Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models

Abstract

To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44(+) cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.

Fig. 1.

Visualizing BCSCs with optical reporters. (A) BLI of L2T-TN1 tumor cells (10–50,000) implanted into four separate fat pads of NOD/SCID mice. Transduced td-Tomato⁺ cells were sorted from the labeled parental tumor. (B) Stable integration of the optical reporters L2T or L2G into TN1 tumor cells after lentiviral transduction. Reporter expression in harvested cells was measured by flow cytometry in both parental tumors (parental) and passaged tumors (p1–p3). Sample flow cytometry dot plots are shown.

Fig. 2.

Analyzing the early growth of breast cancer cell subsets in vivo via BLI. (A and B) Monitoring tumor growth mediated by TN1 BCSCs (passage 2–5). CD44⁺ cells (1 × 10⁵) were transduced with L2G (MOI 50) or the mock and then transplanted into mouse mammary fat pads. Mice were imaged from day 1 to day 52 after implantation when palpable tumors formed. Quantification (total flux, photons per second, p/s) of the bioluminescent signal from the tumor regions in A is depicted in B. Error bars represent the SD of the mean for three experiments. *P < 0.01 for early-phase growth curve day 7 and P < 0.05 for day 52. (C and D) Analysis of primary breast tumor cell subsets into NOD/SCID or NSG (NOD/SCID with IL2R-γchain^−/−) mice. Primary breast tumor cells were dissociated from clinical patient specimens, and 3,000 CD44⁺ or CD44⁻ cells were sorted for transduction with L2T fusion reporter gene (MOI 10), implanted into mice, and analyzed by BLI over time. Representative images are shown in C and the light emission is quantified in D. Error bars represent SD of the mean for six replicate experiments (three from NSG mice).

Fig. 3.

BLI revealed lung and lymph node metastases. (A) Spontaneous lung metastases detected noninvasively in mice bearing a TN1 tumor (passage 3) in the L4 mammary fat pad (Upper). Focal metastases were observed throughout the lung upon dissection, placed in a plate with PBS (Lower). (B) Residual BLI signal observed in the animal after the tumor section (Upper) localized to lymph node (Lower) (Fig. S6). (C) Representative H&E stains of sections from the breast tumor (BT), dissected lungs in A, and lymph nodes (LN) in B. (Magnification: 200×.) (D) Representative H&E stains of sections from four TN tumors (TN1–TN4) and mouse lungs. (Magnification: 200×.)

Fig. 4.

Invasive cells from TN1 and TN2 breast tumors (passage 3–5) are enriched for CSC markers. (A and B) Invasive TN1 (A) and TN2 (B) cells were collected in response to human EGF (25 nmol/L), compared with Matrigel alone. Results are plotted as average number of cells per needle. Error bars represent the SEM. A, *P = 0.02388, n = 5; B, *P = 0.00359, n = 5, Student's t test. (C) Guava flow cytometry analysis of the CD44⁺ population (percentage) contained in the APTCs and the ITCs isolated from the L2G-TN1 breast tumor. Results are shown as percentage of total GFP⁺ cells. Error bars: SD. *P = 0.00005, n = 4. (D) Representative dot plot from the Guava flow cytometer analysis of CD44 expression on the TN1-APTCs and -ITCs.

Fig. 5.

Flow cytometry analysis and tumorigenic assays of lung metastatic cancer cells. (A) CD44, td-Tomato, and SSC-A side scatter profiles of transduced TN2 parental breast tumor cells (Upper) and lung metastatic cells (Lower), isolated from xenografts at passage 4. (B) Upper: Flow cytometry analysis of human CD44 expression on lung metastatic cells (gated viable H2K^d− cells) of untransduced TN1 (passage 5) and TN2 (passage 3) tumors. The CD44⁺ and CD44⁻ gates used for sorting in tumorigenicity assays are shown. Lower: Table of tumorigenic assay data derived from bulk lung cells from TN1, TN2, or normal control mice as well as sorted CD44⁺ or CD44⁻ tumor cells (viable H2K^d− nonmouse cells). Results are shown as the number of tumors formed over the number of injections performed. These data indicate that the metastatic CSCs are enriched in CD44⁺ tumor cells.