Intratumoral oxygen gradients mediate sarcoma cell invasion

Abstract

Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O2 depletion. Here, we report the impact of hypoxic O2 gradients on sarcoma cell invasion and migration. O2 gradient measurements showed that large sarcoma mouse tumors (>300 mm(3)) contain a severely hypoxic core [≤0.1% partial pressure of O2 (pO2)] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1-6% pO2). To analyze tumor invasion, we used O2-controllable hydrogels to recreate the physiopathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O2 gradient accurately, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O2 acts as a 3D physicotactic agent during sarcoma tumor invasion and propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets.

Keywords: gradients; hydrogel; hypoxia; migration; sarcoma.

Fig. 1.

Enhanced invasion of sarcoma tumor grafts in hypoxic hydrogels. (A) In situ DO measurements in KIA tumors. H&E stains (B) and HIF1-α stains (C, Left) and quantification (C, Right) of small and large tumors are shown. (Scale bars: 4× images, 200 μm; 40× images, 20 μm.) (D) Schematic illustration of tumor encapsulation within HI hydrogel matrix. (E) DO levels of HI hydrogels encapsulated with tumor biopsy in hypoxic and nonhypoxic matrices up to day 7 in culture. (F, Left) Light microscope (Left) and fluorescence microscope (Right) images of sarcoma tumors encapsulated within nonhypoxic and hypoxic matrices (phalloidin in green, nuclei in blue). H, hydrogels; T, tumors. (Scale bars: 100 μm.) (F, Right) Quantitative analysis of the sarcoma tumor invasion into hydrogel matrix. (G) Immunofluorescence staining and quantification of collagen deposition by tumor grafts cultured for 7 d (collagen in red, nuclei in blue). (Scale bars: 25 μm.) Significance levels: **P < 0.01; ***P < 0.001.

Fig. S1.

Primary mouse sarcoma tumors. The whole tumor is shown, using tiled micrographs of H&E stains (Left) and HIF-1α stains (Right) of small and large tumors. (Scale bars: 1 mm.)

Fig. 2.

Hypoxia promotes primary sarcoma migration. (A) Light microscope (Left) and fluorescence microscope (Right) images of day 3 images of KIA-GFP sarcoma tumors encapsulated within nonhypoxic and hypoxic matrices. Migrating GFP cells were tracked to determine 3D trajectories of tracked cells (representative trajectories) (scale bars: 100 μm) (B); overall speed (C); and MSD in the x, y, and z directions (D). Plots were created using the position of KIA-GFP cells in the hydrogels. Significance levels: *P < 0.05; ^{^}P < 0.01; ^#P < 0.001.

Fig. S2.

Primary sarcoma tumor migration velocity. KIA-GFP sarcoma tumors were encapsulated within nonhypoxic and hypoxic matrices. Day 3 migrating GFP cells were tracked to determine velocity in the x, y, and z directions.

Fig. S3.

Primary sarcoma migration trajectories. Two-dimensional trajectories of tracked cells in hypoxic (A) and nonhypoxic (B) hydrogels are shown.

Fig. 3.

Sarcoma cells remodel the hypoxic hydrogel. (A) Noninvasive DO readings at the bottom of the hypoxic and nonhypoxic hydrogel sarcoma cell constructs. (B) Sarcoma cells encapsulated within HI hydrogels incorporating DQ Gtn for 3 d. (B, i) Representative fluorescence microscopy images. (Scale bars: 25 μm.) (B, ii) Quantitative analysis of relative fluorescence intensity. (C) Young’s modulus (Pa) of the hypoxic and nonhypoxic hydrogels on day 0 and after 3 d of culture. w/o, without. (D) Real-time RT-PCR analysis of collagen modification genes. (E) Immunofluorescence staining and analysis of collagen deposition by the encapsulated cells (collagen in red, nuclei in blue). (Scale bars: 50 μm.) The effect of HIF-1α suppression in encapsulated sarcoma cells [KIA_scr, a control; KIA_HIF-1α(−), HIF knockdown] after 7 d in culture was analyzed, including collagen modification gene expression (F) and collagen deposition and quantification (G) (collagen in red, nuclei in blue). (Scale bars: 50 μm.) Graphical results are shown as the average value ± SD. Significance levels: *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant.

Fig. S4.

HIF-1α expression. Representative immunofluorescence staining of HIF-1α expression by the encapsulated cells (HIF-1α in red, nuclei in blue). Note the abundant nuclear staining and cytoplasmic staining as they relate to rapid protein turnover in the hypoxic hydrogels. (Scale bars: 25 μm.)

Fig. 4.

Efficient sarcoma cell migration in hypoxic gradients (A, i) Illustration of hypoxic and nonhypoxic O₂ gradients in HI hydrogels. (A, ii) Invasive DO readings showing gradients in the HI hydrogels on days 1 and 3. KIA-GFP cells were tracked on day 3 in hypoxic and nonhypoxic hydrogels to determine 3D trajectories of tracked cells (representative trajectories) (B); overall speed (C); velocity in the x, y, and z directions (D); and MSD in the x, y, and z directions (E). Plots were created using the position of KIA-GFP cells in the hydrogels. Significance levels: *P < 0.05; ^{^}P < 0.01; ^#P < 0.001.

Fig. S5.

DO gradient measurement. Invasive DO readings in the hypoxic (A) and nonhypoxic (B) hydrogels on days 1, 3, 5, and 7.

Fig. S6.

Sarcoma cell migration trajectories. Two-dimensional trajectories of tracked cells in hypoxic (A) and nonhypoxic (B) hydrogels are shown.

Fig. S7.

Sarcoma migration in nongradient gels. KIA-GFP cells were tracked on day 3 in nongradient hypoxic gels and compared with the cell migration in gradient hypoxic and nonhypoxic gels. Overall speed (A); velocity in the x, y, and z directions (B); and MSD in the x, y, and z directions (C) are shown. Plots were created using the position of KIA-GFP cells in the hydrogels.

Fig. 5.

Minoxidil inhibits sarcoma cell migration and matrix remodeling in hypoxic hydrogel. KIA-GFP cells were tracked on day 3 in hypoxic-treated and untreated hydrogel to determine 3D trajectories of tracked cells (representative trajectories) (A); overall speed (B); velocity in the x, y, and z directions (C); and MSD in the x, y, and z directions (D). Plots were created using the position of KIA-GFP cells in the hydrogels. (E) Collagen deposition and quantification (collagen in red, nuclei in blue). (Scale bars: 50 μm.) (F) Proteolytic degradation of HI hydrogels incorporating DQ Gtn for 3 d. (Left) Representative fluorescence microscopy images. (Scale bars: 20 μm). (Right) Quantitative analysis of relative fluorescence intensity. (G) Western blot analyses for PLOD2 in KIA cells cultured in hypoxic conditions with and without minoxidil treatment. Significance levels: *P < 0.05; ^{^}P < 0.01; ^#P < 0.001.

Fig. S8.

Minoxidil treatment effect on sarcoma cell migration trajectories. Two-dimensional trajectories of tracked cells in untreated hydrogels (A) and hydrogels treated with 0.5 mM minoxidil (B) are shown.

Fig. S9.

Minoxidil treatment effect on sarcoma cell migration in nonhypoxic hydrogel. KIA-GFP cells were tracked on day 3 in nonhypoxic-treated and untreated hydrogels to determine 3D trajectories of tracked cells (representative trajectories) (A); overall speed (B); velocity in the x, y, and z directions (C); and MSD in the x, y, and z directions (D). Plots were created using the position of KIA-GFP cells in the hydrogels.