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Cell cycle progression in confining microenvironments is regulated by a growth-responsive TRPV4-PI3K/Akt-p27Kip1 signaling axis

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Science Advances  07 Aug 2019:
Vol. 5, no. 8, eaaw6171
DOI: 10.1126/sciadv.aaw6171
  • Fig. 1 Faster stress relaxation in alginate hydrogels promotes tumor spheroid growth.

    (A) Schematics of cells dividing in physiological tissues such as an epithelial monolayer and a growing tumor. Physiological tissues provide a viscoelastic confining microenvironment to cells. Viscoelastic hydrogels can be used to mimic these microenvironments. (B) Stress relaxation tests on alginate hydrogels exhibiting slow (S), medium (M), and fast (F) relaxation. Relaxation modulus was normalized by the initial modulus in response to 10% compressional strain. (C) Time scale at which the relaxation modulus is relaxed to half of its initial value from stress relaxation tests. (D) The initial modulus of gels in (B). (E) Fluorescence images of spheroids formed by MDA-MB-231 cancer cells in slow-, medium-, and fast-relaxing gels at the indicated days. Here and in all other figures, red and green fluorescence indicate red fluorescent protein (RFP) histones and green fluorescent protein (GFP) microtubules, respectively. (F) The growth curves of MDA-MB-231 spheroids cultured in gels with an initial modulus of 3 or 16 kPa and varying relaxation, over 30 days (n = 10 to 77 spheroids). (G) The diameter of spheroids formed by MDA-MB-231 as a function of relaxation time at 15 days. The diameter of spheroids for (H) MCF7 at 15 days (n = 11 to 47 spheroids) and (I) HT1080 at 14 days (n = 20 to 43 spheroids). (J) Fluorescence images of spheroids formed by MDA-MB-231 for EdU staining at day 10. (K) The fraction of EdU-positive MDA-MB-231 cells cultured in gels with an initial modulus of 3 or 16 kPa and varying relaxation [soft and stiff, n = 3, measured in 16 to 38 cells; one-way analysis of variance (ANOVA) tests; *P < 0.05 and **P < 0.01]. (L) The fraction of EdU-positive MDA-MB-231 cells as a function of relaxation time. Data are shown as means ± SD, except for (G and I), where data are shown as means ± SEM. Scale bars, 10 μm (for all figures).

  • Fig. 2 Hydrogel stress relaxation regulates cell cycle progression from the G1 phase to the S phase.

    (A) Cell cycle analysis of cells cultured in slow-, medium-, and fast-relaxing gels for 10 days, measured by flow cytometry. Inset, fluorescence intensity (FI) versus forward scatter (FSC). Red rectangle indicates a gate of cell population. A.U., arbitrary units. (B) Population of cells in G0/G1, S, and G2/M phases in soft (3 kPa) and stiff (16 kPa) gels with varying relaxation (n = 2 to 4 per each condition). (C) Population of cells in the G0/G1 phase as a function of relaxation time. (D) A schematic of cell cycle progression including a mechanical checkpoint associated with hydrogel relaxation or mechanical confinement identified by the studies. Data are shown as means ± SD.

  • Fig. 3 TRPV4 activation regulates PI3K/Akt-p27Kip1 signaling activation and proliferation.

    (A) Fluorescence images of p27Kip1 in single cells cultured in slow-, medium-, and fast-relaxing gels. Here and in all other figures, yellow fluorescence indicates p27Kip1, unless otherwise specified. (B) The ratio of the nuclear p27Kip1 to the cytoplasmic p27Kip1 in cells in soft (3 kPa) gels with varying relaxation (n = 30 to 88 cells). (C) The fraction of EdU-positive cells (n = 3, measured in 10 to 48 cells). (D) The diameter of spheroids cultured in soft and fast-relaxing gels for 10 days in the presence of the indicated small-molecule inhibitors (n = 22 to 34 spheroids). n.s., not significant. (E) Immunoblot of Akt and phosphorylated Akt (p-Akt) for cells in soft gels with slow and fast relaxation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) The ratio of the nuclear p27Kip1 to the cytoplasmic p27Kip1 in cells in soft and fast-relaxing gels with and without PI3K/Akt inhibitor (n = 44 to 57 cells). (G) The fraction of EdU-positive cells in soft and fast-relaxing gels with and without PI3K/Akt inhibitor (n = 3, measured in 15 to 28 cells). (H) Fluorescence images of p27Kip1 in cells cultured in fast-relaxing gels with and without TRPV4 antagonist. (I) The ratio of the nuclear p27Kip1 to the cytoplasmic p27Kip1 in cells treated with TRPV4 antagonist (n = 51 to 123 cells). (J) The fraction of EdU-positive cells in fast-relaxing gels with and without TRPV4 antagonist (n = 3, measured in 12 to 30 cells). (K) Fluorescence images of p27Kip1 in cells cultured in slow-relaxing gels with and without TRPV4 agonist and PI3K/Akt inhibitor. (L) The ratio of the nuclear p27Kip1 to the cytoplasmic p27Kip1 in cells in the indicated conditions (n = 34 to 92 cells). (M) The fraction of EdU-positive cells (n = 3, measured in 11 to 36 cells). (N) Fluorescence images of p27Kip1 in cells cultured in fast-relaxing gels with and without GsMTx4. (O) The ratio of the nuclear p27Kip1 to the cytoplasmic p27Kip1 in cells treated with GsMTx4 (n = 35 to 51 cells). (P) The fraction of EdU-positive cells in fast-relaxing gels with GsMTx4 (n = 3, measured in 10 to 19 cells). (Q) A proposed mechanism underlying cell cycle progression in confining hydrogels. In (B) to (D), (L), and (M), one-way ANOVA tests were used, and in (F), (G), (I), (J), (O), and (P), Student’s t tests were used; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The box plots show 25th to 75th percentiles, and whiskers show minimum and maximum. Data are shown as means ± SD. Scale bars, 10 μm [for (A), (H), (K), and (N)].

  • Fig. 4 Single-cell growth, regulated by hydrogel stress relaxation or osmotic pressure, is mediated by the Na+/H+ exchanger and controls p27Kip1 localization.

    (A) A schematic of a cell growing in a hydrogel. Cells must exert mechanical stress to allow cell growth in the confining hydrogels. (B) 2D planar view of 3D computational simulations of hydrogel deformation due to cells exerting a constant outward stress for a time scale of 4 hours. Note that the viscoelastic parameters used for the simulations were derived from creep tests, which more closely approximate the case of a cell applying a constant stress to the hydrogel. White dashed lines indicated cell boundaries. (C) The diameter of the simulation models in response to a constant stress for a time scale of 4 hours. (D) Fluorescence images of single cells growing during the G1 phase in soft gels with varying relaxation for 2 days. (E) Quantification of the diameter of single cells. The diameter of single cells before encapsulation is indicated as “in suspension” and right after encapsulation is denoted as “initial” (n = 43 to 65 cells, one-way ANOVA tests and Tukey’s comparisons with respect to the initial condition). (F) Comparison of the experimentally and computationally obtained diameter of single cells as a function of relaxation time. The time scale for the simulations was 2 days for all cases, except for the case of soft gels with fast relaxation, where the time scale was 4 hours. (G) The diameter of single cells cultured in soft and fast-relaxing hydrogels with varying osmotic pressure (n = 31 to 52 cells). (H) Fluorescence images of p27Kip1 in cells under the different osmotic conditions. TRPV4 agonist was additionally added to cells under an osmotic pressure of 170 mOsm/liter. (I) The ratio of the nuclear p27Kip1 to the cytoplasmic p27Kip1 in cells under conditions corresponding to (H) (n = 31 to 58 cells). (J) The fraction of EdU-positive cells with and without an osmotic pressure of 170 mOsm/liter and TRPV4 agonist (n = 3, measured in 12 to 28 cells). (K) The diameter of single cells in fast-relaxing gels with and without TRPV4 antagonist (n = 46 to 97 cells). (L) Fluorescence images of p27Kip1 in cells in soft and fast-relaxing gels with an NHE inhibitor. TRPV4 agonist was additionally added to cells with an NHE inhibitor. (M) The diameter of single cells (n = 44 to 50 cells). (N) The ratio of the nuclear p27Kip1 to the cytoplasmic p27Kip1 in cells under the conditions corresponding to (L) (n = 44 to 50 cells). (O) The fraction of EdU-positive cells (n = 3, measured in 13 to 33 cells). In (E), (G), (I), (J), and (M) to (O), one-way ANOVA tests were used, and in (K), Student’s t tests were used; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are shown as means ± SD. The box plots show 25th to 75th percentiles, and whiskers show minimum and maximum. Scale bars, 10 μm [for (D), (H), and (L)].

  • Fig. 5 A growth-responsive TRPV4-PI3K/Akt-p27Kip1 signaling axis controls the S phase progression in confining 3D microenvironments.

    (A) Florescence images of p27Kip1 and EdU staining of MCF10A cells cultured in alginate-rBM gels with and without TRPV4 antagonist. (B) The ratio of nuclear p27Kip1 to cytoplasmic p27Kip1 (n = 40 to 97 cells) and quantification of EdU-positive cells for MCF10A cells (n = 3, measured in 15 to 36 cells). (C) Florescence images of p27Kip1 and EdU staining of 3T3 fibroblasts cultured in collagen gels with and without TRPV4 antagonist. (D) The ratio of nuclear p27Kip1 to cytoplasmic p27Kip1 (n = 26 to 44 cells) and quantification of EdU-positive cells for 3T3 fibroblasts (n = 3, measured in 21 to 48 cells). In (B) and (D), Student’s t tests were used; *P < 0.05 and **P < 0.01. (E) When mechanical confinement is low, for example, in hydrogels with fast stress relaxation, cells grow through NHE during the G1 phase. Cell growth promotes the activity of SACs, which activate the PI3K/Akt pathway, which, in turn, drives cytoplasmic localization of p27Kip1, thereby promoting the S phase entry and proliferation. In microenvironments with higher confinement, such as hydrogels with slow stress relaxation, cell growth is inhibited so that SACs are not activated, and therefore, nuclear localization of p27Kip1 blocks the S phase entry and proliferation. Data are shown as means ± SD. Scale bars, 10 μm [for (A) and (C)].

  • Table 1 Stress relaxation properties of selected soft tissues that exhibit substantial stress relaxation.

    Tabulated values from stress relaxation tests of selected tissues. ti and tf, respectively, indicate the time when stress relaxation tests started and ended. Ei is the initial modulus measured at ti in units of kilopascal. The stress relaxation time, τ1/2, indicates the time at which the initial stress relaxes to half its original value. Ef /Ei is the normalized relaxation modulus measured at tf. Times and stresses reported here are approximate. Note that, in some stress relaxation tests, the stress reached an equilibrium stress by the end of the tests, while in other tests, the test may not have been sufficiently long for the stress to reach an equilibrium value or zero. For testing method, comp. and indent. represent compression and indentation, respectively. (–) indicates not reported.

    Tissue typeAnimalStrain (%)Initial modulusStress relaxation timeFinal relaxation
    modulus
    Testing methodReferences
    ti (s)Ei (kPa)τ1/2 (s)tf (s)Ef/Ei (norm)
    Bone marrowRat150.1105000.25Comp.(1)
    Breast cancerHuman400.011.81035000.02Indent.fig. S1
    Murine2–100220–501800.2–0.4Indent.(11)
    AdiposePorcine0.10.01350.1500.06Shear(9)
    Rat150.110010000.5Comp.(1)
    BrainBovine10.60.010.715000.21Shear(10)
    Rat100.010.551400.18Shear(2)
    150.110010000.25Comp.(1)
    LiverBovine0.130.0150.12000.08Shear(14)
    Rat2570.06–0.085010000.25Shear(15)
    150.110010000.3Comp.(1)
    Embryonic tissuesChicken015–701700.2–0.5Comp.(12)
    MuscleMurine10101.25706000.3Shear(16)
    HematomaHuman15020010000.3Comp.(13)
    SkinRat165015000.47Tension(7)
    Swine5–150200–120012000.3–0.5Tension(8)
    Human300120090000Radial tension(6)

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaaw6171/DC1

    Fig. S1. Mechanical characterization of alginate hydrogels and human breast cancer tissue.

    Fig. S2. Tumor growth in hydrogels with varying stress relaxation.

    Fig. S3. Cell cycle progression and cell cycle entry are enhanced in hydrogels with faster relaxation.

    Fig. S4. The inhibition of the PI3K/Akt pathway diminishes the diameter of spheroids formed by HT1080 and MCF7 cells.

    Fig. S5. Cells did not secrete and deposit collagen and laminin-5, and the inhibition of actomyosin contractility did not affect cell cycle progression.

    Fig. S6. Impact of TRPV4 inhibition on proliferation.

    Fig. S7. 3D computational simulations of hydrogel deformation due to cell exerting a constant outward stress in hydrogels with varying relaxation.

    Fig. S8. The growth of single cells in hydrogels with varying stress relaxation.

    Fig. S9. Increasing osmotic pressure and NHE inhibition regulate cell cycle progression.

    Fig. S10. Human patient samples show that cancer cells with cytoplasmic and nuclear p27Kip1 exhibit larger cell size than cells with nuclear p27Kip1.

    Table S1. List of calcium concentrations and corresponding viscoelastic properties.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Mechanical characterization of alginate hydrogels and human breast cancer tissue.
    • Fig. S2. Tumor growth in hydrogels with varying stress relaxation.
    • Fig. S3. Cell cycle progression and cell cycle entry are enhanced in hydrogels with faster relaxation.
    • Fig. S4. The inhibition of the PI3K/Akt pathway diminishes the diameter of spheroids formed by HT1080 and MCF7 cells.
    • Fig. S5. Cells did not secrete and deposit collagen and laminin-5, and the inhibition of actomyosin contractility did not affect cell cycle progression.
    • Fig. S6. Impact of TRPV4 inhibition on proliferation.
    • Fig. S7. 3D computational simulations of hydrogel deformation due to cell exerting a constant outward stress in hydrogels with varying relaxation.
    • Fig. S8. The growth of single cells in hydrogels with varying stress relaxation.
    • Fig. S9. Increasing osmotic pressure and NHE inhibition regulate cell cycle progression.
    • Fig. S10. Human patient samples show that cancer cells with cytoplasmic and nuclear p27Kip1 exhibit larger cell size than cells with nuclear p27Kip1.
    • Table S1. List of calcium concentrations and corresponding viscoelastic properties.

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