Hyaluronic acid hydrogel stiffness and oxygen tension affect cancer cell fate and endothelial sprouting

Abstract

Three-dimensional (3D) tissue culture models may recapitulate aspects of the tumorigenic microenvironment in vivo, enabling the study of cancer progression in vitro. Both hypoxia and matrix stiffness are known to regulate tumor growth. Using a modular culture system employing an acrylated hyaluronic acid (AHA) hydrogel, three hydrogel matrices with distinctive degrees of viscoelasticity - soft (78±16 Pa), medium (309± 57 Pa), and stiff (596± 73 Pa) - were generated using the same concentration of adhesion ligands. Oxygen levels within the hydrogel in atmospheric (21 %), hypoxic (5 %), and severely hypoxic (1 %) conditions were assessed with a mathematical model. HT1080 fibrosarcoma cells, encapsulated within the AHA hydrogels in high densities, generated nonuniform oxygen distributions, while lower cell densities resulted in more uniform oxygen distributions in the atmospheric and hypoxic environments. When we examined how varying viscoelasticity in atmospheric and hypoxic environments affects cell cycles and the expression of BNIP3 and BNIP3L (autophagy and apoptosis genes), and GLUT-1 (a glucose transport gene), we observed that HT1080 cells in 3D hydrogel adapted better to hypoxic conditions than those in a Petri dish, with no obvious correlation to matrix viscoelasticity, by recovering rapidly from possible autophagy/apoptotic events and alternating metabolism mechanisms. Further, we examined how HT1080 cells cultured in varying viscoelasticity and oxygen tension conditions affected endothelial sprouting and invasion. We observed that increased matrix stiffness reduced endothelial sprouting and invasion in atmospheric conditions; however, we observed increased endothelial sprouting and invasion under hypoxia at all levels of matrix stiffness with the upregulation of vascular endothelial growth factor (VEGF) and angiopoeitin-1 (ANG-1). Overall, HT1080 cells encapsulated in the AHA hydrogels under hypoxic stress recovered better from apoptosis and demonstrated greater angiogenic induction. Thus, we propose that oxygen tension more profoundly influences cell fate and the angiogenic potential of 3D cultured HT1080 fibrosarcoma cells than does matrix stiffness.

Figure 1. Acrylated HA hydrogels

(A) Schematic representation of AHA hydrogels, which are formed by reacting AHA macromers with RGD-containing peptides and MMP-sensitive crosslinkers, where the crosslinker concentration controls viscoelasticity. (B) Rheology frequency sweeps of AHA hydrogel with varying MMP crosslinker concentrations, showing that the modulus depends on crosslinker concentration. (C) Elastic modulus following polymerization and swelling: soft (78±16 Pa); medium (309± 57 Pa), and stiff (596± 73 Pa).

Figure 2. HT1080 cells encapsulated in AHA hydrogels with defined stiffnesses

(A) Light microscopy images of three-day culture of HT1080 cells in stiff, medium, and soft AHA hydrogels. (B) Viscoelasticity measurements of the different HT1080 construct types (left) or hydrogels alone (right) along the three-day culture period.

Figure 3. Predicting DO levels in AHA hydrogels

(A) Oxygen consumption rate of HT1080 cells following Michaelis-Menten kinetics. Plots were calibrated according to the residual sum of squares method to find the best fit between theoretical and experimental values. V_max and K_m values of HT1080 cells were 83.19 × 10⁻¹⁸ mol/s and 66 µM, respectively. (B) Schematic of the 3D HT1080-AHA construct in culture comprises three individual layers: (i) AHA gel containing HT1080 cells (middle layer), (ii) growth medium (surrounding layer), and (iii) air (top layer). Model prediction of DO levels within the hydrogels at (C) 21 %, (D) 5 %, and (E) 1 % O₂, with HT1080 cells encapsulated at concentrations ranging from 1 to 10 × 10⁶ cells/ml during the first hours (left panel: lines show DO levels at the center of the hydrogel; broken lines, the DO levels at the edge of the hydrogel), and DO gradient profile throughout the hydrogels’ depth after an hour (right panel).

Figure 4. HT1080 cell fate in AHA hydrogels with varying stiffnesses in atmospheric and hypoxic conditions

(A) Proliferation of HT1080 cells encapsulated in stiff hydrogels over the three-day experiment in atmospheric and hypoxic conditions. (B) Representative cell cycle flow analysis of HT1080 cells encapsulated in stiff hydrogels over the three-day experiment in atmospheric and hypoxic conditions. (C) Quantification of the percentage of apoptotic cells (sub-G₁) in the various conditions. (Di, ii) Quantitative RT-PCR for BNIP3 and GLUT-1 expression in HT1080 cells encapsulated in hydrogels of medium stiffness (3D) and in Petri dishes (plates) in atmospheric and hypoxic conditions. Significance levels (n=3) were set at: *p<0.05, **p<0.01, and ***p<0.001.

Figure 5. Effect of AHA-HT1080 on ECFC sprouting and invasion

(A) Schematic of study strategy for examining the angiogenic potential of AHA-HT1080 constructs: HT1080 cells, SDF1α, and S1P were encapsulated in AHA hydrogels of different stiffnesses and cultured for 24 hours in hypoxic or atmospheric conditions, followed by seeding of ECFCs as a monolayer on top of the hydrogels and culturing for an additional 24 hours. (B) Lectin stain (in red) and side-view imaging of endothelial sprouting in medium hydrogel in atmospheric and hypoxic conditions. (C) Quantification of sprouting length of ECFCs in the different stiffness and oxygen tension conditions. Significance levels (n=3) were set at: *p<0.05, **p<0.01, and ***p<0.001.

Figure 6. Angiogenic gene expression

(A) Quantitative RT-PCR for the expression of MMP-1 in HT1080 cells cultured in Petri dishes (plates) or encapsulated in the different hydrogels for 24 hours. (B) Viscoelasticity measurements of the different construct types along the three-day hypoxic culture period. (D-C) Quantitative RT-PCR for the expression of VEGF, and ANG-1 in HT1080 cells cultured in Petri dishes (plates) or encapsulated in the different hydrogels for 24 hours. Significance levels (n=3) were set at: *p<0.05, **p<0.01, and ***p<0.001.