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. 2024 Apr 18;81(1):188.
doi: 10.1007/s00018-024-05217-z.

Vascular mimicry as a facilitator of melanoma brain metastasis

Olivia K Provance #  1 , Victor O Oria #  2 , Thuy T Tran  1 , Jasmine I Caulfield  1 , Christopher R Zito  1   3 , Adam Aguirre-Ducler  4 , Kurt A Schalper  4 , Harriet M Kluger  1 , Lucia B Jilaveanu  5
Affiliations

Vascular mimicry as a facilitator of melanoma brain metastasis

Olivia K Provance et al. Cell Mol Life Sci. .

Abstract

Melanoma has the highest propensity among solid tumors to metastasize to the brain. Melanoma brain metastases (MBM) are a leading cause of death in melanoma and affect 40-60% of patients with late-stage disease. Therefore, uncovering the molecular mechanisms behind MBM is necessary to enhance therapeutic interventions. Vascular mimicry (VM) is a form of neovascularization linked to invasion, increased risk of metastasis, and poor prognosis in many tumor types, but its significance in MBM remains poorly understood. We found that VM density is elevated in MBM compared to paired extracranial specimens and is associated with tumor volume and CNS edema. In addition, our studies indicate a relevant role of YAP and TAZ, two transcriptional co-factors scarcely studied in melanoma, in tumor cell-vasculogenesis and in brain metastasis. We recently demonstrated activation of the Hippo tumor suppressor pathway and increased degradation of its downstream targets YAP and TAZ in a metastasis impaired cell line model. In the current study we establish the utility of anti-YAP/TAZ therapy in mouse models of metastatic melanoma whereby treatment effectively inhibits VM and prolongs survival of mice with MBM. The data presented herein suggest that VM may be an important and targetable mechanism in melanoma and that VM inhibition might be useful for treating MBM, an area of high unmet clinical need, thus having important implications for future treatment regimens for these patients.

Keywords: Brain metastasis; Melanoma; Vascular mimicry; YAP/TAZ.

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Conflict of interest statement

HMK holds institutional research grants from Merck, Bristol-Myers Squibb and Apexigen. HMK personal fees are associated with Iovance, Celldex, Merck, Bristol-Myers Squibb, Clinigen, Shionogi, Chemocentryx, Calithera, Signatero, Gigagen, GI Reviewers, Seranova, Pliant Therapeutics, and Esai. KAS is a consultant, advisor or speaker for Clinica Alemana Santiago, Shattuck Labs, AstraZeneca, EMD Serono, Takeda, Torque/Repertoire Therapeutics, Moderna Inc., Tesaro/GSK, Agenus, Genmab, OnCusp, Parthenon Therapeutics, Abbvie, Sanofi, Janssen, Bristol-Myers Squibb, Roche, Regeneron, PeerView, Forefront collaborataive, Molecular Templates and Merck. KAS holds research funding from Tesaro/GSK, Takeda, Surface Oncology, Merck, Bristol-Myers Squibb, AstraZeneca, Ribon Therapeutics, Eli Lilly, Boehringer-Ingelheim, Roche and Akoya Biosciences. However, this research has been conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest with these studies.

Figures

Fig. 1
Fig. 1
Intracranial melanoma metastases have higher levels of vascular mimicry than extracranial metastases: a Representative images of immunohistochemical staining for CD34 and PAS in extracranial and intracranial tumors isolated from patients at Yale University. Black arrow: CD34 + /PAS + , red arrow: CD34-/PAS + . Magnification of images are 20x with scale bars representing 50 µM. b Analysis of CD34-/PAS + vessels and CD43 + /PAS + vessels c in matched intracranial and extracranial metastasis patient samples. Black is intracranial, blue is extracranial, and the grey line connects the matched pairs. Analysis of CD34 + /PAS + and CD34-/PAS + vessels in intracranial metastases (d) and in extracranial metastases (e). The grey line connects values from the same tumor. Significance for all panels assessed by paired-Student’s t-test. ****p < 0.0001, ***p < 0.001, **p < 0.01
Fig. 2
Fig. 2
Vascular mimicry in melanoma brain metastases is associated with melanoma brain metastasis tumor volume and edema. a Two-sample unpaired t-test showing that high VM staining (continuous tumor CD34-/PAS+ scores) is significantly associated with high brain metastasis tumor volume (tumor volume scores dichotomized by the median value) (p = 0.0124). b Two-sample unpaired t-test showing that high blood vessel staining (continuous tumor CD3 + /PAS + scores) is not significantly associated with high brain metastasis tumor volume (tumor volume scores dichotomized by the median value) (p = 0.2402). c Continuous scores for VM staining (CD34-/PAS+) were plotted against intracranial edema scores dichotomized by the median value and significance assessed via two-sample unpaired T-test (p = 0.0415). d Two sample t-test of continuous BV scores (CD34+ /PAS+) and dichotomized edema scores (P = 0.1996)
Fig. 3
Fig. 3
Melanoma cell lines form vascular networks in vitro. Quantification of the number of meshes (a), junctions (b) and tube length (c), in a melanoma cell line panel and the representative images (d). Blue represents short-term cultured patient derived cell lines from intracranial melanoma metastases, black represents short-term cultured patient derived cell lines from extracranial metastases, red represents short-term cultures derived from extracerebral metastasis in patients who developed overt brain metastases (cerebrotropic) (d; 10 × magnification). Quantification of the number of meshes (e), junctions (f) and tube length (g), in primary and brain metastatic cell lines. Black represents primary, blue represents cerebrotropic derivatives. B16 variants and YUMM1.1 variants are mouse cell lines. h Representative images of VM structures formed by the cell lines in panels e–g (10 × magnification). All data are expressed as mean ± SD of three biological replicates and statistical significance is determined using an unpaired Student’s T-test. ****p < 0.0001
Fig. 4
Fig. 4
The effect of YAP/TAZ inhibitors on VM and growth in cerebrotropic derivatives of melanoma cell lines. CL.2A (a), YUMM1.1Br (b), and B16-F10 (c) cells were treated with increasing doses of CA3 and VP for 6 hrs at which point VM formation was assessed by mesh quantification. The number of meshes were counted for each treatment group. Black = DMSO, Blue = CA3, Green = VP. d CL.2A cells were treated with the active dose of CA3 (top, 1 µM) and VP (bottom, 1 µM), (e) YUMM1.1Br were treated with the active dose of CA3 (top, 4 µM) and VP (bottom, 4 µM), (f) and B16.F10 cells were treated with the active dose of CA3 (top, 1 µM) and VP (bottom, 0.5 µM), for 72 hrs and cell proliferation was assessed by Cell Titer Glo every 24 hrs. The 0 h timepoint represents cells assessed prior to drug treatment. Representative Relative Luminescent Units (RLU) are normalized by subtracting the 0 h values from subsequent measurements. All data are expressed as mean ± SD of at least three biological replicates and statistical significance is determined using an unpaired Student’s T-test at each time point. ****p < 0.0001, **p < 0.01, *p < 0.05
Fig. 5
Fig. 5
The effect of YAP/TAZ inhibitors on VM and growth in patient derived melanoma brain metastasis cultures. a YUGANK, YUVENA, YUKRIN and YUSIK were treated with 0.5 µM CA3 (blue, left) or 0.5 µM VP (green, right) for 6 h at which point VM formation was assessed by mesh quantification. YUGANK (b), YUVENA (c), YUKRIN (d) and YUSIK (e), were treated with the active dose of CA3 (0.5 µM) and VP (0.5 µM) for 72 hrs and cell proliferation was assessed by Cell Titer Glo every 24 hrs. The 0 h timepoint represents cells assessed prior to drug treatment. Representative Relative Luminescent Units (RLU) are normalized by subtracting the 0 h values from subsequent measurements. All data are expressed as mean ± SD of at least three biological replicates and statistical significance is determined using an unpaired Student’s T-test at each time point. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05
Fig. 6
Fig. 6
CA3 prolongs overall and brain metastasis survival in a murine model of metastasis. a–c Kaplan–Meier curves for mice receiving 1 mg/kg to 10 mg/kg treatment in a left ventricle injection murine model of brain metastasis. KM curves demonstrating the correlation between CA3 treatment and time overall survival (a, log-rank test, p = 0.022), the time to death from first metastasis diagnosis (b, log rank test, p = 0.126) and from brain metastasis diagnosis (c, log rank test, p = 0.004). d Representative images of immunohistochemical staining for YAP, TAZ, BIRC5, and IgG in Cl.2A brain tumors developed after left-ventricle injection in nude mice treated as controls, or with 1 mg/kg CA3. Scale bars represent 200 µM. e Representative images of immunohistochemical staining for CD34 and PAS in the same tumors. Black arrows point to BV, red arrows point to VM structures. Scale bars represent 50 µM. Quantification of VM (f; CD34-/PAS+) and BV (g; CD34+/PAS +) in nine areas from two tumors of each treatment group. Students t-test was used to assess significance between vascular density in control and CA3 treated mice. **p < 0.001
Fig. 7
Fig. 7
Assessment of VM in subcutaneous and brain metastatic mouse models of melanoma treated with anti-angiogenic drugs. a Representative images of immunohistochemical staining for CD34 and PAS in B16.F10 subcutaneous tumors harvested from C57BL/6 mice treated as controls, with 5 mg/kg αVEGF, or with 10 mg/kg lenvatinib. Black arrow: BV; CD34 + /PAS + , red arrow: VM; CD34-/PAS + . Magnification: 20×. b Quantification of the number of BV and c Quantification of VM vessels in three areas from two tumors of each treatment group. d Representative images of immunohistochemical staining for CD34 and PAS in YUMMER1.7 subcutaneous tumors harvested from C57BL/6 mice treated as controls, with 5 mg/kg αVEGF, or with 10 mg/kg lenvatinib. Black arrow: BV; CD34 + /PAS + , red arrow: VM; CD34-/PAS + . Magnification: 20×. e Quantification of the number of BV and VM (f) in three areas from two tumors of each treatment group. g Representative images of immunohistochemical staining for CD34 and PAS in YUMMER1.7 brain tumors developed after left-ventricle injection in C57BL/6 mice treated as controls, with 5 mg/kg αVEGF, or with 10 mg/kg lenvatinib h Quantification of the number of BV, VM (i) in three areas from two tumors of control and αVEGF, and one tumor from lenvatinib. All data are expressed as mean ± SD showing all points. All scale bars represent 200 µM. Statistical significance is determined using a one-way ANOVA ****p < 0.0001, **p < 0.001, *p < 0.05
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