The contribution of stem cells to epidermal and hair follicle tumours in the dog
Background – Although cutaneous stem cells have been implicated in skin tumourigenesis in humans, no studies have been conducted to elucidate the presence and the possible role of stem cells in hair follicle tumours in the dog.
Hypothesis – Stem cell markers are expressed in canine epidermal and follicular tumours and can be used to better understand the biology and origin of these tumours.
Animals and Methods – In the present study, normal skin sections and 44 follicular tumours were retrospectively investigated for the immunohistochemical expression of keratin 15 (K15) and nestin. In addition, 30 squamous cell carcinomas were evaluated for K15 expression.
Results – In normal skin, K15 and nestin were expressed in the outer root sheath cells of the isthmic portion of the hair follicle (bulge region), and K15 expression was also scattered in the basal cell layer of the epidermis. Infundibular keratinizing acanthomas, pilomatricomas and squamous cell carcinomas were mostly negative for K15, trichoblastomas were moderately to strongly positive, tricholemmomas were either negative or strongly positive, and trichoepitheliomas had heterogeneous staining. Nestin expression was generally faint in all follicular tumours.
Conclusions and clinical importance – Our results show that K15 can be a reliable marker for investigating the role of stem cells in hair follicle tumours of the dog, while nestin was judged to be a nonoptimal marker. Furthermore, our study suggests that hair follicle stem cells are present in the bulge region of hair follicles and could possibly play a role in tumourigenesis of canine tumours originating from this portion of the follicle, namely trichoblastomas, tricholemmomas and trichoepitheliomas. The loss of K15 expression in squamous cell carcinomas compared with normal skin suggests that this event could be important in the malignant transformation.
During the last 30 years, stem cells have been the subject of many studies aimed at defining their location, biological activity and functions. A detailed explanation of the functions and biology of stem cells is beyond the scope of this paper; interested readers are referred to reviews in this area.1–6 In human skin, adult stem cells are located in the hair follicle bulge, in the interfollicular areas of the surface epidermis and in the sebaceous glands.1 As with other somatic stem cells, epidermal stem cells express well-defined markers,7,8 and several studies have demonstrated the differential expression of stem cell markers in human hair follicles and interfollicular epidermis.9–11 In dogs, CD34, nestin and keratin 15 (K15) have been found to be useful hair follicle stem cell markers.12–15 In addition to studying the role of cancer stem cells in the development of tumours, the differential expression of stem cell markers has been used as a tool to classify or reclassify several neoplasias. Using morphology alone, it is often difficult to determine the cell of origin of skin tumours.16,17 The more commonly used stem cell markers for these studies are CD34, K15 and nestin, which have been demonstrated in normal canine hair follicles.12–15
The goals of this study were as follows: (i) to investigate the expression of stem cell markers in canine epidermal and hair follicle tumours; and (ii) to clarify the origin of follicular tumours in the dog according to tumour immunophenotype.
Materials and methods
Thirty cutaneous squamous cell carcinomas (SCCs) and 44 follicular tumours were selected from the archives of the Department of Biopathological Sciences and Hygiene of Animal and Alimentary Productions, Perugia and from the archives of the Department of Comparative Biomedical Sciences, Teramo. For the follicular tumours, four cases of tricholemmomas (TLs) and 10 cases each of infundibular keratinizing acanthomas (IKAs), trichoblastomas (TBs), pilomatricomas (PMs) and trichoepitheliomas (TEs) were used.
All specimens were routinely fixed in 10% neutral buffered formalin, paraffin embedded, and cut into 4- to 5-μm-thick sections. The slides were stained with haematoxylin and eosin (H&E) and examined by light microscopy.
Immunohistochemistry was performed using a commercially available mouse monoclonal antibody against K15 (1:75 dilution; Keratin15 Ab-1, clone LHK15; Thermoscientific, Fremont, CA, USA) and a rabbit polyclonal antibody against nestin (1:500 dilution; ab7659; Abcam, Cambridge, UK). Formalin-fixed and paraffin-embedded tissue sections were deparaffinized, rehydrated, and washed in distilled water. Antigen retrieval was performed by microwave treatment for 20 min at the highest power in a preheated Tris-EDTA buffer solution (10 mmol/L Tris base and 1 mmol/L EDTA, pH 9.0) for the K15 antibody and in sodium citrate buffer (10 mmol/L sodium citrate, pH 6.0) for the nestin antibody. Endogenous peroxidase was blocked using 3% H2O2 for 5 min at room temperature. Slides were then washed for 10 min in TBS (Tris-phosphate-buffered saline) and incubated with primary antibodies for 1 h in a humidified chamber at room temperature. After incubation, slides were washed in TBS for 10 min and incubated with secondary biotinylated anti-rabbit, anti-mouse and anti-goat immunoglobulins in phosphate-buffered saline followed by sequential incubation with peroxidase-labelled streptavidin (LSAB+/System-HRP; Dakocytomation, Glostrup, Denmark). 3-Amino-9ethylcarbazole was used as the chromogen (AEC + Substrate-Chromogen Ready-to-use; Dakocytomation) and Carazzi’s haematoxylin as the counterstain. Coverslips were mounted with Faramount Mounting Medium (Dakocytomation). Negative controls were treated in the same manner, omitting the primary antibody and incubating tissue sections with TBS.
In a preliminary phase of our experimental setting, we tested the bulge-specific expression of CD34 (biotinylated mouse anti-canine CD34; BD Biosciences, San Diego, CA, USA) in canine hair follicles, as previously described.13 However, no reaction was observed in any of the control and tumour samples, for any dilution or antigen retrieval method used.
The expression of the K15 and nestin was evaluated by determining cytoplasmic reactivity. Any nuclear staining was considered a background artifact. The percentage of immunopositive neoplastic cells in the tumour mass showing cytoplasmic expression of K15 or nestin was semiquantitatively evaluated and scored as negative (score 0; <5% of positive tumour cells), mild (score 1; 6-25% of positive tumour cells), moderate (score 2; 26-50% of positive tumour cells) or high (score 3; >50% of positive tumour cells). The intensity of the reaction was also evaluated as absent (0), weak (1), moderate (2) or strong (3). For each tumour, the location of positive cells was also recorded (i.e. central lobules versus peripheral part of the tumour lobules; basal versus differentiated cells).
Data were analysed using descriptive statistics.
Tumour samples and histopathological examination
Tumours were classified independently by two pathologists (C.B. and L.B.) according to Gross et al.18 When applicable, tumours were subclassified into the subtypes recognized by the current classification.18 When a discrepancy emerged in the assessment/reassessment of tumours, the two pathologists reviewed the cases and came to a final agreement. Of the follicular tumours, five cases were malignant (three trichoepitheliomas and two pilomatricomas). The remaining tumours were benign. The desmoplastic reaction, the degree of invasiveness into the surrounding tissue, the presence of ulceration and the degree of inflammatory reaction were recorded as additional features. See Table S1 for the signalment, location and stem cell marker results for individual tumours.
Expression of K15 and nestin in normal epidermis and hair follicle.
Figure 1 shows K15 and nestin expression in the normal canine skin. Keratin 15 was detected in the cytoplasm of cells of the external root sheath of the hair follicle, in the isthmic region close to the insertion of the arrector pili muscle and multifocally in the basal layer of the epidermis of control dogs and in normal skin adjacent to tumours. Nestin expression was less pronounced, more diffuse, and confined to the middle portion of the hair follicle.