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other·ophthalmology, dermatology, oncology, epidemiology, public health·PMC10783873

Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

eLife · 18 authors, 14 centres

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TL;DR

This study used single-cell RNA sequencing of 138,982 cells from 13 samples (normal skin, actinic keratosis, squamous cell carcinoma in situ, and invasive cutaneous squamous cell carcinoma) to map the full progression from precancer to cancer. Key findings include identification of malignant basal cell subpopulations in SCCIS, monotonically changed driver genes (e.g., KLF6, FOSL1), and functional validation of LGALS1, IFITM3, FTH1, BST2, and SAT1 in regulating proliferation, apoptosis, migration, and invasion. The results provide a valuable resource for understanding cSCC initiation and progression and identifying potential therapeutic targets.

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**Background:** Cutaneous squamous cell carcinoma (cSCC) is the second most common keratinocyte malignancy, with actinic keratosis (AK) as a precancerous lesion. The molecular mechanisms driving progression from normal skin to AK to squamous cell carcinoma in situ (SCCIS) to invasive cSCC remain poorly understood. This study aimed to comprehensively characterize the transcriptomic landscape at single-cell resolution across these stages to identify key driver genes, malignant cell subpopulations, and tumor microenvironment (TME) interactions.

**Methods:** The authors performed single-cell RNA sequencing (scRNA-seq) on 138,982 cells from 13 samples of six patients, including three AK, one SCCIS, three cSCC (low-risk and high-risk), and six patient-matched normal skin samples. Single-cell ATAC sequencing (scATAC-seq) was also performed on one poorly differentiated cSCC sample. Cell types were identified using known markers, and copy number variation (CNV) analysis was used to define malignant cells. Differentially expressed genes (DEGs) were identified across stages, and key candidates were validated by immunohistochemistry (IHC) and immunofluorescence (IF) in independent cohorts (20 AK vs. normal pairs; 15 normal, 15 SCCIS, 60 cSCC). Functional experiments (siRNA knockdown, CCK8, apoptosis, migration, invasion assays) were performed in HaCaT, A431, SCL-I, and SCL-II cell lines. Cell-cell communication was analyzed using CellChat.

**Key Results:** - Nine major cell types were identified in normal skin, with subtypes in basal (Basal1, Basal2), spinous (Spinous1, Spinous2), and follicular (Follicular1, Follicular2) cells. Basal1 expressed stemness markers (COL17A1, TP63, ITGB1), while Basal2 showed increased inflammatory genes (KRT16, S100A8, S100A9). - From normal to AK, 549, 305, and 434 up-regulated DEGs were found in Basal1, Basal2, and Pro KCs, respectively. ALDH3A1 and IGFBP2 were significantly up-regulated in AK (IF: 85% and 75% of AK tissues positive, respectively; p<0.001) but not in SCCIS or cSCC. IGFBP2 overexpression promoted proliferation and invasion in HaCaT and A431 cells (p<0.05). - In the P2 patient (AK and SCCIS), 21 monotonically up-regulated genes were identified in basal cells from normal to AK to SCCIS, including KLF6, FOSL1, JUND, SQSTM1, and RHOB. Monotonically down-regulated genes included SPRR2A, S100A14, and ENO1. - In SCCIS, basal cell proportion increased significantly. CNV analysis identified a malignant basal subpopulation (Basal-SCCIS-tumor) with higher CNV scores, enriched for DNA damage response genes (PCNA, MCM7), heat shock proteins (HSPA1A/B, HSP90AA1, HSPA6), activated keratins (KRT6A/B/C, KRT16, KRT17, KRT19), and S100 family genes (S100A7, S100A8, S100A9). MAGEA4 was strongly positive in 73.33% of SCCIS and 76.67% of cSCC (p<0.001 vs. normal). ITGA6 was up-regulated in SCCIS (p<0.05) and cSCC (p<0.001). siRNA silencing of MAGEA4 in A431 cells reduced proliferation, migration, and invasion (p<0.01) and increased apoptosis (p<0.01). ITGA6 silencing similarly reduced proliferation, migration, and invasion in A431, SCL-I, and SCL-II cells (p<0.01) and increased apoptosis (p<0.01). - In cSCC, CNV scores correlated with malignant grade (well-differentiated: low; moderately differentiated: moderate; poorly differentiated: high). LGALS1, IFITM3, and FTH1 were significantly overexpressed in cSCC vs. normal (IHC: p<0.001 for all; IFITM3 and FTH1 also higher in poorly differentiated vs. well-differentiated, p<0.05). siRNA silencing of LGALS1, IFITM3, FTH1, BST2, and SAT1 inhibited proliferation, increased apoptosis (p<0.01), and reduced migration (except FTH1) and invasion (p<0.01) in cSCC cell lines. - TME analysis revealed 11 lymphocyte subpopulations, 8 DC/Mac subpopulations, and 6 fibroblast subpopulations. Cell-cell communication strength increased with malignancy (p<0.0001). MHC-II, laminin, and TNF signaling pathways were enhanced in poorly differentiated cSCC.

**Clinical Implications:** This study provides a comprehensive single-cell atlas of cSCC progression, identifying early malignant cells in SCCIS and key driver genes (ALDH3A1, IGFBP2, MAGEA4, ITGA6, LGALS1, IFITM3, FTH1, BST2, SAT1) that may serve as biomarkers or therapeutic targets. The findings highlight the importance of basal cell subpopulations and TME interactions in cSCC development, offering potential avenues for early detection and targeted therapy.

STUDY TYPE

other

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ophthalmology

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88% · B

ORIGINAL PAPER

PMC10783873

Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

eLife

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ABSTRACT

Cutaneous squamous cell carcinoma (cSCC) is the second most frequent of the keratinocyte-derived malignancies with actinic keratosis (AK) as a precancerous lesion. To comprehensively delineate the underlying mechanisms for the whole progression from normal skin to AK to invasive cSCC, we performed single-cell RNA sequencing (scRNA-seq) to acquire the transcriptomes of 138,982 cells from 13 samples of six patients including AK, squamous cell carcinoma in situ (SCCIS), cSCC, and their matched normal tissues, covering comprehensive clinical courses of cSCC. We identified diverse cell types, including important subtypes with different gene expression profiles and functions in major keratinocytes. In SCCIS, we discovered the malignant subtypes of basal cells with differential proliferative and migration potential. Differentially expressed genes (DEGs) analysis screened out multiple key driver genes including transcription factors along AK to cSCC progression. Immunohistochemistry (IHC)/immunofluorescence (IF) experiments and single-cell ATAC sequencing (scATAC-seq) data verified the expression changes of these genes. The functional experiments confirmed the important roles of these genes in regulating cell proliferation, apoptosis, migration, and invasion in cSCC tumor. Furthermore, we comprehensively described the tumor microenvironment (TME) landscape and potential keratinocyte-TME crosstalk in cSCC providing theoretical basis for immunotherapy. Together, our findings provide a valuable resource for deciphering the progression from AK to cSCC and identifying potential targets for anticancer treatment of cSCC.

CITATION EXPORT

Dan-Dan Zou, Ya-Zhou Sun, Xin-Jie Li et al.. Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma. eLife. (2023). https://doi.org/10.7554/eLife.85270

AUTHORS

18 · 14 CENTRES

Dan-Dan Zou, Ya-Zhou Sun, Xin-Jie Li, Wen-Juan Wu, Dan Xu, Yu-Tong He, Jue Qi, Ying Tu, Yang Tang, Yun-Hua Tu, Xiao-Li Wang, Xing Li, Feng-Yan Lu, Ling Huang, Heng Long, Li He, Xin Li, Beate Maria Lichtenberger

1 https://ror.org/02g01ht84Department of Dermatology, First Affiliated Hospital of Kunming Medical University Yunnan China
2 https://ror.org/00c099g34Department of Dermatology, The Affiliated Hospital of Kunming University of Science and Technology, The First People's Hospital of Yunnan Province, Kunming Yunnan China
3 https://ror.org/00rfd5b88Clinical Big Data Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University Shenzhen, Guangdong China
4 https://ror.org/0064kty71School of Medical, Shenzhen Campus of Sun Yat-sen University Shenzhen, Guangdong China
5 https://ror.org/0103dxn66Department of Dermatology, Changzheng Hospital, Naval Medical University Shanghai China
6 Department of Dermatology, People's Hospital of Chuxiong Yi Autonomous Prefecture, Chuxiong Yunnan China
7 Department of Dermatology, Qujing Affiliated Hospital of Kunming Medical University, The First People’s Hospital of Qujing Yunnan China
8 Department of Dermatology, First Affiliated Hospital of Dali University, Dali Yunnan China
9 Wenshan Zhuang and Miao Autonomous Prefecture Dermatology Clinic, Wenshan Zhuang and Miao Autonomous Prefecture Specialist Hospital of Dermatology, Wenshan Yunnan China
10 https://ror.org/00rfd5b88Guangdong Provincial Key Laboratory of Digestive Cancer Research, The Seventh Affiliated Hospital of Sun Yat-sen University Guangdong China
https://ror.org/05n3x4p02Medical University of Vienna Austria
https://ror.org/0220mzb33King's College London United Kingdom
https://ror.org/05n3x4p02Medical University of Vienna Austria
https://ror.org/05n3x4p02Medical University of Vienna Austria

EDITOR / REVIEWER

Beate Maria Lichtenberger, Tony Ng, Beate Maria Lichtenberger