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. 2018 Sep 24;13(9):e0198464.
doi: 10.1371/journal.pone.0198464. eCollection 2018.

Impact of MMP-2 and MMP-9 enzyme activity on wound healing, tumor growth and RACPP cleavage

Dina V Hingorani  1 , Csilla N Lippert  2 , Jessica L Crisp  2 , Elamprakash N Savariar  2 , Jonathan P C Hasselmann  3 , Christopher Kuo  3 , Quyen T Nguyen  4   5 , Roger Y Tsien  1   2   4 , Michael A Whitney  2 , Lesley G Ellies  3   4
Affiliations

Impact of MMP-2 and MMP-9 enzyme activity on wound healing, tumor growth and RACPP cleavage

Dina V Hingorani et al. PLoS One. .

Abstract

Matrix metalloproteinases-2 and -9 (MMP-2/-9) are key tissue remodeling enzymes that have multiple overlapping activities critical for wound healing and tumor progression in vivo. To overcome issues of redundancy in studying their functions in vivo, we created MMP-2/-9 double knockout (DKO) mice in the C57BL/6 background to examine wound healing. We then bred the DKO mice into the polyomavirus middle T (PyVmT) model of breast cancer to analyze the role of these enzymes in tumorigenesis. Breeding analyses indicated that significantly fewer DKO mice were born than predicted by Mendelian genetics and weaned DKO mice were growth compromised compared with wild type (WT) cohorts. Epithelial wound healing was dramatically delayed in adult DKO mice and when the DKO was combined with the PyVmT oncogene, we found that the biologically related process of mammary tumorigenesis was inhibited in a site-specific manner. To further examine the role of MMP-2/-9 in tumor progression, tumor cells derived from WT or DKO PyVmT transgenic tumors were grown in WT or DKO mice. Ratiometric activatable cell penetrating peptides (RACPPs) previously used to image cancer based on MMP-2/-9 activity were used to understand differences in MMP activity in WT or knockout syngeneic tumors in WT and KO animals. Analysis of an MMP-2 selective RACPP in WT or DKO mice bearing WT and DKO PyVmT tumor cells indicated that the genotype of the tumor cells was more important than the host stromal genotype in promoting MMP-2/-9 activity in the tumors in this model system. Additional complexities were revealed as the recruitment of host macrophages by the tumor cells was found to be the source of the tumor MMP-2/-9 activity and it is evident that MMP-2/-9 from both host and tumor is required for maximum signal using RACPP imaging for detection. We conclude that in the PyVmT model, the majority of MMP-2/-9 activity in mammary tumors is associated with host macrophages recruited into the tumor rather than that produced by the tumor cells themselves. Thus therapies that target tumor-associated macrophage functions have the potential to slow tumor progression.

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Conflict of interest statement

M. Whitney and Q. Nguyen are non-employee scientific advisors to Avelas Biosciences, which has licensed the ACPP technology from the University of California Regents. This affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. The remaining authors declare that they have no other competing interests.

Figures

Fig 1
Fig 1. Delayed wound healing in DKO mice.
A. Representative WT and DKO mice on Day 11 following the creation of bilateral, vertical, 8-mm wounds. The initial wounds were located as indicated by the arrows in the WT panel, with aberrant healing in the DKO apparent B. Quantitation of the day 11 skin surface wound area (n = 6 mice and 12 wounds per group). C. H&E stained cross-sections of skin from WT and DKO mice at day 11 after wounding with wound margins indicated by dashed lines. These skin tissue of the wounded area was collected [dotted black box, A and C (enlarged)] and sectioned perpendicular to the length on the wound (white stipple) to determine distance between hair folicles on either side of the incision. D. Quantitation of the distance between healthy hair follicles adjacent to the wound (n = 2–3 sections per wound; 12 wounds per group). Data are means ± SEM, analyzed by Student’s t test. *** p < 0.001.
Fig 2
Fig 2. Modest effect of DKO on mammary tumorigenesis.
A. Comparison of tumor burden in PyVmT;WT (N = 10) and PyVmT;DKO mice (N = 5) aged 22–25 weeks. B. Tumor burden by mammary gland site from pectoral (#1, 2, 3) to inguinal (#4, 5). Data are means ± SEM analyzed by Student’s t test, * p < 0.05. C. Whole mounts of the #4 inguinal mammary glands indicate delayed tumorigenesis in the DKO.
Fig 3
Fig 3. The tumor cell genotype contributes more than the stromal genotype to MMP-2/-9 activity in cleaving the MMP-2/-9-cleavable RACPP.
A. C57BL/6 mice (WT and DKO) with orthotopic WT and DKO tumors (T) in their bilateral mammary fat pads. After 2 h incubation with an intravenously administered MMP-2/-9-cleavable RACPP, the tumors were imaged. B. The tumor ratios (Cy5 emission/Cy 7 emission, corresponding to cleaved/uncleaved ACPP) were quantified (N = 5 mice/group; N = 7 tumors/group). The data from two sets of independent experiments, which had the same relative comparison between tumor groups with different overall ratio ranges, were normalized so the sets could be combined. Each set was normalized to its lowest ratio (all values divided by the lowest ratio value) such that the lowest ratio for each set was re-mapped onto the value one. C. The tumor weights from one of the C57BL/6 mouse strain experiments; the weight was highest for the WT-tumor (T) in WT-mouse (M). Data are means ± SEM analyzed by one-way ANOVA and Holm-Sidak’s multiple comparisons test. D. Ratiometric images Cy5/Cy7 2 h after intravenous injection of MMP-2 selective RACPP, with cleavable sequence TLSLEH, in WT, 2KO (N = 4 mice per WT or KO group; N = 8 tumors/group) and DKO mice (N = 3 mice/group; N = 6 tumors/DKO group). In each mouse, the WT tumor is on the left and the DKO tumor is on the right. E. Quantified tumor ratios of Cy5 emission/Cy7 emission for the cohort of 11 mice imaged with the MMP2-selective RACPP, stratified by tumor type and mouse strain.
Fig 4
Fig 4. At the microscopic level, the host stroma enhances the WT-T ratio.
A. The ratios for tumor sections (10 μm; WT-T in WT-M and DKO-M and DKO-T in WT-M and DKO-M) were evaluated with confocal microscopy and then the tissue sections were stained with H&E to examine the morphology. B. Quantitation of the ratios corresponding to 4–6 confocal images per group. Data are means ± SEM analyzed by one-way ANOVA and Sidak’s multiple comparisons test.
Fig 5
Fig 5. RACPP ratios are higher in macrophages than tumor cells.
A. Immunofluorescence staining with F4/80 pan macrophage antibody marker (yellow) on WT Py8119-GFP (green) tumor tissue excised from WT C57Bl6-albino mice injected with MMP cleavable RACPP (Cy5: red). B. Macrophage infiltration surrounding tumor cells. C. Macrophage distribution in the tissue and D. overlay with Cy5 from cleaved RACPP due to MMP-2/-9 activity. E,F. Much of the Cy5 from cleaved RACPP is seen in stromal region surrounding tumor cells with dimmer puncta seen on the Cy5 image alone. G,H. Higher magnification images demonstrating high Cy5 signal in the macrophages at the tumor periphery rather than I, J. those at the tumor center. K,L. Higher magnification showing accumulation of Cy5 from cleaved RACPP in the tumor cells.
Fig 6
Fig 6. Tumor associated macrophage (TAM) infiltration is modulated by MMP-2/-9.
Panels A-F are representative sections stained with F4/80 to identify TAM infiltration in the various tumor cell/host mouse genotype combinations. A. WT-T in WT mouse. B. WT-T in 2KO mouse. C. WT-T in DKO mouse. D. DKO-T in WT mouse. E. DKO-T in 2KO mouse. F. DKO-T in DKO mouse. Scale bar 100 μm. G. Macrophage infiltration at the periphery (0.5 mm into the tumor). H. Macrophage infiltration into the center of the tumor (1 mm in from the tumor boundary). N = 6–8 tumors per condition. Data are means ± SEM.
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