Migration and invasion of cancer cells constitute fundamental processes in tumor progression and metastasis. Migratory cancer cells commonly upregulate expression of plasminogen activator inhibitor 1 (PAI1), and PAI1 correlates with poor prognosis in breast cancer. However, mechanisms by which PAI1 promotes migration of cancer cells remain incompletely defined. Here we show that increased PAI1 drives rearrangement of the actin cytoskeleton, mitochondrial fragmentation, and glycolytic metabolism in triple-negative breast cancer (TNBC) cells. In two-dimensional environments, both stable expression of PAI1 and treatment with recombinant PAI1 increased migration, which could be blocked with the specific inhibitor tiplaxtinin. PAI1 also promoted invasion into the extracellular matrix from coculture spheroids with human mammary fibroblasts in fibrin gels. Elevated cellular PAI1 enhanced cytoskeletal features associated with migration, actin-rich migratory structures, and reduced actin stress fibers. In orthotopic tumor xenografts, we discovered that TNBC cells with elevated PAI1 show collagen fibers aligned perpendicular to the tumor margin, an established marker of invasive breast tumors. Further studies revealed that PAI1 activates ERK signaling, a central regulator of motility, and promotes mitochondrial fragmentation. Consistent with known effects of mitochondrial fragmentation on metabolism, fluorescence lifetime imaging microscopy of endogenous NADH showed that PAI1 promotes glycolysis in cell-based assays, orthotopic tumor xenografts, and lung metastases. Together, these data demonstrate for the first time that PAI1 regulates cancer cell metabolism and suggest targeting metabolism to block motility and tumor progression.Implications: We identified a novel mechanism through which cancer cells alter their metabolism to promote tumor progression.

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 clinicallydetectable 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 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms

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<div>Abstract<p>Cancer cells reprogram energy metabolism through metabolic plasticity, adapting ATP-generating pathways in response to treatment or microenvironmental changes. Such adaptations enable cancer cells to resist standard therapy. We employed a coculture model of estrogen receptor–positive (ER⁺) breast cancer and mesenchymal stem cells (MSC) to model interactions of cancer cells with stromal microenvironments. Using single-cell endogenous and engineered biosensors for cellular metabolism, coculture with MSCs increased oxidative phosphorylation, intracellular ATP, and resistance of cancer cells to standard therapies. Cocultured cancer cells had increased MCT4, a lactate transporter, and were sensitive to the MCT1/4 inhibitor syrosingopine. Combining syrosingopine with fulvestrant, a selective estrogen receptor degrading drug, overcame resistance of ER⁺ breast cancer cells in coculture with MSCs. Treatment with antiestrogenic therapy increased metabolic plasticity and maintained intracellular ATP levels, while MCT1/4 inhibition successfully limited metabolic transitions and decreased ATP levels. Furthermore, MCT1/4 inhibition decreased heterogenous metabolic treatment responses versus antiestrogenic therapy. These data establish MSCs as a mediator of cancer cell metabolic plasticity and suggest metabolic interventions as a promising strategy to treat ER⁺ breast cancer and overcome resistance to standard clinical therapies.</p>Implications:<p>This study reveals how MSCs reprogram metabolism of ER⁺ breast cancer cells and point to MCT4 as potential therapeutic target to overcome resistance to antiestrogen drugs.</p></div>

Cancer cells reprogram energy metabolism through metabolic plasticity, adapting ATP-generating pathways in response to treatment or microenvironmental changes. Such adaptations enable cancer cells to resist standard therapy. We employed a co-culture model of estrogen receptor-positive (ER+) breast cancer and mesenchymal stem cells (MSCs) to model interactions of cancer cells with stromal microenvironments. Using single-cell endogenous and engineered biosensors for cellular metabolism, co-culture with MSCs increased oxidative phosphorylation, intracellular ATP, and resistance of cancer cells to standard therapies. Co-cultured cancer cells had increased MCT4, a lactate transporter, and were sensitive to the MCT1/4 inhibitor syrosingopine. Combining syrosingopine with fulvestrant, a selective estrogen receptor degrading drug, overcame resistance of ER+ breast cancer cells in co-culture with MSCs. Treatment with antiestrogenic therapy increased metabolic plasticity and maintained intracellular ATP levels, while MCT1/4 inhibition successfully limited metabolic transitions and decreased ATP levels. Furthermore, MCT1/4 inhibition decreased heterogenous metabolic treatment responses versus antiestrogenic therapy. These data establish MSCs as a mediator of cancer cell metabolic plasticity and suggest metabolic interventions as a promising strategy to treat ER+ breast cancer and overcome resistance to standard clinical therapies. Implications: This study reveals how MSCs reprogram metabolism of ER+ breast cancer cells and point to MCT4 as potential therapeutic target to overcome resistance to anti-estrogen drugs.

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<div>Abstract<p>Cancer cells reprogram energy metabolism through metabolic plasticity, adapting ATP-generating pathways in response to treatment or microenvironmental changes. Such adaptations enable cancer cells to resist standard therapy. We employed a coculture model of estrogen receptor–positive (ER⁺) breast cancer and mesenchymal stem cells (MSC) to model interactions of cancer cells with stromal microenvironments. Using single-cell endogenous and engineered biosensors for cellular metabolism, coculture with MSCs increased oxidative phosphorylation, intracellular ATP, and resistance of cancer cells to standard therapies. Cocultured cancer cells had increased MCT4, a lactate transporter, and were sensitive to the MCT1/4 inhibitor syrosingopine. Combining syrosingopine with fulvestrant, a selective estrogen receptor degrading drug, overcame resistance of ER⁺ breast cancer cells in coculture with MSCs. Treatment with antiestrogenic therapy increased metabolic plasticity and maintained intracellular ATP levels, while MCT1/4 inhibition successfully limited metabolic transitions and decreased ATP levels. Furthermore, MCT1/4 inhibition decreased heterogenous metabolic treatment responses versus antiestrogenic therapy. These data establish MSCs as a mediator of cancer cell metabolic plasticity and suggest metabolic interventions as a promising strategy to treat ER⁺ breast cancer and overcome resistance to standard clinical therapies.</p>Implications:<p>This study reveals how MSCs reprogram metabolism of ER⁺ breast cancer cells and point to MCT4 as potential therapeutic target to overcome resistance to antiestrogen drugs.</p></div>

<p>Figure S5. Representative images of glucose and ATP FRET reporters in MSCs. Pseudocolor scale depicts high (red) and low (blue) CFP lifetime. Scale bar = 50 µm. Representative images of intracellular glucose (A) and ATP (B) in HS5 MSCs when in co-culture with ER+ breast cancer cells. Cancer cells are unlabeled in pseudocolor images.</p>