Extracellular Mechanical Stimuli Alters the Metastatic Progression of Prostate Cancer Cells within 3D Tissue Matrix

Abstract

This study aimed to understand extracellular mechanical stimuli's effect on prostate cancer cells' metastatic progression within a three-dimensional (3D) bone-like microenvironment. In this study, a mechanical loading platform, EQUicycler, has been employed to create physiologically relevant static and cyclic mechanical stimuli to a prostate cancer cell (PC-3)-embedded 3D tissue matrix. Three mechanical stimuli conditions were applied: control (no loading), cyclic (1% strain at 1 Hz), and static mechanical stimuli (1% strain). The changes in prostate cancer cells' cytoskeletal reorganization, polarity (elongation index), proliferation, expression level of N-Cadherin (metastasis-associated gene), and migratory potential within the 3D collagen structures were assessed upon mechanical stimuli. The results have shown that static mechanical stimuli increased the metastasis progression factors, including cell elongation (p < 0.001), cellular F-actin accumulation (p < 0.001), actin polymerization (p < 0.001), N-Cadherin gene expression, and invasion capacity of PC-3 cells within a bone-like microenvironment compared to its cyclic and control loading counterparts. This study established a novel system for studying metastatic cancer cells within bone and enables the creation of biomimetic in vitro models for cancer research and mechanobiology.

Keywords: EQUicycler; actin; bone; cancer; cyclic; cytoskeleton; elongation; extracellular; invasion; loading; mechanical; metastasis; prostate; static; stimuli.

Figure 1

Schematic of the 3D tissue matrix synthesis and mechanical stimulation loadings applied. Created with Biorender.com.

Figure 2

Schematic representation of EQUicycler system (A) before mechanical loading and (B) during the mechanical loading of the PC-3 embedded collagen construct. The displacement of the moving plate against the fixed plate causes compression of the silicon post, which creates mechanical strain on the cell-embedded collagen ring surrounding the post. (C) Schematic representation of the PC-3 cell-embedded collagen constructs before and during the mechanical loading. (D) Profile of applied strain during the mechanical loading associated with different experimental groups. “Design and Validation of Equiaxal Mechanical Strain Platform, EQUicycler, for 3D Tissue Engineered Constructs” BioMed Research International, Hindawi (2017) [11].

Figure 3

(A) The longitudinal section of the PC3-embedded collagen construct matrix (B) The longitudinal section of the invasion model assay (collagen matrix and acellular insert) (C) Schematic representation of penetrating PC3 cells into acellular collagen insert (invasion model).

Figure 4

The confocal images demonstrate the changes in the cytoskeleton organization of PC-3 cells under static, cyclic, and no mechanical stimuli conditions over three days. Green: F-actin, red: G-actin, blue: nucleus. Images were taken with 63× objective. For day 0, only one representative image is shown, as no mechanical stimulation was applied. Scale bar = 10 µm for the control group (day 1,2,3) and cyclic loading (day 1) and 25 µm for cyclic and static mechanical stimuli group (day 1,2,3).

Figure 5

(A) Confocal images of PC-3 cells embedded within the 3D collagen matrix following the mechanical stimuli. The green color is the cell cytoplasm. The scale bar is 50 µm. (B) Cell elongation index (EI) analysis for PC-3 cells within the 3D collagen matrix at day 3. The EI is calculated by dividing the cell’s long axis by the cell’s short axis, where EI = 1 designates an amoeboid cell, and EI ≥ 2 is designated as an elongated cell. Mean represented by the black line. The gradient scale to the right designates the spectrum of cell measurements with corresponding representative images of cell morphologies. * p < 0.001 relative to unloaded control. At least 3 fields of view from a set of images were selected from each group for quantification (n = 3).

Figure 6

F-actin (green) and G-actin (red) presence in PC-3 cells within the 3D collagen matrix under control (unloaded), cyclic, and static loading conditions. Scale bar = 25 µm.

Figure 7

(A) Single-cell actin polymerization analysis for actin ratio (F/G) value plot per experimental group (mean represented by black line). Ratios were not standardized to any group * p < 0.001 relative to control. Actin polymerization analysis for (B) cellular F-actin levels within each sample group and (C) cellular G-actin levels within each sample group. The error bar represents mean ± SD (n = 60). * p < 0.001 relative to control.

Figure 8

The changes in the number of cells over three days following the mechanical stimuli. The data on the number of cells on each characterization day were normalized with respect to the number of cells on day 1 to track the possible cell growth. Each bar represents the mean ± SD of the normalized number of cells for experimental groups (n = 4).

Figure 9

Normalized fold change in N-Cad gene expression following three-day static, cyclic, and no mechanical stimuli (control). GAPDH was used as the housekeeping gene. Values are shown as the mean ± SE (n = 3). * p < 0.001 relative to control, # p < 0.001 relative to experimental group.

Figure 10

Immunofluorescence images of PC-3 cells (green) at t = 0 h, t = 12 h, and t = 24 h after 3 days of mechanical stimuli. The images demonstrate that the number of PC-3 cells invaded into the acellular construct and their longest distance translocation into the acellular construct increased in the static loading group (static loading group t = 24 h). The white line is a hypothetical line representing the border between cellular and acellular constructs. Scale bar 250 µm.