<p>1. Quantified metabolic shifts in response to antiestrogenic therapy and MCT1/4 inhibition. Change in median values in picoseconds of NADH or CFP lifetime (ATP) after addition of each compound. Labels at the top of the table denote the relevant comparison in each column.</p>
<p>Figure S7. Inhibition of monocarboxylate transporters regulates intracellular and extracellular lactate. Western blot shows expression of MCT4 in monocultures of MCF7, T47D, HS5, and HS27a cells. Images are from adjacent lanes on one gel and non-adjacent lanes from a second gel. We show β-actin as a loading control. B,C) Graphs show mean values ± standard deviation for relative concentrations of intracellular (B) and extracellular (C) lactate in monocultures of MCF7 cells or co-cultures of MCF7 and HS5 cells treated with 10 µM syrosingopine, an inhibitor of MCT1/4, or vehicle for 3 days. *** p<0.0001 using paired t tests for each comparison.</p>
<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<sup>+</sup>) 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<sup>+</sup> 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<sup>+</sup> breast cancer and overcome resistance to standard clinical therapies.</p>Implications:<p>This study reveals how MSCs reprogram metabolism of ER<sup>+</sup> breast cancer cells and point to MCT4 as potential therapeutic target to overcome resistance to antiestrogen drugs.</p></div>
<p>Figure S2. NADH lifetime and ROS quantification for ER+ breast cancer cells with CM and MSCs in monoculture and co-culture. Cell type of interest is bolded in co-culture comparisons. A-B) Histograms of NADH lifetime for HS5 (A) and HS27a (B) in monoculture or co-culture with MCF7, T47D, and HCC1428 (HCC) ER+ breast cancer cells. C-E) Histograms of NADH lifetime for MCF7 (C), T47D (D), and HCC1428 (HCC, E) cells in co-culture and monoculture with control or CM from HS5 or HS27a MSCs. Dashed lines represent cancer cells in CM and solid lines represent cancer cells in monoculture or co-culture with MSCs. N>100 cells.</p>
<p>Figure S8. Single-cell metabolic responses over time. Cell type of interest is bolded in co-culture comparisons. Cumulative AUC changes in NADH lifetime over 30 minutes post-treatment in 10-minute intervals. For a single cell, each change is normalized to the initial NADH lifetime. Table shows changes in median values in picoseconds of NADH or CFP lifetime (ATP) after addition of each compound. Labels at the top of the table denote the relevant comparison in each column.</p>
<p>Figure S1. Representative images of NADH lifetimes for various experimental conditions. Cell type of interest is bolded in co-culture comparisons. Top row of images denotes cancer cells marked with nuclear mCherry. Pseudocolor scale depicts high (red) and low (blue) NADH lifetime. Scale bar = 50 µm. A) HS5 and HS27a MSCs in monoculture. B-C) MCF7 (B) or T47D (C) cells in monoculture or with conditioned media (CM) from listed MSCs. D) HCC1428 (HCC) cancer cells in monoculture, CM from MSCs, or direct co-culture with MSCs.</p>
<p>Figure S2. NADH lifetime and ROS quantification for ER+ breast cancer cells with CM and MSCs in monoculture and co-culture. Cell type of interest is bolded in co-culture comparisons. A-B) Histograms of NADH lifetime for HS5 (A) and HS27a (B) in monoculture or co-culture with MCF7, T47D, and HCC1428 (HCC) ER+ breast cancer cells. C-E) Histograms of NADH lifetime for MCF7 (C), T47D (D), and HCC1428 (HCC, E) cells in co-culture and monoculture with control or CM from HS5 or HS27a MSCs. Dashed lines represent cancer cells in CM and solid lines represent cancer cells in monoculture or co-culture with MSCs. N>100 cells.</p>
The prostate-specific membrane antigen (PSMA) is a validated target for detection and management of prostate cancer (PC). It has also been utilized for targeted drug delivery through antibody–drug conjugates and polymeric micelles. Polyamidoamine (PAMAM) dendrimers are emerging as a versatile platform in a number of biomedical applications due to their unique physicochemical properties, including small size, large number of reactive terminal groups, bulky interior void volume, and biocompatibility. Here, we report the synthesis of generation 4 PSMA-targeted PAMAM dendrimers [G4(MP-KEU)] and evaluation of their targeting properties in vitro and in vivo using an experimental model of PC. A facile, one-pot synthesis gave nearly neutral nanoparticles with a narrow size distribution of 5 nm in diameter and a molecular weight of 27.3 kDa. They exhibited in vitro target specificity with a dissociation constant (Kd) of 0.32 ± 0.23 μm and preferential accumulation in PSMA+ PC3 PIP tumors versus isogenic PSMA– PC3 flu tumors. Positron emission tomography-computed tomography imaging and ex vivo biodistribution studies of dendrimers radiolabeled with 64Cu, [64Cu]G4(MP-KEU), demonstrated high accumulation in PSMA+ PC3 PIP tumors at 24 h post-injection (45.83 ± 20.09% injected dose per gram of tissue, %ID/g), demonstrating a PSMA+ PC3 PIP/PSMA– PC3 flu ratio of 7.65 ± 3.35. Specific accumulation of G4(MP-KEU) and [64Cu]G4(MP-KEU) in PSMA+ PC3 PIP tumors was inhibited by the known small-molecule PSMA inhibitor, ZJ-43. On the contrary, G4(Ctrl), control dendrimers without PSMA-targeting moieties, showed comparable low accumulation of ∼1%ID/g in tumors irrespective of PSMA expression, further confirming PSMA+ tumor-specific uptake of G4(MP-KEU). These results suggest that G4(MP-KEU) may represent a suitable scaffold by which to target PSMA-expressing tissues with imaging and therapeutic agents.
<p>Figure S3. ROS quantification for MSCs in monoculture and co-culture. Flow cytometry data for ROS detected by CellROX Green in HS5 and HS27a MSCs in monoculture or co-culture with MCF7 (A), T47D (B), or HCC1428 (C) ER+ breast cancer cells. Solid and dashed lines depict MSCs in monoculture co-culture, respectively. Cell type of interest is bolded in co-culture comparisons. N>10,000 cells.</p>
