IL-8 regulates the stemness properties of cancer stem cells in the small-cell lung cancer cell line H446

Abstract

Purpose: Cancer stem cells (CSCs) are a small population of cancer cells located within a tumor that are highly tumorigenic, capable of tumor initiation, and resistant to cancer therapies. We identified the potential genes involved in regulating stemness properties and investigated the mechanisms in small-cell lung cancer (SCLC).

Materials and methods: Whole transcriptome sequencing technology was used to screen the potential genes involved in regulating stemness properties from SCLC-SCs (uPAR⁺) and differentiated cells (uPAR^-) in the H446 cell line. The selected genes were validated by quantitative reverse transcription PCR and ELISAs. The effect of IL-8 on stemness of sphere-forming cells was determined through tumor sphere formation, wound healing migration, and in vivo tumorigenesis assays.

Results: In our study, uPAR⁺ and uPAR^- cells showed different gene expression profiles. IL-8 was upregulated in SCLC sphere-forming cells. Blocking IL-8 expression with siRNA led to loss of stemness, including the self-renewal capability, migration, expression of stemness-related genes, and in vivo tumorigenicity, in sphere-forming cells. Consistently, exogenously added IL-8 enhanced stemness properties in parental cells.

Conclusion: IL-8 was upregulated in SCLC sphere-forming cells, and critical for the acquisition and/or maintenance of the stemness features in the SCLC cell line H446. Our results suggest that blocking IL-8 signaling may provide a novel therapeutic approach for targeting SCLC-SCs and improve treatment and outcomes in SCLC.

Keywords: IL-8; cancer stem cells; small-cell lung cancer; stemness; tumor sphere; uPAR.

Figure 1

Upregulation of IL-8 in sphere cells. Notes: (A) The expression levels of selected genes were determined in sphere cells and adherent cells from the H446 cell line by qRT-PCR. For each target gene, expression levels are calculated relative to the expression levels in adherent cells, arbitrarily set as 1. Each qRT-PCR assay was performed at least three times. ^**P<0.01. (B) IL-8 secretion and (C) CXCL2 secretion by sphere cells and adherent cells were analyzed after 24 h and 48 h by ELISAs. Data are expressed as the mean±standard error of the mean of three independent experiments. ^*P<0.05, ^**P<0.01. Abbreviations: ns, not significant; qRT-PCR, quantitative reverse transcription PCR.

Figure 2

The effect of IL-8 on self-renewal of CSCs in the H446 cell line. Notes: (A) The expression of IL-8 in sphere cells transfected with siIL-8 or negative control (siNC) was detected by qRT-PCR. ^*P<0.05, ^***P<0.001, compared with control. (B) IL-8 secretion by sphere cells transfected with siIL-8 or siNC was analyzed by ELISAs. Data are expressed as the mean ± standard error of the mean (sem) of three independent experiments. ^***P<0.001. (C) The sphere formation capability was evaluated in sphere cells transfected with siIL-8 or siNC. Data are expressed as the mean ± sem of three independent experiments. ^**P<0.01. (D) Representative micrographs of spheres formed from cells transfected with siIL-8 or siNC. Scale bar 100 µm. (E) The sphere formation capability of H446 cells treated with 0 ng/mL or 10 ng/mL human recombinant IL-8 (rh-IL-8). Data are expressed as the mean ± sem of three independent experiments. ^***P<0.001. (F) Representative micrographs of spheres formed by H446 cells treated or not with rh-IL-8. Scale bar 50 µm. Abbreviations: siIL-8, IL-8 siRNA; siNC, negative control oligonucleotide.

Figure 3

The effect of IL-8 on expression of stemness genes. Notes: (A) The levels of stemness-related genes were analyzed in sphere cells transfected with siIL-8 or siNC by qRT-PCR. ^**P<0.01, compared with control. (B) The mRNA levels of the indicated stemness-related genes in H446 cells treated with 100 ng/mL rh-IL-8. Data are expressed as mean ± standard error of the mean of three independent experiments. ^*P<0.05, ^**P<0.01, compared with control. Abbreviations: ns, not significant; rh-IL-8, human recombinant IL-8; siIL-8, IL-8 siRNA; siNC, negative control oligonucleotide; uPAR, urokinase plasminogen activator receptor; qRT-PCR, quantitative reverse transcription PCR.

Figure 4

IL-8 increases the migration of H446 cells. Notes: (A) The wound healing assay indicated that human recombinant IL-8 (rh-IL-8) treated H446 cells had a significant reduction in the width of the wound compared with the control after 48 h. (B) The migration distances of H446 cells treated by 10 ng/mL rh-IL-8 are shown. Data are expressed as mean ± standard error of the mean of three independent experiments. ^**P<0.01.

Figure 5

siIL-8 treatment inhibits tumor growth. Notes: (A) Subcutaneous implantation of sphere cells in nude mice led to tumor formation. (B) Growth curve of xenograft tumors volume of siIL-8 treatment group and siNC control group. ^*P<0.05. Abbreviations: siIL-8, IL-8 siRNA; siNC, negative control oligonucleotide.