Chemotherapy elicits pro-metastatic extracellular vesicles in breast cancer models

Abstract

Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca²⁺-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C⁺CCR2⁺ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.

Figure 1. PTX enhances pulmonary metastasis in mammary tumour-bearing mice

a. Drug scheduling in tumour-bearing mice. b. Cumulative weight of multifocal mammary tumours (mean ± s.e.m.) in MMTV-PyMT mice. CREMO, n=14 mice; PTX, n=16. Each dot represents one mouse carrying several tumours. Data show two independent experiments combined (EPFL cohort). c. Volume of 4T1 or 4T1-mCh tumours (mean ± s.e.m.; n=7 mice/group) in untreated Rag1^−/− mice. d. Weight of 4T1-mCh tumours (mean ± s.e.m.) in Rag1^−/− mice. CREMO, n=12 mice; PTX, n=14. Data show two independent experiments combined. e. Weight of 4T1 (n=4) or 4T1-mCh/HER2 tumours (mean ± s.e.m.) in Swiss nu/nu mice. PBS, n=7 mice; CREMO, n=8; PTX, n=9. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. f. Representative hematoxylin/eosin (H&E) images of lung sections of MMTV-PyMT mice from the experiment shown in (b). Scale bars, 1 mm. Data are quantified in (g) and (h). g-i. Number (g) and mean area (h, i) of pulmonary metastases (mean ± s.e.m.) in MMTV-PyMT mice. (g, h): CREMO, n=14 mice; PTX, n=16; two independent experiments combined (EPFL cohort). (i): PBS, n=21; PTX, n=49; five independent experiments combined (OHSU cohort). Statistical analysis in (g) and (i) by unpaired two-tailed Student’s t-test. j. Fluorescence-activated cell sorting (FACS) analysis of mCh⁺CD31^– cancer cells (mean ± s.e.m.; relative to viable CD45⁻ lung-derived cells) in lungs of 4T1-mCh tumour-bearing mice. CREMO, n=8 mice; PTX, n=9. Statistical analysis as in (g). The FACS panels on the right show the gating strategy. k. Representative confocal immunofluorescence images showing mCh⁺ (red) 4T1 cancer cells in lung sections of mice from the experiment in (j). Nuclei are stained with DAPI (blue). Scale bars, 200 μm (left and middle panel) and 50 μm (right panel). l. FACS analysis of mCh⁺CD31⁻ cancer cells (mean ± s.e.m.) in lungs of 4T1 (n=4) or 4T1-mCh/HER2 tumour-bearing mice. PBS, n=7 mice; CREMO, n=8; PTX, n=9. m. FACS analysis of mCh⁺HER2⁺CD45^– cancer cells (mean ± s.e.m.; absolute cell counts) in blood of 4T1-mCh/HER2 tumour-bearing mice. PBS, n=7 mice; CREMO, n=8; PTX, n=9. The FACS panels on the right show the gating strategy. Source data are shown in Supplementary Table 5.

Figure 2. Chemotherapy-elicited EVs are pro-metastatic in mouse and zebrafish tumour model

a. TEM images of CREMO-EV (n=1 biological sample) and PTX-EV (n=2 independent biological samples) isolated from 4T1 cells. One representative image is shown for each EV type. Bottom panels show magnified fields of upper panels. Scale bars, 200 nm (upper panels) and 100 nm (bottom panels). b. Schematics illustrating lung pre-conditioning and tumour colonization assays. c. Number of 4T1 metastatic nodules (mean ± s.e.m.; n=8 mice/group) in lungs of pre-conditioned Balb/c mice. Statistical analysis by unpaired two-tailed Student’s t-test. Right panels show representative H&E images of lung sections (magnified fields below). Scale bars, 1 mm. d. Procedure to isolate tumour-derived EVs from chemotherapy-treated MMTV-PyMT mice. e, f. Number of MMTV-PyMT metastatic nodules (mean ± s.e.m.) in lungs of pre-conditioned FVB/n mice; CREMO-EV, n=13 mice; PTX-EV, n=14; PBS-EV, n=8; DOX-EV, n=9. Statistical analysis as in (c). Lower panels in (e) show representative H&E images of lung sections. Scale bars, 1 mm. g. Number of MMTV-PyMT metastatic nodules (mean ± s.e.m.) in lungs of pre-conditioned FVB/n mice. PBS, n=9 mice; CREMO 1, n=9; CREMO 10, n=8; PTX 1 mg/kg, n=9; PTX 10 mg/kg, n=9; DOX, n=9. CREMO 1 and 10 are the vehicle controls for 1 and 10 mg/kg PTX, respectively. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. h. Concentration (mean ± s.e.m.; n=5 acquisitions of one sample/condition) and size distribution of EVs isolated from plasma of 4T1-mCh tumour-bearing mice treated as indicated, determined by NTA. i. Schematics illustrating experiments in zebrafish embryos. j. Volume of tumour deposits (mean ± s.e.m.) in embryos injected with CREMO-EVs (n=24), PTX-EVs (n=22), CREMO (n=12) or PTX (n=12), determined by confocal imaging analysis. Statistical analysis as in (g). k. Confocal image of a representative zebrafish embryo injected with CFP⁺ MDA-MB-453 cells (blue). Blood vessels are GFP⁺ (green). The right panel shows the caudal haematopoietic area (CHA) with CFP⁺ tumour deposits. Scale bar, 0.5 mm. l. Representative confocal images of the CHA of zebrafish embryos, imaged as in (k). Scale bar, 70 μm. Quantitative data are shown in (j). Source data are shown in Supplementary Table 5.

Figure 3. PTX enriches ANXA6 in EVs in a Ca²⁺-dependent manner

a. Unsupervised clustering of proteins in CREMO-EV and PTX-EV (n=6 independent EV preparations/condition) from 4T1 cells, determined by LC-MS/MS analysis. b. LC-MS/MS analysis of CREMO-EV and PTX-EV (n=6 independent EV preparations) showing total spectrum count (mean ± s.d.) of ANXA6, CD9 and CD81. Statistical analysis by two-way ANOVA with Sidak’s multiple comparison test. c. Western blotting analysis of ANXA6, calnexin (CANX), GAPDH and RAB7 in 4T1 cells treated with CREMO or PTX for 24h before subcellular fractionation. The “membranes” fraction encompasses early and late endosomes, endoplasmic reticulum and mitochondria. The experiment was performed once. d. Western blotting analysis of ANXA6 and CD9 in CREMO- or PTX-treated 4T1 cells, or matched CREMO-EV or PTX-EV. Additional experiments are shown in Fig. 3e, f, h; Fig. 4c; and Supplementary Fig. 1b. e. Western blotting analysis of ANXA6 and CD81 in the indicated EV preparations isolated from either 4T1 or PyMT-IK1 cells. f. Western blotting analysis of the indicated proteins in PyMT-IK1 cells and matched EVs 48h after treatment of the cells with PBS, CREMO, PTX, DMSO or DOX, with or without the calcium chelator BAPTA-AM. One representative experiment is shown of three performed for EVs and one for cells. g. ANXA6 band intensity (mean ± s.d.; n=3 independent experiments, one of which is shown in (f) above) in the indicated EV preparations analysed by Western blotting. Statistical analysis as in (b). h. Western blotting analysis of EVs from 4T1 cells treated for 48h with PBS, CREMO or PTX, with or without BAPTA-AM. One representative experiment is shown of three performed. i. ANXA6 band intensity (normalized to CD81; mean ± s.d.; n=3 independent experiments, one of which is shown in (h) above) in the indicated EV preparations. Statistical analysis as in (b). j. Protein content by BCA (left panel) and concentration by NTA (right panel) of the indicated EVs (mean ± s.d.; n=3 independent EV preparation/condition) obtained from 4T1 cells treated with or without BAPTA-AM. Statistical analysis as in (b). Source data are shown in Supplementary Table 5. Unprocessed blots are shown in Supplementary Fig. 9.

Figure 4. EV-associated ANXA6 promotes mammary tumour metastasis

a. Schematics of the lentiviral vectors used to disrupt the expression of Anxa6, Rab27a or Rela in cells. b, c. Western blotting analysis of ANXA6, GAPDH and CD81 in 4T1-mCh cells (b) or secreted EVs (c). Anxa6-WT (parental line) and two independent Anxa6-KO clones are shown, either untreated (b) or treated as indicated (c). The experiments were performed once. d. mCh mean fluorescence intensity (MFI; left y axis) and concentration by NTA (right y axis) of EVs (mean ± s.d.; n=3 independent cell cultures/condition) released by Anxa6-KO 4T1-mCh cells (clone #1) treated for 72h with CREMO or PTX. Statistical analysis by unpaired two-tailed Student’s t-test. e. Representative wide-field TEM images of the indicated EVs isolated from Anxa6-WT (top panels) or Anxa6-KO (clone #1, bottom panels) 4T1-mCh cells. Scale bars, 200 nm. The experiment was performed once. f. Mode size (mean values ± s.d.) of the indicated EVs isolated from Anxa6-WT (top panel) or Anxa6-KO (clone #1, bottom panel) 4T1-mCh cells, determined by NTA. Data show results from n=5 (Anxa6-WT) and n=3 (Anxa6-KO) independent EV preparations. g. Concentration (mean ± s.e.m.; n=3 acquisitions of one sample/condition) and size distribution of EVs isolated from medium conditioned by CREMO- or PTX-treated Anxa6-KO 4T1-mCh cells (clone #1), determined by NTA. h. mCh⁺ metastatic area fraction per lung section (mean ± s.e.m.) in Rag1^-/- mice pre-conditioned with the indicated EVs. CREMO-EV/Anxa6-WT, n=7 mice; PTX-EV/Anxa6-WT, n=7; CREMO-EV/Anxa6-KO (clone #1), n=7; PTX-EV/Anxa6-KO (clone #1), n=7. Statistical analysis by two-way ANOVA with Tukey’s multiple comparison test. Representative images of lung sections stained with DAPI (white) are shown on the right; the mCh signal (red) was acquired as direct fluorescence. Scale bars, 1 mm. i. mCh⁺ metastatic area fraction per lung section (mean ± s.e.m.) in Rag1^-/- mice pre-conditioned with EVs. DMSO-EV/Anxa6-WT, n=7 mice; DOX-EV/Anxa6-WT, n=8; DMSO-EV/Anxa6-KO (clone #1), n=8; DOX-EV/Anxa6-KO (clone #1), n=8. Statistical analysis as in (h). Source data are shown in Supplementary Table 5. Unprocessed blots are shown in Supplementary Fig. 9.

Figure 5. PTX induces CCL2 expression and Ly6C⁺ monocyte expansion in lungs of mammary tumour-bearing mice

a. Quantitative polymerase chain reaction (qPCR) analysis of the indicated genes (mean ± s.e.m.) in lungs of either tumour-free (TF) or 4T1 tumour-bearing (TB) mice treated as indicated. TF + CREMO, n=5 mice; TF + PTX, n=5; TB + CREMO, n=8; TB + PTX, n=9. Statistical analysis by two-way ANOVA with Tukey’s multiple comparison test. b. ELISA-based CCL2 protein (mean ± s.e.m.) in lungs of mice treated as in (a). Tumour-free + CREMO, n=5 mice; Tumour-free + PTX, n=5; Tumour-bearing + CREMO, n=6; Tumour-bearing + PTX, n=6. Statistical analysis as in (a). c. FACS analysis of CD45⁺CD11b⁺Ly6C⁺Ly6G^–F4/80⁺ monocytes (Ly6C⁺ Mo) in lungs of tumour-bearing mice. A representative sample is shown to illustrate the gating strategy. FMO, fluorescence minus one. d-g. FACS analysis of Ly6C⁺ Mo (mean ± s.e.m.) in tumour-bearing mice treated as indicated. Data show the frequency of Ly6C⁺ monocytes in the CD45⁺ population, relative to control (CREMO or PBS). (d): CREMO, n=7 mice; PTX, n=8. (e): PBS, n=5; CREMO, n=5; PTX, n=5. (f): PBS, n=7; CREMO, n=8; PTX, n=9. (g): CREMO, n=13; PTX, n=14. Statistical analysis by unpaired two-tailed Student’s t-test (d, g) or one-way ANOVA with Tukey’s multiple comparison test (e, f). Data in (g) show three independent experiments combined. h, i. FACS analysis of Ly6C⁺ Mo (mean ± s.e.m.) in lungs of tumour-free FVB/n (h) or Rag1^-/- (i) mice treated as indicated and analysed two days after treatment. (h): PBS, n=12 mice; CREMO, n=6; PTX 1 mg/kg, n=8; PTX 10 mg/kg, n=6; DOX, n=6. (i): CREMO, n=5; PTX, n=5. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test (h) or unpaired two-tailed Student’s t-test (i). Source data are shown in Supplementary Table 5.

Figure 6. Ly6C⁺ monocytes mediate the pro-metastatic activity of chemotherapy-elicited EVs

a. Schematics of EV pre-conditioning studies in tumour-free mice. b-d. qPCR analysis of Ccl2 (mean ± s.e.m.) in lungs of tumour-free mice that received EVs. (b): CREMO-EV, n=8; PTX-EV, n=9. (c): CREMO-EV, n=8; PTX-EV, n=7. (d): CREMO-EV, n=10; PTX-EV, n=9. Statistical analysis by unpaired two-tailed Student’s t-test. e-g. FACS analysis of Ly6C⁺ Mo (mean ± s.e.m.) in lungs of tumour-free mice that received EVs. (e): CREMO-EV, n=8; PTX-EV, n=9. (f): CREMO-EV, n=6; PTX-EV, n=7. (g): PBS (no EVs), n=11; CREMO-EV, n=14; PTX-EV, n=15. Statistical analysis by unpaired two-tailed Student’s t-test (e, f) or one-way ANOVA with Tukey’s multiple comparison test (g). h. qPCR analysis of Ccr2 (mean ± s.e.m.) in lungs of mice that received MMTV-PyMT tumour-derived CREMO-EV (n=10) or PTX-EV (n=9). Statistical analysis as in (b). i. Correlation between Ccl2 and Ccr2 transcript levels in lungs of FVB/n mice shown in (d) and (h). The Pearson correlation coefficient (r) is indicated. j, k. qPCR analysis of Ccl2 (j) and FACS analysis of Ly6C⁺ Mo (k) in the lungs of tumour-free mice (mean ± s.e.m.) that received PBS-EV (n=5) or DOX-EV (n=5). Statistical analysis as in (b). l. Bioluminescence (BL) analysis (total photon flux; mean ± s.e.m.) of Ccr2 WT or KO mice pre-conditioned with EVs, analysed at day 10 post-cell injection. The right panel shows representative mice. CREMO-EV/Ccr2-WT, n=10; PTX-EV/Ccr2-WT, n=11; CREMO-EV/Ccr2-KO, n=7; PTX-EV/Ccr2-KO, n=8. Statistical analysis by two-way ANOVA with Tukey’s multiple comparison test. Data show three independent experiments combined. m. qPCR analysis of Ccr2 (mean ± s.e.m.) in myeloid cells (Ly6C⁺ Mo; Ly6C^low Mo; and non-alveolar macrophages, Mac) FACS-sorted from lungs of tumour-free mice that received MMTV-PyMT tumour-derived CREMO-EV (n=5; except for Mac, n=4) or PTX-EV (n=6). Statistical analysis as in (l). n, o. qPCR analysis of Ccl2 and Ccr2 (n) and FACS analysis of Ly6C⁺ Mo (o) in lungs of mice (mean ± s.e.m; n=6 mice) that received CREMO-EV or PTX-EV from either Anxa6-WT or Anxa6-KO 4T1-mCh cells. Statistical analysis by two-way ANOVA with Sidak’s multiple comparison test. Source data are shown in Supplementary Table 5.

Figure 7. Chemotherapy-elicited EVs promote inflammatory EC activation through ANXA6 transfer

a. FACS of mCh⁺ ECs (mean ± s.e.m.) in lungs of 4T1 (n=6) or 4T1-mCh (CREMO, n=8; PTX, n=9) tumour-bearing Rag1^−/− mice. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. Right panels show gating strategy. b. Representative confocal images of anti-CD31 endothelial (green) and anti-mCh (magenta) immunostaining of lung sections from 4T1-mCh tumour-bearing mice treated as in (a); nuclei are stained with DAPI (blue). Scale bars, 10 μm. c. FACS of mCh⁺ ECs (mean ± s.e.m.) in lungs of 4T1 (n=4) or 4T1-mCh/HER2 (PBS, n=7; CREMO, n=8; PTX, n=9) tumour-bearing Swiss nu/nu mice. Statistical analysis as in (a). d. Western blotting analysis of the indicated proteins in Anxa6-KO bEnd.3 cells. The experiment was performed twice; Supplementary Fig. 6b shows a replicate experiment. e. Duolink staining of Anxa6-KO bEnd.3 cells showing ANXA6/p65 proximity (number of white dots/cell; mean ± s.e.m.; n=8 randomly selected images, each containing at least 12 cells). Statistical analysis as in (a). Right panels show representative images; nuclei are stained with DAPI (blue). Scale bars, 30 μm. Data show one experiment of two performed. f. Western blotting analysis of p65 (left), NF-kB activity (middle), and qPCR of Ccl2 (right) in bEnd.3 cells (mean ± s.d.; n=3 independent cell cultures/condition). Statistical analysis by unpaired two-tailed Student’s t-test. g, h. NF-kB activity (g) and qPCR of Ccl2 (h) in bEnd.3 cells (mean ± s.d.; n=3 independent cell cultures/condition). Statistical analysis by two-way ANOVA with Sidak’s multiple comparison test. Data show one experiment of two (g) or three (h) performed. i, j. qPCR analysis of Ccl2 (i) and NF-kB activity (j) in bEnd.3 cells (mean ± s.d.; n=3 independent cell cultures/condition). Statistical analysis by two-way ANOVA with Tukey’s multiple comparison test (i, left panel) or unpaired two-tailed Student’s t-test (i, right panel; and j). k. qPCR of Ccl2 in mCh⁺CD31⁺CD45^– ECs sorted from lungs of FVB/n mice (mean ± s.e.m.; n=5 mice). Statistical analysis by unpaired one-tailed Student’s t-test. Right panel shows the purity of the sorted ECs. Source data are shown in Supplementary Table 5. Unprocessed blots are shown in Supplementary Fig. 9.

Figure 8. ANXA6 is detected in circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy

a. Western blotting analysis of EVs isolated from MDA-MB-231 cells treated as indicated. The experiment was performed three times for PTX and once for DOX. b. Schematic of the treatment timeline and time-points of blood collection in breast cancer patients (n=6). AC, anthracycline (DOX) plus cyclophosphamide. c. LC-MS/MS-based quantification of ANXA6 in EVs isolated from plasma of breast cancer patients (n=6) before chemotherapy (pre-treatment), after AC, and after PTX. The data show quantitative values of ANXA6 presented as fold-change versus pre-treatment level. Note that the amount of EVs that could be isolated from patient #38 after PTX was insufficient to perform LC-MS/MS analysis. Tumour response was assessed at the time of surgery. d. Western blotting analysis of ANXA6 in plasma EVs of one patient (#56), analysed at the indicated time-points. The experiment was performed once. Source data are shown in Supplementary Table 5. Unprocessed blots are shown in Supplementary Fig. 9.