Tumor cell-organized fibronectin maintenance of a dormant breast cancer population

Abstract

Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, nonproliferative state before reactivation and outgrowth. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created an in vitro cell culture system with carefully controlled ECM substrates to observe entrance into and exit from dormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle-arrested, and actively proliferating cells. Cell populations capable of entering dormancy formed an organized, fibrillar fibronectin matrix via α_vβ₃ and α₅β₁ integrin adhesion, ROCK-generated tension, and TGFβ2 stimulation, and cancer cell outgrowth after dormancy required MMP-2-mediated fibronectin degradation. We propose this approach as a useful, in vitro method to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation.

Fig. 1. Serum withdrawal induces a reversible dormant phenotype in breast cancer cells.

(A) ZR-75-1 breast cancer cell line number and proliferation (Ki67 staining) over time with serum withdrawal (red) and recovery in full serum (blue). (B) Survival of breast cell lines on TCPS (TC), collagen I coverslips (Col), or a mixture of ECM proteins inspired by those found in the bone marrow (BM)–functionalized coverslips. Average time to decrease confluence from 100 to 25% (scored by visual inspection by the same observer) is displayed on the left in red, and conditions where viable cells were detected at 8 weeks in culture are labeled on the right in black. (C) Best-performing cell lines were cultured on TCPS for 6 weeks and stimulated with varying media conditions (GF, growth factor cocktail; CM, conditioned medium from mesenchymal stem cells). SF, serum-free. Heat map shows increase in confluence over 7-day stimulation in blue. (D) Ki67 quantification of BT549 and ZR-75-1 plated on TCPS and (E) of HCC 1954 on TCPS or collagen. Black, 10% serum control; blue, day 28 serum-free culture; green, 7-day recovery. (F) Immunofluorescent staining for phospho-p38 (red; Thr¹⁸⁰/Tyr¹⁸²) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) in HCC 1954 on collagen coverslips after 2 and 28 days of serum starvation (scale bar, 100 μm). (G) Population quantification of HCC 1954 via costaining of Ki67, p21, and senescence-associated β-galactosidase (β-gal). (H) BrdU- and EdU-labeling experiment schematic and results for HCC 1954 on collagen. Black, double negative; red, BrdU positive; green, EdU positive; blue, double BrdU and EdU positive. P < 0.05 was considered statistically significant. P < 0.05 is denoted with *P < 0.05, and ****P ≤ 0.0001.

Fig. 2. Dormant cells up-regulate extracellular fibronectin specifically during serum withdrawal.

(A) Top, immunofluorescence for matrix proteins and DAPI in HCC 1954 on collagen in serum-free medium for 2 and 28 days. Bottom, immunofluorescence and for fibronectin in HCC 1954s grown for 28 days or serum-starved for 28 days and recovered in situ for 1 week (blue, DAPI; green, fibronectin; pink, laminin; red, collagen I, vitronectin, or osteopontin, as labeled). Scale bar, 100 μm. (B) Total MMP activity of HCC 1954 cells on collagen coverslips (C) Number of nuclei resulting from reactivation of dormant cells treated with a pan MMP inhibitor for 7 days (MMPi; GM6001, 25 μM) or control (serum-containing media, no inhibitor). (D) HCC 1954 day 7 survival on collagen, fibronectin (FN), or HCC 1954 day 28 decellularized ECM (decell ECM) coverslips.

Fig. 3. TGFβ2 stimulates fibronectin matrix production that mediates survival during mitogen withdrawal.

(A and B) Survival of HCC1954 under control, TGFβ receptor inhibition (LY-364947, 5 μM), and TGFβ1 (1 ng/ml) or TGFβ2 (2 ng/ml) stimulation over (A) 7 days or (B) 28 days. (C) Immunofluorescence for fibronectin (green) and DAPI (blue) at day 28. (D) Expression of secreted TGFβ1, TGFβ2, or TGFβ3 in serum-starved cultures at 2 and 28 days in culture. P < 0.05 is denoted with *, P ≤ 0.01 with ** and P ≤ 0.0001 with ****.

Fig. 4. Dormancy-associated fibronectin is assembled via α₅β₁ integrin and ROCK to mediate survival.

(A) Experimental timeline of inhibitor dosing. Day 7 experiments were dosed continually through a day 7 endpoint; day 28 experiments were dosed continually through a day 28 endpoint; separate cultures were established for 21 days, where inhibitor dosing was initiated for an additional 7 days (“dose-established culture”). (B) Day 7 survival of HCC 1954 on collagen with inhibitors dosed at seeding and every medium change. (C) Survival of cells with inhibitors, dosed after establishment of dormant culture for 21 days and then subsequent dosing at every medium change through days 21 to 28. (D) Day 7 survival of HCC 1954 on decellularized matrix with inhibitors dosed at every medium change. (E) Day 28 survival of cells with inhibitors dosed at seeding and every medium change throughout the entirety of the experiment. Green, α₅ integrin function affecting antibody; blue, Y-27632 (ROCK inhibitor at various concentrations). (F) Immunofluorescence for fibronectin (green) and DAPI (blue) at day 28 on collagen with inhibitors dosed for the entire 28-day time period. Scale bar, 100 μm. (G) Immunofluorescence for fibronectin (green) and DAPI (blue) at day 28 on collagen with exogenous fibronectin (10 μg/ml) or fibronectin with Y-27632 (1 μM). (H) Day 28 survival of cells under control, exogenous fibronectin, or fibronectin with Y-27632. P < 0.05 is denoted with *, P ≤ 0.001 with *** and P ≤ 0.0001 with ****.

Fig. 5. Survival is mediated via adhesion-FAK-ERK signaling.

(A) Experimental timeline of inhibitor dosing. Day 7 experiments were dosed continually through a day 7 endpoint; day 28 experiments were dosed continually through a day 28 endpoint; separate cultures were established for 21 days, where inhibitor dosing was initiated for an additional 7 days (dose-established culture). (B) HCC 1954 survival at day 7 on collagen coverslips with selected inhibitors dosed for the duration of the experiment. (C) Survival of HCC 1954 cells after establishment of dormant culture for 21 days and then subsequent dosing with inhibitors through days 21 to 28. (D) HCC 1954 survival at day 7 with inhibitors when seeded onto HCC 1954 decellularized ECM. Black, control; blue, anti-β₁ (MAB17781; 1 μg/ml); green, FAK inhibitor 14 (10 μM); purple, PD0325901 (MEK inhibitor, 10 μM); red, FR180204 (ERK inhibitor, 20 μM); gray, cilengitide (100 μM). P < 0.05 is denoted with *, P ≤ 0.001 with *** and P ≤ 0.0001 with ****.