Long noncoding RNA MALAT1 is dynamically regulated in leader cells during collective cancer invasion

Abstract

Cancer cells collectively invade using a leader-follower organization, but the regulation of leader cells during this dynamic process is poorly understood. Using a dual double-stranded locked nucleic acid (LNA) nanobiosensor that tracks long noncoding RNA (lncRNA) dynamics in live single cells, we monitored the spatiotemporal distribution of lncRNA during collective cancer invasion. We show that the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is dynamically regulated in the invading fronts of cancer cells and patient-derived spheroids. MALAT1 transcripts exhibit distinct abundance, diffusivity, and distribution between leader and follower cells. MALAT1 expression increases when a cancer cell becomes a leader and decreases when the collective migration process stops. Transient knockdown of MALAT1 prevents the formation of leader cells and abolishes the invasion of cancer cells. Taken together, our single-cell analysis suggests that MALAT1 is dynamically regulated in leader cells during collective cancer invasion.

Keywords: biosensor; bladder cancer; lncRNA; metastasis; single cell analysis.

Fig. 1.

A dual double-stranded LNA nanobiosensor for probing lncRNA dynamics during collective cancer invasion. (A), Schematics of the FRET-based LNA nanobiosensor for detecting lncRNA in leader and follower cells. (B), Intracellular distributions of β-actin, MALAT1, and UCA1 RNA expressions detected by the nanobiosensors in live cancer cells (5,637). (Scale bars, 10 µm.) Images are representative of eight experiments. (C), Time-lapse images for MALAT1 dynamics in leader cells (marked by white arrows) during collective cell migration. (Scale bars, 30 µm.) Images are representative of five experiments. (D), Detection of MALAT1 expression in 3D tumor spheroids derived from patients with muscle-invasive bladder cancer. Blue dashed squares indicate the zoom-in regions (Right). Black arrows indicate protruding structures with leader cells sprouting from the spheroids. [Scale bars, 200 µm (Left) and 40 µm (Right).] Images are representative of six (Stage II) and ten (Stage IIIB) tumor spheroids. (E), 3D rendering of a cancer spheroid with leader cells (white arrows) and dissociated cancer cells (white arrowheads). (Scale bar, 200 µm.)

Fig. 2.

LNA nanobiosensors detect TGF-β-induced MALAT1 upregulation in cell nuclei and cytoplasm. (A–C), Confocal images of β-actin mRNA, MALAT1, and UCA1 in live bladder cancer cells (5,637). The cells were treated with buffer control and TGF-β. FRET channels (Left), merged FRET and brightfield channels (Middle), and zoom-in views of single cells (Right) are shown to illustrate the expression distributions. Images are representative of five experiments. [Scale bars, 50 μm (Left and Middle), 10 μm (Right).] (D and E), Relative expression of β-actin, MALAT1, and UCA1 RNA (D) in whole cells and (E) colocalized with the nuclei. One-way ANOVA followed by Tukey’s post hoc test was used to compare transcript numbers between control and TGF-β treatment (n = 5, NS not significant, *P < 0.05, ****P < 0.0001).

Fig. 3.

MALAT1 is up-regulated in leader cells during collective cancer migration. (A), Detection of β-actin mRNA, MALAT1, and UCA1 in leader cells and follower cells. Collective cell migration was induced by scratching the cell monolayer. Cells at the protrusion tip with an aggressive morphology, exhibiting significant amount of lamellipodia and filopodia, are considered leader cells (white arrows), and cells behind the leader cells were considered follower cells. Images are representative of four experiments. (Scale bars, 50 µm.) Student’s t test was used to compare the expression between the leader and follower cells (n ≥ 25, NS not significant, *P < 0.05). (B), Collective migration of cancer cells treated with control siRNA, MALAT1 siRNA, and UCA1 siRNA at 1 h and 8 h. Black dotted lines illustrate the boundary of the migrating cells. Images are representative of four experiments. (Scale bars, 300 µm.) (C), Cell migration rate estimated by the average distance between the cell boundaries. One-way ANOVA followed by Tukey’s post hoc test was used to compare the migration rates (n = 4, NS not significant, ****P < 0.0001). (D and E), Single-molecule tracking of MALAT1 transcripts in leader cells and follower cells. (Left) Representative images from eight experiments. (Scale bars, 5 µm.) (Middle) Traces of MALAT1 transcripts. Black and blue dashed lines indicate the cell boundary and the nucleus, respectively. (Scale bars, 5 µm.) (Right) Comparison of cytoplasmic and nuclear diffusivities of MALAT1. Student’s t test was used to compare the diffusivities of MALAT1 RNA in the nucleus and cytoplasm (n ≥ 5, NS not significant, *P < 0.05).

Fig. 4.

MALAT1 is up-regulated in invading sprouts of 3D bladder cancer spheroids. (A), Bladder cancer spheroids (5,637) were treated with TGF-β (40 ng/mL) and allowed to invade the invasion matrix for 3 d. Black arrows indicate invading protrusions from the spheroid. Yellow dashed squares highlight a protruding structure in the zoom-in views (Bottom). [Scale bars, 250 µm (Top) and 80 µm (Bottom).] Images are representative of five experiments. (B), The expression of β-actin mRNA and MALAT1 at protrusions. The value was normalized to the initial intensity. One-way ANOVA was used to analyze the RNA expression level, indicating an increasing MALAT1 expression (P = 5.82 × 10⁻⁵) and a decreasing β-actin expression (P = 0.0023) (n ≥ 3). (C), MALAT1 siRNA and control siRNA treated spheroids in the 3D invasion assay. (Scale bars, 150 µm.) Images are representative of three experiments. (D), Comparison of the number of protrusions per spheroid with control siRNA and MALAT1 siRNA on day 3. (E), Comparison of MALAT1 expression of cancer spheroids with control and MALAT1 siRNA. Student’s t test was used to compare the number of protrusions and the MALAT1 expression (n = 6 for control siRNA and n = 5 for MALAT1 siRNA, *P < 0.05). (F), The length and width of protrusions from the cancer spheroids on day 3. Two-way ANOVA followed by Tukey’s post hoc test was used to compare the dimensions of protrusions from spheroids with MALAT1 siRNA and control siRNA (n = 4 for MALAT1 siRNA and n = 12 for control siRNA, *P < 0.05, **P < 0.01). (G), MALAT1 expression at the protrusions of spheroids treated with MALAT1 siRNA and control siRNA on day 3. Student’s t test was used to compare the expression. (n = 7 for MALAT1 siRNA and n = 19 for control siRNA, *P < 0.05).

Fig. 5.

MALAT1 expression is associated with the clinical stage of bladder cancer patient-derived cancer cells. (A), β-actin mRNA and MALAT1 expressions in cancer cells derived from bladder cancer patients. (Scale bars, 300 µm.) (B and C), MALAT1 is up-regulated in the stage IIIB sample compared to the stage II sample. (D), The portion of cells with a high level of MALAT1. Student’s t test was used to compare between samples (n ≥ 1,148 for gene expression and n = 5 for cell portions with an enhanced MALAT1 expression).

Fig. 6.

MALAT1 is associated with leader cells in tumor spheroids derived from muscle-invasive bladder cancer patients. (A–G), MALAT1 expression in 3D human tumor spheroids derived from bladder cancer patients. Clinical samples of stage II (DT2101 and DT2334), Stage IIIA (DT2092 and DT2148), Stage III (DT2153 and DT2115), and Stage IV (DT2296) were included to cover the spectrum of muscle-invasive bladder cancer. Blue dashed squares highlight the zoom-in views (Bottom) with leader cells (black arrows) or dissociated cells (black arrowheads). [Scale bars, 200 µm (Top) and 100 µm (Bottom).] Images are representative of at least six spheroids. (H and I), The number of detached cells and protrusions per spheroid on day 3 (n = 5). (J–L), Correlation of the spheroid volume, the number of detached cells, and the number of leader cells with MALAT1 expression on day 3. At least five spheroids were analyzed for each sample, and all detectable leader cells and detached cells were analyzed.

Fig. 7.

MALAT1 is dynamically regulated during the formation and termination of leader cells. (A), Time lapse images of cancer cells (5673) during collective cell migration induced by the scratch assay. (Scale bars, 50 µm.) (B), Time-lapse images tracking the switching and termination of a leader cell (yellow dashes) near the migrating front. A follower cell was initially behind the cell boundary. The cell migrated to the front, acquired the leader cell role, and displayed an aggressive morphology. Then, the leader cell reached the other boundary and was surrounded by other cells in the monolayer. (Scale bars, 20 µm.) (C), Tracking of nuclear MALAT1 of the leader cell and follower cells outlined in (B). Data represent the portion of nuclear MALAT1 in the cell. MALAT1 increased when the cell assumed the role of leader cell and decreased when the boundary merged. (D), Examples of nuclear MALAT1 in leader cells during the merging of cell boundaries. Time zero (or peak) is when the leader cells reached and contacted the other boundary. MALAT1 returned to the basal level (or steady state) in 30 to 60 min. (E), Peak-steady ratio of nuclear MALAT1 in leader cells and follower cells. (F), Peak-steady ratio of nuclear MALAT1 and β-actin mRNA in leader cells. Student’s t test was used to compare the Peak-steady ratio (n = 5, **P < 0.01).