Journal: bioRxiv

Article Title: Tumor cell-organized fibronectin is required to maintain a dormant breast cancer population

doi: 10.1101/686527

Figure Lengend Snippet: 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 coated coverslips (Col), or a mixture of ECM proteins inspired by those found in the bone marrow (BM)-coated 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 MSCs). Heat map shows increase in confluence over 7 days stimulation in blue. d. Ki67 quantification of BT549, and ZR-75-1 plated on TCPS and e. of HCC1954 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 DAPI (blue) in HCC1954 on collagen coverslips after 2 and 28 days of serum-starvation (Scale bar 100 μm). g. Population quantification of HCC 1954 via co-staining of Ki67, p21, and senescence-associated β-galactosidase. 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 *, ≤ 0.01 with **, ≤ 0.001 with ***, and ≤ 0.0001 with ****.

Article Snippet: ECM proteins (collagen I and natural mouse laminin were purchased from Life Technologies, human plasma fibronectin from Millipore, human collagen IV from Sigma-Aldrich, recombinant human tenascin C, osteopontin, thrombospondin-1, and vitronectin from R&D Systems, and laminin 511 and laminin 521 from Biolamina) were covalently bound to the glass surface by incubation at room temperature for three hours, then with 10 mg/cm 2 MA(PEG)24 (Thermo Fisher Scientific) for two hours to block non-specific protein adsorption, rinsed three times in PBS, and UV-sterilized prior to cell seeding.

Techniques: Staining, Labeling, Cell Culture

Journal: bioRxiv

Article Title: Tumor cell-organized fibronectin is required to maintain a dormant breast cancer population

doi: 10.1101/686527

Figure Lengend Snippet: a. Top rows, immunofluorescence for matrix proteins and DAPI in HCC 1954s 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. Scale: 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, 25μm) 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.

Article Snippet: ECM proteins (collagen I and natural mouse laminin were purchased from Life Technologies, human plasma fibronectin from Millipore, human collagen IV from Sigma-Aldrich, recombinant human tenascin C, osteopontin, thrombospondin-1, and vitronectin from R&D Systems, and laminin 511 and laminin 521 from Biolamina) were covalently bound to the glass surface by incubation at room temperature for three hours, then with 10 mg/cm 2 MA(PEG)24 (Thermo Fisher Scientific) for two hours to block non-specific protein adsorption, rinsed three times in PBS, and UV-sterilized prior to cell seeding.

Techniques: Immunofluorescence, In Situ, Activity Assay

Journal: bioRxiv

Article Title: Tumor cell-organized fibronectin is required to maintain a dormant breast cancer population

doi: 10.1101/686527

Figure Lengend Snippet: 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 21-28. d. HCC1954 survival at day 7 with inhibitors when seeded onto HCC 1954 decellularized ECM. Black: control, blue: anti- β 1 (MAB17781, 1 µg/ml), green: FAK inhibitor 14 (10 µM), purple: PD0325901 (MEK inhibitor, 10 µM), red: FR180204 (ERK inhibitor 20µM), gray: cilengitide (100 µM). p < 0.05 is denoted with *, ≤ 0.01 with **, ≤ 0.001 with ***, and ≤ 0.0001 with ****.

Article Snippet: ECM proteins (collagen I and natural mouse laminin were purchased from Life Technologies, human plasma fibronectin from Millipore, human collagen IV from Sigma-Aldrich, recombinant human tenascin C, osteopontin, thrombospondin-1, and vitronectin from R&D Systems, and laminin 511 and laminin 521 from Biolamina) were covalently bound to the glass surface by incubation at room temperature for three hours, then with 10 mg/cm 2 MA(PEG)24 (Thermo Fisher Scientific) for two hours to block non-specific protein adsorption, rinsed three times in PBS, and UV-sterilized prior to cell seeding.

Techniques: