Short-term cellular memory tunes the signaling responses of the chemokine receptor CXCR4

Abstract

The chemokine receptor CXCR4 regulates fundamental processes in development, normal physiology, and diseases, including cancer. Small subpopulations of CXCR4-positive cells drive the local invasion and dissemination of malignant cells during metastasis, emphasizing the need to understand the mechanisms controlling responses at the single-cell level to receptor activation by the chemokine ligand CXCL12. Using single-cell imaging, we discovered that short-term cellular memory of changes in environmental conditions tuned CXCR4 signaling to Akt and ERK, two kinases activated by this receptor. Conditioning cells with growth stimuli before CXCL12 exposure increased the number of cells that initiated CXCR4 signaling and the amplitude of Akt and ERK activation. Data-driven, single-cell computational modeling revealed that growth factor conditioning modulated CXCR4-dependent activation of Akt and ERK by decreasing extrinsic noise (preexisting cell-to-cell differences in kinase activity) in PI3K and mTORC1. Modeling established mTORC1 as critical for tuning single-cell responses to CXCL12-CXCR4 signaling. Our single-cell model predicted how combinations of extrinsic noise in PI3K, Ras, and mTORC1 superimposed on different driver mutations in the ERK and/or Akt pathways to bias CXCR4 signaling. Computational experiments correctly predicted that selected kinase inhibitors used for cancer therapy shifted subsets of cells to states that were more permissive to CXCR4 activation, suggesting that such drugs may inadvertently potentiate pro-metastatic CXCR4 signaling. Our work establishes how changing environmental inputs modulate CXCR4 signaling in single cells and provides a framework to optimize the development and use of drugs targeting this signaling pathway.

Enhanced mitochondrial fission suppresses signaling and metastasis in triple-negative breast cancer

“…To identify the activity of ERK and Akt, we used the kinase translocation reporter (KTR) for ERK and Akt as previously described [17,18]. This KTR construct contains H2B fused to mCherry (H2B-mCherry), the Akt-KTR reporter (Aquamarine), the ERK-KTR reporter (mCitrine), and a puromycin selection marker all separated by P2A linker sequences cloned into the Piggyback transposon vector as described previously (pHAEP) [18]. To stably express PISD in cells with the KTR construct, we generated a construct with unlabeled PISD and a hygromycin selection marker separated by a P2A sequence (PISD-hygromycin).…”

“…After 1 h, we washed the construct with PBS and placed it in a 35-mm dish with a 20-mm glass bottom. We covered the construct with 2 mL of Fluoro-Brite DMEM containing the same additives as described in the "Cell culture" section and placed the dish in the incubator for 3 h. After 3 h, we imaged the dish every 20 min for 3 h on an Olympus FVMPE-RS upright twophoton microscope using settings as described previously [18]. We identified and tracked cells using custom MATLAB code that tracked the H2B-mCherry nucleus of cells on the construct.…”

“…We analyzed kinase translocation reporter images for activation of Akt and ERK and identified mitochondrial morphology using an adaptive thresholding method in MATLAB as described previously [17,18]. For individual fluorescence microscopy images of mitochondria, we used MATLAB to skeletonize the mitochondria, measure total pixel area and major axis length using the regionprops command, and assign each mitochondrion a pixel ID.…”

“…Immediately after euthanizing mice, we imaged lung metastases ex vivo by two-photon microscopy. We used the excitation and emission filters described in our previous publication and quantified activation of Akt and ERK using custom MATLAB code [18]. For analysis of metastases to the bone marrow from intracardiac injection, we flushed the bone marrow with PBS from the femur and tibia and measured bioluminescence from cancer cells as described previously [34].…”

“…For imaging Akt and ERK activation in the bone marrow, we euthanized mice, immediately used a Dremel tool to think cortical bone of excised tibias, and imaged KTR cells ex vivo by twophoton microscopy as described for lung metastases. We quantified the activation of ERK and Akt in single cells using custom MATLAB code [18].…”