PURPOSE
To investigate the effects of mitomycin-C (MMC) and 5-fluorouracil (5-FU) on the viability, proliferation, and migratory capacity of cultured ocular adnexal sebaceous carcinoma (SC) cells.
DESIGN
Laboratory investigation.
METHODS
Human SC cell lines (Bascom Palmer 50 and 52 [BP50 and BP52]) and human limbal stem cells (LSCs) were treated with various concentrations of MMC and 5-FU. Cytotoxicity was assessed with the tetrazolium MTT colorimetric viability assay on normal corneal vs tumor cells. Growth curves and scratch assays were performed to characterize the effects of these chemotherapeutic agents on SC proliferation and migration, respectively.
RESULTS
MMC decreased BP52 cell viability in a dose-dependent manner with a half-maximal effective dose (EC 50 ) of 11.8 μM after 72 hours. SC viability decreased >50% at 80 mM 5-FU after 72 hours. MMC reduced LSC viability in a dose-dependent manner with an EC 50 value of 3.24 μM, and 5-FU decreased LSC viability >50% at 160 μM. MMC decreased SC cell proliferation and migration in a dose-dependent manner. 5-FU displayed antiproliferative effects but did not affect cell migration at concentrations below 1000 μM.
CONCLUSIONS
Our in vitro data corroborate clinical observations that MMC is efficacious for treating ocular adnexal SC, albeit at the expense of LSC viability. Our findings also demonstrate that topical 5-FU exhibits antiproliferative effects that supersede its cancer-killing and antimigratory effects on cultured SC cells.
Sebaceous carcinoma (SC) is a malignant neoplasm that most commonly arises from the sebaceous glands of the ocular adnexa. It often masquerades as benign lesions, inflammatory conditions, or other malignancies, leading to delayed diagnosis, misdirected therapy, and higher morbidity and mortality. SC frequently exhibits diffuse pagetoid (intraepithelial) spread and multicentric origins, which are difficult to treat and generally require orbital exenteration to control. No standardized treatment exists, but surgical excision is the mainstay of therapy.
Various medical therapies have been attempted to treat the affected ocular surface and preserve the globe in cases of locally advanced SC. Topical chemotherapeutic agents like mitomycin-C (MMC) and 5-fluorouracil (5-FU) are used for conjunctival melanoma and ocular surface squamous carcinoma. This has translated to their empirical use for advanced SC as primary or adjuvant treatments. Yet, there is a dearth of scientific evidence on the efficacy of these topical agents for SC due in part to the lack of appropriate experimental models.
Recently, we reported the establishment and characterization of an ocular adnexal SC cell line named Bascom Palmer 50 (BP50) and demonstrated the effectiveness of MMC on its viability. In this present study, we further characterize the effects of MMC and 5-FU on 3 clinically salient cancer hallmarks: resistance to cell death, sustained proliferation, and cell migration. We also investigated the effects of these medications on the viability of cultured limbal stem cells (LSCs) to gauge their toxicity on normal corneal structures. Together, these findings may help inform clinical decision making about topical chemotherapy for advanced ocular adnexal SC.
METHODS
The University of Miami Institutional Review Board approved this study. Written informed consent for the establishment of the cell lines was obtained from the patients.
SC CELL CULTURE
BP50 and BP52 SC cell lines were used for this study. They were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; Gibco; Thermo Fisher Scientific) containing 10% fetal bovine serum (Thomas Scientific; Premium U.S. Source Heat Inactivated) supplemented with 1% antibiotic/antimycotic at 37°C in humidified 5% CO 2 . The cells were validated by short tandem repeat profiling.
LSC ISOLATION AND CULTURE
Sclerocorneal rims from 2 deceased donors were obtained from the Florida Lions Eye Bank (Miami) as excess tissue from cornea transplantations. Sclerocorneal rims were processed (no later than 12 hours postcollection) to remove undesired tissue components, including conjunctiva, iris, and Tenon capsule. The cleansed tissues were cut into equal size (2 mm × 2 mm) explants and subjected to overnight digestion (12-16 hours) in 1 mg/mL of collagenase A in DMEM/F-12 GlutaMAX medium (Thermo Fisher Scientific) with 50 µg/mL gentamicin at 37°C. The tissue was then gently disaggregated, and cells were passed through a 100-µm cell strainer and centrifuged at 2000 rpm for 5 minutes. LSCs were cultured in StemPro hESC serum-free medium (Thermo Fisher Scientific) at 37 °C in humidified 5% CO 2 .
DRUGS
MMC prepared for topical administration (0.4 mg/mL) and 5-FU (Roche) prepared for intravenous administration (50 mg/mL) were diluted in fresh complete media before each assay.
CELL VIABILITY ASSAY
SC and LSC viabilities after 72-hour treatment with MMC or 5-FU were assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Biotium) according to the manufacturer’s protocol, as previously described. Optical density was quantified with a SpectraMax MiniMax 300 Imaging Cytometer (Molecular Devices) at 570 nm, and background absorbance was measured at 630 nm. Quadruplicate readings were recorded. Values were normalized to the untreated control. The half-maximal effective doses (EC 50 ) on cell viability were determined with Combenefit software.
CELL PROLIFERATION ASSAY
Proliferation was measured with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) following the manufacturer’s protocol. SC cells were seeded with complete medium then treated with MMC or 5-FU the next day. Media with drug were replaced every 48 hours. Luminescence measurements were taken every 24 hours on the SpectraMax MiniMax 300 Imaging Cytometer (Molecular Devices). The mean percentage luminance change compared to the baseline was graphed. Each treatment was performed in triplicate. Growth curves were analyzed by calculating the area under the curves (AUCs), which provide estimates of percentage inhibition throughout the entire experiment. Ordinary 1-way ANOVAs with Dunnett post hoc comparison tests were performed to determine differences between the control and treated groups.
MIGRATION ASSAY
SC cells were grown to confluence and then pretreated with MMC or 5-FU for 24 hours. A uniform scratch was made in the center of each well with the 0.50-mm-width SPLScar Scratcher (Kisker Biotech). Baseline phase-contrast images were taken with the EVOS FL Auto Imaging System (Thermo Fisher Scientific). After 24 hours with treatment, cells were fixed, stained with crystal violet (Sigma-Aldrich), and reimaged. To measure SC cell migration without the confounding influence of proliferation, the experiment was limited to 24 hours after the scratch. The surface areas of the gap at baseline and after 24 hours were determined via manual tracing on software Fiji, and the percentage closure was calculated. Each treatment was performed in triplicate. The mean percentage wound closure was graphed. Ordinary 1-way ANOVAs with Dunnett post hoc comparison tests were performed.
Statistics
GraphPad Prism 6, version 9.0.2, was used for statistical data analysis. Values are presented as the mean ± SD.
RESULTS
EFFECTS OF MMC AND 5-FU ON SC AND LSC VIABILITY
The effects of MMC and 5-FU on SC and LSC viability were evaluated quantitatively with the colorimetric MTT assay, which measures cellular metabolic activity as an indicator of cell viability.
MMC decreased BP52 viability in a dose-dependent manner with a half-maximal effective dose (EC 50 ) of 11.8 μM after 72 hours of treatment ( Figure 1 , A), similar to what was reported in BP50 previously (EC 50 = 10.4 μM). 5-FU did not display potent acute toxic effects on cultured SC cells. SC viability decreased >50% with approximately 80 mM 5-FU (3 orders of magnitude greater concentration than MMC) after 72 hours ( Figure 1 , B).
MMC decreased LSC viability in a dose-dependent manner with an EC 50 of 3.24 μM ( Figure 2 , A). LSC viability decreased >50% with 160 μM 5-FU after 72 hours ( Figure 2 , B).
SC CELL PROLIFERATION AFTER MMC OR 5-FU TREATMENT
We measured the effects of MMC and 5-FU on SC cell proliferation by quantifying adenosine triphosphate (ATP) levels over time. The amount of cellular ATP is directly proportional to the number of viable cells.
MMC decreased SC cell proliferation in a dose-dependent manner according to the area under the curves (AUCs) quantification. Concentrations less than or equal to the MMC EC 50 were tested. BP50 demonstrated a 28.92% ± 2.32% ( P < .0001), 38.61% ± 1.09% ( P < .0001), and 57.87% ± 2.70% ( P < .0001) AUC reduction with 1.1, 3.3, and 10 μM MMC treatment compared to the untreated control group ( Figure 3 , A). BP52 demonstrated a 19.74% ± 3.64% ( P < .01), 37.78% ± 1.70% ( P < .0001), and 53.20% ± 1.09% ( P < .0001) decrease in the AUCs compared to the control after treatment with 1.1, 3.3, and 10 μM MMC, respectively ( Figure 3 , B). Significant cytotoxicity was observed following 5-day treatment with 10 μM MMC in both SC cell lines.
