Hydrogel microenvironments for cancer spheroid growth and drug screening

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Science Advances  27 Apr 2018:
Vol. 4, no. 4, eaas8998
DOI: 10.1126/sciadv.aas8998


  • Fig. 1 MCS growth in hydrogel ECMs.

    (A) Scanning electron microscopy image of Matrigel. Scale bar, 1 μm. (B) Phase-contrast microscopy image of MCSs grown from MCF-7 breast cancer cell lines in Matrigel for 14 days. Scale bar, 100 μm. (C) PEG hydrogels are formed by a transglutaminase Factor XIIIa–catalyzed cross-linking reaction between two multi-arm PEG-peptide conjugates: n-PEG-MMP-Lys and n-PEG-Gln. Phase-contrast microscopy (D) and confocal microscopy (E) images of MCSs grown from OV-MZ-6 ovarian cancer cell lines in PEG hydrogels shown in (C). Scale bars, 100 μm. Cell actin filaments were stained with rhodamine phalloidin (red), and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). (F) Scanning electron microscopy image of the hydrogel formed from poly(N-isopropylacrylamide)–modified CNCs. Scale bar, 10 μm. (G) MCS growth from MCF-7 breast cancer cell lines in hydrogels shown in (F). Scale bar, 100 μm. Figures were reproduced with permission from Poincloux et al. (44) (A), Ehrbar et al. (66) (C), Loessner et al. (64) (D and E), and Li et al. (14) (F and G).

  • Fig. 2 Formation of MCSs in fibrin hydrogels with varying stiffness.

    (A) An individual B16-F1 cell grows into an MCS in fibrin gel with stiffness of 90 Pa within 4 days. (B) MCSs formed after 5-day culture from an individual B16-F1 cell in fibrin hydrogels with stiffness of 90 Pa (left), 420 Pa (middle), and 1050 Pa (right). Scale bars, 50 μm (A and B). (C) Variation in the MCS number with cell culture time in the fibrin hydrogels. (D) Increase in MCS size plotted as a function of culture time, plotted for hydrogels with different stiffness. The stiffness of 3D fibrin gels with concentrations of 1, 4, and 8 mg ml−1 is 90, 420, and 1050 Pa, respectively. Figures were reproduced with permission from Liu et al. (15) (A to D).

  • Fig. 3 MCS release from p-CNC hydrogel.

    (A) Schematic of encapsulation, growth, and release of MCF-7 MCSs from the hydrogel formed by poly(N-isopropylacrylamide)–modified CNCs. (B) Phase-contrast microscopy images of MCF-7 MCSs released from the hydrogel as in (A) after 15-day culture. Scale bar, 500 μm. (C) Immunostaining of the representative MCF-7 MCS released from the hydrogel as in (A) by Alexa Fluor 488 E-cadherin rabbit monoclonal antibody (green), DAPI (blue), Alexa Fluor 568 phalloidin (red), and the merged fluorescence image of the MCS. Scale bars, 50 μm. Figures were reproduced with permission from Li et al. (14) (A to C).

  • Fig. 4 Emerging microtechnologies for MCS growth in 3D microscale hydrogels.

    (A) Schematic of the MF platform for MCS formation, which is composed of an external fluidic injection system, coextrusion microdevice, and off-chip gelation bath. An enlarged view of the chip (right) shows the three-way configuration, with cell suspension (CS), intermediate solution (IS), and alginate solution (AL), respectively, flowing into the coaligned capillaries. The inlets of the chip are connected to three syringes controlled by two syringe pumps. The compound liquid microdroplets fall into a 100 mM calcium bath. Calcium-mediated gelation of the alginate shell freezes the structure of the capsule, and cells remain encapsulated. (B) Growth of a representative spheroid encapsulated in alginate capsule. Time on top of the images is recorded from encapsulation. Scale bars, 50 μm. (C) Bright-field images (left) of MCSs in a 96-well plate by bioprinting. Individual spheroid obtained from 4T1–green fluorescent protein (GFP) orthotopic mouse breast tumor. Scale bars, 1 mm (left image) and 500 μm (fluorescent images). Figures were reproduced with permission from Alessandri et al. (59) (A and B) and Truong et al. (37) (C).

  • Fig. 5 MCSs in the hydrogels as models for drug screening and NP transport.

    Morphology of MCSs formed by coculture of the human hepatocellular liver carcinoma cell line HepG2 and fibroblasts in collagen hydrogel without (A) and with (B) 10 μM doxorubicin treatment for 4 days. Scale bars, 100 μm. (C) Change in diameter of MCSs (breast cancer cell line MDA-MB-231) in the collagen hydrogels, plotted as a function of duration of the paclitaxel treatment. The drug was delivered via bolus dose or NPs for 24 hours. (D) Image of the MF device (left) with MCS (MDA-MB-435 melanoma cell line) in Matrigel, which was used to study NP transport in the tumor tissue model. Scale bar, 1000 μm. The spheroid (right) stained with anti-Laminin–FITC (fluorescein isothiocyanate). Scale bar, 100 μm. (E) Schematic (left) and image (right) of 40 (top) and 110 nm (bottom) fluorescent PEG-Au NPs administered for 1 hour at the flow rate of 50 ml/h. The 40-nm NPs entered the MCS and accumulated in the interstitial spaces (arrows in top image), and the 110-nm NPs were excluded from the MCS. Scale bars, 100 μm. Images in (E) represent an overlay of fluorescence (excitation, 633 nm; emission, 650 to 700 nm) and differential interference contrast images. (F) NP penetration depth in MCSs treated with 40-nm PEG-Au NPs at various flow rates. Figures were reproduced with permission from Yip et al. (107) (A and B), Charoen et al. (35) (C), and Albanese et al. (114) (D to F).

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