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. 2016 Dec 7;11:6533-6545.
doi: 10.2147/IJN.S118065. eCollection 2016.

Nanomechanical Measurement of Adhesion and Migration of Leukemia Cells With Phorbol 12-myristate 13-acetate Treatment

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Free PMC article

Nanomechanical Measurement of Adhesion and Migration of Leukemia Cells With Phorbol 12-myristate 13-acetate Treatment

Zhuo Long Zhou et al. Int J Nanomedicine. .
Free PMC article

Abstract

The adhesion and traction behavior of leukemia cells in their microenvironment is directly linked to their migration, which is a prime issue affecting the release of cancer cells from the bone marrow and hence metastasis. In assessing the effectiveness of phorbol 12-myristate 13-acetate (PMA) treatment, the conventional batch-cell transwell-migration assay may not indicate the intrinsic effect of the treatment on migration, since the treatment may also affect other cellular behavior, such as proliferation or death. In this study, the pN-level adhesion and traction forces between single leukemia cells and their microenvironment were directly measured using optical tweezers and traction-force microscopy. The effects of PMA on K562 and THP1 leukemia cells were studied, and the results showed that PMA treatment significantly increased cell adhesion with extracellular matrix proteins, bone marrow stromal cells, and human fibroblasts. PMA treatment also significantly increased the traction of THP1 cells on bovine serum albumin proteins, although the effect on K562 cells was insignificant. Western blots showed an increased expression of E-cadherin and vimentin proteins after the leukemia cells were treated with PMA. The study suggests that PMA upregulates adhesion and thus suppresses the migration of both K562 and THP1 cells in their microenvironment. The ability of optical tweezers and traction-force microscopy to measure directly pN-level cell-protein or cell-cell contact was also demonstrated.

Keywords: adhesion; cell-to-cell contact; migration; optical trapping; protein micropillar matrix; traction-force microscopy.

Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Results of the adhesion assay. Notes: (A) Optical images of K562 and THP1 cells with and without (control) PMA treatment; (B) cell-adhesion assay tested by protein-coated 96-well microplates using MTT (n=3); (C) rearrangement of the data in (B) for the K562 control and THP1 control groups without PMA treatment. The experiments were repeated twice. Results of two-population t-tests are indicated in (B and C). Abbreviations: PMA, phorbol 12-myristate 13-acetate; BSA, bovine serum albumin.
Figure 2
Figure 2
Binding forces between leukemia cells and protein-coated spheres, measured using optical tweezers. Notes: (A) SEM images of protein-coated spheres. (B) Optical images of the pulling process between leukemia cells and protein-coated spheres a and d show the initial contact between sphere and cell. b, e and c, f show the separation process of sphere and cell when the stage was moved away from the trapped sphere. (C) Maximum trapping force (pN) vs laser power (mW) of protein-coated spheres (n=3 for each laser-power data point collected). (D) Measured binding forces between leukemia cells and protein-coated spheres (n=7 for each separated group). “SiR-E-cadherin” denotes interaction between cells transfected with SiR-E-cadherin siRNAs and spheres coated with anti-E-cadherin. (E) Rearrangement of data in (D) for the K562 control and THP1 control groups without PMA treatment. Results of two-population t-tests indicated in (D and E). Abbreviations: SEM, scanning electron microscopy; siRNAs, small-interfering RNAs; PMA, phorbol 12-myristate 13-acetate; BSA, bovine serum albumin.
Figure 3
Figure 3
Western blot results showing the expression of E-cadherin (E-cad) and vimentin (Vim) proteins in K562 and THP1 cells. Notes: (A, C) Western blots results of leukemia cells treated with and without PMA; (B, D) quantitative results of (A and B) using ImageJ software by assuming the intensity of the GAPDH proteins (inner control) to be 1. The experiments were performed three times, with the scatter of data indicated by error bars. Results of two-population t-tests indicated in (B and D). Abbreviations: PMA, phorbol 12-myristate 13-acetate; SiR, small-interfering RNA; SiR-E-cad, E-cadherin siRNA sequence CDH1; SiR-ctrl, the negative control of small-interfering RNA sequence.
Figure 4
Figure 4
Binding forces between leukemia and hBMSCs/hFBs measured using optical tweezers. Notes: (A) Maximum trapping force (pN) vs laser power (mW) of the trapped leukemia cells and hBMSCs/hFBs; (B) optical images of the pulling process between leukemia cells and hBMSCs/hFBs; (C) binding forces between leukemia cells and hBMSCs/hFBs (n=7 for each separated cell–cell binding force-measurement group). Results of two-population t-tests are shown in (C). Abbreviations: hBMSCs, human bone marrow stromal cells; hFBs, human fibroblasts; PMA, phorbol 12-myristate 13-acetate.
Figure 5
Figure 5
Results of transwell-migration assay. Notes: (A) Optical images of leukemia cells on the insert membrane layer of the transwell-migration assay; (B) transwell-migration measurement (n=3). The experiments were repeated twice. Results of two-population t-tests shown in (B). Abbreviations: PMA, phorbol 12-myristate 13-acetate; BSA, bovine serum albumin.
Figure 6
Figure 6
Traction-force measurements of K562 and THP1 cells with and without PMA treatment using multiphoton photochemical cross-linking-based fabrication. Notes: (AD) Protein micropillar matrix and leukemia cells at different scales; (E) traction force of K562 and THP1 cells with and without PMA treatment on the BSA protein micropillar matrix. Ten cells for each group were measured. The results of two-population t-tests shown in (E). Abbreviations: PMA, phorbol 12-myristate 13-acetate; BSA, bovine serum albumin.

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