The Skin: A Multi-Cellular Barrier to the External World
The skin is a multi-layered organ that covers the entire body and protects us from the external environment. The outermost layer, the epidermis, is largely composed of keratinocytes, which proliferate in the lower epidermis and migrate out, becoming post-mitotic and losing their nuclei, eventually becoming corneocytes. Corneocytes are shed from the outer part of the skin as new corneocytes migrate in.
The epidermis also contains melanocytes, the pigmented cells from which melanoma develops. Under the epidermis lies the dermis, a layer of connective tissue that contains hair follicles, sweat glands, and the nerve endings that allow us to feel touch and temperature. The dermis also contains dermal fibroblasts, which participate in wound healing, as well as make the extracellular matrix that connects the skin to underlying body structures. Together, the layers of the skin allow us to interact with the world around us, while also protecting our internal organs from exposure.
The catalog of Lifeline® skin cells includes:
• Dermal Microvascular Endothelial Cells (Adult)
• Epidermal Melanocytes (Neonatal)
• Dermal Microvascular Endothelial Cells (Neonatal)
• Epidermal Melanocytes (Adult)
• Epidermal Melanocytes (Neonatal)
• Epidermal Melanocytes (Neonatal, Highly Pigmented)
• Dermal Fibroblasts (Neonatal, Xeno-Free)
• Epidermal Keratinocytes
• Dermal Fibroblasts (Neonatal)
• Epidermal Keratinocytes (Adult)
• Epidermal Keratinocytes (10-Donor Pool)
Recent Studies Using Lifeline® Melanocytes, Dermal Fibroblasts, and Keratinocytes
While melanoma is more rare than other types of skin cancer, it is the most aggressive, with metastasis sites including the brain, lung, liver, and bone. Development of effective targeted treatments for melanoma rely on identifying particular characteristics in melanoma cells that can be targeted without affecting healthy, normal cells. With this in mind, Sakamoto and colleagues set out to examine the therapeutic efficacy of antibodies generated against EPHA2, a receptor tyrosine kinase that is overexpressed in melanoma and correlated with poor prognosis. Interestingly, ligand-stimulated EPHA2 signaling is predicted to have a tumor suppressor effect by reducing proliferation and migration and inducing apoptosis. Therefore, activation of the pathway should have anti-tumor effects.
This group had previously generated two monoclonal antibodies against EPHA2, SHM16 and SHM17, which they confirmed bound to EPHA2 using immunoprecipitation, affinity purification and liquid chromatography/tandem mass spectrometry, and reactivity with human EPHA2 in melanoma cells with and without EPHA2. Using flow cytometry, they next demonstrated that EPHA2 was expressed on the surface of three melanoma cell lines (A375, A2058, and G361), but not expressed on Lifeline® normal human dermal fibroblasts. Compared to the weak reactivity of SHM16, SHM17 reacted well with EPHA2 on the surface of Lifeline® normal human melanocytes.
The researchers next performed a competitive binding experiment with ephrin-A1 (the EPHA2 ligand) to determine where SHM16 and SHM17 bound to EHPA2. They found that while SHM17 did disrupt ephrin-A1 binding to EPHA2, SHM16 did not, suggesting the epitopes of the two antibodies are different, and only SHM16 interferes with ligand binding. To examine the ability of the anti-EPHA2 antibodies to interfere with tumorigenic behavior of melanoma cells, the authors treated melanoma cells with SHM16 or SHM17 and performed a wound scratch assay and a Matrigel invasion assay. They found that SHM16, but not SHM17 reduced migration and invasion of A375 melanoma cells. Finally, they investigated whether either antibody could deliver an immunotoxin to melanoma cells. Using a saporin (a cytotoxin)-conjugated secondary antibody, the researchers found that SHM16, but not SHM17, successfully delivered saporin to A375, A2058, G361, and SK-MEL28 melanoma cells lines. In all but SK-MEL28 cells, SHM16 treatment caused significant cytotoxicity.
Together, the results of this study suggest that the anti-EPHA2 agonistic antibody, SHM16, is a potential therapeutic approach to treating melanoma.
Psoriasis is a common skin condition that results from the hyperproliferation of keratinocytes. In particular, the pro-inflammatory interleukin-36 (IL-36) family of cytokines has been implicated in disease pathogenesis. Given its role in promoting keratinocyte proliferation in psoriasis, Jiang and colleagues wondered whether IL-36 could also regulate keratinocyte proliferation in the context of wound healing. To this end, they found that compared to unwounded skin, IL-36α, IL-36β, and IL-36γ transcript expression, as well as IL-36γ protein expression was increased in the skin of wounded mice. Furthermore, using mice lacking Tlr3 or Trif (components of a toll-like receptor signaling pathway initiated upon skin injury), they found that induction of IL-36 cytokine expression depended on Tlr3-Trif signaling. Then, using Lifeline® normal human epidermal keratinocytes treated with UVB-damaged cells, the authors illustrated that IL-36γ induction upon exposure to damaged cells was dependent on TLR3. Additionally, they found that the TLR3 ligand, poly(I:C), induced IL-36γ expression in Lifeline® keratinocytes in a TLR3-dependent fashion.
To determine whether the Vitamin D receptor (VDR) is involved in regulating expression of IL-36γ through TLR3, the researchers treated Lifeline® keratinocytes with poly(I:C) and found that in fact, VDR expression was suppressed. Furthermore, VDR silencing increased IL-36γ expression, while VDR overexpression reduced IL-36γ expression. The authors then identified VDR binding sites in the IL-36γ promoter, illustrating the direct regulation of IL-36γ by VDR. Using overexpression and knockdown studies, they also demonstrated that activation of SLUG (a transcription factor that negatively regulates VDR transcription) by TLR3 inhibits VDR expression, which in turn, releases the brake on IL-36γ expression.
The researchers then found that poly(I:C)-induced IL-36γ expression results in stimulation of REG3A (a regulator of keratinocyte proliferation) expression in Lifeline® keratinocytes, mouse keratinocytes, and in the skin wounds of mice. Using skin wounding in mice, the researchers demonstrated that IL-36γ and REG3A inhibited keratinocyte differentiation to promote proliferation. Finally, using Lifeline® keratinocytes and a scratch assay, the group showed that the TLR3-IL-36γ-REG3A axis promotes wound healing, suggesting this signaling axis might be a viable target for wound healing therapy.
Make sure to keep up with us here on the blog every other week to learn more about how researchers around the world are using Lifeline® cells to answer their scientific questions!
Melanoma information from www.melanoma.org