Background: While significant advancements have been made in breast cancer treatment, metastasis remains a negative outcome which drastically worsens the patient prognosis. Common metastatic sites include the bones, lungs, and liver, with secondary brain metastases seen in advanced disease. Treatments for disseminated tumor cells often carry risks of systemic toxicity without guaranteeing complete eradication. It is vital that we identify key molecular mechanisms underlying the systemic metastatic cascade so that we can disrupt this process. Our team has created a unique mouse model utilizing the 4T1 murine mammary tumor cell line, generating lung/brain metastases with a 100% success rate. This model will help identify the conditions necessary for the development of primary and secondary breast cancer metastases.
Methods: 4T1 parental cells (4T1-Ps) were genetically labeled with firefly luciferase and EGFP for imaging. A highly brain-metastatic subpopulation (4T1-BR) was then established via serial in vivo passage and isolation from the brains of BALB/C mice following iv 4T1-P administration. Brain metastasis was confirmed through bioluminescent imaging, the examination of neurological symptoms, and light sheet microscopy. Genes selectively upregulated in 4T1-BR cells were identified through RNA transcriptomic analysis. Twenty-eight potential candidates encoding for surface-expressed proteins were selected. Surface expression was evaluated using flow cytometry and immunofluorescent staining. Upregulated candidates were knocked out using CRISPR-Cas9 in 4T1-BR cells. Changes in the metastatic biodistribution and survival were assessed in vivo following iv administration.
Results: 4T1-BR cells demonstrate increased surface CD151 expression, as observed using flow cytometry and immunofluorescent staining. Mice transplanted with CD151-/- 4T1-BR cells demonstrated a decreased total bioluminescent signal but no change in their survival, the brain metastasis incidence, or the lung/brain tumor burden.
Conclusions: Surface CD151 expression is increased in brain-metastatic 4T1-BR populations compared to 4T1-Ps. While not directly related to survival or the brain metastatis potential, CD151 may play a functional role in altering systemic biodistribution.
