Chemical Tools for Selective Activity Profiling of Endogenously Expressed MMP-14 in Multicellular Models

Abstract

Matrix metalloproteases (MMPs) are a large family of zinc-dependent endopeptidases involved in a diverse set of physiological and pathological processes, most notably in cancer. Current methods for imaging and quantifying MMP activity lack sufficient selectivity and spatiotemporal resolution to allow studies of specific MMP function in vivo. Previously, we reported a strategy for selective targeting of MMPs by engineering a functionally silent cysteine mutation that enables highly specific covalent modification by a designed activity-based probe. Here, we describe the translation of that technology into a mouse model of breast cancer and subsequent demonstration of the utility of the approach for studies of MMP-14 activation in the tumor microenvironment. Using this approach, we find that MMP-14 is active in late stage tumors and is predominantly associated with stromal cell populations that have been activated by specific signaling molecules (e.g., TGFβ) produced by tumor cells. Our data demonstrate the applicability of this approach for studies of MMP function in whole organisms and identify important regulatory mechanisms for MMP-14 activity in the tumor microenvironment.

Figure 1:

Specific labeling of active MMP-14 using protein engineering coupled with ABPs. a) Engineered MMP-14 contains a cysteine nucleophile that is irreversibly labeled by the ABP. Covalent binding occurs only with the active form of MMP-14 following affinity targeting of the catalytic zinc by the hydroxamate-containing ABP, but not with the proenzyme or the inhibited form in which the zinc is inaccessible. b) ABP labeling is selective to the probe sensitive MMP while any interactions with native MMPs result in reversible weak inhibition.

Figure 2:

Chemical structure and optimized synthesis of an ABP that targets the cysteine-mutant MMP-14. a) ABPs TND124 and NAP8 are designed based on the core scaffold of GM6001 and comprise its high affinity isobutylsuccinylhydroxamic acid motif (red) with the addition of a chloromethyl amide electrophilic warhead (blue) and a Cy5 fluorescent tag (green). NAP8 has an additional propylamide to improve its metabolic stability. b) Optimized synthesis strategy to prepare double-protected hydroxylamine derivative 6, synthesis procedures were adopted from Overkleeft et al. c) Improved synthesis scheme for NAP8, employing an on-resin click reaction to add the electrophile 12 in the penultimate reaction step.

Figure 3:

Active MMP-14 is selectively labeled by ABPs in stimulated fibroblasts. a) Recombinant MMP-14 F260C catalytic domain was incubated with TND124 or NAP8 over a range of concentrations. Enzyme activity was measured with the MMP FRET substrate MCA-PLGL-Dap(Dnp)-AR-NH₂ (Anaspec). Activity is relative to a DMSO control. Each data point represents an average of three replicates, error bars ± SD. b) Primary mouse fibroblasts derived from the MMP14-F260C knock in mice and immortalized WT fibroblasts were serum starved for 12 hours followed by 24 hours incubation in the presence or absence of growth factors (all at 10 ng/ml), GM6001 (100 μM) and NEM (100 μM). Cell cultures were then incubated with NAP8 (10 μM) for 60 minutes and washed. Cell were lysed in sample buffer and resolved on SDS-PAGE followed by in-gel fluorescence scanning for Cy5 (Fl-Chnl). Western blot analysis was performed with antibodies for MMP-14 and α-tubulin. Locations for pro-MMP14 (60kDa) and mature MMP14 (57kDa) are marked. c) Serum starved primary mMmp14-F260C fibroblasts were incubated with TGFβ1 for 24 hours then NAP8 over a range of concentrations as indicated. Cell lysates were analyzed by SDS-PAGE followed by in-gel fluorescence scanning for Cy5 (Fl-Chnl). Western blot analysis was performed with MMP-14 antibody. d) Same as c) except cells were labeled with 10 μM NAP8 for the indicated incubation times. e) Serum starved WT fibroblasts were incubated with TGFβ1 over a range of concentrations for 24 hours. Western blot analysis was performed with antibodies for MMP-14 and α-tubulin. f) Serum starved primary mMmp14-F260C fibroblasts were incubated in the presence or absence of TGFβ1 (50 ng/ml) for 24 hours, then treated with NAP8 (10 μM). Cell lysates were analyzed by SDS-PAGE followed by in-gel fluorescence scanning for Cy5 (Fl-Chnl). Western blot analysis was performed with antibodies for MMP-14, α-smooth muscle actin (SMA) and α-tubulin.

Figure 4:

Active MMP-14 is detected only in M1 activated macrophages. Primary bone marrow-derived macrophages (BMDMs) from the WT or mMmp14-F260C knock in mice were primed in the presence of a) LPS (100 ng/ml) and b) other pro-inflammatory cytokines; IFNγ (40 ng/ml), IL6 (50 ng/ml), IL4 (10 ng/ml), TGFβ1 (10 ng/ml) for 16 hours, and treated with GM6001 (10 μM) or DMSO vehicle control. Cell cultures were treated with TND124 (10 μM) and lysed cells were analyzed by SDS-PAGE followed by in-gel fluorescence scanning for Cy5 (Fl-Chnl). Western blot analysis was performed with antibodies for MMP-14 and α-tubulin. Locations for pro-MMP-14, mature MMP-14 and an inactive fragment are marked. c) Primary mMmp14-F260C and WT BMDMs were primed with different TLR agonists; LPS (100 ng/ml), S. aureus (heat inactivated, 1×10⁷ cells/ml), Poly I:C (1 μg/ml), R-848 (1 μg/ml), IFNγ (50 ng/ml) for 16 hours. Cell cultures were treated with TND124 (5 μM) and lysed cells were analyzed by SDS-PAGE followed by in-gel fluorescence scanning for Cy5 (Fl-Chnl). Western blot analysis was performed with MMP-14 antibody. d) Western blot analysis for BMDMs primed with LPS (100 ng/ml) or R-848 (1 μM) over 24 hours.

Figure 5:

Active MMP-14 is not detected on the surface of primary epithelial cells. a) Primary mMmp14-F260C epithelial cells were serum starved for 12 hours followed by 24 hours incubation in the presence of absence of growth factors (10 ng/ml). Cell cultures were treated with NAP8 (10 μM) and lysed cells were analyzed by SDS-PAGE followed by in-gel fluorescence scanning for Cy5 (Fl-Chnl). Western blot analysis was performed with antibodies for MMP-14 and α-tubulin b) Confocal microscopy of mammary spheroids in 3D-cultures. Primary organoids isolated from WT and mMmp14-F260C 14-week virgin females were cultured on Matrigel for 10 days. At day 10 spheroids were treated with NAP8 (10 μM) fixed, stained with the molecular marker DAPI and visualized by confocal microscopy (scale bar 50 μm). c) Magnified images (64X) of sections of mMmp14-F260C spheroids treated with NAP8 (10 μM, purple) and stained with antibodies for MMP-14 (green), E-cadherin (red) and DAPI (blue). The magnified section is marked with a white box (scale bar 5 μm) on the white light image of the spheroid (bottom right). d) Crude primary organoid extract, grown in 2D-cultures were pretreated with GM6001 (10 μM), NEM (100 μM) or DMSO and then treated with NAP8(10 μM). Cells were fixed and stained with anti-MMP-14 and DAPI and visualized by confocal microscopy. NAP8 (red), MMP14 (green), DAPI (blue), cells that have active MMP14 are marked with a white arrow (scale bar 20 μm).

Figure 6:

The majority of MMP-14 activity originates from stromal cells in the microenvironment of late-stage carcinoma that respond to stimulation by tumor cells. a) Tumors harvested from the mammary glands of mMmp14(F260C)^WTMMTV-PyMT⁺ and mMmp14(F260C)^+/+MMTV-PyMT⁺ mice were cultured in 2D-cultures and treated with NAP8 (10 μM) at the first passage or after 10 passages. Whole cell lysates were analyzed by SDS-PAGE and scanned for in-gel fluorescence of Cy5 (Fl-Chnl). Western blot analysis was performed with MMP-14 antibody. b) Primary tumor cultures, passaged tumor cultures (neoplastic epithelial cells) and primary fibroblasts from mMmp14(F260C)^+/+MMTV-PyMT⁺ mice were incubated with TGFβ1 (50 ng/ml) or supernatant of neoplastic epithelial cells (24 hours, serum free medium) for 24 hours, then treated with NAP8 (10 μM). Whole cell lysates were analyzed by SDS-PAGE and scanned for in-gel fluorescence of Cy5 (Fl-Chnl). Western blot analysis was performed with antibodies for MMP-14, α-smooth muscle actin (SMA) and α-tubulin. c) 2D-cultures of mMmp14(F260C)^WTMMTV-PyMT⁺ and mMmp14(F260C)^+/+MMTV-PyMT⁺ passaged tumor cells, fibroblasts and co-cultures of passaged tumor cells and fibroblasts were pretreated with DMSO or GM6001 (10 μM), or pre-incubated with supernatant of neoplastic epithelial cells (24 hours, serum free medium), then treated with NAP8 (10 μM). Cells were fixed and stained with anti-MMP-14 and DAPI and visualized by confocal microscopy. NAP8 (red), MMP14 (green), DAPI (blue), each experiment was performed in three independent biological repeats (scale bar 20 μm).