An epigenetically distinct breast cancer cell subpopulation promotes collective invasion

Abstract

Tumor cells can engage in a process called collective invasion, in which cohesive groups of cells invade through interstitial tissue. Here, we identified an epigenetically distinct subpopulation of breast tumor cells that have an enhanced capacity to collectively invade. Analysis of spheroid invasion in an organotypic culture system revealed that these "trailblazer" cells are capable of initiating collective invasion and promote non-trailblazer cell invasion, indicating a commensal relationship among subpopulations within heterogenous tumors. Canonical mesenchymal markers were not sufficient to distinguish trailblazer cells from non-trailblazer cells, suggesting that defining the molecular underpinnings of the trailblazer phenotype could reveal collective invasion-specific mechanisms. Functional analysis determined that DOCK10, ITGA11, DAB2, PDFGRA, VASN, PPAP2B, and LPAR1 are highly expressed in trailblazer cells and required to initiate collective invasion, with DOCK10 essential for metastasis. In patients with triple-negative breast cancer, expression of these 7 genes correlated with poor outcome. Together, our results indicate that spontaneous conversion of the epigenetic state in a subpopulation of cells can promote a transition from in situ to invasive growth through induction of a cooperative form of collective invasion and suggest that therapeutic inhibition of trailblazer cell invasion may help prevent metastasis.

Figure 10. Model of the commensal relationship between trailblazer and opportunist subpopulations.

Heterogenous tumors may contain subpopulations of trailblazer (green) and opportunist (red) cells. Over time, the trailblazer subpopulation can invade, creating paths through which the opportunist cells migrate away from the primary tumor. Once into the vasculature, both populations may seed organs (such as the lung) and form metastatic lesions.

Figure 9. Cooperation between tumor cell subpopulations during collective invasion in vivo.

(A) Immunostaining of primary tumors composed of MCFDCIS cells alone or a mixture of MCFDCIS and SUM159 trailblazer cells (1:1 ratio). Representative images are shown. Arrows indicate representative areas of MCFDCIS invasion (MCFDCIS, n = 10 mice; MCFDCIS+SUM159 trailblazer, n = 10 mice). (B) Representative images of patient samples immunostained with anti-CD44 antibody. The dashed line shows the boundary of the DCIS tumor. Solid arrows indicate CD44⁺ tumor cells. The dashed arrow indicates CD44⁺ stromal cells. Scale bar: 50 μm.

Figure 8. Trailblazer cells induce the collective invasion of opportunist cells through a commensal relationship.

(A) Representative images of SUM159 spheroids composed of 100% trailblazer, 100% opportunist, or 25% trailblazer/75% opportunist cells in organotypic culture. Spheroids were formed in hanging drops for 24 hours before plating in organotypic culture. Arrows identify invasive projections. Scale bar: 50 μm. Graph shows the percentage of invasive spheroids (mean ± range of 30 spheroids per condition, n = 2). (B) Representative x-z views of the invasion into ECM of 100% trailblazer, 100% opportunist, or 25% trailblazer/75% opportunist SUM159 cells. Fluorescent nuclei are shown. Scale bar: 50 μm. Graph shows the number of SUM159 opportunist cells that invaded ≥50 μm into the ECM (mean ± SD, n = 3). Unpaired Student’s t test. (C) Representative time-lapse images of spheroids composed of 100% trailblazer, 100% opportunist, or 25% trailblazer/75% opportunist cells. Solid arrows indicate cells leading invasion. Arrows with dashed tails indicate cells following into an existing projection (100 spheroids per condition total, n = 3). Scale bar: 25 μm. (D) SUM159 trailblazer cells were transfected with the indicated siRNAs for 24 hours before clustering with untransfected SUM159 opportunist cells in spheroids at 1:4 ratio. Spheroid clusters grown for 48 hours in organotypic culture and representative images are shown. Arrow identifies invasive projection. Scale bar: 50 μm. Graphs show the percentage of invasive spheroids (mean + SD, 50 spheroids per condition, n = 3). *P < 0.05, **P < 0.01, ****P < 0.0001, unpaired Student’s t test.

Figure 7. The trailblazer signature gene DOCK10 is required for metastasis.

(A) Representative bioluminescence imaging of control and DOCK10 shRNA–expressing SUM159 trailblazer-Luc-GFP primary tumors. Graph shows the relative photon flux of the primary tumors (mean ± SD, control = 7 mice, DOCK10 shRNA = 8 mice). NS, unpaired Student’s t test. (B) Representative bioluminescence imaging (BLI) and fluorescent imaging of the lungs from mice bearing control and DOCK10 shRNA–expressing SUM159 trailblazer-Luc-GFP primary tumors. Images show bioluminescence imaging of lungs immediately after mice were sacrificed and GFP expression of SUM159 trailblazer cells in the lungs immediately after mice were sacrificed (control = 7 mice, DOCK10 shRNA = 8 mice; scale bar: 100 μm) as well as lungs immunostained with anti-GFP antibody and counterstained with phalloidin. (control = 7 mice, DOCK10 shRNA = 8 mice; scale bar: 50 μm). Graphs show the relative photon flux and number of micrometastases in the lungs normalized to the photon flux of the corresponding primary tumor (mean ± SD, control = 7 mice, DOCK10 shRNA = 8 mice). ***P < 0.001, Mann Whitney U test.

Figure 6. Multiple trailblazer genes are necessary for LCP formation.

(A) SUM159 trailblazer cells transfected with the indicated siRNAs were plated onto a layer of ECM for 24 hours and stained. Scale bar: 50 μm. Graph shows the relative length of LCPs (mean + SD of ≥20 cells, n = 3). (B) Invasion of SUM159 trailblazer cells transfected with the indicated siRNAs. Scale bar: 50 μm. Graph shows relative invasion (mean + SD, n = 3). (C) Collective invasion of SUM159 opportunist CDC42^Q61L cells, as shown in B. Graph shows relative invasion compared with control SUM159 trailblazer cells (mean + SD, n = 3). (D) Relative invasion of SUM159 trailblazer and SUM159 trailblazer CDC42^Q61L cells transfected with the indicated siRNAs (mean + SD, n = 3). (E) SUM159 trailblazer cells transfected with the indicated siRNAs were plated onto a layer of ECM for 24 hours and stained. Scale bar: 50 μm. Graph shows the relative length of LCPs (mean ± SD of ≥20 cells, n = 3). Horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate minimum and maximum. (F) Representative images of the movement of SUM159 trailblazer cells transfected with the indicated siRNAs over a 14-hour time period in monolayer culture. The color indicates the time period within the 14 hours of imaging. Scale bar: 50 μm. Graph shows the displacement of cells over time (mean ± SD, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired Student’s t test.

Figure 5. The elevated expression of genes required for trailblazer cell invasion correlates with poor patient outcome.

(A) Patients were classified as Trailblazer high and Trailblazer low using the 7 genes that were required for invasion in the SUM159 trailblazer and SUM229 trailblazer cells (DOCK10, DAB2, LPAR1, PPAP2B, ITGA11, VASN, and PDGFRA). Kaplan-Meier curves were drawn for both the Trailblazer-high and Trailblazer-low groups. Survival differences were compared using the log-rank (Mantel-Cox) test. (B) Kaplan-Meier survival curves for Trailblazer-high and Trailblazer-low TNBC patients. Survival differences were compared using the log-rank (Mantel-Cox) test.

Figure 4. Trailblazer cells express higher levels of genes required for collective invasion.

(A) Heat map showing the expression of the 26 genes that are specifically elevated in SUM159 and HCC1143 trailblazer cells. Expression is the mean of 2 biological replicates. (B) The relative invasion of SUM159 trailblazer cells ≥50 μm into the ECM. The number of invasive cells is normalized to the total cell number in the field of view for each condition. Relative invasion is the normalized invasive value of the test condition divided by the normalized invasion value of the control cells from the experimental replicate (median ± range, n = 3). The dashed red box indicates genes that were further investigated. Images are representative x-z views of SUM159 trailblazer invasion into ECM after transfection with the indicated siRNAs. For images of additional siRNA-transfected cells, see Supplemental Figure 5. Scale bar: 50 μm. (C) The relative invasion of SUM229 trailblazer cells ≥40 μm into the ECM normalized to the total cell number and compared with control cells (mean ± SD, n = 3). (D) Heat map showing that genes required for trailblazer cell invasion are highly expressed in 578T cells. Expression is the mean of 2 biological replicates. (E) The relative invasion of 578T cells ≥50 μm into the ECM normalized to the total cell number and compared with control cells (mean ± SD, n = 3).

Figure 3. The expression of canonical mesenchymal traits is not sufficient to induce the trailblazer phenotype.

(A) Representative FACS analysis of EpCAM and CD49f expression in the indicated cells (n = 3). (B) Representative FACS analysis of EpCAM and CD49f expression in parental SUM149 and SUM229 cells (n = 3). (C) HCC1143, SUM149, and SUM229 cells were sorted into daughter subpopulations based on the level of EpCAM expression. Sorted EpCAM^hi and EpCAM^lo cells grown in organotypic culture and stained with phalloidin and the percentage of invasive spheroids for the EpCAM^hi and EpCAM^lo subpopulations derived from the HCC1143, SUM149, and SUM229 cells (mean + SD, n = 3) are shown. Scale bar: 50 μm. (D) Heat map showing the mRNA expression of EMT-related genes, including epithelial markers (CDH1, CLDN3, CLDN7, and EPCAM) and mesenchymal markers (FOXC1, FOXC2, SNAI1, SNAI2, TWIST1, VIM, ZEB1, and ZEB2). Expression is the mean of 2 biological replicates. (E) Heat map showing the mRNA expression of basal cytokeratins (KRT14 and KRT5), the basal transcription factor p63, and epithelial cytokeratins (KRT8 and KRT18). Expression is the mean of 2 biological replicates. T, trailblazer; O, opportunist; P, parental.

Figure 2. The trailblazer and opportunist phenotypes are heritable.

(A) Model depicting the methodology for generating the trailblazer and opportunist subpopulations (also see the Methods). (B) Quantification of invasion (mean + SD, n = 3). **P < 0.01, ***P < 0.001, unpaired Student’s t test. (C) Representative images of 4T1, SUM159, and HCC1143 parental, daughter trailblazer, and daughter opportunist subpopulation spheroids stained with phalloidin (n = 3). Masks show the outline of the spheroids. Arrows identify invasive spheroids. Scale bar: 50 μm. (D) Representative time-lapse imaging of SUM159 trailblazer (SUM159-T) and normal mammary gland explants (n = 3). Imaging began 24 hours after plating explants in organotypic culture. Arrow shows an example area of collective invasion. Scale bar: 50 μm.

Figure 1. Breast cancer cell lines contain subpopulations of invasive trailblazer and noninvasive opportunist cells.

(A) Representative images of breast cancer spheroids in organotypic culture stained as indicated (n = 3). Masks show the outline of the spheroids. Inset regions are indicated by dashed boxes. Solid arrows identify representative invasive trailblazer spheroids. Arrows with dashed tails identify representative noninvasive opportunist spheroids. Scale bar: 50 μm. PR, progesterone receptor. (B) The percentage of invasive trailblazer spheroids in each cell line. Error bars indicate SD, n = 3. *P < 0.05, ***P < 0.001, unpaired Student’s t test, compared with T47D. (C) Representative H&E-stained primary breast tumors. Arrows indicate patterns of tumor organization that are consistent with collective invasion. Scale bar: 50 μm. (D) Representative images of SUM159 spheroids (day 5) stained as indicated (n = 3). Inset regions are indicated by dashed boxes. Solid arrows indicate where collagen I is being reorganized into parallel tracks by trailblazer cells. Arrows with dashed tails indicate where collagen I is arranged perpendicular to the edge of the noninvasive opportunist spheroid. Scale bar: 50 μm. (E) Time-lapse phase images of the SUM159 spheroids and cell displacement over 18 hours (mean ± SD, n = 3, 15 spheroids total per condition). Solid arrows indicate a leading cell. The arrow with a dotted tail indicates a following cell. Scale bar: 50 μm. **P < 0.01, unpaired Student’s t test.