Single-cell sequencing depicts tumor architecture and empowers clinical decision in metastatic conjunctival melanoma

Conjunctival melanoma (CoM) is a potentially devastating tumor that can lead to distant metastasis. Despite various therapeutic strategies for distant metastatic CoM, the clinical outcomes remain unfavorable. Herein, we performed single-cell RNA sequencing (scRNA-seq) of 47,017 cells obtained from normal conjunctival samples (n = 3) and conjunctival melanomas (n = 7). Notably, we noticed a higher abundance of cancer-associated fibroblasts (CAFs) in tumor microenvironment (TME), correlated with enhanced angiogenic capacity and increased VEGFR expression in distal metastatic CoM. Additionally, we observed a significant decrease in the proportion of total CD8+ T cells and an increase in the proportion of naive CD8+ T cells, contributing to a relatively quiescent immunological environment in distal metastatic CoM. These findings were confirmed through the analyses of 70,303 single-cell transcriptomes of 7 individual CoM samples, as well as spatially resolved proteomes of an additional 10 samples of CoMs. Due to the increase of VEGFR-mediated angiogenesis and a less active T cell environment in distal metastatic CoMs, a clinical trial (ChiCTR2100045061) has been initiated to evaluate the efficacy of VEGFR blockade in combination with anti-PD1 therapy for patients with distant metastatic CoM, showing promising tumor-inhibitory effects. In conclusion, our study uncovered the landscape and heterogeneity of the TME during CoM tumorigenesis and progression, empowering clinical decisions in the management of distal metastatic CoM. To our knowledge, this is the initial exploration to translate scRNA-seq analysis to a clinical trial dealing with cancer, providing a novel concept by accommodating scRNA-seq data in cancer therapy.


Materials and Methods
Benign lesions Melanoma lesions Epithelial cells

Fig
Fig. S1 to S8 for multiple supplementary figures Fig. S2 Fig. S3 UMAP plot showing diverse cell distribution colored by patient origin.(d) Chromosomal copy-number variations (CNVs) in each single cell from different samples were inferred according to the averaged expression profiles across chromosomal intervals in validation cohort 1. Supplementary Figure S4.Transcriptional trajectory analysis of CoM.(a) Transcriptional trajectory of different process colored by clusters.(b) Pseudotime state of all melanoma cells from both the ST and DMT groups colored by patient origin.(c-f) Transcriptional trajectory analysis showing different process of melanoma cells by color, states (c), clusters (d), ST or DMT group origin (e-f).
Fig. S5 Violin plots indicating the expression of angiogenesis-associated genes in CAFs (orange) and myofibroblasts (blue) in discovery cohort.(d) Dot plot showing the expression levels of the specific cell marker genes in different cell types.The size of the dot suggests the proportion of cells expressing the marker genes.The spectrum of colors indicates the mean expression levels of the marker genes.(e) Cell phone analysis showing the interaction of melanoma cells, CAFs and the immune microenvironment.The spectrum of colors indicates the correlation between two cell types.(f) UMAP plot showing the subtypes of CAFs derived from ST and DMT patients in validation cohort 1, colored by cell type, myofibroblasts in blue and CAFs in orange.(g) UMAP plot in validation cohort 1 was stained by the expression of marker genes of myofibroblasts (TAGLN, MYL9), and CAFs (FAP, MMP2).(h) Bar plots showing the proportion of CAFs (orange) and myofibroblasts (blue) in ST (left) and DMT (right) samples.Cell type annotations are provided in the figure (right).The difference was statistically significant.All statistical analyses are Chi-square tests.(i) Violin plots indicating the expression of angiogenesis-associated genes in CAFs (orange) and myofibroblasts (blue) in validation cohort 1. (j) Isolation of CAF subtypes (FAP positive) from CoM tumor tissues via FACS.The clinical distal metastatic tissues were derived from Mel0618.(k) IF staining with FAP (up) and MYL9 (bottom) antibody in CAFs (left) and myofibroblast (right).Scale bars, 50 μm.Supplementary Figure S6.Molecular characteristics of immune cells in discovery cohort and validation cohort 1 of CoM samples.(a) Proportion of each immune cell subset in the ST group (blue) and DMT group (red) from discovery cohort.Two-tailed unpaired Student's t-test.(b) Proportion of each immune cell subset in the ST group (blue) and DMT group (red) from validation cohort 1. One-tailed unpaired Student's t-test.(c) The proportions of specific subsets of CD8 + T cells between the ST group and DMT group were analyzed (left).Pie charts showing the proportion of CD8 + Tn cells (red) in ST and DMT groups (right) from validation cohort 1.(d) The infiltration percentage of specific subsets of CD8 + T cells between the ST group (blue, n=3) and DMT group (red, n=4) were analyzed in discovery cohort.Two-tailed Fig. S6 a b (a) Heatmap plot showing the different expression in ST group (blue) and DMT group (orange), upregulated proteins in red and down regulated proteins in blue.

Table S1 .
Patient and tumor characteristics at presentation for single cell RNA sequencing

Table S2 .
Conjunctival melanoma Patient and Tumor Characteristics in validation cohort.

Table S3 .
Cell number of assigned cell clusters in CoM and Benign conjunctival samples