Melanoma Brain Metastasis Model Research Articles

Abstract C030: Timing of melanoma brain metastasis seeding dictates the immunomodulatory effect and efficacy of systemic anti-CD40 agonist therapy

Abstract Introduction: Melanoma brain metastasis (MBM) affects nearly 50% of patients with advanced melanoma. Despite progress in systemic therapies, particularly immune checkpoint inhibitors, intracranial responses remain highly variable. The CD40/CD40L axis and its pleiotropic effects on the anti-tumoral immune response has the potential to improve MBM treatment. Our aim was to investigate whether the timing of MBM seeding, in a two-site mouse model of MBM, impacts the intracranial efficacy of anti-CD40 agonism (aCD40). Methods: 2 x 105 B16F1-cOVA murine melanoma cells were subcutaneously (s.c.) injected in the right flank in C57BL/6 mice (5F, 5M). After 3 days, 1 x 105 B16F1-cOVA cells were intracerebrally (i.c.) injected into the right striatum under stereotaxic guidance (early MBM’ cohort). A separate cohort of C57BL/6 mice (5F, 5M) had s.c. injection of B16F1-cOVA cells, followed by an i.c. injection of B16F1-cOVA cells after 5 days (‘late MBM’ cohort). In both experimental cohorts, the mice were randomly allocated to receive either an aCD40 antibody or an isotype IgG antibody (control) intraperitoneally on days 4 (D4) and 7 (D7) post-i.c. injection (D0). S.c. tumor volume and survival data were serially collected. Separately, tumor-bearing mice treated with either aCD40 or IgG (n = 4 per early/late MBM groups) were euthanized day 9 (D9) post-i.c. injection and tissues (blood, brain tumor [BT], and flank tumor [FT]) were collected for immune profiling by spectral flow cytometry. Results: The early MBM cohort showed a significant s.c. tumor response to aCD40 therapy with a mean D9:D4 s.c. tumor volume ratio of 2.81 (SD±2.05) vs 16.51 (±13.10) in the IgG group (unpaired t-test; p=0.0497). The late MBM cohort showed no significant difference in s.c. tumor response between aCD40 or IgG (D9:D4 ratio 1.52±1.00 vs 1.20±0.83; p=0.5999). Survival increased in early MBM following aCD40 vs IgG (median 20 days vs 13 days; logrank test, p=0.0078), which was not replicated in late MBM (median 21 days vs 19 days; p=0.7717). Systemic aCD40 led to reduced circulating B cells, most expressing CD11b, and increased monocytes, compared to IgG treatment. In the BT, there were higher infiltration of CD8 T cells (normalized to microglia), including OVA+ CD8 T cells, and CD4 T cells in early MBM treated with aCD40 vs IgG. Systemic aCD40 induced more infiltrating SIRP1a+ cDC2 cells than XCR1+ cDC1 cells and vice versa in the IgG group, in both the BT and FLT. Meanwhile, infiltrating B cells were predominantly CD11b+ in aCD40 groups in the BT, regardless of MBM timing. Conclusion: Systemic aCD40 therapy improved survival of MBM-bearing mice, with a shorter interval between s.c. and i.c. implantation; an effect abrogated by later MBM seeding. Systemic aCD40 therapy induced specific changes in the BT infiltrative immune profile, which may explain the distinct response between the early and late BM cohorts. These findings have implications on the experimental modelling of MBM and provide insight into variability of MBM response to immunomodulating therapies. Citation Format: Vinton W. T. Cheng, Victoria R. Breza, Beata Chertok, Natasha D. Sheybani, Timothy N. J. Bullock, Richard J. Price. Timing of melanoma brain metastasis seeding dictates the immunomodulatory effect and efficacy of systemic anti-CD40 agonist therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C030.

TMIC-12. LITT RESULTS IN ROBUST T CELL INFILTRATION WITHIN THE TUMOR MICROENVIRONMENT IN A BRAIN METASTASIS MURINE MODEL

Abstract INTRODUCTION Laser interstitial thermal therapy (LITT) is a novel minimally invasive neurosurgical technique used to ablate intra-axial brain tumors with real-time thermometry. The impact of LITT on the brain metastatic tumor microenvironment (TME) is unknown in patients due to limitations of tumor tissue collection post-LITT. The primary objective of this study was to describe the kinetics of immunologic infiltration within the TME in a melanoma brain metastasis model. METHOD: C57BL6 mice underwent intra-cranial implantation of B16F10-OVA tumor cells and treated with LITT using a custom-made murine LITT system developed in our laboratory. The tumors were treated to 42°C (near infra-red 1064 nm DPSS laser system) 7 days post-surgery. Tumors were collected 2 days and 10 days post-LITT for immune-phenotyping using flow cytometry. Blood was collected 6 h and 48 h post-LITT to perform the ELISA for IFN-g and TNF-a. RESULTS LITT treatment triggered a cascade of local infiltration of immune cells at different time points. 2-days post-LITT resulted in increased CD8 T cells (p<0.05), macrophage (p<0.05) and microglia infiltration (p<0.01) within the TME compared to control and sham surgery animals. By 10 days after LITT, significant infiltration by CD8 T cells (p<0.0001) and M1 macrophages (p<0.0001) was observed. The CD8 T cells were T central memory (TCM) and T effector memory (TEM). Moreover, comparison between 2 days and 10 days after LITT revealed significant increase of TEM (p<0.0001) and TCM (p<0.001) 10 days post-LITT. In an ELISA assay, non-significant differences were observed in IFN-g and TNF-a level at 48 h. CONCLUSIONS LITT enhances the recruitment of cytotoxic CD8 T cells within TME in a time-dependent manner. The ability of LITT to recruit TCM and TEM to the TME strongly support its potential therapeutic benefit, especially in the context of immunotherapy.

Antipsychotic Zuclopenthixol Inhibits Melanoma Growth and Brain Metastasis by Inducing Apoptosis and Cell Cycle Arrest.

The incidence of melanoma brain metastasis (MBM) is high and significantly compromises patient survival and quality of life. Effective treatment of MBM is made difficult by the blood-brain barrier (BBB), since it restricts the entry of drugs into the brain. Certain anti-psychotic drugs able to cross the BBB have demonstrated efficacy in suppressing brain metastasis in preclinical studies. However, the activity of zuclopenthixol against MBM is not yet clear. Cell viability assays were employed to investigate the potential of zuclopenthixol in the treatment of MBM. Subsequently, the mechanism of action was investigated by RNA-sequencing (RNAseq), flow cytometry-based cell cycle and apoptosis assays, protein expression analysis, and autophagy flux detection. Additionally, the efficacy of zuclopenthixol against tumor growth was investigated in vivo, including MBM models. Zuclopenthixol inhibited the proliferation of various melanoma cell lines at minimal doses by causing cell cycle arrest in the G0/G1 phase and mitochondrial-mediated intrinsic apoptosis. Zuclopenthixol also induced cytoprotective autophagy, and inhibition of autophagy enhanced the anti-melanoma effects of zuclopenthixol. Furthermore, zuclopenthixol inhibited the growth of human melanoma tumors in nude mice, as well as the growth of intracranial metastases in a mouse model of MBM. These results demonstrate that zuclopenthixol has significant potential as an effective therapeutic agent for MBM.

TMOD-05. EXTRACRANIAL TUMORS INFLUENCE INTRACRANIAL RESPONSE TO IMMUNE CHECKPOINT INHIBITORS IN PRE-CLINICAL MODELS OF MELANOMA BRAIN METASTASIS

Abstract Melanomas frequently spread to the brain, with up to 75% of patients developing brain metastases. Treatment of metastatic melanoma has been revolutionized by the advent of immune checkpoint inhibitors (ICI) targeting PD-1 and CTLA-4. However, responses to ICI within the immune-specialized brain environment are not well understood owing to the lack of immunocompetent animal models for the investigation of intracranial responses to checkpoint blockade. We studied the effect of ICI in a syngeneic mouse model of melanoma brain metastasis with concurrent intracranial and subcutaneous tumors. Either D3UV3 cells, derived from a UVB irradiated clone of D4M.3A melanoma cell line, or vehicle were subcutaneously injected into the flank of C57BL/6 mice. 3 days later, D3UV3 cells tagged with firefly luciferase were implanted into the striatum using stereotactic injection. Mice were then treated with anti-PD-1 antibody, anti-CTLA-4 antibody, a combination of anti-PD-1 and anti-CTLA-4 antibodies, or isotype controls. We observed striking differences in survival between mice with concurrent intracranial and subcutaneous tumors compared to intracranial tumors alone. Mice with intracranial tumors alone experienced no benefit in survival following monotherapy with anti-PD-1 (p=1) or anti-CTLA-4 (p=0.1) compared to isotype-treated mice, and only a slight benefit with combination treatment (p=0.049). In contrast, mice with concurrent tumors experienced a significant increase in overall survival with anti-CTLA-4 monotherapy (p=0.01) and combination anti-CTLA-4 and anti-PD-1 treatment (p=0.01) compared to isotype-treated mice, although treatment with anti-PD-1 alone did not increase survival (p=0.28). These results indicate that the presence of extracranial disease can modulate intracranial immune responses following checkpoint blockade. We have therefore established a pre-clinical model with concurrent intracranial and extracranial tumors to better recapitulate the clinically observed context of brain metastases. We hope this model will lead to a better understanding of the setting in which ICI is effective for patients with melanoma brain metastases.

62. PRESENCE OF EXTRACRANIAL TUMORS INFLUENCES RESPONSE TO IMMUNE CHECKPOINT INHIBITORS IN A PRE-CLINICAL MODEL OF MELANOMA BRAIN METASTASIS

Up to 75% of patients with melanoma develop brain metastases. While immune checkpoint inhibitors (ICI) targeting PD-1 and CTLA4 have revolutionized the treatment of metastatic melanoma, responses within the immune-specialized microenvironment of the brain are not well understood and there is a paucity of animal models to investigate the effect of ICI intracranially. We characterized responses to checkpoint inhibitors in a syngeneic mouse model of melanoma brain metastasis with concurrent intracranial and subcutaneous melanoma. D3UV3 cells (obtained from David Fisher’s laboratory) were derived using UVB irradiation from D4M.3A melanoma cell line and implanted into the striatum using stereotactic injection or subcutaneously injected into the flank of C57BL/6 mice. Mice were then treated with anti-PD-1 antibody, anti-CTLA4 antibody, a combination of anti-PD-1 and anti-CTLA4, or isotype controls. While mice with intracranial melanoma alone had no response to monotherapy with anti-PD-1 or anti-CTLA4 antibody (p=1 and 0.1, respectively), and only a slight response to combination therapy (p=0.049), mice with concurrent subcutaneous tumors had significantly improved responses to anti-PD-1, anti-CTLA4 and combination treatment (p=0.002, 0.01 and 0.01 respectively compared to mice with intracranial tumors alone with equivalent treatment). These results demonstrate that the presence of an extracranial tumor influences response to ICI in pre-clinical mouse models of melanoma brain metastasis. We have therefore established a pre-clinical model with concurrent intracranial and extracranial tumors to better recapitulate the clinically observed context of melanoma brain metastases and lead to a better understanding of the setting in which ICI are effective for patients with this devastating complication.

Upregulation of cell surface GD3 ganglioside phenotype is associated with human melanoma brain metastasis.

Melanoma metastasis to the brain is one of the most frequent extracranial brain tumors. Cell surface gangliosides are elevated in melanoma metastasis; however, the metabolic regulatory mechanisms that govern these specific changes are poorly understood in melanoma particularly brain metastases (MBM) development. We found ganglioside GD3 levels significantly upregulated in MBM compared to lymph node metastasis (LNM) but not for other melanoma gangliosides. Moreover, we demonstrated an upregulation of ST8SIA1 (GD3 synthase) as melanoma progresses from melanocytes to MBM cells. Using RNA‐ISH on FFPE specimens, we evaluated ST8SIA1 expression in primary melanomas (PRM) (n = 23), LNM and visceral metastasis (n = 45), and MBM (n = 39). ST8SIA1 was significantly enhanced in MBM compared to all other specimens. ST8SIA1 expression was assessed in clinically well‐annotated melanoma patients from multicenters with AJCC stage III B‐D LNM (n = 58) with 14‐year follow‐up. High ST8SIA1 expression was significantly associated with poor overall survival (HR = 3.24; 95% CI, 1.19–8.86, P = 0.02). In a nude mouse human xenograft melanoma brain metastasis model, MBM variants had higher ST8SIA1 expression than their respective cutaneous melanoma variants. Elevated ST8SIA1 expression enhances levels of cell surface GD3, a phenotype that favors MBM development, hence associated with very poor prognosis. Functional assays demonstrated that ST8SIA1 overexpression enhanced cell proliferation and colony formation, whereby ST8SIA1 knockdown had opposite effects. Icaritin a plant‐derived phytoestrogen treatment significantly inhibited cell growth in high GD3‐positive MBM cells through targeting the canonical NFκB pathway. The study demonstrates GD3 phenotype associates with melanoma progression and poor outcome.

Verification of 5-Aminolevurinic Radiodynamic Therapy Using a Murine Melanoma Brain Metastasis Model.

Melanoma is a highly aggressive cancer with a propensity for brain metastases. These can be treated by radiotherapy, but the radiation-resistant nature of melanoma makes the prognosis for melanoma patients with brain metastases poor. Previously, we demonstrated that treatment of mice with subcutaneous melanoma with 5-aminolevurinic acid (5-ALA) and X-rays in combination, (“radiodynamic therapy (RDT)”), instead of with 5-ALA and laser beams (“photodynamic therapy”), improved tumor suppression in vivo. Here, using the B16-Luc melanoma brain metastasis model, we demonstrate that 5-ALA RDT effectively treats brain metastasis. We also studied how 5-ALA RDT damages cells in vitro using a B16 melanoma culture. Cell culture preincubated with 5-ALA alone increased intracellular photosensitizer protoporphyrin IX. On X-ray irradiation, the cells enhanced their ∙OH radical generation, which subsequently induced γH2AX, a marker of DNA double-strand breaks in their nuclei, but decreased mitochondrial membrane potential. After two days, the cell cycle was arrested. When 5-ALA RDT was applied to the brain melanoma metastasis model in vivo, suppression of tumor growth was indicated. Therapeutic efficacy in melanoma treatment has recently been improved by molecular targeted drugs and immune checkpoint inhibitors. Treatment with these drugs is now expected to be combined with 5-ALA RDT to further improve therapeutic efficacy.

Abstract 1977: Chemokines expression in an in vitro model of melanoma brain metastasis

Abstract Melanoma brain metastasis is still a great challenge. This work aimed to characterize the secretion profile and understand the possible role of chemokines in a xenogeneic in vitro model of melanoma brain metastasis. Brain primary mixed cultures from cerebral cortex were prepared from the neonates Wistar rats and maintained at 37°C, 5% CO2until 80% of confluency and maturity. Melanoma lineages used were UACC62 and SKMEL-28. Co-cultures were performed to study the direct interactions between melanoma and cortical cells. The indirect effects in the cerebral microenvironment were evaluated through conditioned medium (CM) from brain cells on melanoma lineages. It was used an early CM collected at 24h (CM24) and a late CM collected after 48h (CM48), whose both were incubated during more 24h (24CM24 and 24CM48) or 48h (48CM24 and 48CM48) on melanoma cultures. The chemokines IP10, CCL5 and IL8 were studied through Cytometric Bead Array (CBA),MIG and CCL2 also, however their values were undetectable. After the characterization of the model, IP10 was silenced to evaluate its influence in the model. CBA demonstrated that in SKMEL-28 the late soluble factors during 48h (48CM48) were capable of rase IP10 levels (p=0,0286). On the other hand, direct and indirect contact with brain cells reduced IL8 levels, compared to SKMEL28 alone with respectively p = 0.02 and p=0,0286, where both chemokines were not detected. On UACC62, IP10 levels were higher only in the co-culture (p=0,02), suggesting the need of contact between the different cells. Also, IL8 was decreased in all conditions compared to UACC62. After IP10 silencing, SKMEL-28 presented higher levels of IL8 in 48CM48. On UACC62, the IP10 silencing resulted in elevation of IL8 levels when the cells were in co-culture. RANTES did not present alterations in its levels in any of the conditions after IP10 knock-down. The IP10 silencing was also capable of impair the transwell invasion of SKMEL-28 in the presence of CM24 and CM48. Together, these results confirmed that IP10 alters IL8 levels, but no RANTES in a mimetic microenvironment of melanoma brain metastasis. Furthermore, IP10 is capable of influencing SKMEL-28 migration in this xenogeneic model. More studies to better understand the role of IP10 and IL8 in the establishment of melanoma brain metastasis in this model are in progress. Citation Format: Renata Santos, Maria Clara Sampaio, Amanda Albuquerque, Michelly Pereira, Maira Pitta, Moacyr Rêgo. Chemokines expression in an in vitro model of melanoma brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1977.

Ultrasound-mediated delivery and distribution of polymeric nanoparticles in the normal brain parenchyma of a metastatic brain tumour model.

The treatment of brain diseases is hindered by the blood-brain barrier (BBB) preventing most drugs from entering the brain. Focused ultrasound (FUS) with microbubbles can open the BBB safely and reversibly. Systemic drug injection might induce toxicity, but encapsulation into nanoparticles reduces accumulation in normal tissue. Here we used a novel platform based on poly(2-ethyl-butyl cyanoacrylate) nanoparticle-stabilized microbubbles to permeabilize the BBB in a melanoma brain metastasis model. With a dual-frequency ultrasound transducer generating FUS at 1.1 MHz and 7.8 MHz, we opened the BBB using nanoparticle-microbubbles and low-frequency FUS, and applied high-frequency FUS to generate acoustic radiation force and push nanoparticles through the extracellular matrix. Using confocal microscopy and image analysis, we quantified nanoparticle extravasation and distribution in the brain parenchyma. We also evaluated haemorrhage, as well as the expression of P-glycoprotein, a key BBB component. FUS and microbubbles distributed nanoparticles in the brain parenchyma, and the distribution depended on the extent of BBB opening. The results from acoustic radiation force were not conclusive, but in a few animals some effect could be detected. P-glycoprotein was not significantly altered immediately after sonication. In summary, FUS with our nanoparticle-stabilized microbubbles can achieve accumulation and displacement of nanoparticles in the brain parenchyma.

Stem cell-released oncolytic herpes simplex virus has therapeutic efficacy in brain metastatic melanomas

The recent Food and Drug Administration approval of immunogenic oncolytic virus (OV) has opened a new era in the treatment of advanced melanoma; however, approximately 50% of patients with melanoma develop brain metastasis, and currently there are no beneficial treatment options for such patients. To model the progression of metastases seen in patients and to overcome the hurdles of systemic delivery of OV, we developed melanoma brain metastasis models in immunocompromised and immunocompetent mice, and tested the fate and efficacy of oncolytic herpes simplex virus (oHSV)-armed mesenchymal stem cells (MSCs). Using brain-seeking patient-derived melanoma cells and real-time in vivo imaging, we show a widespread distribution of micrometastases and macrometastases in the brain, recapitulating the progression of multifoci metastases seen in patients. We armed MSCs with different oHSV variants (MSC-oHSV) and found that intracarotid administration of MSC-oHSV, but not of purified oHSV alone, effectively tracks metastatic tumor lesions and significantly prolongs the survival of brain tumor-bearing mice. In a syngeneic model of melanoma brain metastasis, a combination of MSC-oHSV and PD-L1 blockade increases IFNγ-producing CD8+ tumor-infiltrating T lymphocytes and results in a profound extension of the median survival of treated animals. This study thus demonstrates the utility of MSCs as OV carriers to disseminated brain lesions, and provides a clinically applicable therapeutic platform to target melanoma brain metastasis.

Therapeutics for Brain Metastases, v3.

The role of blood-brain barrier (BBB) permeability in the efficacy of brain metastasis therapeutics is debated. Both BBB-permeable and BBB-impermeable compounds were compared in a melanoma brain metastasis model using imaging through a cranial window. Only the BBB-permeable compound inhibited both the ∼30% permeable metastases and the ∼70% impermeable metastases. Clin Cancer Res; 22(24); 5953-5. ©2016 AACRSee related article by Osswald et al., p. 6078.

Abstract 2073: Focused ultrasound-mediated transport of poly(alkyl) cyanoacrylate nanoparticles across the blood-brain barrier in a melanoma brain metastasis model

Abstract Drug delivery into the brain is impeded by the blood-brain barrier (BBB) - a dynamic interface that protects brain homeostasis, but also screens the brain from the vast majority of large and/or hydrophilic drug molecules. Focused ultrasound (FUS) has emerged as one of the promising methods to open the BBB safely and reversibly. We have previously reported FUS-mediated BBB opening using a novel platform consisting of microbubbles surrounded by poly(alkyl cyanoacrylate) nanoparticles that could prospectively be used for FUS- and nanoparticle-mediated drug delivery into the brain (1,2). Here, we have been investigating FUS-mediated BBB opening using a novel ultrasound system capable of generating FUS at two frequencies, 1.1 MHz and 7.8 MHz during the same experiment. This system allows a very precise selection of the exposure area. We used FUS exposure at 1 MHz to open the BBB by cavitation, while exposure at 7.8 MHz was employed to enable the action of acoustic radiation force. This force is caused by a transfer of momentum from acoustic waves to the tissues in which they propagate, and can facilitate nanoparticle transport in the extracellular matrix. FUS-mediated BBB opening was performed in NOD/SCID mice with melanoma brain metastases developed after intracardiac injection of human melanoma brain metastasis cells (3). The cells were fluorescently labeled, which enabled their detection in the brain. Poly(isohexyl) cyanoacrylate (PIHCA) nanoparticle-microbubbles similar to those reported in our earlier works (1,2) were injected immediately before the FUS exposure. Successful opening of the BBB was verified by MRI using a gadolinium-based contrast agent. An optimal window of exposure intensities that allow BBB disruption was found to be close a mechanical index of 0.3. Location of fluorescently labeled nanoparticles relative to blood vessels and tumor cells was determined in frozen sections using confocal microscopy. Tissue damage and FUS-induced changes at the cellular and molecular level were studied using histological and molecular techniques. In conclusion, the dual-frequency ultrasound transducer setup and our novel nanoparticle-microbubble platform showed promising results which will be used to develop a novel treatment of brain metastasis combining dual-frequency FUS with drug delivery using microbubbles. 1. Nanoparticle-stabilized microbubbles for multimodal imaging and drug delivery. Mørch Ý et al. Contrast Media Mol Imaging. 2015 Sep;10(5):356-66 2. Nanoparticle delivery to the brain - By focused ultrasound and self-assembled nanoparticle-stabilized microbubbles. Åslund AK et al. J Control Release. 2015 Oct 28;220(Pt A):287-294 3. Automated tracking of nanoparticle-labeled melanoma cells improves the predicted power of a brain metastasis model. Sundstrøm T et al. Cancer Res. 2013 Feb;73(8):2445-2456. Citation Format: Habib Baghirov, Andreas Åslund, Sofie Snipstad, Sigrid Berg, Rune Hansen, Frits Thorsen, Yrr Mørch, Catharina de Lange Davies. Focused ultrasound-mediated transport of poly(alkyl) cyanoacrylate nanoparticles across the blood-brain barrier in a melanoma brain metastasis model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2073.

Convection-enhanced delivery of sorafenib and suppression of tumor progression in a murine model of brain melanoma through the inhibition of signal transducer and activator of transcription 3.

OBJECT Despite recent advances, metastatic melanoma remains a terminal disease, in which life-threatening brain metastasis occurs in approximately half of patients. Sorafenib is a multikinase inhibitor that induces apoptosis of melanoma cells in vitro. However, systemic administration has been ineffective because adequate tissue concentrations cannot be achieved. This study investigated if convection-enhanced delivery (CED) of sorafenib would enhance tumor control and survival via inhibition of the signal transducer and activator of transcription 3 (Stat3) pathway in a murine model of metastatic brain melanoma. METHODS Melanoma cells treated with sorafenib in vitro were examined for signaling and survival changes. The effect of sorafenib given by CED was assessed by bioluminescent imaging and animal survival. RESULTS The results showed that sorafenib induced cell death in the 4 established melanoma cell lines and in 1 primary cultured melanoma cell line. Sorafenib inhibited Stat3 phosphorylation in HTB65, WYC1, and B16 cells. Accordingly, sorafenib treatment also decreased expression of Mcl-1 mRNA in melanoma cell lines. Because sorafenib targets multiple pathways, the present study demonstrated the contribution of the Stat3 pathway by showing that mouse embryonic fibroblast (MEF) Stat3 +/+ cells were significantly more sensitive to sorafenib than MEF Stat3 -/- cells. In the murine model of melanoma brain metastasis used in this study, CED of sorafenib increased survival by 150% in the treatment group compared with animals receiving the vehicle control (p < 0.01). CED of sorafenib also significantly abrogated tumor growth. CONCLUSIONS The data from this study indicate that local delivery of sorafenib effectively controls brain melanoma. These findings validate further investigation of the use of CED to distribute molecularly targeted agents.

Development and characterization of a clinically relevant mouse model of melanoma brain metastasis

Development and characterization of a clinically relevant mouse model of melanoma brain metastasis

Abstract A39: Multimodal imaging of blood-brain barrier disruption during brain metastatic progression in a relevant experimental mouse model

Abstract Introduction: Efficient drug delivery to brain metastases is difficult due to an often intact blood-brain barrier (BBB) around smaller lesions, and very few therapeutic drugs penetrate an intact BBB. Relevant experimental brain metastatic models are needed to study biological mechanisms and therapeutic responses of early brain lesions in relation to the BBB. Previous studies injecting small molecules such as sodium fluorescein (MW 376D) have indicated that the BBB is disrupted in experimental brain metastasis larger than 0.25 mm (1). However, little is known regarding at which time point in experimental brain metastasis development the BBB is disrupted, and to what extent larger sized molecules at that time are able to penetrate tumor tissue. We addressed these issues by performing multimodal imaging studies, after injecting contrast agents of various sizes into an established melanoma brain metastasis model (2, 3). Materials and Methods: All studies were approved by the UCD Animal Care and Use Committee. As in (3), we injected 5X105 human melanoma brain metastasis cells harboring the Luciferase and GFP genes, intracardially into 32 NOD/SCID mice. All animals were followed by bioluminescence imaging (BLI) weekly for 6 weeks. MRI (Bruker Biospec 70/30, RARE sequence, RARE factor=2, TE/TR/ST=9 ms/750 ms/1mm, FOV=2x2 cm, Matrix=256x256, NA=4) was performed at weeks 3, 4, 5 and 6 after tumor cell inoculation, before and after i.v. injections of either Gd-HPDO3A (Prohance, MW 559D, 0.5 μmol/g, 20 mice) or a newly synthetized Gadolinium contrast agent with 3 Gd(III) chelates, termed C3, (MW 2kD, 0.167 μmol/g, 5 mice). 64Cu-Albumin (MW 66.5kD) and 18F-FLT (MW 244D) was also injected i.v. prior to PET imaging at weeks 4 and 6. Results and Discussion: BLI visualized tumor cell spread in all animal brains 15mins after injections, and a gradual increase in tumor burden between weeks 1 to 6. T1w MRI at week 3 did not show any tumors, however BLI confirmed presence of metastasis. MRI detected tumors at week 4, and the mean, total number of tumors increased from 26 tumors at week 4, to 89 tumors at week 6. There was an exponential increase in tumor burden from week 4 to 6. The number of leaky tumors also increased exponentially, with mean diameters increasing from around 600 μm to around 700 μm. The smallest leaky tumors detectable by MRI were ~200 μm (week 5). The number of non-leaky tumors peaked at 5 weeks, and their mean diameters increased from 260 μm to 450 μm. Metastatic tumor cell proliferation was shown by 18F-FLT, which accumulated in the animal brains at weeks 4 and 6. 64Cu-Albumin PET also showed leakage of albumin into the metastatic lesions at week 6. Conclusions: Our study shows that T1w 7T MRI can detect leaky brain metastases down to ~200 μm in diameter. Molecules up to 2kD in size may penetrate brain tumor tissue early in tumor development. Our PET study indicates that larger molecules such as albumin may leak out of the metastatic lesions later in tumor development. References (1) Fidler IJ et al. Lancet Oncol 3:53-57, 2002. (2) Sundstrøm T et al, Cancer Res 2012 (In Press). (3) Wang J et al. Neuropathol Appl Neurobiol 37:189-205, 2011. Citation Format: Frits Thorsen, Brett Fite, Lisa Mahakian, Victoria Harrison, Sarah Johnson, Elizabeth Ingham, Shengping Qin, Jai W. Seo, Thomas Meade, Terje Sundstrøm, Katherine W. Ferrara. Multimodal imaging of blood-brain barrier disruption during brain metastatic progression in a relevant experimental mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A39.

Abstract 3790: Preclinical profile of LGX818: A potent and selective RAF kinase inhibitor

Abstract Selective RAF inhibitors have significant activity in patients with metastatic melanoma whose tumors express BRAFV600E. However, not all patients respond equally well to treatment and the duration of response is often limited to less than 6 months. LGX818 was developed with the hypothesis that a more potent inhibitor with excellent pharmacological properties would maximize the degree and duration of patient response. LGX818 is a highly potent RAF inhibitor with selective anti-proliferative and apoptotic activity in cells expressing BRAFV600E. In the A375 (BRAFV600E) human melanoma cell line LGX818 suppresses phospho-ERK (EC50 = 3 nM) leading to potent inhibition of proliferation (EC50 = 4 nM). No significant activity was observed against a panel of 100 kinases (IC50 &amp;gt; 900 nM) and LGX818 did not inhibit proliferation of &amp;gt; 400 cell lines expressing wild-type BRAF. Contributing to the high potency of LGX818 is the extremely slow off-rate from BRAFV600E which is not observed with other RAF inhibitors. In biochemical assays the dissociation half-life was &amp;gt;24 hours which translated into sustained target inhibition in cells following drug wash-out. Single dose PK/PD studies in human melanoma xenograft models (BRAFV600E) indicated that LGX818 treatment at oral doses as low as 6 mg/kg resulted in strong (75%) and sustained (&amp;gt;24 hours) decrease in phospho-MEK, even following clearance of drug from circulation. Decreases in phospho-ERK were consistent with phospho-MEK but markers of downstream transcriptional output (DUSP6 and SPRY4) appeared to provide a more sensitive measure of pathway activation. LGX818 induced tumor regression in multiple BRAF mutant human tumor xenograft models grown in immune compromised mice and rats at doses as low as 1 mg/kg. Consistent with the in vitro data, LGX818 was inactive against BRAF wild-type tumors at doses up to 300 mg/kg bid, with good tolerability and linear increase in exposure. Efficacy was also achieved in a more disease-relevant spontaneous metastatic melanoma and a model of melanoma brain metastasis. LGX818 is a potent and selective RAF kinase inhibitor with unique biochemical properties that contribute to an excellent pharmacological profile. A Phase I clinical trial in patients with BRAF mutant tumors is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3790. doi:1538-7445.AM2012-3790

The metastatic microenvironment: Brain‐residing melanoma metastasis and dormant micrometastasis

Brain metastasis occurs frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. We generated a reproducible melanoma brain metastasis model, consisting of brain-metastasizing variants and local, subdermal variants that originate from the same melanomas thus sharing a common genetic background. The brain-metastasizing variants were obtained by intracardiac inoculation. Brain metastasis variants when inoculated subdermally yielded spontaneous brain dormant micrometastasis. Cultured cells from the spontaneous brain micrometastasis grew very well in vitro and generated subdermal tumors after an orthotopic inoculation. Expression analysis assays indicated that the brain metastasis and micrometastasis cells expressed higher levels of angiopoietin-like 4, prostaglandin-synthesizing enzyme cyclooxygenase-2, matrix metalloproteinase-1 and preferentially expressed antigen in melanoma and lower levels of claudin-1 and cysteine-rich protein 61 than the corresponding cutaneous variants. The reproducible models of human melanoma metastasizing experimentally and spontaneously to the brain will facilitate the identification of novel biomarkers and targets for therapy and contribute to the deciphering of mechanisms underlying melanoma metastasis.

Abstract 1946: Identification of miRNAs that contribute to melanoma brain metastasis

Abstract Brain metastasis occurs in a large proportion of metastatic melanoma patients and is associated with a dismal prognosis. However, the molecular mechanisms that govern melanoma tropism to the brain remain poorly understood. MicroRNAs (miRNAs) play essential roles in many physiological and pathological processes, and have been recently shown to exert key roles during cancer metastasis. In this study we find that specific miRNAs may be important mediators of melanoma dissemination to the brain. First, we conducted a miRNA microarray analysis of metastatic melanoma tissues that revealed a subset of miRNAs differentially expressed in brain metastases (n=11) relative to other sites (n=48). A brain-specific signature comprised of seven miRNAs was further validated in an independent cohort of metastatic melanoma samples (n=36; 9 brain metastatic specimens). Then, we analyzed the trend of expression of those miRNAs during tumor progression by comparing their levels in primary tumors and their paired metastasis from patients with or without recurrence in the brain (n=18). Differential expression of some miRNAs was already evident at diagnosis in primary tumors that recurred in the brain, while for others it was acquired in the transition from primary to metastasis, suggesting that it may be a later event in tumor progression. Furthermore, in vitro modulation of specific signature miRNAs significantly altered the ability of melanoma cells to execute processes such as adhesion and transmigration through human brain endothelial cells and proliferation in human astrocytes conditioned media. Additionally, using an in vivo model of melanoma brain metastasis, we confirmed the capacity of specific miRNA alterations to promote melanoma cells’ competence to reach the brain. Finally, the analysis of potential downstream mediators of select miRNAs revealed the involvement of immuno-suppressive molecules, as well as inflammatory and chemotactic mediators in this process. Collectively, our results expand our understanding of the mechanisms that control melanoma brain metastasis, potentially revealing novel therapeutic avenues for patients for whom no viable approaches are currently available. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1946.

The sodium pump α1 sub‐unit: a disease progression–related target for metastatic melanoma treatment

Melanomas remain associated with dismal prognosis because they are naturally resistant to apoptosis and they markedly metastasize. Up-regulated expression of sodium pump α sub-units has previously been demonstrated when comparing metastatic to non-metastatic melanomas. Our previous data revealed that impairing sodium pump α1 activity by means of selective ligands, that are cardiotonic steroids, markedly impairs cell migration and kills apoptosis-resistant cancer cells. The objective of this study was to determine the expression levels of sodium pump α sub-units in melanoma clinical samples and cell lines and also to characterize the role of α1 sub-units in melanoma cell biology. Quantitative RT-PCR, Western blotting and immunohistochemistry were used to determine the expression levels of sodium pump α sub-units. In vitro cytotoxicity of various cardenolides and of an anti-α1 siRNA was evaluated by means of MTT assay, quantitative videomicroscopy and through apoptosis assays. The in vivo activity of a novel cardenolide UNBS1450 was evaluated in a melanoma brain metastasis model. Our data show that all investigated human melanoma cell lines expressed high levels of the α1 sub-unit, and 33% of human melanomas displayed significant α1 sub-unit expression in correlation with the Breslow index. Furthermore, cardenolides (notably UNBS1450; currently in Phase I clinical trials) displayed marked anti-tumour effects against melanomas in vitro. This activity was closely paralleled by decreases in cMyc expression and by increases in apoptotic features. UNBS1450 also displayed marked anti-tumour activity in the aggressive human metastatic brain melanoma model in vivo. The α1 sodium pump sub-unit could represent a potential novel target for combating melanoma.