Mechanical Stress Impairs Mitosis Progression in Multi-Cellular Tumor Spheroids

<div><p>Growing solid tumors are subjected to mechanical stress that influences their growth rate and development. However, little is known about its effects on tumor cell biology. To explore this issue, we investigated the impact of mechanical confinement on cell proliferation in MultiCellular Tumor Spheroids (MCTS), a 3D culture model that recapitulates the microenvironment, proliferative gradient, and cell-cell interactions of a tumor. Dedicated polydimethylsiloxane (PDMS) microdevices were designed to spatially restrict MCTS growth. In this confined environment, spheroids are likely to experience mechanical stress as indicated by their modified cell morphology and density and by their relaxation upon removal from the microdevice. We show that the proliferation gradient within mechanically confined spheroids is different in comparison to MCTS grown in suspension. Furthermore, we demonstrate that a population of cells within the body of mechanically confined MCTS is arrested at mitosis. Cell morphology analysis reveals that this mitotic arrest is not caused by impaired cell rounding, but rather that confinement negatively affects bipolar spindle assembly. All together these results suggest that mechanical stress induced by progressive confinement of growing spheroids could impair mitotic progression. This study paves the way to future research to better understand the tumor cell response to mechanical cues similar to those encountered during in vivo tumor development.</p></div

Mechanical confinement does not impair mitotic cell rounding within MCTS.

<p>(A) Cryosections of a control MCTS and a mechanically confined MTCS (6 days in the PDMS device), stained for DNA (blue) and E-Cadherin (green) (scale bar, 10 µm). The cell outlines are drawn manually (green dashed line) to extract the area and the circularity of cells. (B) Area values of interphase cells (blue) and mitotic cells (orange) in control MCTS and in the body and tips of mechanically confined MCTS (6 days in the PDMS microdevice). Lines correspond to the mean ± SD. (C) Circularity values of interphase cells (blue) and mitotic cells (orange) in control MTCS and in the body and tips of mechanically confined MCTS (6 days in the PDMS microdevice). The error bars represent the mean ± SD. For control MCTS, 167 interphase cells and 175 mitotic cells were analyzed from 8 MCTS from 2 experiments. For confined MCTS, 307 interphase cells and 125 mitotic cells were analyzed in the body, and 146 interphase cells and 91 mitotic cells were analyzed in the tip, both from 10 MCTS from 4 experiments. (D) Cryosections of a mechanically confined MCTS stained for DNA (blue), E-Cadherin (grey), EdU (green) and pH3 (red) (scale bar, 10 µm). (E) Area (left panel) and circularity (right panel) values of pH3-positive mitotic cells in the body of mechanically confined MCTS relative to EdU incorporation (EdU+/pH3+: 79 cells analyzed, EdU−/pH3+: 43 cells analyzed, from 3 MCTS from 2 independent experiments). The lines correspond to the mean ± SD.</p

Mechanically confined growth impairs the regionalization of mitotic cells in MCTS.

<p>(A) Transmitted light images of a control MCTS and of a MCTS grown in a PDMS microdevice for 6 days. (B) Upper panels: Detection by immunofluorescence of mitotic cells (anti-phosphorylated Histone H3 antibody, pH3; in green) in cryosections of a control MCTS and a mechanically confined MCTS. The orientation of mechanically confined MCTS cryosections is parallel to the bottom of the channel (middle, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080447#pone-0080447-g001" target="_blank">Fig 1C</a>) and perpendicular to the bottom of the channel (right, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080447#pone-0080447-g001" target="_blank">Fig 1C</a>). Nuclei are stained with DAPI (blue). Lower panels: mean fluorescence intensity of pH3 staining in 8 cryosections from 6 control MCTS (3 independent experiments), 11 parallel cryosections from 11 mechanically confined MCTS (4 independent experiments) and 8 perpendicular cryosections from 6 mechanically confined MCTS (3 independent experiments). Dashed lines represent the walls of the PDMS channel. White lines indicate the width of the area where mitotic cells are localized (scale bar, 100 µm). (C) Percentages of mitotic cells (pH3-positive cells) in the peripheral (P) and the central (C) areas of control MCTS (n = 14 areas analyzed, from 7 MCTS from 3 experiments) and in the peripheral (P) and central (C) areas and the tips (Tips) of confined MCTS (n = 29 areas analyzed, from 12 MCTS from 6 experiments). The bars correspond to the mean ± SEM.</p

Growth-associated external mechanical stress leads to accumulation of cells arrested in mitosis.

<p>(A) Upper panel: Immunodetection of EdU incorporation (green) and mitotic cells (pH3-positive, red) in a cryosection from MCTS grown in PDMS microdevices for 6 days. Nuclei are stained using DAPI (blue). Lower panel: High-contrast image of the immunodetection of EdU incorporation (white) (scale bar, 100 µm). (B) Analysis of EdU incorporation in mitotic cells. Images correspond to magnifications of the regions indicated by the white squares in A from the tip (top panels) and the body (bottom panels) of a mechanically confined MCTS. The white arrow indicates a pH3- positive cell that is not EdU-positive. This cell is next to a pH3-positive/EdU-positive cell. (C) Percentage of pH3-positive/EdU-negative cells in the body (589 mitotic cells from 48 cryosections) and in the tips (331 mitotic cells from 95 cryosections) of mechanically confined MCTS (20 MCTS from 4 independent experiments) and in control (CTL) MCTS (358 mitotic cells from 34 cryosections from 15 MCTS from 4 independent experiment). Bars correspond to the mean ± SEM. (D) Map showing the localization of pH3-positive/EdU-negative cells in 8 cryosections from 8 mechanically confined MCTS from 4 independent experiments. The white line represents the outline of the MCTS and the red dots the localization of the pH3-positive/EdU-negative cells. The dashed lines indicate the microdevice PDMS walls.</p

Proliferation, hypoxia and apoptosis within MCTS grown in PDMS microdevices.

<p>(A) Transmitted light images of control and confined MCTS grown in a PDMS microdevice for 6 days. (B) Detection by immunofluorescence of Ki67 staining (marker of proliferative cells). Mean fluorescence intensity of 10 cryosections from 4 different control MCTS (from 3 independent experiments) and of 6 cryosections from 4 mechanically confined MCTS (6 days in the PDMS microdevice; from 3 independent experiments). (C) Detection by immunofluorescence of Cyclin A staining. Mean fluorescence intensity of 6 cryosections from 4 different control MCTS (from 3 independent experiments) and of 6 cryosections from 4 mechanically confined MCTS (from 3 independent experiments). (D) Detection by immunofluorescence of hypoxia (pimonidazole; in green). Mean fluorescence intensity of 8 cryosections from 8 different control MCTS (from 3 independent experiments) and of 8 cryosections from 6 mechanically confined MCTS (from 4 independent experiments). The grey circle indicates the MCTS margins. (E) Cleaved PARP staining (apoptosis marker). Mean fluorescence intensity of 6 cryosections from 6 control MCTS (two independent experiments) and of 6 cryosections from 6 mechanically confined MCTS (6 days in the PDMS device; 4 independent experiments). c-PARP, cleaved PARP. The grey circle indicates the MCTS margins. (A–E) The dashed lines indicate the PDMS walls. (A–D) The color scale indicates the fluorescence intensity (scale bar, 100 µm.). Nuclei are stained by DAPI (blue).</p

Mechanically confined MCTS show bipolar spindle defects.

<p>(A) Maximal projection of two mitotic cells from two z-stacks of images of a cryosection from a mechanically confined MCTS (6 days in the PDMS microdevice) incubated with an anti-γTubulin antibody (green). Nuclei were stained with DAPI (blue) (scale bar, 5 µm). (B) Distribution (percentage) of mitotic cells as a function of the number of spindle poles in control MCTS (CTL, 37 mitotic cells analyzed from 10 MCTS from 3 independent experiment) and in the body of mechanically confined MCTS (Confined, 66 mitotic cells analyzed from 8 MCTS from 4 independent experiment). (C) Distribution of the pole-to-pole distance (in µm) in bipolar mitotic cells from the control (CTL) and mechanically confined (Confined) MCTS analyzed in (B). The lines correspond to the mean ± SD.</p

Known genotoxic compounds identified with the DDR-Act-Fp reporter system amongst 1,280 pharmacologically compounds of the LOPAC library.

<p>Results are expressed as percentage of the fluorescence intensity measured in cells treated with 10μM Etoposide. ID number, compound name and putative mode of action are indicated.</p

Pharmacological manipulation of the DDR-Act-FP reporter using ATM kinase inhibitors.

<p>HCT116 cells expressing the DDR-Act reporter were treated with Etoposide 20 μM (E20) for 24 hours, in the absence or in the presence of KU-55933 or CP-466722 at the indicated concentration. (A) Western blot analysis of p53 and actin as control. (B) Fluorescence level was monitored after 24h. Bar graph represents the average fluorescence intensity+/-SD from 6 samples (500 cells analysed/sample with Cellomics scan software) for each condition. *:P<0.05; **:P<0.01; ***:P<0.005 (Unpaired t-test, Prism).</p

Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model

<div><p>Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.</p></div

A Fluorescent assay to monitor DDR pathway activation.

<p>(A) Schematic representation of the principle of the DDR-Act-FP reporter. Upon DNA-damage dependent accumulation of p53, transcriptional activation of the p21 promoter leads to the expression of the fluorescent protein (FP). (B) Principle of the assay. A cell line stably expressing the DDR-Act-FP reporter is used to produce 3D spheroids. DDR pathway activation can be monitored globally on 2D monolayer cultured cells and on 3D spheroids grown in 96 well plates using a High Content Screening approach.</p