Establishment and Characterization of Three Human Ocular Adnexal Sebaceous Carcinoma Cell Lines

Abstract

Ocular adnexal sebaceous carcinoma (SebCA) represents one of the most clinically problematic periocular tumors, often requiring aggressive surgical resection. The pathobiology of this tumor remains poorly understood, and few models exist that are suitable for preclinical testing. The aim of this study was to establish new cell lines to serve as models for pathobiological and drug testing. With patient consent, freshly resected tumor tissue was cultured using conditional reprogramming cell conditions. Standard techniques were used to characterize the cell lines in terms of overall growth, clonogenicity, apoptosis, and differentiation in vitro. Additional analyses including Western blotting, short tandem repeat (STR) profiling, and next-generation sequencing (NGS) were performed. Drug screening using mitomycin-C (MMC), 5-fluorouricil (5-FU), and 6-Diazo-5-oxo-L-norleucine (DON) were performed. JHH-SebCA01, JHH-SebCA02, and JHH-SebCA03 cell lines were established from two women and one man undergoing surgical resection of eyelid tumors. At passage 15, they each showed a doubling time of two to three days, and all could form colonies in anchorage-dependent conditions, but not in soft agar. The cells contained cytoplasmic vacuoles consistent with sebaceous differentiation, and adipophilin protein was present in all three lines. STR profiling confirmed that all lines were derived from their respective patients. NGS of the primary tumors and their matched cell lines identified numerous shared mutations, including alterations similar to those previously described in SebCA. Treatment with MMC or 5-FU resulted in dose-dependent growth inhibition and the induction of both apoptosis and differentiation. MYC protein was abundant in all three lines, and the glutamine metabolism inhibitor DON, previously shown to target high MYC tumors, slowed the growth of all our SebCA models. Ocular adnexal SebCA cell lines can be established using conditional reprogramming cell conditions, and our three new models are useful for testing therapies and interrogating the functional role of MYC and other possible molecular drivers. Current topical chemotherapies promote both apoptosis and differentiation in SebCA cells, and these tumors appear sensitive to inhibition or MYC-associated metabolic changes.

Keywords: cell line; eyelid cancer; mitomycin C; next-generation sequencing; sebaceous carcinoma.

Figure 1

Appearance of surgical specimens and corresponding cultured cells. (A) Hematoxylin and eosin-stained sebaceous carcinomas from the three patients showing basaloid nodules of tumor in the eyelid dermis (upper panels, representative nodules marked by asterisks). A higher-magnification view of SebCA02 (second row, middle panel) highlights markedly atypical nuclei and cytoplasmic vacuoles, which were seen in all three tumors, as well as mitotic figures (circles) and apoptotic bodies (arrows). Representative immunohistochemical staining for adipophilin and Ki67 shows sebaceous differentiation and high levels of proliferation. Tumor cells were also positive for p53 and p16, while the surrounding stroma was negative. (B) Sebaceous carcinoma cell lines grown in conditional reprogramming cell conditions were adherent and polypoid to spindled with short processes; variable numbers of vacuoles were noted in their cytoplasm (insets). P: passage number. Scale bar: 20 μm.

Figure 2

Analysis of sebaceous carcinoma cell growth and differentiation. (A) Western blot analysis confirmed strong adipophilin expression in all three sebaceous carcinoma cell lines, while this marker of sebaceous differentiation was not abundant in two established medulloblastoma cell lines (D283 and D425). Sebaceous carcinoma cells had similar MYC protein levels to the medulloblastoma lines, which are known to be dependent on high MYC expression. (B) Quantification of adipophilin and MYC protein expression levels (normalized to actin expression). **** p ≤ 0.0001. (C) All three sebaceous carcinoma cell lines grew robustly when evaluated with Cell-Titer blue assays (**** p ≤ 0.0001; n = 6 wells per line). (D) The three lines were capable of anchorage-dependent colony formation when seeded at single cell density, with images taken after 14 days.

Figure 3

Effects of chemotherapy on sebaceous carcinoma cells. (A) Dose-dependent inhibition in viable cell mass growth was seen on MTT assay after 5 days in all three lines with increasing concentrations of mitomycin C (**** p ≤ 0.0001, n = 6 wells per concentration). (B) Induction of cleaved PARP (Cl. PARP) was identified on Western blot after 48 h of treatment, supporting apoptotic effects of the therapy. (C,D) Growth inhibition (n = 6 wells per concentration) and apoptotic induction was also seen following 5-FU treatment. * p < 0.05, *** p < 0.001, **** p ≤ 0.0001.

Figure 4

Sebaceous differentiation after therapy. (A) Mitomycin-C treatment increased adipophilin protein expression in all three lines, suggesting that it can promote sebaceous differentiation of these carcinoma cells. (B) While 5-FU increased adipophilin expression in SebCA01 and SebCA03, a decrease was seen in SebCA02. Numbers between the blots with 1 and 10 μM treatment represent adipophilin expression normalized to actin.

Figure 5

Targeting metabolic changes associated with elevated MYC. Inhibition of glutamine metabolism using DON for 5 days slowed the growth of all three lines in a dose-dependent fashion on MTT assay (**** p ≤ 0.0001, n = 6 wells per concentration).