table 1. Description of clinicopathologic characteristics

Table 2. IHC results of CSCs markers

                                                 Figure 1.

Evaluation of LVI in all cases showed that 72.5% of cases are positive (n=29), while only 27.5% of cases were negative (n=11). Tumor classification based on ag- gressiveness highlighted that grade II had the  highest  percentage  of  LVI positivi-  ty (55.1% grade II), followed by grade I (31.0%) and grade III (13.7%).

IHC results:

CSCs markers

Among 40 cases of this study ALDH1+ was observed in 80% of them, and ALDH1-

, CD44+, CD44-, CD24+  and CD24-  were as

follows: 20%, 17.5%, 82.5%, 17.5% and 82.5%, respectively. More details regarding IHC results are provided in Table 2 and Fig- ures (B, C and D). In addition, 15% of cases were positive for both ALDH1 and CD44,

from which 83.3% and 16.6% were grade II and III, respectively. Surprisingly, all grade I cases only showed ALDH1 positivity.

Further analysis illustrated that 17.5% of cases were enriched with cancer stem cell, in which 71.4% and 28.5% of them were diagnosed as grade II and III, respectively (P≤ 0.05). Interestingly, despite high oc- currence of grade I, we could not  detect any CSCs positive cells.

Association between tumor histology types and cancer-initiating cells was demon- strated in Table 3. Furthermore, all positive CSCs cases accounted  for  LVI  positive  (7 of 29; 24.1%). More details regarding these correlations are provided in Table 4. Also, associations between tumor histology and LVI status are summarized in Table 5.

Table 3. Frequency of histology types enriched with CSCs

Table 4. Correlation between LVI status and CSCs existence

Table 5. Description of Tumor histology and LVI status

Discussion

This study was designed to determine the presence of CSCs in canine malignant mam- mary gland tumors, and to evaluate CSCs associations with clinical and biological features of these malignancies. Based  on the literature, despite possessing a rare pop- ulation, CSCs are suspected for their prob- able roles in tumor aggressiveness, therapy resistance and recurrence (Queiroga et al., 2011; Aleskandarany et al., 2015; Rogez et al., 2018). In medical oncology, CSCs have been postulated as an independent prog- nostic factor in different solid tumors such as HBC (Mohamed et al., 2018). The final goal of most recent studies is achieving the precise and detectable factor to predict can- cer fate in both human and animal patients (Horimoto et al., 2016; Case et al., 2017; Flynn et al., 2019). In an attempt to pursue this goal, we proposed the prognostic role for CSCs in CMGC. In the current work, 17.5% of tumor specimens contained CSCs that was the most interesting result in accor-

dance with findings in HBC which demon- strated an average range from 2% to almost 27% (Camerlingo et al., 2014; Im et al., 2015; Parmiani, 2016).

High frequency of CD44+/CD24-/Low/ ALDH1+ has significant impacts on patient outcome, since these populations are asso- ciated with poor prognostic variables which cause aggressive behavior and high met- astatic potential (Magalhães et al., 2013; Rogez et al., 2018).

To support the significance of these cells, early studies have found circulated cancer cells enriched with CD44+/CD24- population in bone marrow of patients with metastatic breast cancers, which enhances the value of this phenotype as an appropriate prognostic factor (Marsden et al., 2012; Horimoto et al., 2016; Salvador et al., 2019).

Another important finding was that the whole CD44+/CD24- populations were ALDH1 positive, which is in  contrast  to the results of other studies that  showed  less than 1% coverage between CD44+/

CD24- cells and ALDH1+ (Horimoto et al., 2016). This discrepancy is related to dif- ferent study designs; the latter was carried out on breast cancer cell lines.

On the other hand,  aggressive  behav-  ior is exacerbated by increased number of expressed markers by cancer cells (Lee et al., 2011; Horimoto et al., 2016; Strati et al., 2019). In agreement with this hypoth- esis, we could demonstrate all CSCs-pos- itive cases are expressed by both CD44+/ CD24- and ALDH1+. Additionally, they all belonged to grade II and III, in contrast to grade I cases that were CSCs-negative and expressed only ALDH1. On the other side, our results also illustrated significant cor- relation between CSCs and tumor grade (P≤0.05). These results differ from Rab- inovich et al. (2018) finding that depicted ALDH1-positivity in high percentage of grade III tumors. Horimoto et al. (2016) regarding these discrepancies showed that these markers should be used together in an attempt to detect more population of CSCs. Since in this study we showed CSCs have   a significant association with grade (P≤ 0.008), our result highlights the importance of this metastasis-initiating population.

In this research in  order  to  evaluate  LVI as a prognostic characteristic, it is interesting to note that all seven  CSCs  cell- rich samples were LVI positive (P≤ 0.025). LVI status can predict patient out- come even in cases which are diagnosed lymph node-negative both clinically and histopathologically, but their tumor recurs after some years despite primary success- ful treatment (Aleskandarany et al., 2015; Bartosh et al., 2016). One of  the  possi-  ble explanations might be  the  separation  of a fraction of tumor mass called circu- lating CSCs, that express CD44+/CD24-/

ALDH1+ markers which, as a potential source of originating metastatic colonies  are proliferatively quiescent (Mohamed et al., 2018; Strati et al., 2019). Furthermore, circulating CSCs act as dormant cancer cells and they are generated before the first evolution of clinically evident tumors. So they could escape from routine therapies and they are able to find their favorable niches. After many years or a long time, these cells can reactivate themselves and make metastatic tumors, all of these fea- tures lead to therapy failure (Marsden  et al., 2012; Payne et al., 2012; Bartosh et al., 2016; Fu et al., 2017; Flynn et al., 2019).

There was a significant association be- tween tumor grade and LVI (P≤0.05). Also, tumor grade II and III had a higher percent- age of vascular invasion compared to grade I, which is supported by Aleskandarany et al. (2015) results, presenting that there is     a strong correlation between these two pa- rameters and the least occurrence of LVI positivity in grade 1 tumors. Other studies provided corresponding results regarding strong prognostic efficacy of histologic grading to predict metastatic relapse (Case et al., 2017; Rakha et al., 2018).

The most common histotypes  observed in this study were tubular carcinoma, tubu- lopapillary carcinoma and intraductal car- cinoma (Figure. A), respectively, which is in agreement with several studies (Rasotto et al., 2012; Patel et al., 2019).

One cohort study confirmed the accurate value of different CMGC histotypes in pre- dicting patient outcome, while three com- mon histotypes in this study do not have prognostic value compared to other tumor types such as comedocarcinoma and ade- nosquamous carcinoma (Canadas et al., 2018). In present work only one case was