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. 2017 Apr 25;19(4):774-784.
doi: 10.1016/j.celrep.2017.04.005.

Blocking Surgically Induced Lysyl Oxidase Activity Reduces the Risk of Lung Metastases

Chen Rachman-Tzemah  1 , Shelly Zaffryar-Eilot  2 , Moran Grossman  3 , Dario Ribero  4 , Michael Timaner  1 , Joni M Mäki  5 , Johanna Myllyharju  5 , Francesco Bertolini  6 , Dov Hershkovitz  7 , Irit Sagi  3 , Peleg Hasson  8 , Yuval Shaked  9
Affiliations

Blocking Surgically Induced Lysyl Oxidase Activity Reduces the Risk of Lung Metastases

Chen Rachman-Tzemah et al. Cell Rep. .

Abstract

Surgery remains the most successful curative treatment for cancer. However, some patients with early-stage disease who undergo surgery eventually succumb to distant metastasis. Here, we show that in response to surgery, the lungs become more vulnerable to metastasis due to extracellular matrix remodeling. Mice that undergo surgery or that are preconditioned with plasma from donor mice that underwent surgery succumb to lung metastases earlier than controls. Increased lysyl oxidase (LOX) activity and expression, fibrillary collagen crosslinking, and focal adhesion signaling contribute to this effect, with the hypoxic surgical site serving as the source of LOX. Furthermore, the lungs of recipient mice injected with plasma from post-surgical colorectal cancer patients are more prone to metastatic seeding than mice injected with baseline plasma. Downregulation of LOX activity or levels reduces lung metastasis after surgery and increases survival, highlighting the potential of LOX inhibition in reducing the risk of metastasis following surgery.

Keywords: breast cancer; host response; hypoxia; lysyl oxidase; metastasis; pre-metastatic niche; surgery.

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Figures

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Graphical abstract
Figure 1
Figure 1
Increased Mortality Rate in Post-surgery Mice (A) A 1 cm incision in the abdomen of non-tumor-bearing 8- to 10-week-old BALB/c mice was performed. Control mice did not undergo any surgical procedure (n = 5 mice /group). After 24 hr, the mice were injected with 2.5 × 104 EMT/6-GFP+ cells through the tail vein to obtain an experimental lung metastasis model. Mouse survival was monitored daily, and a Kaplan-Meier survival curve was plotted (p = 0.025). (B) Plasma drawn from control or post-surgery mice was intraperitoneally injected into naive 8- 10-week-old BALB/c mice (100 μL/mouse; n = 6 mice/group). After 24 hr, 2.5 × 104 EMT/6-GFP+ cells were injected through the tail vein to obtain experimental lung metastases, and survival was monitored. Kaplan-Meier survival curve is shown (p = 0.043). (C and D) Mice (n = 3 mice/group) were injected with plasma as in (B). After 24 hr, mice were injected with the EMT/6-GFP+ (2.5 × 104) cells, and 15 min later, PuMA was performed. Subsequently, lungs were removed and sectioned. Lung slices were cultured in medium for 6 days, and GFP+ cells were detected by fluorescence microscopy. Red arrows indicate GFP+ cells. Scale bar, 200 μm (C). Subsequently, lung sections were prepared as single-cell suspensions. The percentage of GFP+ cells was quantified by flow cytometry (D). LOX activity was assessed by paired Student’s t test for three biological replicates. The survival experiments were repeated twice, and representative Kaplan-Meier curves are provided. ∗p < 0.05, as assessed by Student’s t test. All error bars represent SD.
Figure 2
Figure 2
High Expression of LOX following Surgery Correlates with Increased Pulmonary Metastasis (A–C) A 1 cm incision in the abdomen of non-tumor-bearing 8- to 10-week-old BALB/c mice was performed. Control mice did not undergo any surgical procedure (n = 5 mice /group). After 24 hr, mice were injected with pimonidazole, and 90 min later, the peritoneum was excised, embedded in OCT, and cryosectioned. (A) Sections were immunostained for LOX (green) and hypoxia (red). Nuclei were stained with DAPI (blue). Dashed line marks the incision site. Scale bar, 200 μm. (B) Quantification of the percentage of LOX- and hypoxia-positive pixels is presented (n > 15 field/group). (C) Peritoneum lysates (n = 3 mice/group) were evaluated for LOX activity. (D–H) Lungs from 8- to 10-week-old BALB/c control mice or 24 hr after mice underwent surgery (n = 3 mice/group) were sectioned and immunostained for LOX (red). Nuclei were stained with DAPI (blue). Scale bar, 200 μm (D). Quantification of LOX expression in lung sections by means of percentage of positive pixels is presented (E). Lung lysates (n = 3 mice/group) were evaluated for LOX activity (F) and expression (G). LOX activity was assessed by paired Student’s t test for three biological replicates. (H) Lungs from 8- to 10-week-old BALB/c control mice or 72 hr after surgery (n = 3 mice/group) were assessed for new collagen formation. (I) Two-photon second harmonic generation (SHG) imaging depicting fibrillary collagens (red) in the lungs of control and post-surgery mice. Scale bar, 50 μm. Relative collagen intensity was determined using densitometric analysis (ImageJ). (J) In a parallel experiment to (D)–(H), lungs were removed and embedded in paraffin. Lung sections were immunostained for LOX (brown). Counterstaining was performed using hematoxylin. Images were captured at 100× (large micrograph) and 400× (small micrograph) magnifications. Scale bar, 100μm. (K and L) 8- to 10-week-old BALB/c mice (n = 3 mice/group) were injected with recombinant LOX (rLOX, 25 μg/kg) or PBS. After 24 hr, the mice were intravenously injected with EMT/6-GFP+ cells (2.5 × 104 cells/mouse) and processed for PuMA. Lung slices were cultured in medium for 6 days, and GFP+ cells were detected by fluorescence microscopy (Olympus SZX9 fluorescence stereo microscope). Red arrows indicate GFP+ cells. Scale bar, 200 μm (K). Subsequently, all lung slices were prepared as single-cell suspensions, and the percentage of GFP+ cells was quantified using flow cytometry (L). ∗p < 0.05 and ∗∗p < 0.01 using Student’s t test. All error bars represent SD.
Figure 3
Figure 3
Plasma from Mice that Undergo Surgery Induces Focal Adhesion Signaling in Tumor Cells (A–D) MCF7 cells were cultured in the presence of collagen, fibronectin, or laminin that were primed with (A) plasma from control or post-surgery mice or (C) plasma from post-surgery mice that was either untreated or depleted of LOX (Surgery-LOX). The levels of total paxillin (pax) and p-pax in cell lysates were evaluated by western blotting. α-Tubulin (A) or actin (C) served as loading controls. The ratios between p-pax and α-tubulin or actin (loading controls) were calculated by densitometry analysis (B and D, respectively). The western blot represents three biological repeats. (E and F) Lungs from 8- to 10-week-old BALB/c control or post-surgery mice (n = 3 mice/group) that were treated with PBS or BAPN for three sequential days were sectioned and immunostained for p-pax (red). Nuclei were stained with DAPI (blue). Scale bar, 200 μm (E). Quantification of p-pax expression in lung sections by means of positive pixels is shown in (F). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 using one-way ANOVA followed by Tukey post hoc test. All error bars represent SD.
Figure 4
Figure 4
Blocking LOX Activity in Mice that Undergo Surgery Decreases Tumor Cell Seeding in the Lungs and Increases Survival (A–C) 8- to 10-week-old BALB/c mice were treated with PBS or BAPN (LOX inhibitor, 100 mg/kg) daily for seven consecutive days. On day 2, the mice were injected with 100 μL plasma from control or post-surgery mice. On day 3, EMT/6-GFP+ cells (2.5 × 104) were injected through the tail vein to obtain an experimental lung metastasis assay. A scheme of the experimental procedure is shown in (A). On day 13 after tumor cell injection, lungs were removed and prepared as single-cell suspensions (n = 4 mice/group). The percentage of GFP+ cells was quantified by flow cytometry (B). In a parallel experiment, survival of control and BAPN-treated mice injected with plasma from mice that underwent surgery or control mice was monitored. A Kaplan-Meier survival curve is shown (n = 6 mice/group, p = 0.049) (C). (D–F) 8- to 10-week-old BALB/c mice were treated with PBS control or 20 mg/kg anti-LOX antibodies (n = 4 mice/group). After 6 hr, the mice were intraperitoneally injected with plasma from control or post-surgery mice (100 μL/mouse). On day 2, mice were injected through the tail vein with 2.5 × 104 EMT/6-GFP+ cells to obtain an experimental lung metastasis model. (D) A scheme of the experimental procedure is shown. (E) On day 13 after tumor cell injection, lungs were removed and prepared as single-cell suspensions. The number of GFP+ cells in the lungs was evaluated by flow cytometry. (F) In a parallel experiment, lung sections (n = 5 mice/group) were counterstained with DAPI and analyzed by fluorescence microscopy for the presence of GFP+ cells. Scale bar, 200μm. (G–I) Lungs from 8- to 10-week-old BALB/c mice 72 hr after surgery (n = 3 mice/group) that were treated with anti-LOX antibodies (α-LOX; 20 mg/kg) or BAPN (100 mg/kg) were assessed for new collagen formation (G). (H) Two-photon second harmonic generation (SHG) imaging depicting fibrillary collagens (red) in the lungs of post-surgery mice treated with anti-LOX or BAPN. Scale bar, 50 μm. (I) Relative collagen intensity was determined using densitometric analysis (ImageJ). ∗p < 0.05 and ∗∗p < 0.01 using one-way ANOVA followed by Tukey post hoc test. All error bars represent SD.
Figure 5
Figure 5
LOX-Depleted Plasma of Post-surgery Mice Inhibits Tumor Cell Seeding in the Lungs and Increases Survival (A) Plasma obtained from control non-tumor-bearing BALB/c mice, or 24 hr after the mice underwent abdominal surgery was evaluated for LOX expression (n = 4 mice/group). ∗p < 0.05 as assessed by Student’s t test. (B and C) LOX was depleted from plasma drawn from control and post-surgery mice as described in Experimental Procedures. The plasma was injected into 8- to 10-week-old naive BALB/c mice (n = 3 mice/group), and 24 hr later, EMT/6-GFP+ cells (2.5 × 104) were intravenously injected and processed for PuMA. Lung sections were cultured for 6 days. (B) The percentage of GFP+ cells was quantified by flow cytometry after lung tissues were prepared as single-cell suspensions. (C) Fluorescent images of lung slices were captured by fluorescence microscopy system. Scale bar, 200μm. ∗p < 0.05; ∗∗p < 0.01 using one-way ANOVA followed by Tukey post hoc test. (D and E) C57BL/6-LOX+/− mice and their wild-type counterparts were injected with BAPN (100 mg/kg) and PBS, respectively, for four consecutive days. On day 3, half of the mice from each group underwent abdominal surgery. After 24 hr, plasma from each group of mice was drawn and pooled. The plasma was then injected to naive 8- to 10-week-old C57BL/6 mice, and 24 hr later, LLC-GFP+ cells (2.5 × 104) were intravenously injected through the tail vein to generate lung metastases (n = 6 mice/group). (D) A scheme of the experimental procedure is shown. (E) A Kaplan-Meier survival curve is shown. Statistical significance was achieved only when comparing surgery and surgery + LOX inhibition groups (p = 0.043). All error bars represent SD.
Figure 6
Figure 6
LOX Inhibition in Clinically Relevant Tumor Models Increases Survival and Reduces Tumor Cell Seeding in the Lungs (A–D) 8- to 10-week-old BALB/c mice were used as recipients for orthotopic transplantation of 4T1-mCherry+ cells into the mammary fat pad. When primary tumors reached 150–200 mm3 (day 12), they were resected, and treatment with BAPN (100 mg/kg) or PBS was initiated. The treatment was administered for seven consecutive days. A scheme of the experimental procedure is shown (A). Mouse survival was monitored, and a Kaplan-Meier survival curve was plotted (n = 7 mice/group) (B). In a parallel experiment, 26 days after primary tumor resection, lungs were removed and either prepared as single-cell suspensions or sectioned. The percentage of mCherry+ cells in the lung suspensions was quantified by flow cytometry (n = 5 mice/group) (C). Lung sections were counterstained with DAPI and analyzed by fluorescence microscopy (n = 5 mice/group). Representative images are shown (mCherry+ cells, red; nuclei, blue). Scale bar, 200 μm (D). ∗p < 0.05, as assessed by Student’s t test. (E and F) Plasma samples obtained from colorectal cancer patients at baseline or 24 hr after abdominal surgery (n = 6 patients) were intraperitoneally injected into 8- to 10-week-old CB.17 SCID mice (n = 3 mice/plasma specimen). After 24 hr, mice were intravenously injected with 2.5 × 104 EMT/6-GFP+ cells. After 12 days, lungs were removed and either prepared as single-cell suspensions or sectioned. The percentage of GFP+ cells in the lung suspensions was quantified by flow cytometry (E). Lung sections were stained with DAPI and analyzed by fluorescence microscopy. Representative images of lung sections from mice injected with the indicated patient plasma are shown (GFP+ cells, green; nuclei, blue). Scale bar, 200 μm (F). (G) Two-photon second harmonic generation mode (SHG) imaging depicting fibrillary collagens (red) in the lungs of mice injected with control plasma and plasma from patients 24 hr after surgery. Scale bar, 50 μm. Relative collagen intensity was determined using densitometric analysis (ImageJ) as presented in Figure S6A. (H) Plasma (pooled) from colorectal cancer patients (n = 6) at baseline or 24 hr after surgery was depleted of LOX as described in Experimental Procedures. Subsequently, the plasma was injected peritoneally into naive SCID CB.17 mice (n = 3 mice/group), and 24 hr later, EMT/6-GFP+ cells (2.5 × 104) were intravenously injected for PuMA. Lungs were cultured for 6 days and then visualized by fluorescence microscopy as shown in Figure S6C. Subsequently, lung sections were prepared as single-cell suspensions, and the percentage of GFP+ cells in the lungs was quantified by flow cytometry. ∗∗∗p < 0.01 using one way ANOVA followed by Tukey post hoc test. All error bars represent SD.
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