Longitudinal bioluminescence imaging to monitor breast tumor growth and treatment response using the chick chorioallantoic membrane model

Abstract

The development of successful treatment regimens for breast cancer requires strong pre-clinical data generated in physiologically relevant pre-clinical models. Here we report the development of the chick embryo chorioallantoic membrane (CAM) model to study tumor growth and angiogenesis using breast cancer cell lines. MDA-MB-231 and MCF7 tumor cell lines were engrafted onto the chick embryo CAM to study tumor growth and treatment response. Tumor growth was evaluated through bioluminescence imaging and a significant increase in tumor size and vascularization was found over a 9-day period. We then evaluated the impact of anti-angiogenic drugs, axitinib and bevacizumab, on tumor growth and angiogenesis. Drug treatment significantly reduced tumor vascularization and size. Overall, our findings demonstrate that the chick embryo CAM is a clinically relevant model to monitor therapeutic response in breast cancer and can be used as a platform for drug screening to evaluate not only gross changes in tumor burden but physiological processes such as angiogenesis.

Figure 1

Method to study breast tumor growth using the chick embryo model and longitudinal bioluminescence imaging. (A) Generation of firefly luciferase expressing cell lines and (B) their relative bioluminescence using an increasing cell number for each cell line: MDA231, MCF7, MCF10A lines (5 k, 10 k, 15 k, and 20 k). (C) Schematic diagram representing the experimental timeline from spheroid generation, chick CAM BLI and tumor removal.

Figure 2

Engineered MDA-MB-231luc spheroids growth profile on the chick embryo chorioallantoic membrane (CAM). (A) Tumor spheroid growth curve of engineered MDA-MB-231luc spheroid (2.5 × 10⁵ cells/spheroid) on CAM post engrafting (n = 16). Error bars represents SEM, asterisk denotes significance, Tukey’s test. (B) Representative image of MDA-MB-231luc spheroid on CAM measured via IVIS. (C) Representative image of MDA-MB-231luc spheroid on CAM observed via light microscope. (D) Representative image of H/E staining of MDA-MB-231luc spheroid on CAM.

Figure 3

Engineered MCF7luc spheroids growth profile on the chick embryo chorioallantoic membrane (CAM). (A) Tumor spheroid growth curve of engineered MCF7luc spheroid (0.5 × 10⁵ cells/spheroid) on the chick CAM post engrafting (n = 14). Error bars represent SEM, asterisk denotes significance, Kruskal–Wallis test. (B) Representative image of MCF7luc spheroid on CAM measured via IVIS. (C) Representative image of MCF7luc spheroid on CAM observed via light microscope. (D) Representative image of H/E staining of MCF7luc spheroid on CAM.

Figure 4

Engineered MCF10Aluc spheroids growth profile on the chick embryo chorioallantoic membrane (CAM). (A) Tumor spheroid growth curve of engineered MCF10Aluc spheroid on CAM post engrafting (5 × 10⁴ cells/spheroid N = 4). Error bar represents SEM, asterisk denotes significance, Tukey’s test. (B) Representative image of MCF10Aluc spheroid on CAM measured via IVIS. (C) Representative image of MCF10Aluc spheroid on CAM observed via light microscope.

Figure 5

Axitinib treatment in MDA-MB-231luc and MCF7luc spheres. (A, B) Axitinib treatment of (A) MDA-MB-231luc and (B) MCF7luc tumor spheres in vitro (n = 10 per group). (C, D) Axitinib treatment of (C) MDA-MB-231luc and (D) MCF7luc tumor spheres in vivo, xenografted onto the chick embryo CAM (n = 7–10 per group). Error bars in both graphs represents SEM, asterisk denotes significance, Two-way Anova Multiple Comparison Test. Black arrows demonstrate the days of the treatment (Day 4 and Day 7). (E, G) Representative image of treated, or untreated, tumors on Day 4 and Day 9 for (E) MDA-MB-231luc cells and (G) MCF7luc cells when observed via a light microscope. (F, H) Comparison of control (left) and treatment (right) tumor size when resected at endpoint (Day 9) for (F) MDA-MB-231luc cells and (H) MCF7luc. (I) Total angiogenic area in the tumor periphery of MDA-MB-231 and MCF7 tumors spheres xenograft onto the chick embryo CAM at endpoint (Day 9) treated with axitinib or DMSO control (n = 7 per group). Error bars represents SEM, p-value denotes significance, Tukey’s test. (J) Representative H/E staining of MDA-MB-231luc and MCF7luc control and axitinib-treated tumors harvested from the CAM. (K, L) Representative Ki67 staining of tumor sections (left row) and zoom insets (right row) for: (K) MDA-MB-231luc and MCF7luc control and (L) axitinib-treated tumors harvested from the CAM.

Figure 6

Bevacizumab treatment in MDA-MB-231luc and MCF7luc spheres. (A and B) Bevacizumab treatment of (A) MDA-MB-231luc and (B) MCF7luc tumor spheres in vitro (n = 5 per group). (C, D) Bevacizumab treatment of (C) MDA-MB-231luc and (D) MCF7luc tumor spheres in vivo, xenografted onto the chick embryo CAM (n = 5–7 per group). Error bars in both graphs represents SEM, asterisk denotes significance, Two-way Anova Multiple Comparison Test. Black arrows indicate the days of the treatment (Day 4 and Day 7). (E, G) Representative image of treated, or untreated, tumors on Day 4 and Day 9 for (E) MDA-MB-231luc cells and (G) MCF7luc cells when observed via a light microscope. (F, H) Comparison of control (left) and treatment (right) tumor size when resected at endpoint (Day 9) for (F) MDA-MB-231luc cells and (H) MCF7luc. (I) Total angiogenic area in the tumor periphery of MDA-MB-231 and MCF7 tumors spheres xenograft onto the chick embryo CAM at endpoint (Day 9) treated with axitinib or DMSO control (n = 7 per group). Error bars represents SEM, p-value denotes significance, Tukey’s test.