In a recent publication, we developed and directly compared two in vivo models of breast cancer brain metastasis (BCBM) using non‑invasive imaging modalities and immunohistochemistry.

Establishment of BCBM by stereotactic implantation and intracarotid inoculation

BCBM models were induced using human breast ductal carcinoma (BT474, HER2 3+) and adenocarcinoma cell lines (MDA‑MB‑231.Luc2, TNBC). Tumors were established either by stereotactic intracranial implantation or intracarotid inoculation. Both approaches resulted in survival rates exceeding 90%, no significant body‑weight loss, and low clinical severity scores.

BT474 tumor growth dynamics and establishment patterns

Weekly longitudinal MRI monitoring revealed distinct growth profiles between models. Stereotactic implantation reliably produced a single tumor detectable within 14 days, followed by rapid and uniform growth. In contrast, intracarotid inoculation resulted in dispersed micro‑seeding (1–7 metastases per animal), with slower, heterogeneous growth and tumor detection only after about 7 weeks.
Tumor take rates were also different between models: 93% for the stereotactic model (one tumor per animal) versus 62% for the intracarotid model, which showed variable metastatic burden (mean 2.4 ± 0.5) and pronounced growth heterogeneity.

Figure 1. (A) Tumors take rate and (B) number of tumors in stereotactic-implanted and intracarotid-inoculated animals. (C) Accumulated and (D) individual tumor growth of animals bearing stereotactic-implanted and intracarotid-inoculated BCBM tumors. Bars represent mean ± SEM (n = 13–14). From Cold et al, Cancers 2026.

Figure 1. (A) Tumors take rate and (B) number of tumors in stereotactic-implanted and intracarotid-inoculated animals. (C) Accumulated and (D) individual tumor growth of animals bearing stereotactic-implanted and intracarotid-inoculated BCBM tumors. Bars represent mean ± SEM (n = 13–14). From Cold et al, Cancers 2026.

Blood-brain barrier (BBB) permeability in BT474 tumors

BBB integrity was assessed using in vivo MRI and ex vivo analyses. Both models demonstrated BBB disruption within tumor regions and small tumors (< 2 mm³) generated by stereotactic implantation exhibited significantly greater BBB permeability than intracarotid tumors. Ex vivo staining confirmed the presence of a single lesion in the stereotactic model and multiple metastatic foci in the intracarotid model and sodium fluorescein assays further supported the imaging‑based observations of BBB leakiness.

Figure 2. (A) Representative images of animals bearing BCBM established by stereotactic implantation (top row) and intracarotid inoculation (bottom row) of BT474 cells using MRI. (B) Quantitative analysis of BBB leakiness on small metastases (<2 mm3) established by stereotactic implantation and intracarotid inoculation. Bars represent mean ± SEM, n = 8–14/group. ** p < 0.01. From Cold et al, Cancers 2026.

Figure 2. (A) Representative images of animals bearing BCBM established by stereotactic implantation (top row) and intracarotid inoculation (bottom row) of BT474 cells using MRI. (B) Quantitative analysis of BBB leakiness on small metastases (<2 mm3) established by stereotactic implantation and intracarotid inoculation. Bars represent mean ± SEM, n = 8–14/group. ** p < 0.01. From Cold et al, Cancers 2026.

Validation of tumor presence and proliferative activity

Ex vivo immunohistochemistry confirmed successful tumor establishment and proliferation. Ku80 staining verified single tumors in the stereotactic model and multiple lesions in the intracarotid model, while Ki67 staining revealed proliferative activity predominantly at tumor margins. Sodium fluorescein uptake corroborated BBB disruption in both models. Importantly, micro‑metastases consisting of as few as five tumor cells were detected in the intracarotid model ex vivo but remained below the detection threshold of MRI, highlighting the higher sensitivity of histological methods.

BCBM Was Successfully Established with MDA-MB-231.Luc2 Using Both Methods

To test the reproducibility of the developed intracarotid inoculation method, BCBM was successfully established using MDA‑MB‑231.Luc2 with both stereotactic implantation and intracarotid inoculation. Survival was higher after stereotactic implantation (100%) than intracarotid inoculation (70%). Due to diffuse tumor growth and low MRI contrast, BLI was used for monitoring. Tumor establishment occurred earlier with stereotactic implantation (7 days) than intracarotid inoculation (28 days). Stereotactic implantation produced single, localized lesions with faster, more homogeneous growth, while intracarotid inoculation resulted in multifocal, heterogeneous tumors. Tumor take rates were comparable (86% vs 71%), but humane endpoints were reached earlier in the stereotactic model.

Figure 3. (A) Representative bioluminescence imaging of animals inoculated with MDA‑MB‑231.Luc2 cells by the stereotactic (top) or intracarotid (bottom) method. (B) Tumor growth of stereotactic-implanted and intracarotid-inoculated BCBM tumors. (C) Tumor take rates and (D) survival rates of stereotactic and intracarotid inoculation. (E) Bioluminescence signal at euthanasia in animals bearing stereotactic and intracarotid inoculated BCBM. Bars represent mean ± SEM, n = 5–6.

Figure 3. (A) Representative bioluminescence imaging of animals inoculated with MDA‑MB‑231.Luc2 cells by the stereotactic (top) or intracarotid (bottom) method. (B) Tumor growth of stereotactic-implanted and intracarotid-inoculated BCBM tumors. (C) Tumor take rates and (D) survival rates of stereotactic and intracarotid inoculation. (E) Bioluminescence signal at euthanasia in animals bearing stereotactic and intracarotid inoculated BCBM. Bars represent mean ± SEM, n = 5–6.

Conclusion

The intracarotid inoculation model closely recapitulates key clinical features of BCBM, including extravasation, micro‑seeding, and intratumoral heterogeneity, while maintaining comparable animal welfare outcomes. Together, these models form a robust translational platform for investigating therapeutic strategies targeting BBB dysfunction, metastatic diversity, and treatment response in breast cancer brain metastases.

Interested in learning more about our in vitro and in vivo models and how Minerva Imaging can support studies of targeted radionuclide therapies in relevant BCBM or other xenograft models?

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References:

  • Sigrid Cold, Maria Zeiler Alfsen, Brandur Halgirsson, Mads Neergaard Jorgensen, Jacob Hald, Carsten Haagen Nielsen, Andreas Kjaer, Lotte Kellemann Kristensen and Trine Bjoernbo Engel (2026). Evaluating the Translation Value of Two In Vivo Models for Breast Cancer Brain Metastases. Cancers. DOI: https://doi.org/10.3390/cancers18071095