Histone H3 Phosphorylation in Human Skin Histoculture as a Tool to Evaluate Patient's Response to Antiproliferative Drugs

Histoculture conditions

Four fresh human skin explants were purchased from Cureline Inc. The specimens were obtained from healthy female subjects undergoing abdominoplasty surgery. One melanoma specimen taken from a patient with stage IV metastatic skin lesion was obtained from the University of Rochester. Because all tissue samples used in this study were existing, deidentified samples, the research was exempted from seeking Internal Review Board approval under §46.101, b, (4). After removing subcutaneous fat, the skin specimens were cut into small (2–3 mm) pieces, placed onto Matrix 0.4 μm Millicell-CM insert (Millipore), and cultured at the air-liquid interface with the epidermis side facing upward at 37 °C in 5% CO2 incubator in Dulbecco's modified Eagle medium supplemented with 4.5 g/L glucose, l-glutamine, sodium pyruvate (Mediatech, Inc.), 10% fetal bovine serum (SAFC Biosciences), 100 μg/mL penicillin, and 100 μg/mL streptomycin.^2,3,16

The skin explants were incubated with vehicle (dimethyl sulfoxide [DMSO] 0.1%) or SCH2047069 at 0.025, 0.1, 1, 4, 8, and 10 μM concentrations for 16 and 24 hours. Selection of the drug concentration was based on the previously published data. ¹³ Human melanoma histocultures were treated similar to the skin cultures described earlier. Following drug treatment, four pieces per condition were collected and processed into formalin-fixed paraffin-embedded (FFPE) blocks for IHC analysis. To analyze cells in S-phase, 5 μM of 5-ethynyl-2′-deoxyuridine (EDU) was added to the culture 18 hours prior to tissue harvesting.

Immunohistochemistry

FFPE tissue blocks containing human skin or melanoma tissue were sectioned, deparaffinized, and rehydrated with serial passage through changes in xylene, graded ethanols, and deionized water. All slides were subjected to heat-induced epitope retrieval in target retrieval solution (pH 6.1) (cat S1699; Dako). Slides were then incubated with antiphospho H3 – Alexa488 (#9701; Cell Signaling) for 60 minutes at room temperature in a dark room. EDU staining was done using Click-iT EDU imaging kit following the manufacturer's instructions (Life Technologies, Cat# C10337). Slides were washed twice in TBS-T, mounted using Vectashield Mounting Medium with DAPI (Vector Laboratories, Cat# H-1500), and analyzed using Leica DMRE Fluorescent microscope and Nikon Cool-snap HQ camera. Phospho H3-positive cells were manually counted from two skin sections. Each section had ~3 mm² of epidermis surface. Statistical analyses were conducted using a one-way analysis of variance (ANOVA) test with GraphPad Prism software.

Phosphorylation of histone H3 in human melanoma histoculture

Analysis of phospho H3 in a melanoma histoculture treated with DMSO control for 24 hours showed occasional positive cells undergoing mitosis (Fig. 1A). Upon treatment with SCH2047069, a significant increase in the number of phospho H3-positive cells was observed, demonstrating that KSP inhibition can induce cell cycle arrest in mitosis in melanoma tissue cultured ex vivo. Detailed visualization of phospho H3-positive cells (Fig. 1A, right panel) showed a typical rosette chromosome conformation, previously described to be the consequence of KSP deficiency and incorrect chromosome segregation. ¹³

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Figure 1.

KSP inhibitor SCH2047069 induces mitotic arrest in human melanoma histoculture. (A) Phospho H3 immunostaining in melanoma histocultures. Analysis of phospho H3 showed an accumulation of cells arrested in mitosis upon treatment with SCH2047069. Phospho H3-positive cells have impaired chromosome segregation with a distinct rosette conformation (white arrows). (B) Analysis of cell proliferation by EDU incorporation. Cells in S-phase were visualized using EDU incorporation assay following treatment with SCH2047069 and DMSO control. SCH2047069 treatment has no effect on the number of proliferating EDU+ cells under the described conditions. Representative images are shown.

To determine the overall extent of cell proliferation in the melanoma histoculture, changes in total number of cells in S-phase were analyzed using EDU incorporation. The thymidine analog, EDU, was added to the melanoma histoculture for 18 hours, and EDU-positive cells were visualized by immunofluorescence. A substantial number of EDU-positive cells in the melanoma histoculture sample were detected demonstrating active proliferation of cells under histoculture conditions (Fig. 1B). Notably, addition of SCH2047069 to the culture for 24 hours did not impact the amount of EDU-positive cells, confirming that KSP inhibition arrests cells in mitosis and does not affect DNA replication and transition through the S-phase of cell cycle. ⁶

Altogether, these results demonstrate feasibility of using immunostaining of phospho H3 to measure the effects of the KSP inhibitor SCH2047069 on human melanoma samples treated ex vivo.

Assessment of human skin histocultures response to SCH2047069

To examine the feasibility of using phospho H3 immunostaining in skin punch biopsies as a potential pharmacodynamic biomarker, the effect of SCH2047069 on human skin histocultures was evaluated. A significant increase in the number of cells arrested in mitosis was observed after 16 and 24 hours of culture in the presence of SCH2047069 at 0.1, 4, and 8 μM concentrations (Figs. 2A and B). Notably, the assay showed reproducible results in all the four donors tested. The effect of the drug was more prominent after 24 hours than after 16 hours of treatment (2.48-fold increase in phospho H3 at 8 μM concentration of SCH2047069, P < 0.01, Fig. 2A). Whether the effect of SCH2047069 can be quantified after longer exposures to the drug remains to be determined.

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Figure 2.

Quantification of histone H3 phosphorylation in response to KSP inhibition in human skin biopsies. (A) Quantification of phospho H3-positive cells from skin histoculture sections. Human skin histocultures derived from abdominoplasty specimens of four human donors were treated with SCH2047069 or DMSO control, and phospho H3-positive cells were manually counted from two skin sections. Each section had ~3 mm² of epidermis surface. A significant enrichment on phospho H3-positive cells was observed at 100 nM, 4 μM, and 8 μM concentrations of SCH2047069 compared with DMSO control at 16 and 24 hours. Phospho H3 expression at 24 hours post treatment was significantly higher than that at 16 hours with SCH2043079 at concentrations 4 and 8 μM. Statistical analysis was conducted using two-way ANOVA test. Symbols *,** and *** indicate significance at P < 0.05, P < 0.01, and P < 0.001, respectively. Error bars represent standard error of the mean. (B) Representative images of phospho H3 staining after 24 hours of treatment with 8 μM SCH2047069 or DMSO control. Representative images of skin histocultures from four healthy human donors illustrate the accumulation of phospho H3-positive cells following SCH2047069 treatment.

Our study demonstrates the utility of the human skin histoculture model to evaluate the feasibility of using phospho H3 immunostaining in skin punch biopsies as a pharmacodynamic biomarker of response to KSP inhibitors and potentially other chemotherapeutic drugs that affect cell division.