Disturbance in cerebral blood microcirculation and hypoxic-ischemic microenvironment are associated with the development of brain metastasis.

Brain metastases (BM) constitute an increasing challenge in oncology due to their impact on neurological function, limited treatment options, and poor prognosis. BM occur through extravasation of circulating tumor cells across the blood-brain barrier. However, the extravasation processes are still poorly understood. We here propose a brain colonization process which mimics infarction-like microenvironmental reactions, that is dependent on Angiopoietin (Ang-2) and vascular endothelial growth factor (VEGF). In this study, intracardiac BM models were used, and cerebral blood microcirculation was monitored by 2-photon microscopy through a cranial window. BM formation was observed using cranial magnetic resonance, bioluminescent imaging, and post-mortem autopsy. Ang-2/VEGF targeting strategies and Ang-2 gain-of-function (GOF) mice were employed to interfere with BM formation. In addition, vascular and stromal factors as well as clinical outcome were analyzed in BM patients. Blood vessel occlusions by cancer cells were detected, accompanied by significant disturbances of cerebral blood microcirculation, and focal stroke-like histological signs. Cerebral endothelial cells showed an elevated Ang-2 expression both in mouse and human BM. Ang-2 GOF resulted in an increased BM burden. Combined anti-Ang-2/anti-VEGF therapy led to a decrease in brain metastasis size and number. Ang-2 expression in tumor vessels of established human brain metastases negatively correlated with survival. Our observations revealed a relationship between disturbance of cerebral blood microcirculation and brain metastasis formation. This suggests that vessel occlusion by tumor cells facilitates brain metastatic extravasation and seeding, while combined inhibition of microenvironmental effects of Ang-2 and VEGF prevent the outgrowth of macrometastases.

CCT196969 effectively inhibits growth and survival of melanoma brain metastasis cells.

Melanomas frequently metastasize to the brain. Despite recent progress in the treatment of melanoma brain metastasis, therapy resistance and relapse of disease remain unsolved challenges. CCT196969 is a SRC family kinase (SFK) and Raf proto-oncogene, serine/threonine kinase (RAF) inhibitor with documented effects in primary melanoma cell lines in vitro and in vivo. Using in vitro cell line assays, we studied the effects of CCT196969 in multiple melanoma brain metastasis cell lines. The drug effectively inhibited proliferation, migration, and survival in all examined cell lines, with viability IC50 doses in the range of 0.18-2.6 µM. Western blot analysis showed decreased expression of p-ERK, p-MEK, p-STAT3 and STAT3 upon CCT196969 treatment. Furthermore, CCT196969 inhibited viability in two B-Raf Proto-Oncogene (BRAF) inhibitor resistant metastatic melanoma cell lines. Further in vivo studies should be performed to determine the treatment potential of CCT196969 in patients with treatment-naïve and resistant melanoma brain metastasis.

Cabozantinib Is Effective in Melanoma Brain Metastasis Cell Lines and Affects Key Signaling Pathways.

Melanomas have a high potential to metastasize to the brain. Recent advances in targeted therapies and immunotherapies have changed the therapeutical landscape of extracranial melanomas. However, few patients with melanoma brain metastasis (MBM) respond effectively to these treatments and new therapeutic strategies are needed. Cabozantinib is a receptor tyrosine kinase (RTK) inhibitor, already approved for the treatment of non-skin-related cancers. The drug targets several of the proteins that are known to be dysregulated in melanomas. The anti-tumor activity of cabozantinib was investigated using three human MBM cell lines. Cabozantinib treatment decreased the viability of all cell lines both when grown in monolayer cultures and as tumor spheroids. The in vitro cell migration was also inhibited and apoptosis was induced by cabozantinib. The phosphorylated RTKs p-PDGF-Rα, p-IGF-1R, p-MERTK and p-DDR1 were found to be downregulated in the p-RTK array of the MBM cells after cabozantinib treatment. Western blot validated these results and showed that cabozantinib treatment inhibited p-Akt and p-MEK 1/2. Further investigations are warranted to elucidate the therapeutic potential of cabozantinib for patients with MBM.

Current landscape and future perspectives in preclinical MR and PET imaging of brain metastasis.

Brain metastasis (BM) is a major cause of cancer patient morbidity. Clinical magnetic resonance imaging (MRI) and positron emission tomography (PET) represent important resources to assess tumor progression and treatment responses. In preclinical research, anatomical MRI and to some extent functional MRI have frequently been used to assess tumor progression. In contrast, PET has only to a limited extent been used in animal BM research. A considerable culprit is that results from most preclinical studies have shown little impact on the implementation of new treatment strategies in the clinic. This emphasizes the need for the development of robust, high-quality preclinical imaging strategies with potential for clinical translation. This review focuses on advanced preclinical MRI and PET imaging methods for BM, describing their applications in the context of what has been done in the clinic. The strengths and shortcomings of each technology are presented, and recommendations for future directions in the development of the individual imaging modalities are suggested. Finally, we highlight recent developments in quantitative MRI and PET, the use of radiomics and multimodal imaging, and the need for a standardization of imaging technologies and protocols between preclinical centers.

Trifluoperazine prolongs the survival of experimental brain metastases by STAT3-dependent lysosomal membrane permeabilization.

Brain metastasis is a major cause of mortality in melanoma patients. The blood-brain barrier (BBB) prevents most anti-tumor compounds from entering the brain, which significantly limits their use in the treatment of brain metastasis. One strategy in the development of new treatments is to assess the anti-tumor potential of drugs currently used in the clinic. Here, we tested the anti-tumor effect of the BBB-penetrating antipsychotic trifluoperazine (TFP) on metastatic melanoma. H1 and Melmet1 human metastatic melanoma cell lines were used in vitro and in vivo. TFP effects on viability and toxicity were evaluated in proliferation and colony formation assays. Preclinical, therapeutic efficacy was evaluated in NOD/SCID mice, after intracardial injection of tumor cells. Molecular studies using immunohistochemistry, western blots, immunofluorescence and transmission electron microscopy were used to gain mechanistic insight into the biological activity of TFP. Our results showed that TFP decreased cell viability and proliferation, colony formation and spheroid growth in vitro. The drug also decreased tumor burden in mouse brains and prolonged animal survival after injection of tumor cells (53.0 days vs 44.5 days), TFP treated vs untreated animals, respectively (P < 0.01). At the molecular level, TFP treatment led to increased levels of LC3B and p62 in vitro and in vivo, suggesting an inhibition of autophagic flux. A decrease in LysoTracker Red uptake after treatment indicated impaired acidification of lysosomes. TFP caused accumulation of electron dense vesicles, an indication of damaged lysosomes, and reduced the expression of cathepsin B, a main lysosomal protease. Acridine orange and galectin-3 immunofluorescence staining were evidence of TFP induction of lysosomal membrane permeabilization. Finally, TFP was cytotoxic to melanoma brain metastases based on the increased release of lactate dehydrogenase into media. Through knockdown experiments, the processes of TFP-induced lysosomal membrane permeabilization and cell death appeared to be STAT3 dependent. In conclusion, our work provides a strong rationale for further clinical investigation of TFP as an adjuvant therapy for melanoma patients with metastases to the brain.

Effective Treatment of Metastatic Melanoma by Combining MAPK and PI3K Signaling Pathway Inhibitors.

Malignant melanoma is the most aggressive type of skin cancer and is closely associated with the development of brain metastases. Despite aggressive treatment, the prognosis has traditionally been poor, necessitating improved therapies. In melanoma, the mitogen activated protein kinase and the phosphoinositide 3-kinase signaling pathways are commonly altered, and therapeutically inhibiting one of the pathways often upregulates the other, leading to resistance. Thus, combined treatment targeting both pathways is a promising strategy to overcome this. Here, we studied the in vitro and in vivo effects of the PI3K inhibitor buparlisib and the MEK1/2 inhibitor trametinib, used either as targeted monotherapies or in combination, on patient-derived melanoma brain metastasis cell lines. Scratch wound and trans-well assays were carried out to assess the migratory capacity of the cells upon drug treatment, whereas flow cytometry, apoptosis array and Western blots were used to study apoptosis. Finally, an in vivo treatment experiment was carried out on NOD/SCID mice. We show that combined therapy was more effective than monotherapy. Combined treatment also more effectively increased apoptosis, and inhibited tumor growth in vivo. This suggests a clinical potential of combined treatment to overcome ceased treatment activity which is often seen after monotherapies, and strongly encourages the evaluation of the treatment strategy on melanoma patients with brain metastases.

Improved Drug Delivery to Brain Metastases by Peptide-Mediated Permeabilization of the Blood-Brain Barrier.

Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood-brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis.

Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months. Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance. Augmented mitochondrial respiration is a key resistance mechanism in BRAF-mutant melanomas but, as we show in this study, this dependence on mitochondrial respiration may also be exploited therapeutically. We first used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF-mutant melanoma brain metastases. One of the compounds identified was ß-sitosterol, a well-tolerated and brain-penetrable phytosterol. Here we show that ß-sitosterol attenuates melanoma cell growth in vitro and also inhibits brain metastasis formation in vivo. Functional analyses indicated that the therapeutic potential of ß-sitosterol was linked to mitochondrial interference. Mechanistically, ß-sitosterol effectively reduced mitochondrial respiratory capacity, mediated by an inhibition of mitochondrial complex I. The net result of this action was increased oxidative stress that led to apoptosis. This effect was only seen in tumor cells, and not in normal cells. Large-scale analyses of human melanoma brain metastases indicated a significant role of mitochondrial complex I compared to brain metastases from other cancers. Finally, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either ß-sitosterol or a functional knockdown of mitochondrial complex I. In conclusion, based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, ß-sitosterol represents a promising adjuvant to BRAF inhibitor therapy in patients with, or at risk for, melanoma brain metastases.

A Novel Nanoprobe for Multimodal Imaging Is Effectively Incorporated into Human Melanoma Metastatic Cell Lines.

To facilitate efficient drug delivery to tumor tissue, several nanomaterials have been designed, with combined diagnostic and therapeutic properties. In this work, we carried out fundamental in vitro and in vivo experiments to assess the labeling efficacy of our novel theranostic nanoprobe, consisting of glycogen conjugated with a red fluorescent probe and gadolinium. Microscopy and resazurin viability assays were used to study cell labeling and cell viability in human metastatic melanoma cell lines. Fluorescence lifetime correlation spectroscopy (FLCS) was done to investigate nanoprobe stability. Magnetic resonance imaging (MRI) was performed to study T1 relaxivity in vitro, and contrast enhancement in a subcutaneous in vivo tumor model. Efficient cell labeling was demonstrated, while cell viability, cell migration, and cell growth was not affected. FLCS showed that the nanoprobe did not degrade in blood plasma. MRI demonstrated that down to 750 cells/µL of labeled cells in agar phantoms could be detected. In vivo MRI showed that contrast enhancement in tumors was comparable between Omniscan contrast agent and the nanoprobe. In conclusion, we demonstrate for the first time that a non-toxic glycogen-based nanoprobe may effectively visualize tumor cells and tissue, and, in future experiments, we will investigate its therapeutic potential by conjugating therapeutic compounds to the nanoprobe.