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Targeting disseminated estrogen-receptor-positive breast cancer cells in bone marrow

Abstract

Estrogen receptor-positive (ER+) breast cancer can recur up to 20 years after initial diagnosis. Delayed recurrences arise from disseminated tumors cells (DTCs) in sites such as bone marrow that remain quiescent during endocrine therapy and subsequently proliferate to produce clinically detectable metastases. Identifying therapies that eliminate DTCs and/or effectively target cells transitioning to proliferation promises to reduce risk of recurrence. To tackle this problem, we utilized a 3D co-culture model incorporating ER+ breast cancer cells and bone marrow mesenchymal stem cells to represent DTCs in a bone marrow niche. 3D co-cultures maintained cancer cells in a quiescent, viable state as measured by both single-cell and population-scale imaging. Single-cell imaging methods for metabolism by fluorescence lifetime (FLIM) of NADH and signaling by kinases Akt and ERK revealed that breast cancer cells utilized oxidative phosphorylation and signaling by Akt to a greater extent both in 3D co-cultures and a mouse model of ER+ breast cancer cells in bone marrow. Using our 3D co-culture model, we discovered that combination therapies targeting oxidative phosphorylation via the thioredoxin reductase (TrxR) inhibitor, D9, and the Akt inhibitor, MK-2206, preferentially eliminated breast cancer cells without altering viability of bone marrow stromal cells. Treatment of mice with disseminated ER+ human breast cancer showed that D9 plus MK-2206 blocked formation of new metastases more effectively than tamoxifen. These data establish an integrated experimental system to investigate DTCs in bone marrow and identify combination therapy against metabolic and kinase targets as a promising approach to effectively target these cells and reduce risk of recurrence in breast cancer.

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Fig. 1: ER+breast cancer cells in co-culture spheroids with bone marrow stromal cells exhibit cellular quiescence.
Fig. 2: ER+breast cancer cells show increased reliance on OXPHOS versus surrounding bone marrow stromal cells.
Fig. 3: ER+breast cancer cells activate Akt to a greater extent than ERK in co-culture spheroids with bone marrow stromal cells.
Fig. 4: Ex vivo imaging of cancer cells in bone marrow recapitulates metabolic and signaling profiles from 3D co-culture spheroids.
Fig. 5: Simultaneous treatment of co-culture spheroids with inhibitors of OXPHOS and Akt decreases growth of cancer cells while maintaining viability of stromal cells.
Fig. 6: In vivo dual targeting of OXPHOS and Akt signaling of MCF7 cancer cells injected into the femoral artery of mice.

Code availability

All custom MATLAB code including the image processing files require a material transfer agreement from the University of Michigan.

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Acknowledgements

We thank Ayşe J. Muñiz and Max Wicha for paper feedback. We thank Michael Pihalja for assistance with flow cytometry. We acknowledge funding from United States National Institutes of Health grants R01CA238042, R01CA196018, U01CA210152, R01CA238023, R33CA225549, R50CA221807, and R37CA222563. Brock Humphries, Ph.D., was supported by an American Cancer Society - Michigan Cancer Research Fund Postdoctoral Fellowship, PF-18-236-01-CCG. We acknowledge support to the University of Michigan Rogel Cancer Center through National Institutes of Health grant P30CA046592 for flow cytometry and animal imaging studies. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE 1256260 to Johanna Buschhaus.

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JMB, KEL, and GDL conceptualized and designed the study. BAH and KEL provided reagents. JMB, SSE, SR, BAH, THR, HRH, ASB, and ACC performed experiments. JMB and KEL wrote MATLAB code. JMB, BAH, KEL, and GDL acquired funding. JMB, SR, and THR analyzed data. JMB and GDL wrote the paper. All authors reviewed the paper before submission.

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Correspondence to Gary D. Luker.

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Buschhaus, J.M., Humphries, B.A., Eckley, S.S. et al. Targeting disseminated estrogen-receptor-positive breast cancer cells in bone marrow. Oncogene 39, 5649–5662 (2020). https://doi.org/10.1038/s41388-020-01391-z

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