Epidermal growth factor receptor activation and inhibition in 3D in vitro models of normal skin and human cutaneous squamous cell carcinoma

Abstract

The transmembrane tyrosine kinase epidermal growth factor receptor (EGFR) is considered a key player in the development of cutaneous squamous cell carcinoma (SCC), which is the second most common malignancy in white populations. Inhibition of EGFR with the small molecule tyrosine kinase inhibitor erlotinib is currently under clinical investigation in cutaneous SCC patients. In this study, we investigated the effects of EGFR activation and inhibition on normal and malignant in vitro human skin equivalents (HSEs). In healthy HSEs, increasing EGF concentrations ranging from 5 to 50 ng/mL resulted in a dramatic decrease in epidermal proliferation as immunohistochemically assessed by Ki67 and increased epidermal stress as assessed by K17 after 2 weeks of air-exposed culture. Also, higher concentrations of EGF induced remarkable epidermal disorganization with loss of proper stratification. Similar effects were observed in HSEs generated with cutaneous SCC cell lines SCC-12B2 and SCC-13. Treatment of both healthy and SCC-HSEs with 10 μM erlotinib resulted in efficient reduction of epidermal thickness from 10 to 3 viable cell layers and counteracted EGF-induced epidermal stress. Remarkably, erlotinib treatment caused severe desquamation in healthy HSEs, reminiscent of xerosis as a known side-effect in patients treated with erlotinib. The presented three-dimensional organotypic SCC models appear suitable for further investigations on the morphological and functional impacts of modifying EGFR signaling in cutaneous SCC, without burdening patients or mice. The effective inhibition of epidermal growth by erlotinib in our HSEs confirms the therapeutic potential of this tyrosine kinase inhibitor for cutaneous SCC patients.

Figure 1

Epidermal growth factor (EGF) affects epidermal morphology, proliferation and differentiation in human skin equivalents (HSEs). (a) Shown are cross sections of HSEs chronically supplemented with increasing concentrations of EGF. Epidermal growth factor‐treated healthy HSEs stained with hematoxylin & eosin (H&E) showed global epidermal disorganization increasing with EGF‐concentration. Early differentiation of HSEs was disturbed upon exposure to increasing EGF concentrations, as reflected by a disorganized staining pattern of K10 at higher EGF concentrations. Terminal differentiation, as reflected by involucrin staining, slightly expanded over multiple cell layers in HSEs exposed to increasing concentrations of EGF. Arrows and asterisks indicate examples of significant staining patterns. Scale bar holds for all panels and indicates 200 μm. (b) Proliferation of HSEs first increased and then significantly decreased upon chronic exposure to increasing concentrations of EGF, as quantified after Ki67 staining. Representative Ki67 stainings are shown for all conditions. Arrows indicate examples of significantly stained nuclei.

Figure 2

Epidermal growth factor (EGF) increases epidermal stress in human skin equivalents (HSEs). Shown are cross sections of HSEs chronically supplemented with increasing concentrations of EGF. Epidermal activation and stress in HSEs increased upon chronic exposure to increasing concentrations of EGF, as indicated by immunohistochemical staining of epidermal stress markers keratin 16 (K16) and keratin 17 (K17). Scale bar holds for all panels and indicates 200 μm.

Figure 3

Erlotinib reduces epidermal thickness and integrity. Shown are hematoxylin & eosin (H&E) stained cross sections of healthy human skin equivalents (HSEs) treated with erlotinib during their air‐exposed (AE) culture phase, either with or without 5 ng/mL epidermal growth factor (EGF). Treatment of healthy HSEs with erlotinib resulted in a lower number of viable cell layers and an absence of stratum corneum (b–d) when compared to untreated control HSEs (a). This effect was most pronounced upon chronic supplementation of erlotinib (b) and less pronounced when co‐treated with EGF (d,e). When treated with erlotinib only in the final stage of the HSE culture (10–14 days AE), epidermal thickness was unaffected (c–e). Chronic supplementation with EGF, combined with erlotinib treatment in the final culture (10–14 days AE) phase affected neither the thickness of the epidermis nor that of the stratum corneum of the HSEs (e). Scale bar holds for all panels and indicates 200 μm.

Figure 4

Erlotinib affects epidermal proliferation, differentiation and activation in healthy human skin equivalents (HSEs). (a) Proliferation of HSEs significantly decreased after chronic erlotinib treatment during the entire air‐exposed (AE) culture phase compared to normal skin and untreated HSEs, as quantified after Ki67 staining. Erlotinib treatment only during the last 4 days of AE culture (10–14 days AE) or simultaneous treatment with epidermal growth factor (EGF) did not significantly affect proliferation. Representative Ki67 stainings are shown for the significantly different conditions. Arrows indicate examples of significantly stained nuclei. (b) Shown are cross sections of HSEs supplemented with erlotinib, either with or without EGF. Erlotinib treatment did not affect early and terminal differentiation of HSEs, as reflected by K10 and involucrin staining, respectively. Erlotinib treatment did not result in epidermal stress, as K17 staining remained negative. Scale bar holds for all panels and indicates 200 μm.

Figure 5

Erlotinib affects epidermal thickness in human skin equivalents (HSEs) generated with squamous cell carcinoma (SCC) cell lines SCC‐12B2 and SCC‐13. Shown are hematoxylin & eosin (H&E) stained cross sections of HSEs generated with cutaneous SCC cell lines SCC‐12B2 and SCC‐13 in the presence of epidermal growth factor (EGF) or erlotinib. SCC‐12B2 incorporated in HSEs resulted in a thick and disorganized epidermis. Human skin equivalents with SCC‐13 showed slight epidermal disorganization and poor dermal‐epidermal attachment. Upon EGF supplementation, epidermis generated with SCC‐12B2 thickened, whereas epidermis generated with SCC‐13 increased in disorganization. Erlotinib treatment resulted in strongly reduced epidermal thickness, leaving a thin disorganized epidermal SCC‐12B2 cell layer and a thin and detached but scaly SCC‐13 cell layer on top of the dermal equivalent. Scale bar holds for all panels and indicates 200 μm.