Characterization of tumor growth and metastasis of cell line xenografts in vivo

There are currently no studies that directly compare the metastatic ability of multiple TNBC cell lines with the same murine xenograft model and experimental parameters. We first directly compared the growth and metastasis of six human TNBC cell lines commonly used to study mechanisms of TNBC metastasis. We used 231, 468, BT549, Hs578T, and SUM159 cell lines, which were isolated from patients with different racial backgrounds and tumor types and which have different mutational profiles and properties (Fig. 1A). We confirmed the receptor status of all 6 cell lines, showing that all cell lines have low levels of HER2, and no expression of progesterone receptor or estrogen receptor α (Additional file 1: Fig S1B). All cell lines except 468 expressed Vimentin and no E-cadherin (Additional file 1: Fig S1C), suggesting most cell lines are more mesenchymal, while 468 are more epithelial, in line with previous reports [34]. We injected the same concentration of cells in a Collagen I solution into the 4th left mammary fat pad of female NOD-SCID mice. Tumors were left to grow for 9 weeks or until ulcers began to form. We found that 468 tumors grew the fastest, followed by 231 and BT549 tumors which had similar sizes. BT20 and SUM159 xenografts grew the smallest tumors, and at 8 weeks the SUM159 tumors had ulcerated and had to be sacrificed (Fig. 1B, C, Additional file 1: Fig S2). The Hs578T xenografts did not grow palpable tumors in this time frame. Based on the tumor latency (Additional file 1: Fig S2G) and the endpoint tumor size (Fig. 1C), the data suggest that 231, 468, and BT549 cell lines are highly tumorigenic, BT20 and SUM159 are intermediate tumorigenic, and Hs578T are poorly tumorigenic since they did not form palpable tumors.

Fig. 1

Tumor growth of human TNBC cell xenografts implanted in immunocompromised female NOD-SCID mice. A Characteristics of six human TNBC cell lines used in the study. IDC, invasive ductal carcinoma; B Tumor volume over time for each cell line 231, 468, BT549, Hs578T, BT20, and SUM159. C Tumor volume at the terminal endpoint. Data are shown as mean with SEM, n = 8–10 animals/group. Significance was determined using a one-way ANOVA with Tukey’s multiple comparison test comparing each cell line to every other cell line. Significance (p < 0.05) is denoted by a letter corresponding to each cell line tested, 231 (a), 468 (b), BT549 (c), BT20 (d), SUM159 (e), Hs578T (f)

Next, we examined lung and liver metastases, by quantifying the number of metastases found in each organ, the average size of each metastatic lesion, and the metastatic index, which is the number of metastases relative to primary tumor size. The 231, 468, and BT549 cell lines had the most lung metastases and similar metastatic index. The BT20, Hs578T, and SUM159 cells had very low numbers of lung metastases, with very low metastatic index (Fig. 2A-D). The size of lung metastases varied between cell lines with 231 s having significantly larger metastases than BT549 and 468 (Fig. 2E). The lung metastatic lesions in BT20, Hs578T, and SUM159 were very small (Fig. 2E). We then examined the relationship between primary tumor size, number of lung metastases, and size of lung metastases. For all cell lines, there was a significant correlation between primary tumor volume and the number of metastatic lesions in the lung (Additional file 1: Fig S3A, B). There was no significant correlation between the number and sizes of lung metastases (Additional file 1: Fig S3C, D) or between the size of the primary tumor and the size of the metastases in the lungs (Additional file 1: Fig S3E, F).

Fig. 2

TNBC cells have different metastatic potentials to the lung A Representative tissue sections stained with H&E, obtained from lungs of mice 9 weeks after injection of TNBC cells in their 4th left mammary gland. Scale bar = 500 μm Inset scale bar = 500 μm. B Number of mice that developed lung metastases for each cell line. C The number of metastases per lung for each cell line and D Metastatic burden in lungs (ratio of the number of metastases in the lungs to the primary tumor volume) E Average size of each lung metastasis. Data are shown as mean with SEM 231 n = 6, 468 n = 10, BT549 n = 8, BT20 n = 9, SUM159 n = 9, Hs578T n = 10 animals/group. Significance was determined using a one-way ANOVA with Tukey’s multiple comparison test comparing each cell line to every other cell line. Significance (p < 0.05) is denoted by a letter corresponding to each cell line tested, 231 (a), 468 (b), BT549 (c), BT20 (d), SUM159 (e), Hs578T (f)

The BT549, 231, and 468 cell lines developed the most liver metastases, with similar levels of metastatic index and size of metastases (Fig. 3A–D). The BT20 xenografts resulted in a high number of liver metastases, which given their small tumors, led to a high liver metastatic index for this cell line, significant relative to 231 cells (Fig. 3A–E). Interestingly, for the BT20 cell line while the number of liver metastases was not significantly different from the 231 cell line the size of the metastases was significantly smaller (Fig. 3A–E). SUM159 and Hs578T xenografts both developed significantly fewer liver metastases than 231s (Fig. 3A–E), which were also smaller. We then examined the relationship between primary tumor size, number of liver metastases, and size of liver metastases. In the livers, there were differences between the highly metastatic and poorly metastatic cell lines. There was a significant correlation between primary tumor volume and the number of metastatic lesions in the liver, and the number and size of liver metastases but only for the least metastatic cell lines (BT20, Hs578T, and SUM159) (Additional file 1: Fig S3G-J). For the highly metastatic cell lines (231, 468, and BT549), there was a negative significant correlation between primary tumor size and liver metastases size (Additional file 1: Fig S3E, F).

Fig. 3

TNBC cells have different metastatic potentials to the liver A Representative tissue sections stained with H&E, obtained from livers of mice 9 weeks after injection of TNBC cells in their 4th left mammary gland. Scale bar is 1 mm; inset scale bar is 500 μm dashed lines represent metastasis boundary, black arrows identify smaller metastases. B Number of mice that developed liver metastases for each cell line. C The number of metastases per liver for each cell line and D Metastatic burden in the liver (ratio of the number of metastases in the lungs to the primary tumor volume) E Average size of each liver metastasis. Data are shown as mean with SEM 231 n = 9, 468 n = 10, BT549 n = 10, BT20 n = 9, SUM159 n = 10, Hs578T n = 10 animals/group. Significance was determined using a one-way ANOVA with Tukey’s multiple comparison test comparing each cell line to every other cell line. Significance (p < 0.05) is denoted by a letter corresponding to each cell line tested, 231 (a), 468 (b), BT549 (c), BT20 (d), SUM159 (e), Hs578T (f)

These data characterize the tumorigenicity and metastatic potential of six human TNBC cell lines, demonstrating that 231, 468, and BT549 cell lines are highly tumorigenic, BT20 and SUM159 are intermediate tumorigenic, and Hs578T are poorly tumorigenic since they did not form palpable tumors. These data characterize 231, 468, and BT549 cell lines as highly metastatic to both the lungs and the livers, BT20 as poorly metastatic to the lungs but metastatic to the liver, and SUM159 and Hs578Ts as poorly metastatic to both the lungs and the liver with slight dissemination to the liver.

TNBC cell lines have distinct differences in morphological characteristics

We next investigated the in vitro characteristics of six TNBC cells. It has been previously shown that features of cell morphology correlate to cell motility and metastatic potential [20, 21]. 2D cell adhesion assays are commonly used to investigate cell phenotypic behavior because they are relatively inexpensive and have low equipment requirements. Cells were left to adhere on either Collagen I, the most abundant ECM protein in breast tissue [35, 36], or tissue culture plastic for 4 or 24 h, and their morphology was analyzed: size (cell area and perimeter), irregularity (solidity and form factor), and elongation (eccentricity and compactness) were quantified (Fig. 4A). On Collagen I for 4 and 24 h the 468 cells were the smallest, as measured by cell area and perimeter (Fig. 4B, G, Additional file 1: Fig S4A, D), they were also least irregular indicating fewer protrusions measured by solidity and form factor, but also rounder and less elongated, measured by eccentricity and compactness. (Fig. 4D, E, H, I, Additional file 1: Fig S4B, S4C, S4E, S4F). Interestingly on uncoated plastic for 4 h 231s were smaller than 468s but after 24 h 231s and 468s were not significantly different in size (Additional file 1: Fig S5 A, S5D, S5G, S5K). After 4 h on plastic 468 s exhibited an irregular and elongated morphology, however, after 24 h they became less irregular and more round, similar to when cultured on Collagen I (Additional file 1: Fig S5 B, C, E, F, H, I, K, L). On Collagen I BT549 and BT20 cells were intermediate in size, after 4 h they both exhibited a more rounded morphology, but after 24 h BT549s were more irregular and elongated(Fig. 4B–I, Additional file 1: Fig S4) with a similar effect on plastic (Fig S5). On Collagen I for 4 and 24 h we found that Hs578Ts were the largest cells with more irregular shapes indicating more protrusions and were also rounder and less elongated (Fig. 4B-I, Additional file 1: Fig S4). However, on plastic SUM159s were the largest after 4 h but Hs578Ts were the largest after 24 h similar to on Collagen I (Additional file 1: Fig S5). SUM159 cells on Collagen I were intermediate in size but were similarly more protrusive and more elongated (Fig. 4C–I Additional file 1: Fig S4). SUM159s were very irregular, with more protrusions, and were the most elongated (Fig. 4C–I, Additional file 1: Fig S4). While the cell shape did vary between 4 and 24 h, the relative differences between the different cell lines did stay consistent.

Fig. 4

Characterization of TNBC cell line morphology. A Schematic depicting experimental procedure. B Representative images of 231, 468, BT549, BT20 SUM159 and Hs578T cell lines plated on Collagen I ECM for 4 h, fixed and stained for nuclei (Blue) and F-actin (red). Scale bar = 50 μm. Quantification of cell shape parameters for each cell line 231 (n = 679 cells), BT549 (n = 799 cells), Hs578T (n = 538 cells), 468 (n = 818 cells), BT20 (n = 882 cells) and SUM159 (n = 892 cells) C Area/Cell, D Solidity, and E Eccentricity. F Representative images of 231, BT549, Hs578T, 468, BT20, and SUM159 cell lines plated on Collagen I for 24 h, fixed and stained for nuclei (Blue) and F-actin (red). Scale bar = 50 μm. Quantification of cell shape parameters for each cell line 231 (n = 178 cells), BT549 (n = 184 cells), Hs578T (n = 91 cells), 468 (n = 205 cells), BT20 (n = 135 cells) and SUM159 (n = 192 cells) G Area/Cell, H Solidity, and I Eccentricity. Data show mean ± SEM. Significance was determined using a one-way ANOVA with Tukey’s multiple comparison test comparing each cell line to every other cell line. Significance (p < 0.05) is denoted by a letter corresponding to each cell line tested, 231 (a), 468 (b), BT549 (c), BT20 (d), SUM159 (e), Hs578T (f)

Proliferation and 2D cell motility

Cell proliferation is critical to support both primary and secondary tumor growth. We quantified the proliferation rate of each cell line by measuring the change in metabolic activity over 48 h (Fig. 5A). BT549 and SUM159 cell lines were significantly more proliferative than 231 s (Fig. 5B). 2D cell migration is a commonly used metric to determine metastatic potential. Cells were seeded on Collagen I and imaged to quantify 2D cell migration speed, which measures how fast the cell is moving over the distance traveled, and persistence which measures the Euclidean distance between the start and finish of the cell's path over the total distance traveled (Fig. 5C). SUM159, 231, and 468 cells migrated the fastest in 2D and were not significantly different from each other. Hs578T, BT20, and BT549 cells all migrated significantly slower than 231 cells (Fig. 5D). 231, 468 and BT20 cells had the lowest 2D persistence with BT594s, Hs578Ts and SUM159s all having significantly higher persistence than 231 s respectively (Fig. 5E,F).

Fig. 5

Cell line-specific proliferation, 2D migration, and persistence. A Schematic depiction of presto blue proliferation assay. B The relative proliferation of cell lines C Schematic depiction of 2D migration assay D 2D cell migration and E 2D persistence of cell lines seeded on Collagen 1 coated glass bottom plates. F representative rose plots of the migration of cells on Collagen I ECM-coated glass coverslips. Data show mean ± SEM. Significance was determined using a one-way ANOVA with Tukey’s multiple comparison test comparing each cell line to every other cell line. Significance (p < 0.05) is denoted by a letter corresponding to each cell line tested, 231 (a), 468 (b), BT549 (c), BT20 (d), SUM159 (e), Hs578T (f)

3D methods to quantify cell invasion

To better recapitulate the native breast microenvironment, 3D assays where breast cancer cells are encapsulated in ECM, are used to mimic the 3D in vivo environment. We first investigated 3D invasion in a single-cell invasion assay, where each cell line was encapsulated in Collagen I ECM and plated in a glass bottom dish then imaged overnight to track cell invasion (Fig. 6A). The 468, SUM159, and Hs578T cells invaded significantly faster than the 231 cells, and BT549 and BT20 cells invaded significantly slower than the 231 cells (Fig. 6B). Interestingly, only SUM159 and BT20 cells were significantly more persistent than 231 cells (Fig. 6 C, D).

Fig. 6

3D single cell invasion and spheroid invasion in Collagen I ECM A Schematic depicting experimental set up of 3D invasion assay. B 3D invasion of breast cancer cell lines invading through Collagen I ECM C 3D persistence of cells invading through collagen I ECM D Representative rose plots of the invasion of cells through the collagen I ECM. E Schematic depiction of spheroid invasion assay. F Fold change in spheroid invasion into Collagen I hydrogels over invasion into media G Representative spheroid images of 231, BT549, Hs578T, and SUM159 spheroids cultured in media (upper) or spheroids cultured in Collagen I solution visualized with cytopainter (red). Scale bar = 500 μm. Data show mean ± SEM. Significance was determined using a one-way ANOVA with Tukey’s multiple comparison test comparing each cell line to every other cell line. Significance (p < 0.05) is denoted by a letter corresponding to each cell line tested, 231 (a), 468 (b), BT549 (c), BT20 (d), SUM159 (e), Hs578T (f)

We then used a 3D spheroid assay, where the cell lines were seeded in low attachment U-bottom dishes and centrifuged to form a densely packed spheroid. After growing for 3 days, the spheroids were either left in the original media or Collagen I was added before an additional 4 days of culture. Growth out of spheroids relies on tumor cell proliferation and invasion. We then measured the fold change in the spheroid outer area in the Collagen I group relative to media only after 7 days (Fig. 6E). 468, BT20, BT549, and SUM159 cells had a significantly higher fold change in spheroid invasion compared to 231s (Fig. 6F-G).

PCA was then used to reduce the dimensionality of the data set to compare 2D migration and persistence and 3D invasion and persistence. The scores of each cell line demonstrate that highly tumorigenic and metastatic cell lines, 231, 468, and BT549 clustered together and the poorly tumorigenic and metastatic SUM159, Hs578T, and BT20 clustered together indicating that highly and poorly tumorigenic and metastatic cell lines have similar motility characteristics (Additional file 1: Fig S6A). The loadings scores represent variations in the motility metrics. Interestingly, 2D migration and persistence and 3D invasion cluster together while 3D persistence is by itself.

Cell shape is most correlated with metastatic potential

Our last goal was to determine which assays and their metrics, cell morphology, proliferation, and 2D or 3D motility, best correlate with in vivo tumor growth and metastasis. To do this we used pairwise comparisons to systematically investigate each in vitro metric in relation to in vivo response for all cell lines. First, we determined if there was a linear relationship between each in vitro metric and in vivo response by calculating the R2 which measures the proportion of variation in the in vivo response that is attributed to each in vitro metric, and the Pearson correlation which measures the strength of the linear relationship. First, comparing only cell morphology metrics on Collagen I and plastic for 4 and 24 h we found that form factor and solidity, which quantify how irregular or protrusive a cell is, correlated the strongest with metastasis to the lungs and liver and tumor volume regardless of culturing method (Additional file 1: Fig S7A-C). We also used partial least squares to calculate the scores and loadings which capture the covariance between the independent and dependent variables [37]. The scores plot describes how each cell line projects on principal components 1 (PC1) and PC2. Interestingly, we found that 231s, BT549s and BT20s clustered together (Additional file 1: Fig S7D). The loading scores represent how the independent variables and dependent in vivo responses project on each principal component(Additional file 1: Fig S7 E). We found that solidity and form factor on Collagen I for 4 h clustered closely with the in vivo response metrics. Thus, for comparing in vivo responses to all the in vitro metrics we only included the cell adhesion on Collagen I for 4 h data.

We found that there were distinct differences in the linear relationship between in vitro metrics and in vivo behaviors. Interestingly, we found that solidity and form factor, which quantify how irregular or protrusive a cell is, had a linear relationship with tumor volume (R2 > 0.5) (Fig. 7A, Additional file 1: Fig S8). Only the persistence of 3D single cell invasion had a linear relationship to the number of lung metastases (R2 > 0.5) (Fig. 7A, Additional file 1: Fig S8). Cell size and cell irregularity, all had linear relationships to the number of liver metastases with solidity and form factor having a strong linear relationship (R2 > 0.7 and p < 0.05) (Fig. 7A, Additional file 1: S8). Next, we used Spearman's correlation to investigate the correlation of each assay to in vivo response. We found that area, perimeter, and 3D persistence were negatively correlated with tumor volume, while solidity and form factor were positively correlated (Spearman Coefficient >  ± 0.6). Area, perimeter, eccentricity, compactness, and 3D persistence demonstrated a negative correlation with the number of lung metastasis while solidity and form factor demonstrated a positive correlation. Perimeter and 3D persistence negatively correlated to the number of liver metastases, while solidity and form factor positively correlated (Fig. 7B).

Fig. 7

Distinct differences in cell line-specific behavior in vitro and in vivo. A pairwise analysis of the strength of the linear relationship (R2) between in vitro metrics and in vivo behavior. Numbers represent p-value and colors represent R2 value where blue is high and white is low indicated by gradient B Spearman correlation between in vitro metrics and in vivo behavior. Numbers represent p-value and colors represent the Spearman correlation coefficient where blue is a high positive correlation, white is no correlation and red is a negative correlation indicated by gradient C X-scores plot of the PLS model D PLS loadings for adhesion metrics, proliferation, 2D and 3D motility, and in vivo tumor volume, lung and liver metastases

To visualize the relationship between the cell lines and assays we used partial least squares to calculate the scores and loadings which capture the covariance between the independent and dependent variables [37]. The scores plot describes how each cell line projects on principal components 1 (PC1) and PC2. Highly metastatic cell lines 231, 468, and BT549 clustered together, and poorly metastatic cell lines SUM159s and Hs578T clustered together. BT20s, which were intermediate tumorigenic but poorly metastatic to the lungs but metastatic to the livers, stood alone (Fig. 7C). The loading scores represent how the independent variables and dependent in vivo responses project on each principal component. As expected, cell shape parameters related to size, elongation, and irregularity clustered together. Other cluster groups include lung metastasis and tumor volume; liver metastasis, form factor, and solidity; eccentricity, compactness, area, perimeter, and 2D persistence; 2D migration and 3D invasion; 2D, 3D persistence and proliferation (Fig. 7D). This analysis demonstrates that there were distinct differences in cell line-specific behaviors in vitro and in vivo and that parameters that measure cell shape and 3D persistence are most correlated with in vivo behaviors.