Collagen-Laponite Nanoclay Hydrogels for Tumor Spheroid Growth

Abstract

The extracellular matrix (ECM) plays an important regulatory role in the development and progression of tumoral tissue. Its functions and properties are crucial in determining tumor cell behavior such as invasion, migration, and malignancy development. Our study explores the role of collagen type I in cancer development and spread using engineered tumor models like multicellular spheroids grown in collagen-based hydrogels to simulate early tumor formation. We employ microfluidic techniques to test the hypothesis that (i) adding Laponite nanoclay to collagen hydrogels modifies mechanical and rheological properties and (ii) changing the stiffness of the collagen microenvironment affects tumor spheroid growth. Our findings support our theories and suggest the use of ECM components and engineered tumor models in cancer research, offering a biocompatible and biomimetic method to tailor the mechanical properties of conventional collagen hydrogels.

Figure 1

Experimental setup: Laponite nanoclay was supplied in powder form. It was diluted at 1% w:v in Milli-Q water and, subsequently, in culture media at 1/10 proportion. This working solution was used to fabricate the supplemented collagen hydrogels at different collagen concentrations. 3D tumor cell cultures were incubated in microfluidics, and the spheroids were formed from single-cell proliferation and cell–cell contact. The hydrogels were characterized by scanning electron microscopy, and in terms of swelling ratio and rheology gelled in wells. Additionally, permeability was characterized inside the microfluidic platform.

Figure 2

(A) TEM images of 1% LapNC stock solution and (B) 0.1% LapNC working solution. (C) Representative SEM images at at 25,000× and 50,000× magnification of the collagen hydrogels (2.5, 4, and 6 mg/mL) with/without LapNC. (D) Representative EDX report figures at 100,000× of a 6 mg/mL collagen hydrogel with/without LapNC.

Figure 3

(A) Single representative gelation curves for each collagen concentration (2.5, 4, and 6 mg/mL). (B) Storage moduli (G′) after the hydrogels gelation. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 4

(A) Swelling ratios (mean ± SD) of the different hydrogels: 2.5 mg/mL, 4 and 6 mg/mL of collagen concentration, with (red) and without (blue) the addition of 0.1% Laponite solution. (B) Darcy’s permeability for collagen hydrogels, with (red) and without (blue) the addition of LapNC. Significance for * p < 0.05, ** p < 0.01, and *** p < 0.005.

Figure 5

(A) Brightfield images of a representative PACA culture sample of each condition (collagen concentrations with/without LapNC) at 4 different time points. Each representative image is accompanied by the spheroid area median (μm²) in that condition. The symbol X denotes that the hydrogel collapsed at this time point or that no cells remained in 3D in this condition (total migration to the bottom surface). Scale bar in black 2 mm. (B) PACA cells spheroid area (μm²) distribution over time (shown as box plot): 3D cultures in different matrix: 2.5, 4, and 6 mg/mL of collagen; with and without the addition of LapNC. Conditions not able to form spheroids at specific time points are marked with colored crosses.

Figure 6

Evolution over time of the different cell lines at each condition: 2.5, 4, and 6 mg/mL of collagen with and without the addition of LapNC. Continuous lines show the median values, while dashed lines represent the 90% percentile.

Figure 7

PACA cell migration and beginning of cluster formation within LapNC supplemented (red) and control (blue) 2.5 mg/mL collagen hydrogels: (A) Relative trajectories of cells collected from control and LapNC samples (60 events per sample, n = 3), X and Y axis in μm. (B) Values of V_mean (left) and V_eff (right) of the cell migration in both types of samples. Statistical significance * p < 0.05, ** p < 0.001. (C) Mean square displacement (MSD) of the tracked trajectories. Three devices were analyzed per condition used to study more than 150 cells individually. (D) 3D projection of representative PACA cells at day 1 and 3 of culture. Images were processed and 3D volume was projected with Zen 3.5 Blue software (ZEISS, Germany).