Basement membrane proteins promote progression of intraepithelial neoplasia in 3‐dimensional models of human stratified epithelium

Abstract

We have developed novel 3-dimensional in vitro and in vivo tissue models that mimic premalignant disease of human stratified epithelium in order to analyze the stromal contribution of extracellular matrix and basement membrane proteins to the progression of intraepithelial neoplasia. Threedimensional, organotypic cultures were grown either on a de-epidermalized human dermis with pre-existing basement membrane components on its surface (AlloDerm), on a Type I collagen gel that lacked basement membrane proteins or on polycarbonate membranes coated with purified extracellular matrix proteins. When tumor cells (HaCaT-II4) were mixed with normal keratinocytes (4:1/normals:HaCaT-II4), tumor cells selectively attached, persisted and proliferated at the dermal-epidermal interface in vitro and generated dysplastic tissues when transplanted to nude mice only when grown in the presence of the AlloDerm substrate. This stromal interface was permissive for tumor cell attachment due to the rapid assembly of structured basement membrane. When tumor cells were mixed with normal keratinocytes and grown on polycarbonate membranes coated with individual extracellular matrix or basement membrane components, selective attachment and significant intraepithelial expansion occurred only on laminin 1 and Type IV collagencoated membranes. This preferential adhesion of tumor cells restricted the synthesis of laminin 5 to basal cells where it was deposited in a polarized distribution. Western blot analysis revealed that tumor cell attachment was not due to differences in the synthesis or processing of laminin 5. Thus, intraepithelial progression towards premalignant disease is dependent on the selective adhesion of cells with malignant potential to basement membrane proteins that provide a permissive template for their persistence and expansion.

E-cadherin loss promotes the initiation of squamous cell carcinoma invasion through modulation of integrin-mediated adhesion

“…First, loss of E-cadherin and increased integrin function enables adhesion specifically to Type I collagen, which was the predominant protein present at the stromal interface when 3D cultures were seeded onto Type I collagen gels. Type IV collagen may also play a role in the retention of E-cadherin-deficient cells, as it is subsequently deposited by II-4 cells in the basal layer several days after cells are seeded into 3D tissues (Andriani et al, 2004). Interestingly, integrin-mediated binding to laminin-1 does not appear to play a role during these events, as integrin-blocking antibodies do not significantly decrease the adhesion of E-cad-deficient cells.…”

“…Collectively, these results outline a novel aspect to loss of E-cadherin function that is linked to the mutually interdependent regulation of cell-cell and cellmatrix adhesion and has significant consequences for the conversion of premalignancy to cancer. or by enabling tumor cells to interact with basement membrane (BM) proteins (Andriani et al, 2004). These observations implied that alterations in the ability of IE tumor cells to adhere to adjacent cells or in their capacity to persist at the stromal interface were of crucial importance to their invasive potential.…”

E-cadherin Suppression Accelerates Squamous Cell Carcinoma Progression in Three-Dimensional, Human Tissue Constructs

“…The II-4 cell line used in our studies has been well characterized as representing an early stage of the malignant transformation process (19,20). Because II-4 cells exhibit lowgrade malignant behavior in vivo and harbor many of the important genetic hallmarks of the premalignant and early, invasive stages of SCC (1), such as mutations in p53 and ras, this cell line is optimal for incorporation into models of early carcinoma progression.…”

“…Organotypic constructs with intact basement membrane were prepared as previously described (19). Early-passage human dermal fibroblasts were added to neutralized bovine type I collagen (Organogenesis, Canton, MA) to a final concentration of 2.5 Â 10 4 cells/mL.…”

“…To accomplish this, we have previously developed three-dimensional, human tissue constructs that mimic premalignant disease of stratified squamous epithelium as it occurs in human tissues (18). These three-dimensional tissues have been generated in the presence of structured basement membrane (19) with a well-characterized cell line (HaCaT-II-4) that represents an early stage in the malignant transformation process in human skin (20). Generating three-dimensional tissues that mimic the essential features of human, premalignant disease provides new opportunities to investigate the early stages of cancer development in a clinically and biologically relevant context (21).…”