The development of leptomeningeal melanoma metastases (LMM) is a rare and devastating complication of the late-stage disease, for which no effective treatments exist. Here, we performed a multi-omics analysis of the CSF from LMM patients to determine how the leptomeningeal microenvironment shapes the biology and therapeutic responses of melanoma cells. A total of 45 serial CSF samples were collected from 16 patients, 8 of these with confirmed LMM. Of those with LMM, 7 had poor survival (<4 months) and one was an extraordinary responder (still alive with survival >35 months). CSF samples were analyzed by mass spectrometry and incubated with melanoma cells, that were subjected to RNA-Seq analysis. Functional assays were performed to validate the pathways identified.Mass spectrometry analyses showed the CSF of most LMM patients to be enriched for pathways involved in innate immunity, protease-mediated damage, and IGF-related signaling. All of these were anti-correlated in the extraordinary responder. RNA-Seq analysis showed CSF to induce PI3K/AKT, integrin, B-cell activation, S-phase entry, TNFR2, TGF-β and oxidative stress responses in the melanoma cells. ELISA assays confirmed that TGF-β expression increased in the CSF of patients progressing with LMM. CSF from poorly responding patients conferred tolerance to BRAF inhibitor therapy in apoptosis assays. These analyses identified proteomic/transcriptional signatures in the CSF of patients who succumbed to LMM. We further showed that the CSF from LMM patients has the potential to modulate BRAF inhibitor responses and may contribute to drug resistance.
Overall design: WM164 cells were plated in a 6-well plate at 200,000 cells/well and allowed to attach overnight in normal culture media. Next day, the media was replaced to serum-free RPMI and cells were incubated overnight. Cells were treated with 3μM vemurafenib or vehicle control in 5% FBS/RPMI, or a 1:1 mixture of serum-free media:CSF from patient 1, patient 2 or patient 3 for 8 hours. RNA was extracted using Qiagen’s RNeasy Kit, with on-column DNase digestion (Hilden, Germany). RNA samples were reviewed for quality on the Agilent TapeStation followed by quantitation using the Qubit fluorometric assay. RNAseq libraries were processed using the Ovation Human FFPE RNA-Seq Multiplex System (NuGEN Technologies, San Carlos, CA). Briefly, 100 ng of RNA was used to generate cDNA and a strand-specific library following the manufacturer’s protocol. BioAnalyzer library size assessment and the Kapa Library Quantification Kit were used for library quantification. The libraries were sequenced on the Illumina NextSeq 500 v2 sequencer with two 75-base paired-end runs in order to generate 25-35 million read pairs per sample. Sequencing reads were subjected to a variety of pre- and post-alignment QC measures before mapped against the hg19 reference genome using TopHat 2.0.13. Gene-level quantification was determined using HTSeq 0.6.1 by summation of raw counts of reads aligned to the region associated with each gene. Normalization and differential expression analysis was performed using R/Bioconductor package DESeq2_1.6.3. Benjamini-Hochberg correction was used to adjust p-values to account for multiple comparisons.
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