Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma

Abstract

Objective: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models.

Design: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras ^+/LSL-G12D;Trp53 ^LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras ^+/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2^{Exon2 fl/fl}) or conditional Loxl2 overexpressing mice (R26Loxl2 ^KI/KI) to generate KPCL2^KO or KCL2^KO and KPCL2^KI or KCL2^KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation.

Results: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment.

Conclusion: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.

Keywords: CELL MATRIX INTERACTION; MACROPHAGES; MOLECULAR ONCOLOGY; PANCREATIC CANCER; PANCREATIC FIBROSIS.

Figure 1

LOXL2 mRNA expression correlates with poor overall survival and EMT in patients with PDAC. (A) Differential expression of LOXL2 in adjacent (Adj.) normal tissue vs PDAC tumours and metastasis (met) in GSE62165, META data set, and GSE71729. Unpaired two-sided Student’s t-test. (B) LOXL2 relative mRNA levels ±SD (n=2 technical replicates) in a panel of surgically resected human PDAC tumours (n=25) and four normal pancreas (nPanc) controls (left). Pooled mean ±SEM analysis including four primary KPC tumours (right). (ns, not significant; ****p<0.0001; two-sided t-test with Mann-Whitney U test). (C) Overall survival of patients with PDAC from the Bailey (n=96) data set, stratified according to the median value of LOXL2 expression. HR=Hazard ratio, Cox proportional hazard regression model. A Log-rank test was performed for survival analysis. (D) Differential expression of LOXL2 in PDAC tumours, subtyped as progenitor, squamous, immunogenic or ADEX from the Bailey et al data set. (**P<0.01, ****p<0.0001, one-way analysis of variance with Dunnett post-test). (E) EMT pathway enrichment plots from transcriptomics analysis (GSE62165, META and GSE1729 data sets) of LOXL2 high vs LOXL2 low patients. FDR <0.25. (F) Pearson correlation matrix of mesenchymal-related and epithelial-related genes in 179 patients with human PDAC (TCGA) sorted for LOXL2 mRNA levels and nearest neighbour. The matrix was subjected to supervised hierarchical clustering (Euclidean distance measurement, average linkage clustering). EMT, epithelial to mesenchymal transition; FDR, false discovery rate; LOXL2, lysyl oxidase-like protein 2; mRNA, messenger RNA; PDAC, pancreatic ductal adenocarcinoma; TCGA, The Cancer Genome Atlas.

Figure 2

Macrophages induce LOXL2 and EMT in PDX-derived PDAC cells via OSM. (A) LOXL2 expression in indicated human PDXs. Tubulin, loading control. Positive (+) control=cell lysate from LOXL2-overexpressing 293 T cells. (B) Light micrographs of PDX-derived cells cultured for 72 hours with control media or conditioned medium from M2-polarised MØs (MCM). Scale bar=200 µm. Insets=2X magnified areas. (C) Light micrographs of Panc354 PDX-derived cells cultured for 72 hours with control media (Ctl) or MCM (left). Scale bar=400 µm. Representative images of migrating Ctl-treated or MCM-treated cells through a 0.8 micron transwell (top, right), and mean fold-change ±SD of MCM-treated invading cells compared with Ctl-treated Panc354, Panc215 or Panc265 cells, set as 1.0. (**P<0.01, ***p<0.001, unpaired Student’s t-test). (D) Mean fold-change ±SD of relative mRNA levels for the indicated genes in Ctl-treated or MCM-treated cells. Values normalised to ß-actin. Ctl-treated samples were set as 1.0. (*P<0.05, **p<0.01, ***p<0.001, unpaired Student’s t-test). (E) LOXL2 expression in Ctl-treated (–) or MCM-treated (+) PDX-derived cells. Tubulin, loading control. (F–H) OSM protein levels (pg/mL) present in (F) unpolarised PBMC-conditioned media or MCM, (G) unpolarised (Ctl) PBMC-conditioned media or conditioned media from TGFß1-treated PBMCs, or (H) serum from healthy controls or patients with PDAC. (*P<0.05, ***p<0.001, ****p<0.0001, unpaired two-sided Student’s t-test). EMT, epithelial to mesenchymal transition; LOXL2, lysyl oxidase-like protein 2; mRNA, messenger RNA; OSM, oncostatin M; PBMC, peripheral blood mononuclear cell; PDAC, pancreatic ductal adenocarcinoma; PDX, patient-derived xenografts; TGF, transforming growth factor.

Figure 3

Loss of Loxl2 improves overall survival and decreases tumour burden. (A) Scheme of the genetic mouse models for pancreatic cancer. The colour code (blue, KPC wild type (wt); red, KPCL2^KO) is used for all results. (B) Mean relative mRNA levels±SEM of indicated genes in tumour-derived cells from the indicated genotypes (nd, not detected; ns, not significant; *p<0.05, ***p<0.001; one-way analysis of variance (ANOVA) with Dunnett’s post-test). (C) Loxl2 expression in tumour homogenates from the indicated KPC genotypes. Gapdh, loading control. (D) Survival of wt (+/+), Het (KPC; Loxl2 ^+/-) and KO KPC (KPC; Loxl2 ^-/-) mice. All mice died of PDAC associated disease at the indicated times (p value is shown; ns, not significant; log-rank (Mantel-Cox) test). Calculated median survivals are: wt (+/+): 19 weeks; Het (KPC; Loxl2 ^+/-): 21 weeks; and KO KPC (KPC; Loxl2 ^-/-): 26 weeks. (E) Percent tumour incidence in wt (+/+), Het (KPC; Loxl2 ^+/-) and KO KPC (KPC; Loxl2 ^-/-) mice at 17–18 weeks post birth. (P values are shown, contingency analysis, two-sided Fisher’s exact test). Tumour incidence was determined as positive if a macroscopic tumour was visible on necropsy. (F) Mean pancreas weight ±SEM in wt (+/+), Het (KPC; Loxl2 ^+/-) and KO KPC (KPC; Loxl2 ^-/-) mice at 17–18 weeks post birth (left). (**P<0.01; ns, not significant; one-way ANOVA with Tukey post-test). Representative images of PDAC tumours/genotype (bottom) at 17–18 weeks post birth. (G) Quantification of tissue area in mouse pancreata from wt (+/+), Het (KPC; Loxl2 ^+/-) and KO KPC (KPC; Loxl2 ^-/-) mice at 17–18 weeks post birth, categorised as severely altered tissue (acinar-to-ductal metaplasia (ADM) and inflammation), pancreatic intraepithelial neoplasias (PanINs I–III), cancer tissue (PDAC) or normal acinar tissue (*p<0.05, contingency analysis, two-sided Fisher’s exact test). (H) Representative H&E-stained sections for the grading of the respective tumours: wt (+/+) KPC (blue, n=14), Het KPC (KPC; Loxl2 ^+/-) (orange, n=21) and KO KPC (KPC; Loxl2 ^-/-) mice (red, n=11). Pie chart insets=percent of differentiated (blue) vs poorly differentiated (yellow) tumours for each genotype. Scale bar=250 µm. Het, heterozygous; KO, knockout; Loxl2, lysyl oxidase-like protein 2; mRNA, messenger RNA; PDAC, pancreatic ductal adenocarcinoma.

Figure 4

Overexpression of Loxl2 worsens overall survival and increases tumour burden. (A) Scheme of the KPC-based Loxl2 overexpression genetic mouse model. The colour code green, KPCL2^KI, is used for all results. (B) Light and EGFP tumour images (left). Loxl2 expression in tumour homogenates from the indicated KPC genotypes (right). Gapdh, loading control. Positive (+) control=cell lysate from murine Loxl2-overexpressing 293 T cells. (C) Mean relative mRNA levels ±SEM of indicated genes in tumour-derived cells from the indicated genotypes (***p<0.001; ns, not significant; one-way analysis of variance (ANOVA) with Dunnett’s post-test). (D) Survival of KPC wild type (+/+), KPC; R26L2^+/KI and KPC; R26L2^KI/KI mice. All mice died of PDAC associated disease at the indicated times (p value is shown; ns, not significant; log-rank (Mantel-Cox) test). Calculated median survivals are: wild type (+/+): 20 weeks; KPC; R26L2^+/KI: 18 weeks; and KPC; R26L2^KI/KI: 17 weeks. (E) Percent tumour incidence in KPC wild type (+/+), KPC; R26L2^+/KI and KPC; R26L2^KI/KI mice at 17–18 weeks post birth. (****P<0.0001; ns, not significant; contingency analysis; two-sided Fisher’s exact test). Tumour incidence was determined as positive if a macroscopic tumour was visible on necropsy. (F) Mean pancreas weight ±SEM in KPC wild type (+/+), KPC; R26L2^+/KI and KPC; R26L2^KI/KI mice at 17–18 weeks post birth. (*P<0.05, **p<0.01; ns, not significant; one-way ANOVA with Tukey post-test). (G) Representative images of PDAC tumours/genotype at 17–18 weeks post birth. (H) Quantification of tissue area in mouse pancreata from wild type (+/+) KPC mice (blue, n=7) and KPCL2^KI (KPC; R26L2^+/KI and KPC; R26L2^KI/KI) mice (green, n=12) determined at 17–18 weeks post birth, categorised as severely altered tissue, PanINs I–III, PDAC or normal acinar tissue (*p<0.05, **p<0.01, contingency analysis, two-sided Fisher’s exact test). (I) Representative H&E-stained sections for the grading of the respective tumours: wild type (+/+) KPC (blue, n=6), KPC; R26L2^+/KI (dark green, n=6) and KPC; R26L2^KI/KI mice (light green, n=6). Pie chart insets=percent of differentiated (blue) vs poorly differentiated (yellow) tumours the indicated genotypes. Scale bar=250 µm. ADM, acinar-to-ductal metaplasia; KI, knock-in; Loxl2, lysyl oxidase-like protein 2; PanINS, pancreatic intraepithelial neoplasias; PDAC, pancreatic ductal adenocarcinoma; wt, wild type.

Figure 5

Loxl2 loss and overexpression affects PDAC metastasis and cancer stem cells properties. (A) PDAC tumours and metastases from indicated genotypes (white arrows, metastases). (B–C) Percent incidence of metastasis in (B) wild type (+/+), Het (KPC; Loxl2 ^+/-) and KO KPC (KPC; Loxl2 ^-/-) mice or (C) KPC wild type (+/+), KPC; R26L2^+/KI and KPC; R26L2^KI/KI mice at 17–18 weeks post birth. (**P<0.01, ****p<0.0001; ns, not significant; contingency analysis, two-sided Fisher’s exact test). (D–E) Left: Representative immunohistochemical stainings for indicated proteins in tumour sections from (D) wild type (+/+) KPC and KO KPC (KPC; Loxl2^-/- ) mice or (E) wild type (+/+) KPC and KPCL2^KI (KPC; R26L2^KI/KI) mice. Scale bar=250 µm. Right: Quantification of per cent positive (+) cells/region of interest (ROI). (****P<0.0001; ns, not significant; unpaired Student’s t-test). (F) Mean number % colony area ±SEM, determined 11 days post seeding in indicated cell lines (n=3 cell lines/genotype, ****p<0.0001, ns, not significant, two-sided t-test with Mann-Whitney U test). (G) Mean number (no.) spheres/mL ±SEM, 7 days post seeding, in tumour cell lines from indicated genotypes (*p<0.05, ***p<0.001, unpaired Student’s t-test). (H) Mean percentage of EpCAM+/CD133+ cells ±SEM, in tumour cell lines from indicated genotypes (***p<0.001, two-sided t-test with Mann-Whitney U test). Het, heterozygous; KI, knock-in; KO, knockout; Loxl2, lysyl oxidase-like protein 2; PDAC, pancreatic ductal adenocarcinoma; wt, wild type.

Figure 6

Loxl2 is necessary for PDAC intravasation. (A–B) Mean lung tumour weight ±SD in (A) wild type (+/+, ID32) KPC and Het KPC (KPC; Loxl2 ^+/-, ID86) cells (blue) and KO KPC (KPC; Loxl2 ^-/-, ID90 and ID98) cells (red) or (B) wild type (+/+, ID15 and ID29) KPC cells (blue) and KPCL2^KI (KPC; R26L2^+/KI, ID63 and KPC; R26L2^KI/KI, ID4) cells (green) at 4 weeks post injection in NOD-SCID immunodeficient mice (left) (ns, not significant; unpaired Student’s t-test). Representative images of extracted lungs (top, right) and H&E images (bottom, right). (C) Experimental set-up for in vivo orthotopic tumour establishment in NOD-SCID immunodeficient mice with H2b-mCherry-labelled KPCL2^KO (KPC; Loxl2 ^-/-) cells or EGFP+KPCL2^KI (KPC; R26L2^KI/KI) cells, alone or at a 1:1 ratio (groups 1, 3 and 2, respectively). (D) Mean tumour weight ±SD, 4 weeks post orthotopic injection, for each group (n=5 mice per group) (left). Representative images of PDAC tumours (right). (E–G) Mean number (no.) of mCherry-positive (red) or EGFP-positive (green) cells in (E) tumour homogenates, (F) blood or (G) liver homogenates, in indicated groups (*p<0.05, **p<0.01, ***p<0.001; ns, not significant; unpaired Student’s t-test). Het, heterozygous; KI, knock-in; KO, knockout; Loxl2, lysyl oxidase-like protein 2; PDAC, pancreatic ductal adenocarcinoma; wt, wild type.

Figure 7

Loxl2 affects collagen fibre orientation and premetastatic niche formation. (A) Mean percent (%) CD11b+, F4/80+ or CD11b+/F4/80+ cells ±SEM in liver homogenates from (top) wild type (+/+) KPC and Het KPC (KPC; Loxl2 ^+/-) mice (blue) and KO KPC (KPC; Loxl2 ^-/-) mice (red) or (bottom) wild type (+/+) KPC mice (blue) and KPCL2^KI (KPC; R26L2^+/KI and KPC; R26L2^KI/KI) mice (green) determined at 14–16 weeks post birth. (*P<0.05, **p<0.01, ***p<0.001; ns, not significant; two-sided t-test with Mann-Whitney U test). (B) Representative picrosirius red-stained images of pancreata. Overlay, representative fibre directionality analysis plots depicting the frequency of fibres in a specific orientation. (C) Mean maximum relative (max rel.) peak frequency ±SEM of directionality analysis plots for picrosirius red-stained images of pancreata shown in (B). (n=5–6 mice per genotype with 4–6 representative images analysed per mouse, *p<0.05, ****p<0.0001, two-sided t-test with Mann-Whitney U test). (D) Experimental set-up for clodronate (Clod) in vivo studies. (E) Mean fold-change in Loxl2 (top) or Osm (bottom) relative mRNA levels ±SEM in pancreata from control liposome-treated vs clodronate liposome-treated KPC wt mice. Values were normalised to Hprt levels and control liposome-treated samples were set as 1.0. (n=8 mice per group, ****p<0.0001, two-sided t-test with Mann-Whitney U test). (F) Representative picrosirius red-stained images of pancreata and overlaid fibre directionality analysis plots from control (Ctl)- and clodronate-treated KPC wt mice. (G) Mean maximum relative (max rel.) peak frequency ±SEM of directionality analysis plots for picrosirius red-stained images of pancreata shown in (F). (n=5 mice per group with three representative images analysed per mouse, ***p<0.001, two-sided t-test with Mann-Whitney U test). (H) Number of tumours and metastases detected in indicated groups. Het, heterozygous; Hprt, hypoxanthine phosphoribosyltransferase 1; KI, knock-in; KO, knockout; Loxl2, lysyl oxidase-like protein 2; mRNA, messenger RNA; Osm, oncostatin M; PDAC, pancreatic ductal adenocarcinoma; wt, wild type.