A restricted cell population propagates glioblastoma growth after chemotherapy

Abstract

Glioblastoma multiforme is the most common primary malignant brain tumour, with a median survival of about one year. This poor prognosis is due to therapeutic resistance and tumour recurrence after surgical removal. Precisely how recurrence occurs is unknown. Using a genetically engineered mouse model of glioma, here we identify a subset of endogenous tumour cells that are the source of new tumour cells after the drug temozolomide (TMZ) is administered to transiently arrest tumour growth. A nestin-ΔTK-IRES-GFP (Nes-ΔTK-GFP) transgene that labels quiescent subventricular zone adult neural stem cells also labels a subset of endogenous glioma tumour cells. On arrest of tumour cell proliferation with TMZ, pulse-chase experiments demonstrate a tumour re-growth cell hierarchy originating with the Nes-ΔTK-GFP transgene subpopulation. Ablation of the GFP+ cells with chronic ganciclovir administration significantly arrested tumour growth, and combined TMZ and ganciclovir treatment impeded tumour development. Thus, a relatively quiescent subset of endogenous glioma cells, with properties similar to those proposed for cancer stem cells, is responsible for sustaining long-term tumour growth through the production of transient populations of highly proliferative cells.

Figure 1

Characterization of the Nes-ΔTK-GFP transgene. a, Diagram of the Nes-ΔTK-GFP transgene. b,c, GCV administration ablates neural stem cells (NSCs) in wild type mice. b, Representative Nissl staining of the subventricular zone (SVZ) region in wild type mice treated with GCV (WT;GCV), Nes-ΔTK transgene mice treated with PBS (Nes-ΔTK;PBS), and Nes-ΔTK transgene mice treated with GCV (Nes-ΔTK;GCV); black arrows indicate the stem cell rostral migratory stream (RMS), which is greatly reduced in the Nes-ΔTK;GCV mice. c, GFP (transgene), GFAP (quiescent neural stem cells) and DCX (committed neural progenitors) immunostaining of the subventricular zone (SVZ) stem cell niche. White arrowheads in middle panel (Nes-ΔTK;PBS) indicate DCX-positive GFP-negative cells more distal in the RMS. White arrowheads in bottom panel (Nes-ΔTK;GCV) indicate GFP-positive/GFAP-positive but DCX-negative quiescent NSCs. d, Representative GFP immunostaining in sections from two untreated gliomas of Mut7;Nes-ΔTK mice. From tumor to tumor, varying numbers of GFP-positive cells were observed. e, Representative GFP and Ki67 co-immunostaining in two untreated gliomas of Mut7;Nes-ΔTK mice. White arrowheads highlight GFP-positive but Ki67-negative cells, demonstrating that many transgene GFP-positive cells are quiescent. f, Percentage of Ki67-positive cells in GFP-positive, GFP-negative, and overall tumor population in gliomas from untreated Mut7;Nes-ΔTK-GFP mice. g, Kaplan–Meier survival curve of untreated Mut7 and Mut7;Nes-ΔTK animals. No difference in percent survival was observed. h, Representative H&E staining of Mut7 and Mut7;Nes-ΔTK brains without treatment. Infiltrative malignant gliomas are present in the cortex (red arrowhead) of both genotypes. *, p<0.05; ***, p<0.001.

Figure 2

TMZ targets proliferating derivatives but not the GFP+ quiescent cell population. a, TMZ injection schema. Mut7 mice were treated with TMZ for five days, injected with BrdU two hours after the final TMZ treatment, and sacrificed two hours later for BrdU immunostaining. b, Representative GFAP/BrdU co-staining of glioma from DMSO- or TMZ-treated mice shows dramatic reduction in number of BrdU-positive cells. c, Quantification of the percentage of maximal BrdU-positive cells in gliomas from Mut7 mice treated with or without TMZ showed a significant decrease in the TMZ-treated mice. (Data are mean ± s.e.m.; N=6 for each treatment; p<0.001.) d–f, Nes-ΔTK-positive cells are resistant to TMZ and produce new tumor cells. d, Schema of TMZ treatment and short-term labeling with BrdU analogs. Mut7;Nes-ΔTK mice were treated with TMZ for 5 days and then injected with the BrdU analogs CldU and IdU, one and three days after the last TMZ treatment, respectively. e, Representative tumor section illustrating that repopulating tumor cells after TMZ treatment express the Nes-ΔTK transgene (GFP+); merged panel: CldU-incorporating (arrowhead) or IdU-incorporating (arrow) cells also express GFP driven by the Nes-ΔTK transgene. (Percentage of GFP-positive cells in the CldU-positive = 77±14; percentage of GFP-positive cells in the IdU-positive population = 83±10. N=5.) Note that the majority of CldU-positive cells and IdU-positive cells are positive for GFP expression, and also that the majority of IdU-positive cells are CldU-positive, indicating the CldU-positive cells gave rise to the IdU-positive cells. ***p<0.001. Student’s t-test.

Figure 3

GCV treatment prolongs survival of Mut7;Nes-ΔTK mice. a, Kaplan–Meier curve of Mut7;Nes-ΔTK mice treated with Saline or GCV for 10 weeks starting at 8 weeks of age showed a clear survival advantage for the GCV-treated mice (n=10 for GCV-treated; n=8 for saline-treated; P values determined using Log-rank test). b, Kaplan–Meier survival curves of Mut7;Nes-ΔTK (left) or Mut7 (right) mice treated with GCV or Saline for 2 months starting at 10 weeks of age. GCV treatment increased survival of Mut7;Nes-ΔTK mice compared to saline treatment but had no such effect on the Mut7 mice. (n=5–7 for the GCV-treated mice; n=4–7 for the saline-treated mice; P values determined using Log-rank test) c, GFP/Nestin co-immunostaining of gliomas from control (Mut7 mice treated with GCV or Mut7;Nes-ΔTK mice treated with PBS) and GCV-treated Mut7;Nes-ΔTK mice. Elimination of GFP and nestin double-positive cells in the Mut7;Nes-ΔTK mice treated with GCV from 10 weeks. d, Representative H&E staining of control and GCV-treated Mut7;Nes-ΔTK brains. The tumors in the GCV-treated Mut7;Nes-ΔTK mice are less infiltrative than in control. Tumors indicated by black arrows. e, Representative H&E and Sox2 staining of tumor edges in control and GCV-treated Mut7;Nes-ΔTK tumors showing the GCV-treated tumors have a defined boundary (dotted line) and lack infiltrative Sox2+ cells. f,g, GCV treatment decreases proliferation index in Mut7;Nes-ΔTK tumors. f, Representative Ki67 staining of control (panels 1 and 2) or GCV-treated Mut7;Nes-ΔTK tumors (panels 3 and 4) showing the dramatic decrease in proliferation in the GCV-treated Mut7;Nes-ΔTK tumors. g, Quantification of the percentage of Ki67-positive cells in tumor regions with highest number of proliferating cells in cortex. The percentage is significantly decreased in GCV-treated Mut7;Nes-ΔTK mice (n=3) versus control (n=4). Data are mean ± s.e.m.; **p<0.01. Student’s t-test.

Figure 4

Combination treatment of Temozolomide (TMZ) and GCV inhibits glioma progression in cerebrum. a,b, Therapeutic schema targeting both CSCs and their proliferating progeny. Mut7;Nes-ΔTK mice were treated with TMZ for 5 days, followed two days after by GCV. c, Kaplan–Meier survival curve of Mut7;Nes-ΔTK mice with different treatments. GCV-treated (n=7; median survival=55 days) and combinationally-treated (n=9; median survival=52.5 days) Mut7;Nes-ΔTK mice had similar survival advantage over TMZ-treated (n=6; median survival=30.5 days) or PBS-treated (n=7; median survival=21 days) mice. P values determined using Log-rank test. d, GFP and Nestin double immunostaining of vestigial tumors in TMZ/GCV-treated Mut7;Nes-ΔTK mice (right panel) versus tumor in control (left panel) demonstrates depletion of CSCs as evidenced by lack of GFP expression. e, Maximal cell density in cortical tumors with different treatment regimens. Untreated Mut7 mice (n=6) were used as control. Tumor density of 10-week-old non-symptomatic Mut7 mice (pre-treatment) (n=8) was used as a reference starting point. Cell density was significantly lower in the combinationally-treated (TMZ+GCV) Mut7;Nes-ΔTK mice (n=6) compared to untreated tumors (p=0.0005), compared to tumors in “pre-treatment” mice (p=0.004), and compared to tumors from mice treated with GCV only (n=4) (Data are mean ± s.e.m.; p=0.001; P values determined using Student’s t-test.).