Spatial transcriptomics analysis identifies a unique tumor-promoting function of the meningeal stroma in melanoma leptomeningeal disease

Abstract

Leptomeningeal disease (LMD) remains a rapidly lethal complication for late-stage melanoma patients. The inaccessible nature of the disease site and lack of understanding of the biology of this unique metastatic site are major barriers to developing efficacious therapies for patients with melanoma LMD. Here, we characterize the tumor microenvironment of the leptomeningeal tissues and patient-matched extra-cranial metastatic sites using spatial transcriptomic analyses with in vitro and in vivo validation. We show the spatial landscape of melanoma LMD to be characterized by a lack of immune infiltration and instead exhibit a higher level of stromal involvement. We show that the tumor-stroma interactions at the leptomeninges activate pathways implicated in tumor-promoting signaling, mediated through upregulation of SERPINA3 at the tumor-stroma interface. Our functional experiments establish that the meningeal stroma is required for melanoma cells to survive in the CSF environment and that these interactions lead to a lack of MAPK inhibitor sensitivity in the tumor. We show that knocking down SERPINA3 or inhibiting the downstream IGR1R/PI3K/AKT axis results in re-sensitization of the tumor to MAPK-targeting therapy and tumor cell death in the leptomeningeal environment. Our data provides a spatial atlas of melanoma LMD, identifies the tumor-promoting role of meningeal stroma, and demonstrates a mechanism for overcoming microenvironment-mediated drug resistance unique to this metastatic site.

Keywords: CSF; brain; leptomeninges; melanoma.

Figure 1.. Spatial atlas of melanoma LMD compared to other sites of disease.

A. Schematic of the spatial transcriptomic analysis workflow, including tissue selection, preparation, sequencing, data quality control, and cell type deconvolution analysis. Created with BioRender.com B. Tissue maps showing spatial cell type deconvolution for each spot. The cell type color key is the same for panels B and C. C. Bar graphs showing the average deconvolved cell proportion for the major cell types identified in each sample, organized by tissue of origin. D. Scatter plots showing the mean proportion of spots predominantly comprised of immune and stromal cells in extra-cranial versus leptomeningeal metastasis samples.

Figure 2.. Spatially driven signaling in melanoma LMD.

A. Schematic showing the basic principles behind the algorithm used to identify KEGG pathway enrichment that have significant spatial patterns (left) and a heatmap highlighting the quantity and similarities/differences of KEGG pathways showing significant spatial pattern within each tissue sample (right). B. Spatial tissue maps showing the position of the stroma, immune, and tumor spots along with tissue maps visualizing the average gene set expression for major pathways important in melanoma biology and drug resistance for each LMD sample. C. A schematic showing the workflow for determining which genes show a correlation between the level of expression and the distance between tumor and stroma spots. D. A heatmap of the genes showing a significant correlation between expression and the distance between tumor and stroma spots across multiple LMD samples. E. Spatial tissue maps showing the position of the stroma, immune, and tumor spots along with tissue maps visualizing the log 2 gene expression for GFAP and SERPINA3, and the log2 max expression for sets of genes that are MAPK targets, mTOR targets, and CAF markers (Supplemental Table 4).

Figure 3.. Meningeal cells are required for melanoma survival in the CSF environment.

A. Representative microscopy images showing GFP-tagged WM164 melanoma cells treated with 3μM vemurafenib (BRAFi) or DMSO control in monoculture versus co-culture with primary meningeal cells (left). Bar graphs show the quantification of GFP+ cells across ten biological replicates relative to the respective vehicle-treated controls (right). B. MTT assay showing the relative growth of WM164 melanoma cells treated with increasing doses of vemurafenib (BRAFi) in control media versus media conditioned by the co-culture of melanoma with primary meningeal cells. C. Representative microscopy images showing GFP-tagged WM164 melanoma cells treated with 3μM vemurafenib (BRAFi) or DMSO control in monoculture versus co-culture with primary meningeal cells in the context of CSF. D. Quantification of the GFP+ WM164 melanoma cells treated with 3μM vemurafenib (BRAFi) or DMSO control in monoculture versus co-culture with primary meningeal cells for ten days in CSF (left). WM164 melanoma cells treated with 3μM vemurafenib (BRAFi) or DMSO control in with regular CSF vs CSF conditioned by co-culture of melanoma with primary meningeal cells over 13 days (right). Significance was calculated between the co-culture or conditioned media arm and the respective monoculture control arm E. Flow cytometry assessment of cell cycle using PI staining in WM164 melanoma cells treated with 3μM vemurafenib or DMSO in the context of direct co-culture with primary meningeal cells or with CSF conditioned by co-culture of melanoma with primary meningeal cells. F. Western blot analysis of WM164 melanoma cells treated with 3μM vemurafenib or DMSO control in fresh CSF vs conditioned CSF showing abundance of SERPINA3, pERK(Thr202/Tyr204), ERK, pAKT (Ser473) and AKT. G. Western blot analysis of primary meningeal cells stimulated with WM164-conditioned CSF showing the abundance of SERPINA3, cyclin D1, TGFβ1, TGFβR, and fibronectin. All statistical significance was assessed using Student’s t-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4.. Meningeal cells promote growth and resistance to MAPKi in vivo.

A. A schematic outlining the experimental cohorts of the animal experiment. Mice were injected subcutaneously with 1 million WM164 cells alone, 1 million WM164 cells with 1 million primary meningeal cells (MC), or 1 million WM164 cells with 1 million dermal fibroblast (DF) cells. Mice were fed a control rodent diet (control) or a rodent diet formulated with 200 mg/kg dabrafenib and 2 mg/kg trametinib (MAPKi). Created with BioRender.com B. Line graph showing the average growth of tumors in each experimental cohort over time, with error bars depicting standard error. Tumor size was fixed following the endpoint of animals until the last animal in the cohort reached the endpoint. C. Square root transformation and slope visualization for each experimental cohort in panel B. D. p-values for each two-way comparison of slope among experimental cohorts (ANCOVA). Icons created with BioRender.com E. Bar graph visualizing the average tumor volume in each experimental cohort on day 21. Data represent mean ± standard error. Statistical significance was assessed using Student’s t-test *p < 0.05, **p < 0.01.

Figure 5.. Abrogating SERPINA3/IGF1R signaling sensitized melanoma to MAPKi in the CSF environment.

A. Representative microscopy images showing GFP-tagged WM164 melanoma cells treated with DMSO control, 100nM dabrafenib/10nM trametinib (MAPKi), 1μM linsitinib (IGF1Ri), or the triple combination in monoculture versus co-culture with primary meningeal cells in the context of CSF. B. Quantification of the GFP+ WM164 melanoma cells treated with 3 DMSO control, 100nM dabrafenib/10nM trametinib (MAPKi), 1μM linsitinib (IGF1Ri), or the triple combination in monoculture versus co-culture with primary meningeal cells for 15 days in CSF. C. Western blots showing knockdown of SERPINA3 with two individual SERPINA3 siRNAs (siRNA1 and siRNA2) in the WM164 melanoma cells and the HMC meningeal cells. Scrambled sequence siRNA was used as control (Con siRNA) D. Representative microscopy images showing GFP-tagged WM164 melanoma cells treated with 3μM vemurafenib (BRAFi) or DMSO (Control) in monoculture versus co-culture (Co) with primary meningeal cells in the context of CSF following knockdown of SERPINA3 (siRNA 1 and siRNA2) or Control siRNA (Con siRNA) in both cell types. E. Quantification of GFP-tagged WM164 cells from panel D. Statistical significance was assessed using Student’s t-tests (panels B, C and E). Significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant