The characteristics of Ishikawa endometrial cancer cells are modified by substrate topography with cell-like features and the polymer surface

Abstract

Conventional in vitro culture studies on flat surfaces do not reproduce tissue environments, which have inherent topographical mechanical signals. To understand the impact of these mechanical signals better, we use a cell imprinting technique to replicate cell features onto hard polymer culture surfaces as an alternative platform for investigating biomechanical effects on cells; the high-resolution replication of cells offers the micro- and nanotopography experienced in typical cell-cell interactions. We call this platform a Bioimprint. Cells of an endometrial adenocarcinoma cell line, Ishikawa, were cultured on a bioimprinted substrate, in which Ishikawa cells were replicated on polymethacrylate (pMA) and polystyrene (pST), and compared to cells cultured on flat surfaces. Characteristics of cells, incorporating morphology and cell responses, including expression of adhesion-associated molecules and cell proliferation, were studied. In this project, we fabricated two different topographies for the cells to grow on: a negative imprint that creates cell-shaped hollows and a positive imprint that recreates the raised surface topography of a cell layer. We used two different substrate materials, pMA and pST. We observed that cells on imprinted substrates of both polymers, compared to cells on flat surfaces, exhibited higher expression of β1-integrin, focal adhesion kinase, and cytokeratin-18. Compared to cells on flat surfaces, cells were larger on imprinted pMA and more in number, whereas on pST-imprinted surfaces, cells were smaller and fewer than those on a flat pST surface. This method, which provided substrates in vitro with cell-like features, enabled the study of effects of topographies that are similar to those experienced by cells in vivo. The observations establish that such a physical environment has an effect on cancer cell behavior independent of the characteristics of the substrate. The results support the concept that the physical topography of a cell's environment may modulate crucial oncological signaling pathways; this suggests the possibility of cancer therapies that target pathways associated with the response to mechanical stimuli.

Keywords: cell culture platforms; cell response; drug targets; mechanical forces; physical microenvironment; surface characteristics.

Figure 1

A schematic illustration of the bioimprinting processes that result in polystyrene culture substrates with cell-like topographies. Abbreviations: (−), negative imprint; (+), positive imprint; (f), flat substrate; pST, polystyrene.

Figure 2

Scanning electron microscope images of bioimprints of Ishikawa endometrial cancer cells. Notes: (A) A negative polystyrene imprint and (B) a positive polystyrene imprint. The images illustrate the retained fidelity of the fine features of cells during fabrication of the imprints.

Figure 3

Preferential growth on polystyrene Bioimprint. Notes: Ishikawa cells were cultured on polystyrene imprints of (A) Ishikawa cells and (B) C2C12 cells. The fractions of cells that could potentially have adhered to the flat area or the imprinted area on each slide were calculated. Data are presented as mean ± SEM of measurements from 25 grid areas on each of 4 incubations per substrate topography (**P<0.01).

Figure 4

Effects of Bioimprint substrate on Ishikawa cell size. Notes: Effects on (A) (f)pMA and (−)pMA imprint; and (B) (f)pST, (−)pST, and (+)pST imprints. Cells were grown on (A) flat and negative-imprinted pMA substrates; and (B) flat, negative-, and positive-imprinted pST substrates; and were stained for cytokeratin-18; images of cells were taken using epifluorescence microscopy. ImageJ software was used to measure each parameter from the images taken. Data are presented as mean ± SEM of measurements from at least 25 cells from five different images (**P<0.01; paired t-test). Abbreviations: (−), negative imprint; (+), positive imprint; (f), flat substrate; pMA, polymethacrylate; pST, polystyrene; SEM, standard error of the mean.

Figure 5

Effects of Bioimprint on expression of β1-integrin. Notes: Densitometry results of β1-integrin bands formed after Western blotting of lysates of cells cultured on (A) flat and negative-imprinted pMA substrates and (B) flat, negative-, and positive-imprinted pST substrates. Data are presented as mean ± SEM from at least six tests (*P<0.05; **P<0.01, paired t-test). Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SEM, standard error of the mean; pMA, polymethacrylate.

Figure 6

Effects of blocking β1-integrin on initial cell adhesion. Notes: Cells were grown on respective substrates with either no treatment or with treatments consisting of RGDS alone, RGES alone, or RGDS + RGES for 3 hours and were stained with Coomassie Blue. The number of adherent cells was quantified from a Coomassie Blue staining standard curve. Data are presented as mean ± SEM of measurements from at least three samples, each in triplicate (*P<0.05; **P<0.01; paired t-test). Abbreviations: RGDS, Arg-Gly-Asp-Ser tetrapeptide; RGES, Arg-Gly-Glu-Ser tetrapeptide; SEM, standard error of the mean.

Figure 7

Colocalization of β1-integrin and pFAK. Notes: Immunofluorescence staining of β1-integrin of cells cultured on (A) (f)pMAand (B) (−)pMA imprint; and on (C) (f)pST, (D) (−)pST, and (E) (+)pST imprints. β1-integrin was localized using mouse anti-β1-integrin, and pFAK was localized with rabbit anti-pFAK, which were then imaged with AlexaFlour 488 (green) and Atto 594 (red), respectively. Colocalization of β1-integrin and pFAK is indicated by the arrows, where merging of green and red tags to yellow coloration occurs. Cell nuclei were stained with Hoechst 33342 (blue) (original magnifications: ×20). Abbreviations: (−), negative imprint; (+), positive imprint; (f), flat substrate; pFAK, phosphorylated focal adhesion kinase; pMA, polymethacrylate; pST, polystyrene.

Figure 8

Effects of Bioimprint on FAK and pFAK expression. Notes: Densitometry results of (A) FAK and (B) pFAK bands formed after Western blotting of lysates of cells cultured on (left panel) flat and negative-imprinted pMA substrates and (right panel) flat, negative-, and positive-imprinted pST substrates. Data are presented as mean ± SEM from at least six tests (*P<0.05; **P<0.01; paired t-test). Abbreviations: FAK, focal adhesion kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; pFAK, phosphorylated FAK; pMA, polymethacrylate; pST, polystyrene; SEM, standard error of the mean.

Figure 9

Actin distribution. Notes: Immunofluorescence staining of actin in cells cultured on (A) (f)pMA, (B) (−)pMA imprint; and on (C) (f)pST, (D) (−)pST, and (E) (+)pST imprints. Cells grown on different surfaces were fixed with 4% paraformaldehyde and permeabilized using Triton X; then actin was probed using Texas Red-X Phalloidin red) (original magnifications: ×20). In cells cultured on pMA Bioimprint the actin filaments were more elongated than in cells cultured on flat pMA. There was no observable difference in the arrangement of actin in cells cultured on (−)pST or (+)pST surfaces relative to actin in cells on (f)pST. Abbreviations: (−), negative imprint; (+), positive imprint; (f), flat substrate; pMA, polymethacrylate; pST, polystyrene.

Figure 10

Effects of Bioimprint on expression of actin. Notes: Densitometry results of actin bands formed after Western blotting of lysates of cells cultured on (A) flat and negative-imprinted pMA substrates and (B) flat, negative-, and positive-imprinted pST substrates. Data are presented as mean ± SEM from at least five tests (*P<0.05; paired t-test). Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; pMA, polymethacrylate; pST, polystyrene; SEM, standard error of the mean.

Figure 11

Effects of Bioimprint on expression of cytokeratin-18. Notes: Immunofluorescence staining of cytokeratin-18 in cells cultured on (A) (f)pMA and (B) (−)pMA imprint; and on (C) (f)pST, (D) (−)pST, and (E) (+)pST imprints. Cells grown on different surfaces were fixed with 4% paraformaldehyde and permeabilized using cold methanol; then cytokeratin-18 was probed using mouse anti-cytokeratin-18 that was labeled with AlexaFluor 488 (green). Cell nuclei were co-stained with Hoechst 33342 (blue) (original magnifications: ×20). Abbreviations: (−), negative imprint; (+), positive imprint; (f), flat substrate; pMA, polymethacrylate; pST, polystyrene.

Figure 12

Effects of Bioimprint on cytokeratin-18 expression. Notes: Densitometry results of cytokeratin-18 bands formed after Western blotting of lysates of cells cultured on (A) flat and negative-imprinted pMA substrates and (B) flat, negative-, and positive-imprinted pST substrates. Data are presented as mean ± SEM from at least 12 tests (*P<0.05; **P<0.01; paired t-test). Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; pMA, polymethacrylate; pST, polystyrene; SEM, standard error of the mean.

Figure 13

Effects of Bioimprint on cell number after 60-hour culture. Notes: Cells were grown on (A) flat and negative-imprinted pMA substrates and (B) flat, negative-, and positive-imprinted pST substrates for 60 hours and were stained with Coomassie Blue. The number of cells was quantified from the standard curve of cell staining with Coomassie Blue. Data are presented as mean ± SEM of measurements from at least three samples, each as triplicate cultures (**P<0.01; *P<0.05; paired t-test). Abbreviations: pMA, polymethacrylate; pST, polystyrene; SEM, standard error of the mean.