Abstract
Abstract Background Brain metastases (BM) are a frequent complication of malignant melanoma (MM), with limited treatment options and poor survival. Prevention of BM could be more effective and better tolerated than treating established BM in various conditions. Methods To investigate the temporospatial dynamics of PI3K/Akt/mTOR (PAM) pathway activation during BM formation and the preventive potential of its inhibition, in vivo molecular imaging with an Akt biosensor was performed, and long-term intravital multiphoton microscopy through a chronic cranial window in mice. Results In vivo molecular imaging revealed invariable PAM pathway activation during the earliest steps of brain colonization. In order to perform a long-term intravascular arrest and to extravasate, circulating MM cells needed to activate their PAM pathway during this process. However, the PAM pathway was quite heterogeneously activated in established human brain metastases, and its inhibition with the brain-penetrant PAM inhibitor GNE-317 resulted in only modest therapeutic effects in mice. In contrast, giving GNE-317 in preventive schedules that included very low doses effectively reduced the growth rate and number of BM in two MM mouse models over time, and led to an overall survival benefit. Longitudinal intravital multiphoton microscopy found that the first, rate-limiting steps of BM formation—permanent intravascular arrest, extravasation, and initial perivascular growth—are most vulnerable to dual PI3K/mTOR inhibition. Conclusion These findings establish a key role of PAM pathway activation for critical steps of early metastatic brain colonization and reveal its pharmacological inhibition as a potent avenue to prevent the formation of clinically relevant BM.
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📋 Methods
To investigate the temporospatial dynamics of PI3K/Akt/mTOR (PAM) pathway activation during BM formation and the preventive potential of its inhibition, in vivo molecular imaging with an Akt biosensor was performed, and long-term intravital multiphoton microscopy through a chronic cranial window in mice.
Material and Methods
For additional Material and Methods, see Supplementary Information .
Cell Lines Six different models of human melanoma
BM were used; 4 primary cell lines isolated from human melanoma BM, H1Dl2, 17 and brain-passaged A2058 cells. 18 For in vivo imaging cells expressing green fluorescent protein (GFP) or red fluorescent protein (RFP) were used. 12 A2058 cells were transduced with F01-Clover Akt biosensor 19 or LeGO-it2-luc2-ires-tdTomato (a kind gift from Jonas Schwickert and Andreas Trumpp).
Assessment of PAM Pathway Activation In Vitro
Melanoma cells were treated with 0.1/1/5 µM GNE-317, pictilisib, or the same volume of dimethyl sulfoxide (DMSO) and stained for phospho-Akt or Annexin V. Flow cytometry data were recorded on a FACS Canto II (BD Biosciences) and analyzed using FlowJo version 10 (FlowJo LLC, USA). For Western Blot, A2058 cells were treated for 10 h with 30 µM GNE-317, 10 µM pictilisib, or DMSO. Protein expression of Akt, S6RP, mTOR, and their phosphorylated forms was analyzed.
Immunohistochemistry
Detection of full protein RPS6, 4EBP1, PRAS40, and their phosphorylated forms were automatically performed as similar as described previously 20 (adjustments outlined in Supplementary Material ) on 3 µm thick slides from FFPE tissue microarrays. The study was endorsed by the local ethical committee of the Goethe University Frankfurt, Germany (GS 4/09; SNO_01-12). Proliferation Assays In Vitro Melanoma cells were treated with different doses of GNE-317 or pictilisib, and were monitored in an xCELLigence Real-Time Cell Analyzer (RTCA) system (Roche Diagnostics) as described before. 12 Intravital Microscopy NOD SCID gamma mice (NSG, >8 weeks, >20 g, inhouse bred) were anesthetized with ketamine/xylazine and a chronic cranial window was implanted as described before. 16 At least 3 weeks later, melanoma tumor cells were suspended in phosphate-buffered saline and injected into the left cardiac ventricle. 16 In vivo imaging was performed using a ZEISS LSM 7MP equipped with a Coherent Chameleon Ultra II laser, as previously described. 16 All animal procedures were performed according to the German Cancer Research Center’s guidelines and were approved by the Regierungspraesidium Karlsruhe, Germany (governmental authority for laboratory animal research). GNE-317 Treatment In Vivo NSG mice with previously implanted chronic cranial window received GNE-317 (Genentech, 2.5/12.5/25 mg/kg/day body weight, in 0.5% methylcellulose/0.2% polysorbate (MCT)) via oral gavage in a preventive schedule from day 4 to day 28 or in a treatment schedule from day 21 to day 28. Control mice received a similar volume of MCT from day 4 to day 28. Akt Biosensor Studies A2058 melanoma cells transduced with Akt biosensor were treated with 30 µM GNE-317, 10 µM pictilisib, or DMSO in medium for 10h, followed by live-cell in vitro imaging. Cytoplasm/nucleus ratio of GFP was calculated, low Akt activity was defined as a ratio below 1 standard deviation (SD) of control cells with high Akt activity. For in vivo studies, low Akt activity was confirmed via fluorescence microscopy. Intravital microscopy was performed
Show full methods section
To investigate the temporospatial dynamics of PI3K/Akt/mTOR (PAM) pathway activation during BM formation and the preventive potential of its inhibition, in vivo molecular imaging with an Akt biosensor was performed, and long-term intravital multiphoton microscopy through a chronic cranial window in mice.
Material and Methods
For additional Material and Methods, see Supplementary Information .
Cell Lines Six different models of human melanoma
BM were used; 4 primary cell lines isolated from human melanoma BM, H1Dl2, 17 and brain-passaged A2058 cells. 18 For in vivo imaging cells expressing green fluorescent protein (GFP) or red fluorescent protein (RFP) were used. 12 A2058 cells were transduced with F01-Clover Akt biosensor 19 or LeGO-it2-luc2-ires-tdTomato (a kind gift from Jonas Schwickert and Andreas Trumpp).
Assessment of PAM Pathway Activation In Vitro
Melanoma cells were treated with 0.1/1/5 µM GNE-317, pictilisib, or the same volume of dimethyl sulfoxide (DMSO) and stained for phospho-Akt or Annexin V. Flow cytometry data were recorded on a FACS Canto II (BD Biosciences) and analyzed using FlowJo version 10 (FlowJo LLC, USA). For Western Blot, A2058 cells were treated for 10 h with 30 µM GNE-317, 10 µM pictilisib, or DMSO. Protein expression of Akt, S6RP, mTOR, and their phosphorylated forms was analyzed.
Immunohistochemistry
Detection of full protein RPS6, 4EBP1, PRAS40, and their phosphorylated forms were automatically performed as similar as described previously 20 (adjustments outlined in Supplementary Material ) on 3 µm thick slides from FFPE tissue microarrays. The study was endorsed by the local ethical committee of the Goethe University Frankfurt, Germany (GS 4/09; SNO_01-12). Proliferation Assays In Vitro Melanoma cells were treated with different doses of GNE-317 or pictilisib, and were monitored in an xCELLigence Real-Time Cell Analyzer (RTCA) system (Roche Diagnostics) as described before. 12 Intravital Microscopy NOD SCID gamma mice (NSG, >8 weeks, >20 g, inhouse bred) were anesthetized with ketamine/xylazine and a chronic cranial window was implanted as described before. 16 At least 3 weeks later, melanoma tumor cells were suspended in phosphate-buffered saline and injected into the left cardiac ventricle. 16 In vivo imaging was performed using a ZEISS LSM 7MP equipped with a Coherent Chameleon Ultra II laser, as previously described. 16 All animal procedures were performed according to the German Cancer Research Center’s guidelines and were approved by the Regierungspraesidium Karlsruhe, Germany (governmental authority for laboratory animal research). GNE-317 Treatment In Vivo NSG mice with previously implanted chronic cranial window received GNE-317 (Genentech, 2.5/12.5/25 mg/kg/day body weight, in 0.5% methylcellulose/0.2% polysorbate (MCT)) via oral gavage in a preventive schedule from day 4 to day 28 or in a treatment schedule from day 21 to day 28. Control mice received a similar volume of MCT from day 4 to day 28. Akt Biosensor Studies A2058 melanoma cells transduced with Akt biosensor were treated with 30 µM GNE-317, 10 µM pictilisib, or DMSO in medium for 10h, followed by live-cell in vitro imaging. Cytoplasm/nucleus ratio of GFP was calculated, low Akt activity was defined as a ratio below 1 standard deviation (SD) of control cells with high Akt activity. For in vivo studies, low Akt activity was confirmed via fluorescence microscopy. Intravital microscopy was performed
📊 Figures
Fig. 4
Impact of a dual PI3K/mTOR inhibitor on melanoma cells in vitro and in vivo . (A) Representative in vivo molecular imaging examples of biosensor labeled A2058 cells, showing a reduction of Akt activit...
Fig. 1
Establishment and validation of an Akt biosensor to measure PAM pathway activation. (A) A2058 melanoma cells expressing the Akt biosensor (green) were seeded in matrigel and incubated with dual PI3K/m...
Fig. 2
PAM pathway activation is an invariable early event in melanoma BM. (A) Intracardiac injection of biosensor labeled A2058 melanoma cells in mice for longitudinal intravital multiphoton microscopy. In ...
Fig. 3
Akt and mTOR signaling in established human BM. (A) Expression of PAM pathway targets in FFPE tissue microarrays of human melanoma BM. (B) Signaling of phosphorylated PAM pathway targets in specimen o...
Fig. 5
Preventive vs therapeutic schedules. (A) Longitudinal intravital multiphoton microscopy investigating GNE-317 treatment in preventive and treatment schedules in A2058 and H1 melanoma models. (B) Diffe...
Fig. 6
The earliest steps of the brain metastatic cascade are most vulnerable to PAM pathway inhibition. (A) Tumor cell survival between days 3 and 28 after intracardiac injection. A2058 cells, intravital mu...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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