Fig 2: Functional impacts of a HSCR-associated SNP (rs2435357) and the deletion of a novel S-HSCR enhancer on RET expression. (A) ATAC-seq and ChIP-seq profiles of hPSC and hNC in intron 1 of RET show that rs2435357 is residing in a hNC-specific ATAC-seq peak. (B) Location of rs2435357 in the RET gene locus and in the sgRNA used for CRISPR-Cas9-mediated HDR for editing the C allele to the HSCR-associated risk allele T. The electrographs of Sanger sequencing show the successful introduction of the risk allele at rs2435357 in the UE-rs2435357 hPSC line. (C) Differentiation strategy to generate human neural crest (hNC) and neuronal progenitor (hNP). HU is encoded by the ELAVL4 gene. Immunostaining of SOX10 and TUJ1 in hNC and hNP of the control and the mutant (UE-rs2435357) lines. Scale bars: (hNC) 100 µm; (hNP) 200 µm. RT-qPCR analysis showing the comparable ELAVL4 expression level in hNP in the control (n = 5) and the mutant (UE-rs2435357) (n = 3) lines. t-test, (ns) not significant. (D) RT-qPCR analysis showing RET expression in the hPSC and hNP stages of the control (n = 5) and the mutant (UE-rs2435357) (n = 3). t-test, (ns) not significant. (E) Hi-C data from neural progenitor cells show that the enhancer in intron 1 of RASGEF1A (marked in yellow on the right) has physical interaction with the promoter of RET (marked in yellow on the left) at 10-kbp bin size. (F) ATAC-seq and ChIP-seq data from hPSC and hNC at the RASGEF1A intron 1 locus. (G) The design of sgRNAs used for the CRISPR-Cas9 system for deleting the DNA fragment in RASGEF1A intron 1. Genotyping reveals the specific deletion of RASGEF1A intron 1 in the UE-RASGEF1A-int1-KO hPSC line. (WT) Wild type, (KO) knockout. (H) Immunostaining of SOX10, TUJ1, and HU in hNC and hNP of the control and the mutant (RASGEF1A-int1-KO) lines, respectively. Scale bars: (hNC) 100 µm; (hNP) 200 µm. (I) RT-qPCR reveals the expression level of RET in the hPSC and hNP stages of the control (n = 4–5) and the mutant (RASGEF1A-int1-KO) (n = 6–7). t-test, (ns) not significant.

Fig 3: JCV T-antigen is expressed in neural crest cells.Fluorescent images of bone marrow cells isolated from JCV T-antigen transgenic mice cultured in neural conditions show that these cells are positive for p75 (A,B), nestin (D,E) and SOX-10 (G,H), which are all markers of the neural crest. JCV T-antigen (J,K) was detected in the nucleus of p75 positive cells. Bone marrow cells cultured in mesenchymal media were negative for p75, nestin and SOX-10, and did not express JCV T-antigen (C, F, I, L). Cell nuclei were visualized with DAPI. (Scale bar: 100 µm). (M) Diagram illustrating expression of JCV T-antigen in bone marrow cells. T-antigen expression was associated with neural crest lineage cells of the bone marrow cultured in serum-free neural stem cell media supplemented with bFGF and EFG (left panel), while mesenchymal cells maintained in a-MEM in the presence of 20% serum were negative for T-antigen (right panel). (N) FACS analysis of neural crest cells and MSCs demonstrates positivity for JCV T-antigen in 99% of neural crest cells and lack of JCV T-antigen expression in MSCs.

Fig 4: (A) Representative regions of interest of patient 35041: RNA in situ hybridization staining for CXCL10 RNA (yellow spots), immunofluorescence staining of CD45+ cells (green), Sox10+ cells (red) and DAPI counterstain (blue). (B) Cell proportions across all 22 samples on a per patient basis: Percentages of CD45+ cells (light green), CD45+ CXCL10+ cells (dark green), Sox10+ cells (pink), Sox10+ CXCL10+ cells (red), DAPI (light gray), and DAPI CXCL10+ (dark gray). (C) Percentage of all CXCL10 positive cells on a per patient basis correlated to RNA sequencing results for CXCL10 transcripts divided into low 0–3.28, medium 3.28–6.56 and high 6.56–9.84 (all RNAseq scores log2-transformed). (D) Percentage of all CXCL10 positive cells on a per patient basis correlated to immunotherapy response groups (PD, SD, PR ad CR). (**=p <0.01, *=p <0.05). CR, complete response; PD, progressive disease; PR, partial response; SD, stable disease.

Fig 5: Representative regions of interest of the response patient 01179 (partial response). (A) Complete panel consisting of CD45+ cells (green), Sox10+ cells (red), CXCL10 transcripts (yellow spots), DAPI nuclear counterstain (blue), (B) Sox10+ cells (red), DAPI (blue), (C) CXCL10 RNA (yellow spots), DAPI (blue), (D) CD45+ cells (green), DAPI (blue).