Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.
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All gene expression array and RNA-seq data have been deposited in GEO and are publicly available. For the stroma and epithelium specific gene expression analysis, the breast cancer datasets from Finak et al.37 and Gruosso et al.36 have been used (GSE9014, GSE58644, GSE88715 and GSE88847). The association between PLOD2 and DMFS was determined using an online tool (http://xena.ucsc.edu) to download GEO data (GSE2034, GSE5327 and GSE7390) from 683 patients analysed on Affymetrix U133A platform as described in Yau et al.47. The association between PLOD2 gene expression and RFS was determined using an online tool (http://kmplot.com/analysis/) from 1,809 patients analysed on Affymetrix platform (HGU133A and HGU133+2 microarrays)48. Affymetrix ID 202619 or 202620 were used for PLOD2 probes (2014 version) in these analyses. The cBioPortal for Cancer Genomics was used to access the publicly available METABRIC dataset to determine the levels of LOX, PLOD2 and LOXL2 gene expression in patients with breast cancer segregated by ER and HER2 status and to assess gene expression associations of LOX, PLOD2 and LOXL2 with CD16363. Macrophage RNA-seq data have been deposited into GEO (GSE157290). Source data are provided with this paper.
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We thank J. Northcott for writing the ImageJ Macro, L. Korets for mouse husbandry and N. Korets for histology support, as well as K. Lövgren and S. Baker for LH2 immunostaining on patient biopsies. We thank the UCSF Genomics core for their support with the RNA-seq analysis on mouse TAMs and C. Stashko for generating the gene expression heat map. The work was supported by investigator grants through the US National Cancer Institute R33 CA183685 (K.C.H. and V.M.W) and R01CA192914 and CA174929 to (V.M.W), and R01CA222508-01 to (V.M.W. and E.S.H.), as well as US DOD Breast Cancer Research Program (BCRP) grant BC122990 (V.M.W.). Trainee support was provided by US DOD BCRP grant BC130501 (O.M.), US NIH grants TL1 TR001081 and US NIH T32 HL007171 (A.S.B.), and US NIH T32 grant CA 108462 (O.M.). Funding from Eastern Star Scholar-Minnesota Masonic Cancer Center (A.C.N.), the Swedish Research Council (S.B.) and US NIH R01 CA057621 (Z.W.) also supported the work.
The authors declare the following competing interests: L.M.C. is a paid consultant for Cell Signaling Technologies, Shasqi Inc., and AbbVie Inc., and received reagent and/or research support from Plexxikon Inc., Pharmacyclics, Inc., Acerta Pharma, LLC, Deciphera Pharmaceuticals, LLC, Genentech, Inc., Roche Glycart AG, Syndax Pharmaceuticals Inc., Innate Pharma and NanoString Technologies, and is a member of the scientific advisory boards of Syndax Pharmaceuticals, Carisma Therapeutics, Zymeworks, Inc., Verseau Therapeutics, Cytomix Therapeutics, Inc. and Kineta Inc. T.N. and K.C.H. are co-founders of Omix Technologies.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Partial assignment of MS2 fragmentation spectra of crosslinked amino acid standards.
MS2 fragmentation spectra of commercially available crosslinked amino acid standards with partial assignment of MS2 fragmentation spectra (LNL, DHLNL, dPyr). Protonated forms of precursor ions are denoted by MH+ labels. Colored circles above fragment ions are matched to their suggested fragment ion structure above the full spectra or are listed above the ion if space permits.
Extended Data Fig. 2 Optimized crosslinking amino acid analysis (xAAA) enables accurate measurement of collagen state across a diverse range of tissue types.
a, xAAA workflow schematic. Clinical specimens are hydrolyzed and enriched by solid phase extraction (SPE). b, The enriched hydrolysate is analyzed by LC-SRM on a hybrid quadrupole orbitrap instrument. MS2 spectra is used to accurately identify crosslinked amino acids (xAAs) such as dihydroxy lysinonorleucine (DHLNL) (c) Bar graphs showing quantification of tissue collagen and (d) total xAA measured in human breast, lung, skin, bone, trachea and tendon (pooled n = 3 each tissue). The calculated amino acid crosslink abundancies are normalized to total tissue collagen content (that is hydroxyproline abundance) which is calculated based on dry tissue weight. The final values have been plotted as relative abundance based on peak area. Bar graphs depict relative abundance of Lysine derived-collagen crosslink (LCC) and hydroxylysine-derived collagen crosslink (HLCC) species.
a, Scatter plot showing the mean and s.e.m. of patient age for normal (n = 4) and tumor tissue (ER+ n = 8, HER2+ n = 6, TN n = 5) samples by subtype. b-f, Scatter plots showing the levels of each LCC and HLCC crosslink measured in normal and tumor breast tissues versus patient age. The total abundance of crosslinks (g) was calculated by summing all individual crosslinks and the total tissue HLCC abundance (h) was calculated by summing DHLNL, Pyr, and d-Pyr. All crosslink values are normalized to total collagen content (that is hydroxyproline abundance) and dry tissue weight and are plotted as log2 transformed normalized peak areas from LC-MS data. The best fit line and its slope and r2 value are displayed on each plot. All n values represent biologically independent human tissue specimens.
Extended Data Fig. 4 Validation of LOX overexpression in doxycycline (DOX) treated MMTV-rtTA+/−; TetO_mLox+/− mice.
a–c, Representative dot plots (n = 2) of GFP expression in mouse mammary epithelial cells analyzed by flow cytometry. GFP and mLox are encoded on the same mRNA transcript with independent translation initiation sites. a, GFP expression is induced in DOX-treated MMTV-rtTA+/−; TetO_mLox+/− mice. b, c, GFP expression is not induced in control mice lacking the MMTV-rtTA promoter (b) or control mice not treated with DOX (c). To select for mammary epithelial cells were gated out cells positive for anti-mouse CD45-APC, anti-mouse CD31-APC, anti-mouse Ter119-APC antibodies. d, Quantification of Lox gene expression in whole mouse mammary gland by RT-qPCR in control MMTV-rtTA−/−; TetO_mLox+/− mice (n = 2) and LOX overexpressing MMTV-rtTA+/−; TetO_mLox+/− (n = 2) mice. e, Western blot of whole tumor lysate from control (n = 4) and epithelial LOX overexpressing (n = 6) PyMT tumors. Scatter plots with mean ±s.e.m. quantify optical density of each band normalized to E-cadherin. Western blot for these lysates was run once. Statistical analyses were performed using two-tailed Mann-Whitney U test Pro-LOX **p = 0.0095, Cleaved LOX *p = 0.0381. All n values represent biologically independent mouse tissue specimens.
Extended Data Fig. 5 Design of inducible lysyl oxidase expression in vivo using a TetOFF system in the background of MMTV-PyMT model.
a, PCR results and sequence of PCR primers of N- and C-terminal ends to confirm TetO_mLox transgene incorporation. b, Diagram of inducible TetOFF system. TetO_mLox transgene is reversibly turned off or on in the presence or absence of doxycycline, respectively.
Extended Data Fig. 6 PyMT collagen crosslinking abundance does not increase between 8- and 11-week tumors.
a–f, Scatter plots showing individual and mean values ±s.e.m. of the levels of total collagen crosslinks (a), total HLCCs (b), LNL (c), HLNL (d), DHLNL (e), and Pyr (f) in 8-week (n = 10) and 11-week-old (n = 9) PyMT tumors. Quantity of crosslinks per tissue was calculated normalizing crosslinks to total collagen content (that is, hydroxyproline abundance) and dry tissue weight. Values were plotted as log2 transformed normalized peak areas as quantified from LC-MS data. Statistical analysis was performed using two-tailed unpaired t-test (a) p = 0.6149 (b) p = 0.7451 (c) p = 0.7235 (d) p = 0.1766 (e) p = 0.7563 (f) p = 0.4687. All n values represent biologically independent mouse tumor specimens.
Extended Data Figure 7 Early profibrotic macrophages express M2a markers and are lost upon anti-CSF1 treatment.
a, Scatter plot showing individual values and mean ±s.e.m. of the number of F4-80 positive cells per field of view in IgG1 treated (n = 6) or anti-CSF1 treated (n = 4) PyMT tumors from 8-week-old mice. b, c, Scatter plots showing individual values and mean ±s.e.m. of the percentage of F4/80 positive cells expressing markers MHCII and CD86 (b) and markers CD163 and RELMα (c) in IgG1 treated (n = 6) or anti-CSF1 treated (n = 4–5) PyMT tumors from 8-week-old mice. d–g, Scatter plots showing individual values and mean ±s.e.m. of the number of cells per field of view (FOV) of other non-macrophage immune populations CD11b+ F4-80− (d), MHCIIhi F4-80- (e), CD45+ CD4+ (f), and CD45+ CD8+ (g) in IgG1 (n = 4 e, g; n = 5 d, f) and anti-CSF1 (n = 6) treated mice. Statistical analysis (a-g) was performed using two-tailed Mann-Whitney U test (a) *p = 0.019 (b) MHCIIhi p = 0.7190, CD86+ p = 0.4286 (c) CD163+ *p = 0.0303, RELMα+ p = 0.0381 (d) p = 0.9719 (e) p = 0.1524 (f) p = 0.3074 (g) p = 1.000. h, Heat map of selected differentially expressed genes from macrophages sorted from 8-week and 11-week PyMT tumors. i, Representative images from 8-week-old IgG1 treated and anti-CSF1 treated PyMT mice stained for cytokeratin 8/18 (green), PDGFRα (magenta), vimentin (yellow), and DAPI (blue). Scale bar is 100 µm. j, Scatter plot showing mean ±s.e.m. of fibroblasts (vimentin+ and PDGFRα+) per field of view in 8-week-old IgG1 treated (n = 6) and anti-CSF1 (n = 5) treated PyMT mice. All n for (a-j) values represent biologically independent mouse tissue specimens. k–m, Scatter plot depicting the Spearman correlation of CD163 gene expression with LOX (k), PLOD2 (l), and LOXL2 (*p = 0.0143) (m) in human breast tumors (n = 1904 biologically independent human tissue specimens).
a, Scatter plot showing mean and individual values of atomic force microscopy (AFM) microindentation measurements of individual human samples categorized as normal breast tissue (black circles) or invasive ductal carcinoma (IDC; red squares). b, Scatter plot showing mean and individual values of AFM microindentation measurements of individual mouse mammary tumors from doxycycline treated MMTV-PyMT+/−; Col1a1-tTA+/−; TetO-mLox control mice (DOX-ind PyMT Con; black circles) or water treated mice overexpressing stromal LOX (Water PyMT LOX OX; red squares). c, Scatter plot showing mean and individual values of AFM microindentation measurements of individual mouse mammary gland tumors from 8-week-old IgG1 treated (IgG Control; red squares) and anti-CSF1 treated (aCSF1 Ab; black circles) PyMT tumors.
Extended Data Figure 9 PLOD2 correlations with distant metastasis free survival and probability of relapse free survival.
a, Log2 scaled data graphs showing relative PLOD2 gene expression levels in patients stratified by estrogen and epidermal growth factor receptor two (ER/HER2) status (ER+ HER2- n = 314; ER- or + /HER2+ n = 73; ER-/HER2- n = 133). Whiskers denote minima and maxima, box denotes 25th and 75th percentiles and median. Statistical analysis was performed to compare PLOD2 expression levels among subtypes using Kruskal–Wallis one-way ANOVA (****p < 0.0001) and two-tailed Mann-Whitney U test without adjustment for multiple comparisons for individual comparisons (****p < 0.0001). b, Line graphs showing distant metastasis-free survival (DMFS) for patients with estrogen receptor positive and epidermal growth factor receptor two negative breast tumors (ER +/− HER2-; low n = 157 & high = 157). c, Line graph of DMFS for patients with estrogen receptor negative or negative and epidermal growth factor receptor two positive breast tumors (ER- or +/HER2+; low n = 36 & high = 37). d, Line graph of DMFS for patients with estrogen receptor negative epidermal growth factor receptor negative breast tumors (ER- HER2-; low n = 66 & high = 67). Statistical analyses were performed to compare PLOD2 and DMFS for each subtype or Log-rank (Mantel-Cox) test (b) p = 0.8051 (c) *p = 0.0144 (d) p = **0.0050. e-g, Kaplan-Meier curves indicating the probability of relapse-free survival assessed in ER+ /PR + (e), HER2+ (f), and triple negative (g) patients with breast cancer up to 16 years after diagnosis. A correlation between PLOD2 (gene encoding LH2) expression and relapse-free survival (RFS) has been determined using an online tool (http://kmplot.com/analysis/) as described in the methods. Top and bottom panels represent two distinct Affymetrix PLOD2 probes (202619 and 202620) from the same database. For Kaplan-Meier curves, statistical analyses were performed using LogRank test.
Extended Data Figure 10 Assessment of LOX and neoplastic epithelial LH2 expression in breast cancer.
a, Neoplastic epithelial LOX staining of the Biomax BC081116c TMA was assessed by a researcher with an intensity score and stratified as negative/low, moderate, or high. b, Stromal LOX staining of the Biomax BC081116c TMA was assessed by a researcher with an intensity score and stratified as negative/low, moderate, or high. Scale bar is 500 µm. c, Tumor samples from incident breast cancer cases were collected, and a tissue microarray (TMA) including two 1-mm cores from each tumor was constructed. Neoplastic epithelial LH2 staining was assessed by a pathologist with semi-quantitative intensity score and stratified as negative, low, or moderate/high. Scale bars are 500 µm. See Supplementary Table 2 for patient information. d, Clinical correlation of neoplastic epithelial LH2 intensity score with tumor grades. e, Kaplan-Meier curves indicating cumulative breast cancer specific survival (BCSS) based on epithelial LH2 intensity assessed in patients with breast cancer up to 10 years after diagnosis (LH2 negative n = 271, weak n = 112, moderate/high n = 77). f, BCSS curves by epithelial LH2 intensity including only axillary lymph node negative patients (LH2 negative n = 175, weak n = 67, moderate/high n = 50). g, BCSS curves by epithelial LH2 intensity including only axillary lymph node positive patients (LH2 negative n = 84, weak n = 42, moderate/high n = 26). For tumor grade and LH2 intensity score, statistical analysis was performed using a linear-by-linear association (***p < 0.001). For Kaplan-Meier curves, statistical analyses were using LogRank test (e) *p = 0.025 (f) *p = 0.038 (g) p = 0.165.
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Maller, O., Drain, A.P., Barrett, A.S. et al. Tumour-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression. Nat. Mater. (2020). https://doi.org/10.1038/s41563-020-00849-5