Physical confinement during cancer cell migration triggers therapeutic resistance and cancer stem cell-like behavior

Abstract

Metastasized cancer cells have an increased resistance to therapies leading to a drastic decrease in patient survival rates. However, our understanding of the cause for this enhanced resistance is lacking. In this study, we report that physically tight confinement during cancer cell migration triggers therapeutic resistance and induces cancer stem cell-like behavior including up-regulation in efflux proteins and in cancer stem cell related markers. Moreover, the re-localization of Yes-associated protein (YAP) to the cell nucleus indicated an elevated level of cytoskeletal tension. The increased cytoskeletal tension suggested that mechanical interactions between cancer cells and tight surroundings during metastasis is one of the factors that contributes to therapeutic resistance and acquisition of cancer stem cell (CSC) like features. With this system and supporting data, we are able to study cells with therapeutic resistance and CSC-like properties for the future purpose of developing new strategies for the treatment of metastatic cancer.

Keywords: Cancer metastasis; Physical confinement; Therapeutic resistance.

Fig. 1.. Confined-migrating cancer cells develop chemotherapeutic resistance by increasing drug efflux

(left panel are G55 results and right panel are MDA-MB-231 results). A-B. Quantitative comparison of Dox intracellular accumulation between 2D culture (2D) and cancer cells that migrated through 5 × 5 μm microchannels (5R). Dox (17μM) was introduced to all cancer cells then removed after 4 hours and replaced with the Dox free medium. After overnight incubation with Dox free medium, Dox intensities were quantified in the nucleus, cytoplasm and whole cell (total). N>20 per group. All experiments were triplicated. †p<0.05 between 2D total and others; *p<0.05 between 2D nucleus and others; #p<0.05 between 2D cytoplasm and others. C-D. Representative images of Dox accumulation in 2D culture and cells which have migrated through 5 × 5 μm microchannels (red arrows). E-F. Changes of Dox intensity over time under different degrees of confinement. Percentage shows intensity changes between 4 hr and overnight. N>20 per group. G and I. Viability after Dox (17 μM) 4 hr treatment plus overnight Dox free incubation. N>20 per group. *p<0.05 between 2D and others. H. G55 cell viability after Temozolomide treatment (0.25 mM, 0.5 mM or 1 mM) for 72 hours. *p<0.05. J. MDA-MB-231 cell viability after 5-Fu (5 μM) treatment for 72 hours. *p<0.05. Experiments were replicated.

Fig. 2.. Confined-migrating cells exhibit more radiation resistance than the 2D cultured cells.

A. G55 cells were seeded and allowed to migrate in 5 μm width microchannel (top row of 6-well plate) and to grow in 2D (bottom row of 6-well plate); then the plate was irradiated in various doses with a PDMS cover on the top of the 2D wells to control for the potential effect of PDMS on viability. B. Quantitative comparison of cell viability between 2D and confined-migrating cells at different doses of radiation; average ± Std. *p<0.05. N>20 images from at least 3 wells for migrating cells per group; N=6 for the 2D cultured cells per group. C. Representative live/dead images of confined-migrating G55 cells (migrating through 5 × 5 μm microchannels) treated with 0 Gy and 10 Gy radiation. Images were taken 48 hour post-treatment. (Red arrows indicate lined confined-migrating cells inside microchannels; Green arrows indicate 2D cultured cells.)

Fig. 3.. Confined-migrating cells express significant increased drug efflux proteins.

A-C. Quantitative comparison of drug efflux proteins (ABCG2, MDR1 and NUP62) between G55 confined-migrating cells (indicated by M) and 2D cells (indicated by C). D-E. Quantitative comparison of drug efflux proteins (ABCG2 and MDR-1) in MDA-MB-231 confined-migrating cells (indicated by M) and 2D cells (indicated by C). All Western blot results were normalized by the total protein concentration. (Average + Std). All experiments were reproduced. *p<0.05. F. Representative immunostaining images of NUP62 and ABCG2 for G55 (left) and MDA-MB-231 cells in 2D culturing and after migrating through 5 × 5 μm microchannels (5R, right). Blue: DAPI; Red: NUP62; and Green: ABCG2.

Fig. 4.. Increased expression of cancer stem cell related markers in confined-migrating cancer cells.

Quantitative comparison of cancer stem cell related markers using Western blot analyses between confined-migrating (M) and 2D cultured (C) cancer cells. A1-A4. G55 cells. B1-B4. MDA-MB-231cells. C-E. Increased expression of CD133 in different types of confined-migrating cancer cells: C25 patient derived GBM cells, A549 lung cancer cell line, and PC3 prostate cancer cell line. (H: hypoxia condition, refer to Fig. S4). All results were normalized as relative intensity to the expression in control group. Average + Std. *p<0.05. All experiments were reproduced. Representative blot images of each marker are shown below their respective graph.

Fig. 5.. Confined-migrating cancer cells exhibit increased cytoskeleton tension with minimal hypoxic stress.

A1. 3D reconstructed MDA-MB-231 cells under different degrees of confinement: 2D, 15×15 and 5×5 μm microchannel (Red: PI; Green: F-actin). A2. Cross section ratio of MDA-MB-231 cells inside microchannel. Average + Std, n=4/condition. (cross section ratio = cross section acreage of cell/cross section acreage of microchannel) A3. Immunohistochemical staining for ABCG2 in the G55 murine xenograft model of glioblastoma multiforme demonstrates high ABCG2 expression in migrating tumor cells within cortical white matter tracts. a: health mouse brain tissue (noncancerous). b-c: In areas bordering G55 tumors (labeled with †), elevated ABCG2 expression can be observed in the migrating tumor cells within cortical white matter tracts (labeled with*). Arrow: blood vessel. Arrowhead: tumor cells highly expressing ABCG2 in a whole cell pattern. A4. Hydraulic resistance inside microchannels with different dimensions. h: height; w: width. Results are normalized by the lowest resistance number (i.e., 15×15 μm microchannel). B and G. Quantitative comparison of YAP expression (N/P; nucleus/cytoplasm) between 2D cultured and confined-migrated cells. 5R: cells migrated through 5 × 5 μm microchannel. C and H. Representative YAP fluorescence images of G55 (C) and MDA-MB-231 (H) cells. Blue: DAPI; Green: YAP. Scale bar: 10μm. D and J. Representative hypoxia dye fluorescence images of cells in the central reservoir (2D), 5 × 5 μm entrance and inside 5 × 5 μm microchannels (MC). Red arrows indicate the confined-migrating cells via the 5 × 5 μm microchannels. E and I. Quantitative comparison of hypoxia dye intensity of the cells in different locations. F and K. Western blot HIF-1α relative intensity. C: 2D cultured cells and M: cells migrated through 5 × 12 μm microchannels. All results were normalized to the total proteins. Average + Std. *p<0.05, **p<0.01 between 2D and others. Scale bar: 5 μm. All experiments were reproduced.

Fig 6.. Protein expression changes induced by confined-migration persist after removal from physical confinement.

A-C. Comparison of selected proteins (ABCG2, ALDH and CD44) expression in G55 cells between 2D culture (2D), confined-migrating (M), and 2 days after reseeding confined-migrating cells on the 2D culture (D2). D-F. Comparison expressions of selected proteins (CD133, ABCG2 and ALDH1) in MDA-MB-231 cells between 2D culture (2D), confined-migrating (M), and 2, 3, 4, and 8 days after reseeding confined-migrating cells (D2, D3, D4, and D8). Representative blot images of each marker are shown below their respective graphs. All results were normalized to the total proteins. Average + Std. *p<0.05. All experiments were reproduced.