The online version of this article (https://doi.org/10.1186/s13058-018-1029-4) contains supplementary material, which is available to authorized users.
Curzio Rüegg and Albert Santamaria-Martínez contributed equally to this work.
Obesity is a strong predictor of poor prognosis in breast cancer, especially in postmenopausal women. In particular, tumors in obese patients tend to seed more distant metastases, although the biology behind this observation remains poorly understood.
To elucidate the effects of the obese microenvironment on metastatic spread, we ovariectomized C57BL/6 J female mice and fed them either a regular diet (RD) or a high-fat diet (HFD) to generate a postmenopausal diet-induced obesity model. We then studied tumor progression to metastasis of Py230 and EO771 grafts. We analyzed and phenotyped the RD and HFD tumors and the surrounding adipose tissue by flow cytometry, qPCR, immunohistochemistry (IHC) and western blot. The influence of the microenvironment on tumor cells was assessed by performing cross-transplantation of RD and HFD tumor cells into other RD and HFD mice. The results were analyzed using the unpaired Student t test when comparing two variables, otherwise we used one-way or two-way analysis of variance. The relationship between two variables was calculated using correlation coefficients.
Our results show that tumors in obese mice grow faster, are also less vascularized, more hypoxic, of higher grade and enriched in CD11b+Ly6G+ neutrophils. Collectively, this favors induction of the epithelial-to-mesenchymal transition and progression to claudin-low breast cancer, a subtype of triple-negative breast cancer that is enriched in cancer stem cells. Interestingly, transplanting HFD-derived tumor cells in RD mice transfers enhanced tumor growth and lung metastasis formation.
These data indicate that a pro-metastatic effect of obesity is acquired by the tumor cells in the primary tumor independently of the microenvironment of the secondary site.
Effects of postmenopausal obesity on primary breast cancer tumoursᅟ
Additional file 2: Figure S1. Scheme of the experimental procedure (A). Body weight comparison between ovariectomized (n = 12 RD, n = 12 HFD) and non-ovariectomized (n = 5 RD, n = 5 HFD) mice (B). Body weight of ovariectomized RD and HFD mouse groups after 13 weeks of diet (C; n = 29 RD and n = 35 HFD). Data distribution of mouse weight in RD (D) and HFD (E) groups. Number of metastases seeded by same-sized tumors in RD and HFD mice (F and G). Error bars in panel B indicate SEM. (TIF 2261 kb)
Additional file 3: Figure S2. CD31 staining in C57BL/6 Rag2−/− mice show no differences between tumors from RD and HFD mice when they are grown in C57BL/6 Rag2−/− hosts (A, scalebar = 100uM). HIF1a staining in wild-type (wt) and C57BL/6 Rag2−/− mice show that hypoxia is increased in tumors from wt obese mice, while it is not changed in tumors in HFD-fed C57BL/6 Rag2−/− mice (B, scalebar 100 uM). HE staining of RD and HFD tumors showing enlarged nuclei and less packed chromatin in the latter (C, scalebar 50 um). IHC analyses in tumor samples show faster progression in HFD compared to RD tumors (D, n = 5, scalebar 50 um). (JPG 3325 kb)
Additional file 4: Figure S3. FACS analyses show lower percentages of F4/80+ macrophages in the CD11b + compartment in Py230 HFD tumors (A, n = 18 RD, n = 20 HFD). Percentages of M1 (F4/80 + CD206-) and M2 macrophages (F4/80 + CD206+) identified in the CD11b + compartment of C57BL/6 and FVB/N mice (B, n = 3 RD, n = 7 HFD for C57BL/6 and n = 4 RD, n = 4 HFD for FVB/N). Normalized western blot analysis for CD11b in Py230-C57BL6 tumors (C; N = 8), Py230-C57BL/6;Rag2−/− tumors (D; N = 9) and PyMT-FVB/N tumors (E; n = 8). Clodronate liposomes treatment increases metastasis (F, n = 5 RD, n = 6 HFD). (TIF 1676 kb)
Additional file 5: Figure S4. Western blot analysis for E-cadherin, N-cadherin, p21, and HIF1a in Py230-C57BL/6 tumor lysates of RD and HFD (A). Western blot analysis for E-cadherin in PyMT tumors grown in FVB/N mice (B). Tumor weight of groups used for qPCR and western blot analyses (C, N = 10). CD11b strongly correlates with N-cadherin and anti-correlates with E-cadherin (D, N = 10). IHC on RD vs HFD tumors show nuclear p53 accumulation in the latter (E, scalebar 100 uM). qPCR analyses on Py230 RD and HFD tumors show significant downregulation of claudins and other cell-cell junction genes (F, n = 5). (TIF 9127 kb)
WCRF/AICR. Food, nutrition, physical activity, and the prevention of cancer: a global perspective. Washington DC: AICR; 2007.
Williams CB, Yeh ES, Soloff AC. Tumor-associated macrophages: unwitting accomplices in breast cancer malignancy. NPJ Breast Cancer. 2016;2
Kuonen F, Laurent J, Secondini C, Lorusso G, Stehle JC, Rausch T, Faes-Van't Hull E, Bieler G, Alghisi GC, Schwendener R, et al. Inhibition of the Kit ligand/c-Kit axis attenuates metastasis in a mouse model mimicking local breast cancer relapse after radiotherapy. Clin Cancer Res. 2012;18(16):4365–74. CrossRefPubMed
Ewens A, Mihich E, Ehrke MJ. Distant metastasis from subcutaneously grown E0771 medullary breast adenocarcinoma. Anticancer Res. 2005;25(6B):3905–15. PubMed
Levin BE, Keesey RE. Defense of differing body weight set points in diet-induced obese and resistant rats. Am J Phys. 1998;274(2 Pt 2):R412–9.
Sieri S, Chiodini P, Agnoli C, Pala V, Berrino F, Trichopoulou A, Benetou V, Vasilopoulou E, Sanchez MJ, Chirlaque MD, et al. Dietary fat intake and development of specific breast cancer subtypes. J Natl Cancer Inst. 2014;106(5)
Sieri S, Krogh V, Ferrari P, Berrino F, Pala V, Thiebaut AC, Tjonneland A, Olsen A, Overvad K, Jakobsen MU, et al. Dietary fat and breast cancer risk in the European prospective investigation into cancer and nutrition. Am J Clin Nutr. 2008;88(5):1304–12. PubMed
Michailidou Z, Turban S, Miller E, Zou X, Schrader J, Ratcliffe PJ, Hadoke PW, Walker BR, Iredale JP, Morton NM, et al. Increased angiogenesis protects against adipose hypoxia and fibrosis in metabolic disease-resistant 11beta-hydroxysteroid dehydrogenase type 1 (HSD1)-deficient mice. J Biol Chem. 2012;287(6):4188–97. CrossRefPubMed
Pasarica M, Sereda OR, Redman LM, Albarado DC, Hymel DT, Roan LE, Rood JC, Burk DH, Smith SR. Reduced adipose tissue oxygenation in human obesity: evidence for rarefaction, macrophage chemotaxis, and inflammation without an angiogenic response. Diabetes. 2009;58(3):718–25. CrossRefPubMedPubMedCentral
Rausch ME, Weisberg S, Vardhana P, Tortoriello DV. Obesity in C57BL/6J mice is characterized by adipose tissue hypoxia and cytotoxic T-cell infiltration. Int J Obes. 2008;32(3):451–63. CrossRef
Cancer Genome Atlas N. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490(7418):61–70. CrossRef
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545–50. CrossRefPubMedPubMedCentral
- Obesity promotes the expansion of metastasis-initiating cells in breast cancer
- BioMed Central
Neu im Fachgebiet Onkologie
Mail Icon II