In the last few years, a growing body of evidence has been reported supporting the notion that the capability to sustain tumor formation and growth exclusively resides in a small population of cells called cancer stem cells (CSCs). Search of markers that demonstrate the stem cell-like phenotype of these tumor initiating cells is an active field of investigation, and several stem cell markers have been described recently in skin tumors [
CD133 is the human homologue of mouse prominin-1, a five transmembrane domain cell surface glycoprotein that has received notable attention because of its expression restricted to various somatic stem cell subpopulations [
In previous studies that report the CD133 immunohistochemical staining of human glandular epithelia, such as salivary glands, lacrimal glands, pancreatic ducts, endocervical glands, and sweat glands, a peculiar pattern of CD133 expression has been described [
The finding of the CD133 antibody staining sweat gland cells prompted us to design a study that we now report, in order to evaluate the expression of CD133 in skin eccrine and apocrine tumors.
A retrospective search retrieved 43 previously diagnosed skin eccrine and apocrine adnexal tumors from the files of the Department of Pathology of the Albacete University Hospital. Glass slides, paraffin blocks, and histopathological reports from all the cases were obtained. Slides were reviewed and adnexal skin tumors were diagnosed according to the criteria of the World Health Organization (WHO) classification [
For culture of skin cancer cells, seven samples from human skin tumors that corresponded to one eccrine porocarcinoma and six squamous cell carcinomas were obtained. Fresh tissue from these cases was included in Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with penicillin/streptomycin and fungizone, immediately after surgical resection.
Formalin fixed, paraffin embedded tissue sections 4
Sections of skin with normal sweat glands were used as positive controls. In addition, when present in the sections, normal sweat glands from the skin adjacent to the neoplasms were used as internal positive controls. Negative controls were performed by omitting the anti-CD133 antibody during the primary antibody incubation.
Immunohistochemical staining of solid areas and of acinar or ductal structures from the tumors was separately evaluated. CD133 staining was graded using a semiquantitative scale. Acinar or ductal structures were evaluated as follows: (−) no staining; (+) staining of secretory material in the lumen of isolated ductules or acini and/or weak staining of the apical or luminal border of few ductules or acini; (++) clear staining of the apical or luminal border of most ductules or acini present in the tumour; (+++) staining of the apical or luminal border of all ductules or acini present in the tumor. CD133 expression was evaluated by two senior pathologists (EP and SYNC) in a blinded fashion without knowledge of clinical and pathological information. In cases of discrepant assessments, slides were reevaluated by both pathologists under a multihead microscope, and an agreement was obtained.
Tumor fragments were mechanically and enzymatically disaggregated by digestion with collagenase type IA (2 mg/mL) (Gibco, Invitrogen) in Hank’s balanced salt solution (HBSS) at 37°C for 2 hrs. Cells were filtered through a 40
All experiments were performed on cell suspensions prepared at first passage of primary culture from tumours. Cells were detached using 0.02% EDTA in phosphate-buffered saline (PBS) for 15 min at 37°C and washed with PBS before staining. Cell suspensions were adjusted to
The patient cohort consisted of 29 males and 23 females, ranging in age from 24 to 90 years (median, 49 years). The site of presentation was variable, most of them localized on the head and neck region. Clinical data are summarised in Table
Summary of clinical and immunohistochemical findings.
Diagnosis | Age | Location | CD133 |
---|---|---|---|
Eccrine spiradenoma ( |
76 | Nasal | +++ |
52 | Neck | +++ | |
58 | Nasal | ++ | |
| |||
Hidradenoma ( |
38 | Unknown | +++ |
77 | Forehead | +++ | |
85 | Face | +++ | |
48 | Head | ++ | |
78 | Unknown | ++ | |
| |||
Eccrine hidrocystoma ( |
76 | Nasal | +++ |
52 | Neck | ++ | |
58 | Nasal | +++ | |
| |||
Poroma ( |
34 | Plantar | ++ |
85 | Forearm | +++ | |
51 | Scalp | +++ | |
31 | Leg | + | |
59 | Unknown | +++ | |
67 | Unknown | ++ | |
| |||
Porocarcinoma ( |
90 | Back | +++ |
| |||
Syringocystadenoma papilliferum ( |
36 | Scalp | ++ |
60 | Pectoral | +++ | |
28 | Scalp | +++ | |
| |||
Syringoma ( |
42 | Unknown | + |
36 | Forearm | ++ | |
32 | Neck | + | |
31 | Eyelid | + | |
67 | Inferior eyelid | − | |
36 | Eyelid | ++ | |
| |||
Cylindroma ( |
51 | Scalp | ++ |
51 | Scalp | +++ | |
80 | Scalp | ++ | |
47 | Unknown | +++ | |
| |||
Hidradenoma papilliferum ( |
75 | Vulvar | + |
31 | Vulvar | + | |
| |||
Chondroid syringoma ( |
38 | Supralabial | ++ |
36 | Nasal | +++ | |
60 | Forehead | +++ | |
53 | Eyelid | +++ | |
| |||
Apocrine hidrocystoma ( |
33 | Eyelid | − |
79 | Eyelid | − | |
40 | Face | − | |
| |||
Apocrine carcinoma ( |
62 | Axilar | − |
| |||
Microcystic adnexal carcinoma ( |
46 | Upper lip | +++ |
53 | Upper lip | +++ |
CD133 staining was graded using a semiquantitative scale. Acinar or ductal structures were evaluated as follows: (−) no staining; (+) staining of secretory material in the lumen of isolated ductules or acini and/or weak staining of the apical or luminal border of few ductules or acini; (++) clear staining of the apical or luminal border of most ductules or acini present in the tumour; (+++) staining of the apical or luminal border of all ductules or acini present in the tumor.
From the three different monoclonal antibodies tested on formalin-fixed, paraffin- embedded tissue sections, and in accordance with previous reports [
CD133 expression was observed at the endoluminal or apical surface of the cells of the acini and terminal ductules of normal eccrine glands (Figure
CD133 in normal eccrine glands stains the endoluminal surface of the cells and the sweat gland secretion (a). Intercellular canaliculi at the lateral membrane of eccrine cells are also observed (b).
CD133 was not expressed in any of the squamous or basal cell carcinomas tested. Using the AC133 antibody the adnexal tumors analyzed in this work showed the following staining pattern (summarized in Table
CD133 positivity at the inner surface of chondroid syringoma branching tubules. No staining of the stromal cells or of outer epithelial cells is noted.
Papillary projections of syringocystadenoma papilliferum lined by pseudostratified columnar epithelium are CD133 positive. The positivity is very intense at the endoluminal surface.
Linear CD133 staining of columnar cells lining tubules of a hidradenoma case. Only cells that are located at the inner surface of the tubules are stained.
Ductal structures of porocarcinoma seen in H&E sections (a) are CD133 positive (b).
Seven samples derived from human skin tumors were processed with the goal of obtaining single cell suspensions. Due to an insufficient number of cells obtained from the tumors or to fungal or bacterial contamination, only four samples were successfully cultured and analyzed for cell surface CD133/1 epitope expression. These successfully cultured biopsies include one eccrine porocarcinoma and three squamous cell carcinomas. Microscopic examination of the cultured cells revealed different morphologies that varied consistently to the histological tumor type. Cells from squamous cell carcinoma showed epithelioid or spindle cell appearance while eccrine porocarcinoma derived cells had more rounded shape and grouped together into islets of small and atypical cells. The presence of epithelial cells in these cultures was confirmed through E-cadherin and EpCam positive expression as epithelial markers.
To determine whether skin tumor cell cultures contained a population of CD133 positive cells, we used flow cytometry to examine the expression of CD133/1 surface marker with the AC133 antibody. All samples from squamous cell carcinoma contained less than 0.1% of positive cells, while nearly 22% of positive cells were detected in eccrine porocarcinoma. Figure
CD133 expression profiles in human skin primary tumour cells. Analysis of CD133/1 expression in samples from eccrine porocarcinoma (b) and from squamous cell carcinoma (a). Flow cytometry of porocarcinoma derived cell cultures show a 22% of CD133/1 epitope positive cells and CD133 immunostaining. Squamous cell carcinoma shows negative staining for CD133 and 0% positive CD133 cells on flow cytometry.
Progress in cutaneous stem cell biology, and in carcinogenesis understanding, depends heavily on the availability of markers specific for distinctive stem cell populations [
The CD133 expression on the cell surface of differentiated tumor cells is an argument against CD133 as a specific cancer stem cell marker in the skin. However, the existence of a small CD133+ CSC population cannot be completely ruled out, as it has been demonstrated in other organs, where CD133 is expressed on the cell surface of both, CSCs, and differentiated tumor cells [
Taken together, our findings demonstrate a specific immunohistochemical expression of the AC133 antibody at the secretory surface of differentiated normal and neoplastic cells of eccrine glands. Experiments that use this antibody as a marker of CSCs in skin should be interpreted with caution. The AC133 antibody is a sensitive and specific marker of cutaneous glandular differentiation, useful for the diagnosis of tumors with possible eccrine or apocrine differentiation.
The authors are grateful to Pedro Javier Benito Castellanos and to Laura Abendaño for technical support.