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Cited by 168Melanoma and MAPK PathwaysCell death mechanisms and regulationCutaneous Melanoma Detection and Management

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Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing

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Vörsmann, H, Groeber, F, Walles, H, Busch, S, Beissert, S, Walczak, H, Kulms, D·2013·Cell Death & Disease
DOI 10.1038/cddis.2013.249PMC PMC3730422
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Abstract

Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.

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Figure 1 Generation of 3D organotypic skin equivalents. Paraffin sections of skin equivalents (a) compared with normal human skin (b) were H&E stained and immunohistochemically analyzed for expression of keratins 14 and 10, involucrin, filaggrin and laminin 5. (c) Melanoma cells of different tumor progression stages, SBCL2 (RGP), WM115 (VGP) and 451-LU (MM), were seeded on top of the dermal layer before addition of keratinocytes. At day 17 of epidermal differentiation, the localization of melanoma cells within the reconstituted skin equivalents was visualized by H&E staining of paraffin sections. Images show one representative out of three independently performed experiments
Figure 1 Generation of 3D organotypic skin equivalents. Paraffin sections of skin equivalents (a) compared with normal human skin (b) were H&E stained and immunohistochemically analyzed for expression of keratins 14 and 10, involucrin, filaggrin and laminin 5. (c) Melanoma cells of different tumor progression stages, SBCL2 (RGP), WM115 (VGP) and 451-LU (MM), were seeded on top of the dermal layer before addition of keratinocytes. At day 17 of epidermal differentiation, the localization of melanoma cells within the reconstituted skin equivalents was visualized by H&E staining of paraffin sections. Images show one representative out of three independently performed experimentsCC BY-NC-ND · PubMed Central
Figure 2 Generation of the organotypic melanoma-spheroid skin model. (a) 250 451-LU melanoma cells per 25 μl drop RPMI medium were placed on the lid of a non-adhesive PBS containing petri dish and incubated for 15 days deriving melanoma spheroids with a size of about 500 μm in diameter. Live/death staining was performed by incubation with 5 μg fluorescein diacetate (FDA) and 45 g propidium iodide (PI)/ml PBS for 20 min at RT in the dark. Living cells stain FDA positive (green) and dead cells stain PI positive (red). (b) Ten 451-LU spheroids were collected, carefully mixed with primary fibroblasts and added to collagen I to from the dermal compartment of the full thickness skin equivalent
Figure 2 Generation of the organotypic melanoma-spheroid skin model. (a) 250 451-LU melanoma cells per 25 μl drop RPMI medium were placed on the lid of a non-adhesive PBS containing petri dish and incubated for 15 days deriving melanoma spheroids with a size of about 500 μm in diameter. Live/death staining was performed by incubation with 5 μg fluorescein diacetate (FDA) and 45 g propidium iodide (PI)/ml PBS for 20 min at RT in the dark. Living cells stain FDA positive (green) and dead cells stain PI positive (red). (b) Ten 451-LU spheroids were collected, carefully mixed with primary fibroblasts and added to collagen I to from the dermal compartment of the full thickness skin equivalentCC BY-NC-ND · PubMed Central
Figure 3 Melanoma spheroids resemble human melanoma metastasis. (a) Paraffin sections of tumor spheroids embedded into skin equivalents were stained for proliferating (Ki67) and apoptotic (TUNEL) cells. (b) Statistical analysis of Ki67-positive cells of peripheral versus central sub-population within the spheroid. Mean±S.D. of three different sections of three different spheroids are shown. (c) Scheme displaying cell sub-populations within a non-vascularized spheroid/tumor. (d) Sections of untreated spheroid-containing skin equivalents and primary human cutaneous metastasis were stained for ABCB5 and JARID1B, respectively. Examples for ABCB5- or JARID1B-positive cells are indicated by black arrows. (e) Statistical analysis of ABCB5- and JARID1B-positive cells of the peripheral sub-population within the spheroid compared with human cutaneous metastasis. Mean±S.D. of three different sections of three different spheroids/metastasis are shown
Figure 3 Melanoma spheroids resemble human melanoma metastasis. (a) Paraffin sections of tumor spheroids embedded into skin equivalents were stained for proliferating (Ki67) and apoptotic (TUNEL) cells. (b) Statistical analysis of Ki67-positive cells of peripheral versus central sub-population within the spheroid. Mean±S.D. of three different sections of three different spheroids are shown. (c) Scheme displaying cell sub-populations within a non-vascularized spheroid/tumor. (d) Sections of untreated spheroid-containing skin equivalents and primary human cutaneous metastasis were stained for ABCB5 and JARID1B, respectively. Examples for ABCB5- or JARID1B-positive cells are indicated by black arrows. (e) Statistical analysis of ABCB5- and JARID1B-positive cells of the peripheral sub-population within the spheroid compared with human cutaneous metastasis. Mean±S.D. of three different sections of three different spheroids/metastasis are shownCC BY-NC-ND · PubMed Central
Figure 4 Combination treatment sensitizes melanoma cells but not primary cells to TRAIL. (a) SBCL2 (RGP), WM-115 (VGP) and 451-LU (MM) were stimulated with TRAIL (100 ng/ml), cisplatin (20 μM) or UVB (200 J/m2) alone, or the combination treatment. After 16 h, apoptosis was determined using a Cell Death Detection ELISA. [R]=resistant; [s]=slightly sensitive; [S]=sensitive. *P<0.01 comparing co-treated with TRAIL-only-treated cells of the same line. (b) 451-LU cells (MM) were stimulated as in (a). After 16 h, protein status of XIAP and cleavage pattern of caspase-3 were determined by western blot analysis. β-Actin served as loading control. One out of three experiments is shown. (c) Primary fibroblasts, keratinocytes and melanocytes as well as 451-LU melanoma cells were treated and analyzed as in (a). Results in (a) and (c) show the mean±S.D. of three independent experiments. (d) Skin equivalents containing 451-LU nests were subjected to either short-term treatment: TRAIL (250 ng/ml), cisplatin (20 μM), UVB (400 J/m2) or in combination applied twice at days 13 and 15; or long-term treatment: TRAIL (250 ng/ml), cisplatin (2.5 μM), UVB (200 J/m2) or in combination applied five times over 10 days. At day 17, tumor volumes determined using ImageJ software. (e) Statistical analysis of tumor nest sizes counting the 10 largest melanoma nests, and overall tumor nest numbers (mean±S.D.) of three sections of skin equivalents derived from five different donors. *P<0.05, **P<0.01 comparing co-treated with TRAIL-only-treated tumor nests
Figure 4 Combination treatment sensitizes melanoma cells but not primary cells to TRAIL. (a) SBCL2 (RGP), WM-115 (VGP) and 451-LU (MM) were stimulated with TRAIL (100 ng/ml), cisplatin (20 μM) or UVB (200 J/m2) alone, or the combination treatment. After 16 h, apoptosis was determined using a Cell Death Detection ELISA. [R]=resistant; [s]=slightly sensitive; [S]=sensitive. *P<0.01 comparing co-treated with TRAIL-only-treated cells of the same line. (b) 451-LU cells (MM) were stimulated as in (a). After 16 h, protein status of XIAP and cleavage pattern of caspase-3 were determined by western blot analysis. β-Actin served as loading control. One out of three experiments is shown. (c) Primary fibroblasts, keratinocytes and melanocytes as well as 451-LU melanoma cells were treated and analyzed as in (a). Results in (a) and (c) show the mean±S.D. of three independent experiments. (d) Skin equivalents containing 451-LU nests were subjected to either short-term treatment: TRAIL (250 ng/ml), cisplatin (20 μM), UVB (400 J/m2) or in combination applied twice at days 13 and 15; or long-term treatment: TRAIL (250 ng/ml), cisplatin (2.5 μM), UVB (200 J/m2) or in combination applied five times over 10 days. At day 17, tumor volumes determined using ImageJ software. (e) Statistical analysis of tumor nest sizes counting the 10 largest melanoma nests, and overall tumor nest numbers (mean±S.D.) of three sections of skin equivalents derived from five different donors. *P<0.05, **P<0.01 comparing co-treated with TRAIL-only-treated tumor nestsCC BY-NC-ND · PubMed Central
Figure 5 Melanoma spheroids respond to combination treatment in vitro. (a) Melanoma spheroids were left untreated or stimulated with TRAIL (100 ng/ml), cisplatin (20 μM), UVB (200 J/m2) or a combination thereof. Twenty hours later, spheroids were stained for apoptosis using FITC-labeled AnnexinV (green) and propidium iodide (PI; red) and analyzed by immunofluorescence microscopy. Nuclei were visualized with Hoechst 33342 (blue). (b) Melanoma spheroids were treated as in (a). Sixteen hours later, the protein status of XIAP and cleavage pattern of caspase-3 were determined by western blot analysis. β-Actin served as loading control. One out of three independently performed experiments is shown
Figure 5 Melanoma spheroids respond to combination treatment in vitro. (a) Melanoma spheroids were left untreated or stimulated with TRAIL (100 ng/ml), cisplatin (20 μM), UVB (200 J/m2) or a combination thereof. Twenty hours later, spheroids were stained for apoptosis using FITC-labeled AnnexinV (green) and propidium iodide (PI; red) and analyzed by immunofluorescence microscopy. Nuclei were visualized with Hoechst 33342 (blue). (b) Melanoma spheroids were treated as in (a). Sixteen hours later, the protein status of XIAP and cleavage pattern of caspase-3 were determined by western blot analysis. β-Actin served as loading control. One out of three independently performed experiments is shownCC BY-NC-ND · PubMed Central
Figure 6 Melanoma spheroids integrated into skin equivalents respond to TRAIL+cisplatin but not to TRAIL+UVB. Organotypic skin equivalents containing 451-LU spheroids were left (a) untreated or subjected to short-term treatment with (b) TRAIL (500 ng/ml), (c) UVB (400 J/m2) or (e) cisplatin (40 μM) alone or in (d) TRAIL+UVB and (f) TRAIL+cispatin combination, respectively, applied twice on days 13 and 15. On day 17, paraffin sections were stained for Ki67, cleaved caspase-3 and TUNEL positivity, respectively. One representative out of three independently performed experiments is shown
Figure 6 Melanoma spheroids integrated into skin equivalents respond to TRAIL+cisplatin but not to TRAIL+UVB. Organotypic skin equivalents containing 451-LU spheroids were left (a) untreated or subjected to short-term treatment with (b) TRAIL (500 ng/ml), (c) UVB (400 J/m2) or (e) cisplatin (40 μM) alone or in (d) TRAIL+UVB and (f) TRAIL+cispatin combination, respectively, applied twice on days 13 and 15. On day 17, paraffin sections were stained for Ki67, cleaved caspase-3 and TUNEL positivity, respectively. One representative out of three independently performed experiments is shownCC BY-NC-ND · PubMed Central

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Metadata

Authors
Vörsmann, H., Groeber, F., Walles, H., Busch, S., Beissert, S., Walczak, H., & Kulms, D.
Affiliations
  • University of Stuttgart (DE)
  • Fraunhofer Institute for Interfacial Engineering and Biotechnology (DE)
  • Technische Universität Dresden (DE)
  • University College London (GB)
Corresponding author
Kulms, D
ORCID
Venue
Cell Death & Disease
Publisher
Springer Science and Business Media LLC
Published
2013-07-11
Volume
4
Issue
7
Pages
e719-e719
ISSN
2041-4889 (ISSN-L)
DOI
10.1038/cddis.2013.249
PMC
PMC3730422
OpenAlex
W2042222655
Cited by
168
References
44
Citations by year
2026: 6 · 2025: 10 · 2024: 14 · 2023: 8 · 2022: 18 · 2021: 12 · 2020: 15 · 2019: 14 · 2018: 16 · 2017: 14 · 2016: 16 · 2015: 13 · 2014: 11 · 2013: 1
FWCI
6.12
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death and Disease (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • UCL Discovery (University College London) (submittedVersion)
  • Fraunhofer-Publica (Fraunhofer-Gesellschaft) (submittedVersion)
Funders
Deutsche Krebshilfe
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Melanoma and MAPK Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
  • Health Sciences › Medicine › Oncology › Cutaneous Melanoma Detection and Management
Keywords
metastatic melanoma, 3D tumor models, TRAIL sensitization, cisplatin, UV-B radiation, combination therapy, drug resistance, in vitro drug testing, tumor necrosis factor-related apoptosis-inducing ligand
MeSH terms
Antineoplastic Agents, Cisplatin, Drug Screening Assays, Antitumor, Drug Synergism, Humans, Melanoma, Skin Neoplasms, Skin, Artificial, Spheroids, Cellular, Coculture Techniques, Cell Line, Tumor, TNF-Related Apoptosis-Inducing Ligand

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