Energetic regulation of coordinated leader-follower dynamics during collective invasion of breast cancer cells

Abstract

The ability of primary tumor cells to invade into adjacent tissues, followed by the formation of local or distant metastasis, is a lethal hallmark of cancer. Recently, locomoting clusters of tumor cells have been identified in numerous cancers and associated with increased invasiveness and metastatic potential. However, how the collective behaviors of cancer cells are coordinated and their contribution to cancer invasion remain unclear. Here we show that collective invasion of breast cancer cells is regulated by the energetic statuses of leader and follower cells. Using a combination of in vitro spheroid and ex vivo organoid invasion models, we found that cancer cells dynamically rearrange leader and follower positions during collective invasion. Cancer cells invade cooperatively in denser collagen matrices by accelerating leader-follower switching thus decreasing leader cell lifetime. Leader cells exhibit higher glucose uptake than follower cells. Moreover, their energy levels, as revealed by the intracellular ATP/ADP ratio, must exceed a threshold to invade. Forward invasion of the leader cell gradually depletes its available energy, eventually leading to leader-follower transition. Our computational model based on intracellular energy homeostasis successfully recapitulated the dependence of leader cell lifetime on collagen density. Experiments further supported model predictions that decreasing the cellular energy level by glucose starvation decreases leader cell lifetime whereas increasing the cellular energy level by AMP-activated kinase (AMPK) activation does the opposite. These findings highlight coordinated invasion and its metabolic regulation as potential therapeutic targets of cancer.

Keywords: bioenergetics; cancer invasion; cancer metabolism; collective migration; leader cell.

Fig. 1.

Dynamic reorganization of collectively invading breast cancer cells in vitro and ex vivo. (A) A representative MDA-MB-231 spheroid expressing the CycleTrak nuclear marker (green/yellow, different colors indicate different cell cycle phases) is invading upward into 4.5-mg/mL collagen with collective strands. (B) Cells dynamically reorganize their relative positions within a typical strand (boxed region from A) during invasion, resulting in leader cell turnovers (asterisks; frames under white lines are enlarged on the Right to highlight leader turnover). Note that the invasion is interrupted when the original leader cells (black arrowheads) pause or move backward before the emergence of the new leaders (red arrowheads). (C) Leader–follower switching during spheroid invasion is more frequent in high-density collagen than in lower-density collagen (n > 50 for each condition; P < 0.0001 from a logarithmic-rank test for trend; shades represent standard error (s.e.); complete leader cell lifetime data are available in Dataset S1). (D) A representative mouse mammary tumor virus–polyomavirus middle T-antigen (MMTV-PyMT) mouse tumor organoid invading into 4.5-mg/mL collagen matrix. (E) A representative strand (boxed region from D) exhibits intermittent short-period pauses or retractions during forward invasion, many of them correlate with leader cell turnover events (as indicated with asterisks; black arrowheads, old leader; red arrowheads, new leader; frames under the white lines are enlarged on the Right to highlight leader turnover). (F) Leader cell lifetime during organoid invasion decreases with increasing collagen density (n > 45 for each condition; P = 0.05 from a logarithmic-rank test for trend; shades represent s.e.). [Scale bar, 50 µm (A and B, Left and D and E, Left), 25 µm (B, Right and E, Right).]

Fig. 2.

Glucose uptake along collectively invading strand in vitro and ex vivo. (A) Glucose uptake along invading strands in a representative spheroid is measured by the glucose analog 2-NBDG (green) and normalized to the CellTracker Orange CMRA dye (red) to obtain a ratiometric normalized glucose uptake value (heat map). (B) MDA-MB-231 leader cells exhibit higher normalized glucose uptake than follower cells as shown by the ratio heat maps alongside with wheat germ agglutinin (WGA) labeling of cell boundaries. (C) Normalized glucose uptake decreases along invading strands from tip to rear in vitro (averaged from n > 10 strands for each condition; shades represent s.e.). (D and E) The difference between leader and follower cells in normalized glucose uptake increases with collagen density in vitro (n = 29, 29, and 27, respectively; P = 0.0059, 0.0037, and <0.0001 from the paired t tests in D; P = 0.0035 from the ANOVA test in E). (F–H) Normalized glucose uptake is higher in leader cells than in follower cells during organoid invasion and exhibits a similar trend as collagen density increases (n = 24, 20, and 24, respectively; P = 0.32, 0.094, and 0.029 from the paired t tests in G; P = 0.78 from the ANOVA test in H). Strand tips orient toward the lower-right corner of the images. *P < 0.05, **P < 0.01, and ****P < 0.0001; n.s., not significant. (Scale bar, 50 µm.)

Fig. 3.

Dynamics of cellular ATP/ADP ratio correlates with leader cell invasion. (A) Time-series images of an leftward invading MDA-MB-231 strand with the PercevalHR ATP/ADP ratio sensor and DRAQ5 nuclear labeling (red) show a follower cell taking over the leader position (as indicated by the asterisk at 700 min; black arrowhead, old leader; white arrowhead, new leader). (B) Heat map of the same strand shows that the normalized ATP/ADP ratio correlates with leader cell invasion. The leader cell invades forward when the ATP/ADP ratio is high (0–160 min). As it invades, the ATP/ADP ratio drops because energy is being consumed (180–320 min). The original leader cell stops invading (340–480 min) and retracts (500–660 min) as the ATP/ADP ratio drops below a certain threshold. Leader–follower switching eventually takes place to sustain continued invasion (680–780 min). (C) Comparison of cumulative invasion distance and frontal ATP/ADP ratio (n = 21 strands; normalized to the range of [0, 1] for each strand) suggests that forward invasion of the leader cell depletes its ATP/ADP ratio (black arrow indicates the ATP/ADP ratio peak), causing invasion to stall (red arrow indicates a major retraction). (D) Rate of change in frontal ATP/ADP ratio and invasion rate of the strands frequently display opposite signs (n = 21 strands; normalized to the range of [0, 1] for each strand, which is then shifted to retain the signs of the variable before normalization). (E) Forward invasion depletes cellular energy as indicated by a negative correlation between the rates (n = 21 strands; P = 0.0043 from the one-sample t test compared with a value = 0). The ATP/ADP ratio in the frontal 25-µm segment of each strand is used to represent the leader cell ATP/ADP ratio. (Scale bar, 50 µm.)

Fig. 4.

Intracellular energy state determines leader cell fate as explained by modeling. (A) An invading strand of MDA-MB-231 cells expressing the CycleTrak sensor in 1.5-mg/mL collagen shows the interaction between the leader cells and the collagen matrix. (B) Illustration of the model shows the leader cell overcoming the energy barrier imposed by the collagen matrix to invade. (C) Model simulation of leader cell lifetime recapitulates its dependence on the collagen density/energy barrier. a.u., arbitrary unit. (Scale bar, 50 µm.)

Fig. 5.

Leader cell lifetime can be modulated by increasing or decreasing cellular energy level. (A) Model predicts that a drop in intracellular energy level by glucose starvation decreases leader cell lifetime and that an elevated energy level due to AMPK activation increases leader cell lifetime. a.u., arbitrary unit. (B) Glucose deprivation decreases the cellular ATP/ADP ratio over time for MDA-MB-231 cells on a glass substrate, whereas AMPK activation by 200-µM 5-aminoimidazole-4-carboxamide riboside (AICAR) increases the ATP/ADP ratio (n > 15 cells for each condition, treatment added at t = 0 min). (C) Glucose starvation decreases total ATP concentration, whereas AMPK activation increases total ATP concentration 20 h after treatment (n = 24, 14, and 16, respectively; P = 0.0052, and <0.0001 from unpaired t tests compared with the control). (D) Experiments verify that leader cell lifetime in spheroids is decreased (P < 0.0001 from the logarithmic-rank test) in glucose-free medium and increased (P < 0.0001 from logarithmic-rank test) when treated with 200-µM AICAR to activate AMPK (n > 100 for each condition; shades represent s.e.). (E) Artificially transferred mitochondria (1 µg/100,000 recipient cells) increases cellular ATP concentration compared with the control (0 µg) 4 d after transfer (n = 5 for each group; P = 0.023 from unpaired t test). (F) Leader cells with artificially transferred mitochondria have increased lifetime compared with control ones within coculture spheroids in 4.5-mg/mL collagen (n > 100 for each condition; P = 0.0051 from the logarithmic-rank test). *P < 0.05 and ****P < 0.0001.