Hypoxia regulates IGF1 expression through HIF1α, and the inhibition of HIF1α or IGF1R decreased CD133- and Oct4-positive GRPs under hypoxia.

<p><b>A.</b> HIF1α expression was suppressed in PC9 or HCC827 hypoxic GRPs by treatment with 50 µM, 100 µM, and 200 µM YC-1 in Lab-Tek chamber slides. Immunofluorescence staining for IGF1, phosphorylated IGF1R (pIGF1R), CD133, and Oct4 was then performed. The numbers of IGF1-, pIGF1R-, CD133-, and Oct4-positive cells were counted, and the ratio of these cells was calculated in five fields from each experiment. **p<0.001. <b>B.</b> PC9 or HCC827 cells were incubated with 1 or 2 µM gefitinib in the presence or absence of 0.01 µM, 0.1 µM, or 1 µM of the IGF1R inhibitor AEW541 under hypoxic conditions for 72 h in Lab-Tek chamber slides. Immunofluorescence staining for CD133 and Oct4 was then performed. The numbers of CD133- and Oct4-positive cells were counted, and the ratio was calculated in five fields for each experiment. **p<0.001. <b>C.</b> PC9 or HCC827 cells were plated in 10-cm plates and allowed to adhere for 24 h. Cells were then incubated with 1 or 2 µM gefitinib in the presence or absence of 0.01 µM, 0.1 µM, or 1 µM of AEW541 under hypoxic conditions for 18 or 11 days. The numbers of colonies were then counted.</p

GRPs were highly enriched for gene expression of stemness, IGF1, and IGF1R.

<p><b>A.</b> Quantitative RT-PCR was performed with primers specific for CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1, which are stemness genes in PC9 or HCC827 parental cells and GRPs. <b>B.</b> Quantitative RT-PCR was performed with primers specific for IGF1 and IGF1R in PC9 or HCC827 parental cells and GRPs. Data were normalized to actin expression. *p<0.01, **p<0.001, ***p<0.0001.</p

IGF1R was phosphorylated on hypoxic GRPs, and knockdown of IGF1 decreased the population of CD133- and Oct4-positive hypoxic GRPs.

<p><b>A.</b> Quantitative RT-PCR was performed with primers specific for IGF1 in PC9 or HCC827 parental cells, GRPs, and hypoxic GRPs. Data were normalized to actin expression. **p<0.001. <b>B.</b> PC9 cells, grown on Lab-Tek chamber slides with or without 1 µM gefitinib and under normoxic or hypoxic conditions for 72 h, fixed, and incubated with the primary antibodies against phosphorylated IGF1R (pIGF1R) and then with secondary antibodies labeled with Alexa Fluor 488 goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Images were obtained using an Axioplan 2 system imaging with AxioVision software. Images used to compare PC9 parental cells, GRPs, and hypoxic GRPs were acquired with the same instrument settings and exposure times, and were processed similarly. The number of pIGF1R-positive cells was counted, and the ratio of these cells was calculated in five fields in each experiment. **p<0.001. <b>C.</b> IGF1 expression was knocked down in PC9 or HCC827 hypoxic GRPs by using small interfering RNA (siRNA) in Lab-Tek chamber slides. Immunofluorescence staining for pIGF1R, CD133, or Oct4 was then performed. Two specific siRNAs and one non-specific control were used. The numbers of pIGF1R-, CD133- and Oct4-positive cells were counted, and the ratio of these cells was calculated in five fields for each experiment. **p<0.001, *p<0.01.</p

Tumor incidence of parental PC9 cells and normoxic and hypoxic GRPs transplanted into NOG mice.

<p>GRPs; Gefitinib resistant persisters.</p><p>NOG mice; NOD/Shi-scid/IL-2Rcnull (NOG) mice.</p>#<p>Tumor incidence was increased significantly in hypoxic GRPs group as compared with parental cell group with 1×10¹ cells/injection, p<0.05.</p><p>To evaluate the <i>in vivo</i> tumorigenic potential, 1×10 cells or 1×10² cells of parental PC9 cells or normoxic and hypoxic PC9 GRPs were mixed with Matrigel and injected into both flanks of NOG mice. Tumor formation was evaluated 33 days after injection.</p

Sphere-forming ability of GRPs was upregulated and hypoxia increased this capacity.

<p>PC9 parental cells, GRPs, and hypoxic GRPs were prepared at densities of 2.5×10³ cells per 2 mL per well in serum-free media supplemented with growth factors and seeded into 6-well ultra-low attachment plates. PC9 parental cells and GRPs were incubated under normoxic conditions, while PC9 hypoxic GRPs were grown under hypoxic conditions. Culture medium was fed every 3 days. The number and size of spheres were recorded and immunofluorescence was performed 7 days after the start of the culture period. Spheres were fixed and incubated with primary antibodies against CD133, Oct4, or phosphorylated IGF1R (pIGF1R), and then with secondary antibody labeled with Alexa Fluor 594 goat anti-mouse IgG (red) or Alexa Fluor 488 goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Images were obtained on an Axioplan 2 imaging system with AxioVision software. <b>A.</b> The number of spheres was significantly increased in PC9 GRPs compared to in parental cells, and was further increased in PC9 hypoxic GRPs. *p<0.01, # p<0.05. <b>B.</b> Sphere size of PC9 hypoxic GRPs was significantly greater than that of parental cells. # p<0.05. <b>C.</b> Immunofluorescent images of control cells of PC9 (left) and spheres of GRPs (right) for CD133, Oct4, and pIGF1R.</p