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Mechanoresponsive stem cells to target cancer metastases through biophysical cues

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Science Translational Medicine  26 Jul 2017:
Vol. 9, Issue 400, eaan2966
DOI: 10.1126/scitranslmed.aan2966
  • Fig. 1. MRCS in vitro validation.

    (A) Schematic of a proposed mechanism of how MRCS works. When the stiffness of ECM increases, YAP/TAZ are activated and localize to the nucleus. Then, YAP/TAZ will bind to the synthetic stiffness-sensing promoter in MRCS and drive the expression of downstream reporters (such as eGFP and Luc) and/or therapeutics. Note: This schematic is simplified to clarify the major components in MRCS mechanism. (B) Representative images of MRCS-eGFP plated on soft (~1 kPa) and firm (~40 kPa) polyacrylamide gels. eGFP (stained with anti-eGFP; green) was turned on in response to higher stiffness. YAP (stained with anti-YAP; red) localization is also regulated by stiffness, such that it concentrates in the nuclei on stiffer substrates. 4′,6-diamidino-2-phenylindole (DAPI) (blue; nuclear counterstain) is displayed. Scale bars, 25 μm. (C) Quantification of fluorescence intensity of eGFP (stained with antibody) from MRCS-eGFP seeded on substrates with different stiffness or on firm (~40 kPa) substrates treated with 10 μM ML-7 (MLCK inhibitor) or 20 μM PF228 (FAK inhibitor). Blebb., blebbistatin. Data are means ± SEM. (D) RT-qPCR analysis of MRCS-eGFP on hydrogels. Expression of eGFP (green) and YAP/TAZ downstream factors (CTGF, purple; ANKRDI, black) was increased on stiff substrate and was down-regulated on soft substrate or with mechanosensing inhibitors, showing that MRCS is stiffness-specific. Quadruplicate samples were used for the analysis. Data are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 2. MRCS-CD activation dependent on substrate stiffness in vitro.

    (A) Quantification of fluorescent signals of CD shows MRCS-CD responding to matrix stiffness in vitro. MRCS-CD were stained with antibody after plating on tunable polyacrylamide gels or glass as indicated, or treated with 50 μM blebbistatin, 10 μM ML-7 (MLCK inhibitors), or 20 μM PF228 (FAK inhibitor). The fluorescent signal of CD was analyzed, and the relative fluorescence intensity is shown. Data are means ± SEM. Triplicate samples were used for the analysis. (B) MRCS-CD kill cancer cells in response to matrix stiffness and 5-FC in vitro. MRCS-CD were cocultured with MDA-MB-231 breast cancer cells (231: MRCS = 2:1) with (800 μg/ml; green) or without (black) 5-FC on substrates with different stiffness. Total cell proliferation (XTT assay) is displayed. The data were normalized to breast cancer only (231: MRCS = 1:0) with or without 5-FC on each stiffness. Triplicate samples were used for the analysis. Data are means ± SD. n.s., not significant. *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Conversion of 5-FC to 5-FU by MRCS-CD in response to matrix stiffness in vitro. MRCS-CD were seeded on substrates with different stiffness, with 5-FC (800 μg/ml) in growth medium for 1, 2, or 5 days. The concentration of 5-FU in the conditioned medium was detected by LC-MS/MS.

  • Fig. 3. MRCS-CD killing cancer cells in vivo.

    (A) Design and timeline of animal experiment to test MRCS-CD with 5-FC in vivo. iv, intravenous; ip, intraperitoneal. (B) Representative images of nude mice that received MRCS-CD treatments show that MRCS-CD decreased lung metastasis signals in vivo. Luciferase imaging was taken before (day 0, left) and after short-term 5-FC treatment (day 9, middle), as well as long-term 5-FC treatment (6 weeks, right). Quantification of luciferase signals in the lungs in vivo after (C) short-term and (D) long-term treatments. (E) Mouse survival after MRCS-CD treatment. In (C), relative growth index = luciferase read on day 9 (after)/luciferase read on day 0 (before). In (D), lung metastasis index = log10 [(luciferase read of the tested mouse)/(luciferase read of average for tumor-free mice)]; the lung metastasis index of tumor-free mice = 0. The differences between “week 0” groups are not statistically significant. n = 9 for each group. *P < 0.05, **P < 0.01, and ***P < 0.001. In (E), P = 0.0382, CD-MSC versus DPBS; P = 0.0429, MRCS-CD versus N-MSC; and P = 0.0211, MRCS-CD versus DPBS. Median survival (days): CD-MSC, 260; MRCS-CD, 260; N-MSC, 141; DPBS, 137.

  • Fig. 4. MRCS-CD killing cancer cells in vivo with minimal side effects.

    (A) Frozen sections of lungs of Luc-RFP-231 tumor-bearing and tumor-free nude mice sacrificed 24 hours after CD-MSC, N-MSC, DPBS, or MRCS-CD infusion were stained with anti–annexin V (green) and DAPI (blue). RFP signal (red) indicates the presence of lung metastasis. Scale bars, 100 μm. Representative images of frozen section samples of tumor-bearing lungs and tumor-free lungs from nude mice treated with CD-MSC, MRCS-CD, N-MSC, or DPBS before and after 5-FC injections by TUNEL assays are shown. Horseradish peroxidase signals (brown) indicate damaged nuclei, and green signals are methyl green counterstain of normal nuclei. Scale bars, 100 μm. (B) Quantification of TUNEL assay data measuring lung tissue damage in vivo. Ten representative images were used per group for quantification. *P < 0.05, **P < 0.01, and ****P < 0.0001. Data are means ± SD.

  • Fig. 5. Specific activation of MRCS in response to mechano-cues in the metastatic niche in vivo.

    (A to C) Frozen sections of lungs of Luc-RFP-231 tumor-bearing NSG mice [cancer region in (A) and noncancer region in (B)] and tumor-free NSG mice (C) sacrificed 24 hours after infusion of MRCS-CD cotransfected with eGFP were stained with anti-Luc (red) to detect lung metastasis, anti-CD (magenta) for CD expressed by MRCS-CD, and anti-eGFP (green) for MRCS-CD tracking. SHG imaging of collagen networks (cyan) was also overlaid on IHC imaging. Scale bars, 50 μm. Multiple images were tiled into a larger composite image. Each representative image was then extracted from the tiled image. (D) Quantification of collagen linearization using displacement-to-length ratio (DLR) of collagen fibers in SHG images. For a line, DLR = 1, and for a curve, DLR < 1. Representative images are shown in fig. S20. Forty-five fibers per group were used for this analysis. Box and whisker plots are shown as minimum, 25th percentile, median, 75th percentile, and maximum. ****P < 0.0001. (E and F) Representative AFM stiffness maps (50 μm × 50 μm) of tumor-bearing (E) and tumor-free (F) lungs. (G and H) Frequency of Young’s modulus values of tumor-bearing (G) and tumor-free (H) lungs from AFM microindentation in the range of 0 to 40 kPa (bin size = 1 kPa), whereas the inset graphs show the frequency within the range of 0 to 10 kPa (bin size = 0.5 kPa). Five hundred measurements per group were analyzed. P < 0.001 (Young’s modulus of tumor-bearing lungs versus tumor-free lungs).

  • Fig. 6. Cross-linking–specific tissue damage by MRCS in response to mechano-cues in the metastatic niche in vivo in spontaneous lung metastasis model.

    Frozen sections of lungs of tumor-bearing NSG mice with Luc-RFP-231 spontaneous lung metastasis from primary tumors [cancer region in (A) and noncancer region in (B)] and tumor-free NSG mice (C) sacrificed after MRCS-CD infusion and 5-FC treatment as indicated (day 9) were stained with anti-PARP p85 (green) for tissue apoptosis. RFP signal (red) indicates the presence of lung metastasis, and colocalization of red and green appears yellow. SHG imaging of collagen networks (cyan) was presented and overlaid with IHC imaging. Scale bars, 100 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/400/eaan2966/DC1

    Materials and Methods

    Fig. S1. Concept of MRCS for targeting breast cancer metastases in the lung.

    Fig. S2. Construction of MRCS.

    Fig. S3. MRCS-eGFP activation in response to substrate stiffness in vitro.

    Fig. S4. MRCS-eGFP in vitro validation with immunostaining.

    Fig. S5. Further MRCS-Luc in vitro validation.

    Fig. S6. CD-MSC able to kill cancer cells in the presence of 5-FC in vitro.

    Fig. S7. MRCS-CD responding to matrix stiffness in vitro.

    Fig. S8. Bystander effect from MRCS-CD starting at 24 hours in vitro on stiff substrate.

    Fig. S9. Bystander effect from MRCS-CD lasting after MSC removal in vitro on stiff substrate.

    Fig. S10. Luc-MSC homing to the metastatic niche in vivo.

    Fig. S11. MRCS homing and specific activation in response to the metastatic niche in vivo.

    Fig. S12. Specific activation of MRCS-eGFP in response to the metastatic niche in vivo.

    Fig. S13. MRCS-CD unable to attenuate cancer growth in the absence of 5-FC in vivo.

    Fig. S14. No detectable side effects in bone marrow cell populations after systemic treatment with MRCS-CD.

    Fig. S15. MRCS-CD causing no detectable side effects in vivo in bone marrow.

    Fig. S16. MRCS-CD causing no detectable side effects in vivo in livers.

    Fig. S17. MRCS-CD causing no detectable side effects in vivo in brains.

    Fig. S18. Up-regulation and colocalization of LOX expression with tumor in tumor-bearing lungs.

    Fig. S19. LOX expression up-regulated with increased collagen expression in the metastatic niche.

    Fig. S20. SHG imaging showing up-regulated and more linearized collagen in tumor-bearing lungs.

    Fig. S21. Split-channel views of MRCS activation in the metastatic niche in vivo.

    Fig. S22. Cross-linking–specific tissue damage by MRCS in response to mechano-cues in the metastatic niche in vivo.

    Fig. S23. Constitutively CD-expressing MSCs causing nonspecific tissue damage in vivo.

    Fig. S24. Spontaneous lung metastasis model establishment.

    Fig. S25. Split-channel views of cross-linking–specific tissue damage by MRCS in the metastatic niche in vivo in spontaneous lung metastasis model.

    Table S1. Primary antibodies.

    Table S2. Secondary antibodies.

    Table S3. Primers used in qPCR.

    References (5768)

  • Supplementary Material for:

    Mechanoresponsive stem cells to target cancer metastases through biophysical cues

    Linan Liu, Shirley X. Zhang, Wenbin Liao, Henry P. Farhoodi, Chi W. Wong, Claire C. Chen, Aude I. Ségaliny, Jenu V. Chacko, Lily P. Nguyen, Mengrou Lu, George Polovin, Egest J. Pone, Timothy L. Downing, Devon A. Lawson, Michelle A. Digman, Weian Zhao*

    *Corresponding author. Email: weianz{at}uci.edu

    Published 26 July 2017, Sci. Transl. Med. 9, eaan2966 (2017)
    DOI: 10.1126/scitranslmed.aan2966

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Concept of MRCS for targeting breast cancer metastases in the lung.
    • Fig. S2. Construction of MRCS.
    • Fig. S3. MRCS-eGFP activation in response to substrate stiffness in vitro.
    • Fig. S4. MRCS-eGFP in vitro validation with immunostaining.
    • Fig. S5. Further MRCS-Luc in vitro validation.
    • Fig. S6. CD-MSC able to kill cancer cells in the presence of 5-FC in vitro.
    • Fig. S7. MRCS-CD responding to matrix stiffness in vitro.
    • Fig. S8. Bystander effect from MRCS-CD starting at 24 hours in vitro on stiff substrate.
    • Fig. S9. Bystander effect from MRCS-CD lasting after MSC removal in vitro on stiff substrate.
    • Fig. S10. Luc-MSC homing to the metastatic niche in vivo.
    • Fig. S11. MRCS homing and specific activation in response to the metastatic niche in vivo.
    • Fig. S12. Specific activation of MRCS-eGFP in response to the metastatic niche in vivo.
    • Fig. S13. MRCS-CD unable to attenuate cancer growth in the absence of 5-FC in vivo.
    • Fig. S14. No detectable side effects in bone marrow cell populations after systemic treatment with MRCS-CD.
    • Fig. S15. MRCS-CD causing no detectable side effects in vivo in bone marrow.
    • Fig. S16. MRCS-CD causing no detectable side effects in vivo in livers.
    • Fig. S17. MRCS-CD causing no detectable side effects in vivo in brains.
    • Fig. S18. Up-regulation and colocalization of LOX expression with tumor in tumor-bearing lungs.
    • Fig. S19. LOX expression up-regulated with increased collagen expression in the metastatic niche.
    • Fig. S20. SHG imaging showing up-regulated and more linearized collagen in tumor-bearing lungs.
    • Fig. S21. Split-channel views of MRCS activation in the metastatic niche in vivo.
    • Fig. S22. Cross-linking–specific tissue damage by MRCS in response to mechano-cues in the metastatic niche in vivo.
    • Fig. S23. Constitutively CD-expressing MSCs causing nonspecific tissue damage in vivo.
    • Fig. S24. Spontaneous lung metastasis model establishment.
    • Fig. S25. Split-channel views of cross-linking–specific tissue damage by MRCS in the metastatic niche in vivo in spontaneous lung metastasis model.
    • Table S1. Primary antibodies.
    • Table S2. Secondary antibodies.
    • Table S3. Primers used in qPCR.
    • References (57–68)

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