In silico-based analysis and in vitro experiments identify SIGMAR1 as a potential marker of putative lung cancer stem cells

Abstract

Background: Lung cancer is the leading cause of cancer-related mortality worldwide; however, despite the development and clinical application of various drugs, the prognosis remains poor. One reason for this is the high rate of recurrence and metastasis. The cancer stem cell (CSC) theory has been proposed to explain their root cause, and removal of CSCs is necessary to cure cancer completely; however, detailed profiles of lung CSCs have not been clarified. Here, we used single-cell RNA sequencing (scRNA-seq) data to identify novel markers for lung CSCs and validated their expression and function in vitro.

Methods: A549-derived tumorspheres were used as a model for lung CSCs. To identify genes upregulated in CSC-like cells, we reanalyzed two publicly available scRNA-seq datasets from human lung cancer tissues. Additionally, trajectory analysis was performed to examine changes in candidate gene expression during CSC differentiation. The role of these candidate genes in CSC regulation was further investigated through functional assays.

Results: Tumorspheres exhibited increased expression of well-established CSC markers. scRNA-seq analysis suggested that SIGMAR1 expression was significantly upregulated in CSC-like cells and decreased with differentiation. Furthermore, siRNA-mediated SIGMAR1 knockdown suppressed tumorsphere self-renewal capacity and reduced CSC marker expression.

Conclusions: We propose that SIGMAR1 serves as a potential functional marker of CSCs and plays a crucial role in regulating self-renewal capacity. Targeting SIGMAR1 may provide a novel therapeutic strategy for preventing metastasis and recurrence-major clinical challenges in lung cancer treatment. Future studies should investigate the underlying mechanisms by which SIGMAR1 modulates CSC properties.

Fig. 1

A549-derived tumorspheres as putative lung CSCs. a Schematic representation of the culture method and b morphological comparison of adherent cells and tumorspheres. c Representative histograms and d expression levels of CD44 and ALCAM in adherent cells and tumorspheres cultured for 7 days (n = 3). e mRNA expression levels of SOX2, NANOG, and PROM1 (n = 3). Scale bar = 50 µm. ^*P < 0.05. ^**P < 0.01. ^***P < 0.001

Fig. 2

Identification of CSC-like cells in GSE131907 with high expression of representative CSC markers. a A schematic of the analysis process. b Heatmap of the results of ssGSEA (Stem^High: n = 4,126; Stem^Low: n = 5,743). c The expression of MYC, ALCAM, EPCAM, and CD44 and d the stemness scores of Stem^Low and Stem^High cells. ^***P < 0.001

Fig. 3

SIGMAR1 is highly expressed in Stem^High cells. a Venn diagram of co-upregulated genes in two sets of scRNA-seq data. b Volcano plot representing the results of GSE131907. c Kaplan–Meier plot showing the results of the survival analysis of the TCGA-LUAD dataset. The blue and red lines represent patients with SIGMAR1^Low and SIGMAR1^High LUAD, respectively (n = 87)

Fig. 4

SIGMAR1 expression decreases as the cells differentiated. a–c UMAP plots representing a stemness, b the stemness score, and c pseudotime. d SIGMAR1 expression in malignant cells mapped onto the differentiation trajectory. e and f Pseudotime kinetics of e ALCAM, MYC, and f SIGMAR1

Fig. 5

SIGMAR1 is highly expressed at both the mRNA and protein levels in A549-derived tumorspheres. a Relative expression of SIGMAR1 mRNA (n = 3). b and c b Representative images of immunoblotting and c the protein expression of CD44 and SIGMAR1 (n = 3). Tumorspheres were obtained by culturing for 12 days. ^*P < 0.05. ^***P < 0.001

Fig. 6

SIGMAR1 knockdown does not influence cell count or viability of adherent cells. a Representative images of immunoblotting and b the protein expression of SIGMAR1 (n = 3). c and d Quantification of cell count and viability in SIGMAR1-knockdown adherent cells (n = 3). N.S., not significant. ^**P < 0.01

Fig. 7

SIGMAR1 silencing reduces the self-renewal capacity of tumorspheres. a Schematic representation of the experimental design. b Representative images of siRNA-transfected tumorspheres. c Quantification of the average tumorsphere size (n = 4). Scale bar = 200 µm. ***P < 0.001

Fig. 8

SIGMAR1 knockdown reduces the mRNA expression of CSC-associated markers in tumorspheres. a The expression of SIGMAR1 in tumorspheres (n = 4). b–d Expression levels of b ALCAM, c MYC, and d ALDH1A1, representative CSC-associated markers (n = 4). ^*P < 0.05. **P < 0.01. ^***P < 0.001

Fig. 9

SIGMAR1 identified as a potential marker of putative lung CSCs through in silico and in vitro analyses. a Reanalysis of two independent human lung cancer scRNA-seq datasets identified Stem^High and Stem^Low cell populations, characterized by high and low CSC properties, respectively. Gene expression analysis demonstrated a significant increase in SIGMAR1 expression in Stem^High cells. Furthermore, the trajectory analysis revealed a progressive decline in SIGMAR1 expression as differentiation advanced. b A549-derived tumorspheres exhibited significantly higher SIGMAR1 expression compared with adherent cells. Moreover, SIGMAR1-knockdown tumorspheres showed a marked reduction in CSC marker expression and self-renewal capacity. diff: differentiation