Spatial transcriptomic analysis of HLA related immune signatures in metastatic cutaneous melanoma patients.

Transcriptomic expression of specific gene signatures within spatial and immune from lymph node metastases in patients with metastatic melanoma.

In the study presented here, a cohort of 55 metastatic melanoma patients was used to acquire expression profiles of specific HLA-related immune signatures for spatial expression analysis in the intersection of immune and tumor segments within the skin tumors and lymph node metastasis.

The Whole Transcriptome Atlas RNA probe kit (NantoString Technologies, Seattle, WA, USA) was used for NanoString GeoMx Digital Spatial profiling analysis of skin tumors and lymph node metastasis of metastatic melanoma patients. Formalin-fixed, paraffin-embedded (FFPE) sections of tumor microarrays of 3,5 µm thickness were deparaffinized, and the antigens retrieved, following the manufacturer's instruction (MAN-10150-01, NanoString Technologies).

In situ hybridization of RNA probes was performed overnight following the manufacturer's instruction (MAN-10150-01, NanoString Technologies).

Fluorophore-tagged antibodies against CD45, S100B/PMEL17, and SYTO13 nuclear dye (Human Melanoma TME morphology kit, NanoString Technologies) were used for staining to enable detection of the regions of interest (ROIs).

The arrays were scanned using the GeoMx instrument. 3-channel immunofluorescent digital images were created of S100B/PMEL17 signal representing tumor cells, CD45 signal representing leukocytes and SYTO13 signal representing nuclei. Based on the digital image, regions of interes (ROI) were selected.

Immune segments were selected from the ROIs. RNA-specific probes coupled with photocleavable oligonucleotides were then collected of the iROIs and subsequently sequenced in a next-generation sequencing pipeline. Gene counts were normalized according the the third-quartile protocol using the GeoMX DSP GEOMX-0061 Version 3.0.0.113.

RAW files include separate patient RAW counts for studied genes. Normalized matrixes include normalized counts using 3rd quartile normalization method.

转移性黑色素瘤患者淋巴结转移灶内空间区域与免疫区域中特定基因特征的转录组表达情况。

本研究纳入55例转移性黑色素瘤患者队列，旨在获取特定人类白细胞抗原（Human Leukocyte Antigen, HLA）相关免疫基因特征的表达谱，用于皮肤肿瘤与淋巴结转移灶内免疫区域与肿瘤区域交界区域的空间表达分析。

本研究采用全转录组图谱RNA探针试剂盒（美国华盛顿州西雅图市NanoString Technologies公司），对转移性黑色素瘤患者的皮肤肿瘤及淋巴结转移灶开展NanoString GeoMx数字空间分析。按照厂商说明书（MAN-10150-01，NanoString Technologies），对厚度为3.5 μm的福尔马林固定石蜡包埋（FFPE）肿瘤微阵列切片进行脱蜡与抗原修复操作。

按照厂商说明书（MAN-10150-01，NanoString Technologies），使用RNA探针开展过夜原位杂交实验。

采用靶向CD45、S100B/PMEL17的荧光标记抗体以及SYTO13核染料（美国NanoString Technologies公司人类黑色素瘤肿瘤微环境形态学试剂盒）进行染色，以实现对感兴趣区域（ROIs）的识别。

使用GeoMx仪器对芯片进行扫描，获取代表肿瘤细胞的S100B/PMEL17信号、代表白细胞的CD45信号以及代表细胞核的SYTO13信号的三通道免疫荧光数字图像。基于该数字图像，选取感兴趣区域（ROIs）。

从选取的ROIs中进一步划分免疫区域，收集结合了光可裂解寡核苷酸的RNA特异性探针标记的免疫感兴趣区域（iROIs），随后通过下一代测序流程进行测序。采用GeoMX DSP GEOMX-0061版本3.0.0.113的三分位标准化方案对基因计数数据进行标准化处理。

原始文件包含各研究基因的单患者原始计数数据；标准化矩阵则包含采用三分位标准化方法得到的标准化计数数据。