Maintaining unperturbed cerebral blood flow is key in the study of brain metastasis and its interactions with stress and inflammatory responses

Abstract

Blood-borne brain metastases are associated with poor prognosis, but little is known about the interplay between cerebral blood flow, surgical stress responses, and the metastatic process. The intra-carotid inoculation approach, traditionally used in animal studies, involves permanent occlusion of the common carotid artery (CCA). Herein we introduce a novel intra-carotid inoculation approach that avoids CCA ligation, namely – assisted external carotid artery inoculation (aECAi) – and compared it to the traditional approach in C57/BL6 mice, assessing cerebral blood flow; particle distribution; blood-brain barrier (BBB) integrity; stress, inflammatory and immune responses; and tumor brain retention and growth. Doppler flowmetry and two-photon imaging confirmed that only in the traditional approach regional and capillary cerebral blood flux were significantly reduced. Corticosterone and plasma IL-6 levels were higher in the traditional approach, splenic numbers of NK, CD3+, granulocytes, and dendritic cells were lower, and many of these indices were more profoundly affected by surgical stress in the traditional approach. BBB integrity was unaffected. Administration of spherical beads indicated that CCA ligation significantly limited brain distribution of injected particles, and inoculation of D122-LLC syngeneic tumor cells resulted in 10-fold lower brain tumor-cell retention in the traditional approach. Last, while most of the injected tumor cells were arrested in extra-cranial head areas, our method improved targeting of brain-tissue by 7-fold. This head versus brain distribution difference, commonly overlooked, cannot be detected using in vivo bioluminescent imaging. Overall, it is crucial to maintain unperturbed cerebral blood flow while studying brain metastasis and interactions with stress and inflammatory responses.

Prophylactic TLR9 stimulation reduces brain metastasis through microglia activation

“…Tumor cells were injected using the assisted external carotid artery inoculation (aECAi; Fig 1A), as previously described [32]. Briefly, mice were anesthetized and the external carotid artery (ECA) uncovered.…”

“…Here, we assessed the efficacy of a systemic administration of CpG-C as a prophylactic treatment for brain metastasis using three preclinical mouse models, including experimental metastasis of syngeneic and of human lung carcinoma, and spontaneous metastasis of syngeneic melanoma. Importantly, the inoculation methodologies and imaging approaches implemented here preserve intact both the neuro-immune niche and brain hemodynamics intact: crucial factors affecting metastatic early stages [32]. We demonstrate that acute and chronic prophylactic treatments result in reduced brain metastasis in both sexes and across ages.…”

Dexmedetomidine promotes metastasis in rodent models of breast, lung, and colon cancers

“…Natural killer (NK) cells play a role in restricting lung metastases in both models, but have no impact on brain tumour retention and brain metastases. 34,35 Thus, given the similar deleterious effects of dexmedetomidine in all organs tested, the impact of dexmedetomidine seems to be mediated, at least partly, by an organ-independent mechanism, as further discussed below.…”

“…This procedure enables a controlled administration of tumour cells directly to the brain without disturbing brain blood flow 34 (see Supplementary material 1.4). This approach and the administration of CT26 cells through the spleen involve surgical procedures of~15 min.…”

“…Notably, the effects of dexmedetomidine were initiated shortly following its administration, and subsided along with cessation of its behavioural effects, indicating quickly inducible and reversible mechanisms. Thus, immune mechanisms might include reduced innate anti-metastatic immunity that can quickly and transiently be modulated, specifically NK cell cytotoxicity, which controls lung and liver metastases in all tumour models used, 30,34,35 but not brain metastasis. Non-immune mechanisms might include changes in vascular dilation and constriction, alteration in the expression of epithelial adhesion molecules, and increased capillary permeability to circulating tumour cells, all of which have been suggested to be modulated by dexmedetomidine, 37,38 as well as to impact metastatic spread.…”