ICAM1 (CD54) Contributes to the Metastatic Capacity of Gastric Cancer Stem Cells

Abstract

Gastric cancer is the fourth leading cause of cancer deaths worldwide. The presence of chemoresistant cells has been used to explain this high mortality rate. These higher tumorigenic and chemoresistant cells involve cancer stem cells (CSCs), which have the potential for self-renewal, a cell differentiation capacity, and a greater tumorigenic capacity. Our research group identified gastric cancer stem cells (GCSCs) with the CD24+CD44+CD326+ICAM1+ immunophenotype isolated from gastric cancer patients. Interestingly, this GCSC immunophenotype was absent in cells isolated from healthy people, who presented a cell population with a CD24+CD44+CD326+ immunophenotype, lacking ICAM1. We aimed to explore the role of ICAM1 in these GCSCs; for this purpose, we isolated GCSCs from the AGS cell line and generated a GCSC line knockout for ICAM1 using CRISPR/iCas9, which we named GCSC-ICAM1^KO. To assess the role of ICAM1 in the GCSCs, we analyzed the migration, invasion, and chemoresistance capabilities of the GCSCs using in vitro assays and evaluated the migratory, invasive, and tumorigenic properties in a zebrafish model. The in vitro analysis showed that ICAM1 regulated STAT3 activation (pSTAT3-ser727) in the GCSCs, which could contribute to the ability of GCSCs to migrate, invade, and metastasize. Interestingly, we demonstrated that the GCSC-ICAM1^KO cells lost their capacity to migrate, invade, and metastasize, but they exhibited an increased resistance to a cisplatin treatment compared to their parental GCSCs; the GCSC-ICAM1^KO cells also exhibited an increased tumorigenic capability in vivo.

Keywords: cancer stem cell; gastric cancer; metastasize; tumorigenic.

Figure 1

The generation of an AGS/iCas9GFP cell line. (A) GFP basal expression in the AGS cell line. (B) GFP expression in the AGS/iCas9GFP cell line Dox−. (C) GFP expression in the AGS/iCas9GFP cell line after inducing with Dox+. (D) The fluorescence microscopy of the AGS/iCas9GFP cell lines after FACS, 20x. Dox+ and Dox−, with or without doxycycline, respectively. Bars indicate 100 µm.

Figure 2

GCSC/ICAM1^KO transduction efficiency 48 h post-induction. (A) AGS/iCas9GFP transduced with sgRNA-SCR Dox+. (B) AGS/iCas9GFP transduced with ICAM1-sgRNA 1/2 Dox−. (C) AGS/iCas9GFP transduced with ICAM1-sgRNA 1/2 Dox+. (D) Merge between blot B (red population) and blot C (blue population) panels. Dox (doxycycline).

Figure 3

The CRISPRiCas9 generation of a stable GCSC/ICAM1^KO cell line. (A) GFP expression in AGS/iCas9GFP Dox+ cells. (B) GCSC/SCR Dox+. (C) GCSC/ICAM1^KO Dox+. (D) GCSC/ICAM1^KO Dox−. The panels represent pass 10 after cell sorting with the respective immunophenotype. Dox (doxycycline).

Figure 4

Regulation of stemness-related transcription factor expression by ICAM1/STAT3 axis. (A) Western blot analysis of total lysates of GCSC/ICAM1^KO and GCSC/SCR cells demonstrated diminished levels of phosphor-STAT3 (p-STAT3-ser727) in GCSC/ICAM1^KO cells. (B) Densitometry of Western blot * p < 0.05. (C) Representative dot plot of cytometry flow analysis; GCSC/ICAM1^KO cells reduced expression of stem cell markers Oct3/4 and Nanog but not Sox2. * p < 0.05. **** p < 0.0001.

Figure 5

GCSC/ICAM1^KO cells lost their ability to migrate and invade but did not alter the expression of EMT markers. (A) Representative images of the migration assay; GCSC/ICAM1^KO cells exhibited a decreased ability to migrate at 24 h and 48 h, 4X. Bars indicate 200 µm. (B,C) A quantitative analysis of the relative number of migrating cells represented in (A) at 24 (B) and 48 h (C); n = 3 per group. (D) Representative images of the invasion assay; GCSC/ICAM1^KO cells exhibited a decreased ability to invade at 24 h, 20X. Bars indicate 100 µm. (E) A quantitative analysis of the invasion assay. The values represent the mean ± SD of three independent experiments and are expressed in relative percentages. Asterisks indicate the comparison made between groups. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (F) A representative dot plot showing the effect of ICAM1 deletion on the expression level of EMT-related proteins.

Figure 6

The tumorigenic and migratory capabilities of xenotransplanted GCSC/ICAM1^KO cells. (A) The experimental design. (B) The percentage of embryos with migrating GCSCs or GCSC/ICAM1^KO cells after 1 and 4 dpi. (C,D) show larvae injected with GCSCs after 1 or 4 dpi, respectively. (E,F) show larvae injected with GCSC/ICAM1^KO cells after 1 or 4 dpi, respectively. (H–J) show larvae after 1 dpi with severe, moderate, or mild phenotypes, respectively. (G) shows a wild-type larva. (K) shows the percentage of larvae with severe, moderate, or mild phenotypes (n = 45). The images were obtained with a Nikon SMZ1500 stereomicroscope. All the bars indicate 100 µm. GCSC = AGS/GCSC, and ICAM1^KO = GCSC/ICAM1^KO. All boxes and arrow heads indicate sites with migrating cells.

Figure 7

GCSCs migrate in groups and establish metastatic tumors. (A) Tg (fli1:EGFP)y1 zebrafish embryos (2 dpf) were given an injection into the yolk sac (red arrow) of 50 fluorescently observed migrating cells (white arrow) inside the posterior cardinal vein (PCV, cyan) over the distal portion of the intestine (I). The insets show five GCSC nuclei inside the PCV, co-stained with Hoechst (white). (C,D) The GCSCs formed a metastatic cell mass that invaded the skeletal muscle at the level of somites 21-21, as observed using a brightfield microscope (C), the nuclei were stained with Hoescht (D) to observe clearly the cell mass. The white arrows show GCSCs, and the white arrowheads indicate zebrafish muscle fiber nuclei. N indicates the notochord, DA the dorsal aorta, and I the intestine. The images were obtained at 63X with an LSM800 confocal microscope. Labeled GCSCs are shown in orange, and 6 µm sections are shown in the (B,C) insets. (B) GCSCs 6 dpi.

Figure 8

GCSC/ICAM1^KO cells exhibited an increased resistance to cisplatin. Viability percentages are shown for AGS (IC50 at 41.6 µM), AGS-GCSC (IC50 at 49.3 µM), GCSC-SCR (IC50 at 55.08 µM), and GCSC-ICAM1^KO (IC50 at 79.2 µM) cells in the presence of different concentrations of cisplatin. n = data from 3 independent assays. The dotted line means the 50% of cell viability.