STAT3 silencing inhibits glioma single cell infiltration and tumor growth

Abstract

Background: Diffuse infiltration remains the fulcrum of glioblastoma's incurability, leading inevitably to recurrence. Therefore, uncovering the pathological mechanism is imperative. Because signal transducer and activator of transcription 3 (STAT3) correlates with glioma malignancy and predicts poor clinical outcome, we determined its role in glioma single cell infiltration and tumor growth.

Methods: STAT3 was silenced in Tu-2449 glioma cells via lentiviral gene transfer. Target gene expression was measured by real-time reverse transcription PCR, Western blotting, and immunohistochemistry. Microvilli were visualized by staining with wheat germ agglutinin. Migration and invasion were measured by Scratch and Matrigel chamber assays. Diffuse infiltration was studied in 350-μm-thick organotypic tissue cultures over 14 days using cells tagged with enhanced green fluorescent protein and live confocal laser scanning microscopy. Survival of tumor-bearing syngeneic, immunocompetent B6C3F1 mice was analyzed by Kaplan-Meier plots.

Results: STAT3 silencing reduced cell migration and invasion in vitro and stopped single cell infiltration ex vivo, while STAT3-expressing cells disseminated through the neuropil at ∼100 µm/day. STAT3 silencing reduced transcription of several tumor progression genes. Mice with intracranial STAT3 knockdown tumors had a significant (P< .0007) survival advantage over controls, yielding 27% long-term survival. STAT3 knockdown reduced podoplanin expression 50-fold and inhibited concurrent microvilli formation. STAT3 knockdown tumors exhibited a weaker podoplanin immunoreactivity compared with controls. Podoplanin staining was diffuse, preferentially at tumor margins, and absent in normal brain.

Conclusions: Our results show compelling evidence that STAT3 is a key driver of diffuse infiltration and glioma growth and might therefore represent a promising target for an anti-invasive therapy.

Keywords: STAT3; brain slices; glioma mouse model; lentiviral gene transfer; single cell infiltration.

Fig. 1.

STAT3 knockdown abrogates STAT3 protein expression and reduces transcription of tumor progression genes. (A) Western blot analysis of Tu-2449 glioma cells stably transduced with either control vector (Con) or STAT3-shRNA (shSTAT3). Cell lysates were subjected to SDS gel electrophoresis and blotted for immunoreaction with antibodies specific to STAT3, cyclin D, Snail, MMP-2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Equal amounts of proteins were loaded. (B) Total RNA was extracted from Tu-2449 glioma cells stably transduced with either control vector (control) or STAT3-shRNA (shSTAT3) and reverse transcribed. Transcripts of the STAT3 target genes Twist, Snail, HIF, MMP-2, MMP-9, and VEGF were quantified by quantitative real-time PCR using gene-specific TaqMan primers and probes. Messenger RNA expression of target genes was normalized to HPRT1. Note that HIF is regulated by STAT3 in spite of normoxic conditions. The data represent 3 independent experiments. Bars = SD of triplicates; *P< .05, significant difference between groups.

Fig. 2.

STAT3 knockdown decreases migration and invasion. (A) Migration of stably transduced Tu-2449 glioma cells was analyzed using the wound healing assay. A single scratch was made in a subconfluent monolayer and photographed after 0 h and 16 h. Cells that migrated into the scratch were counted. (B) Invasion was analyzed in ECM-coated Boyden chambers. Stably transduced cells were seeded in the upper chamber, and transmigrated cells were fixed after 24 h, stained with crystal violet, and counted. Control and STAT3 knockdown cells were statistically compared. The data represent 3 independent experiments. Bars = SD of triplicates; *P< .05, significant difference between groups.

Fig. 3.

STAT3 facilitates diffuse infiltration. Live imaging of fluorescent Tu-2449 glioma cells on organotypic slice cultures. EGFP-tagged cells were seeded on the caudate putamen 2 days after brain slice preparation. (A) Confocal microscopy of wild-type (ie, STAT3-expressing) Tu-2449 glioma cells radially infiltrating into the brain parenchyma 3, 7, and 14 days after application (Z-stack of 16 slices; 200 µm layer thickness). (B) Single diffusely infiltrating wild-type Tu-2449 glioma cells migrate with a velocity of ∼100 µm per day through the neuropil. Note the typical bipolar morphology of infiltrating glioma cells breaching into the cortical layer. Scale bars, 100 µm.

Fig. 4.

STAT3 knockdown stops diffuse infiltration. DsRed- and EGFP-tagged Tu-2249 glioma cells were seeded on the caudate putamen 2 days after brain slice preparation. Infiltration was monitored live by confocal microscopy of red fluorescent wild-type, green fluorescent STAT3 knockdown, and merged yellow fluorescent Tu-2449 glioma cells. Propagation through the neuropil was photographed at days 3, 7, and 14 after application (Z-stacks of 16 slices; 200 µm layer thickness) showing spread of wild-type Tu-2449 glioma cells (red) in contrast to growth arrest of STAT3 knockdown Tu-2449 glioma cells (green). White matter tracts function as guardrails. Scale bars, 200 µm.

Fig. 5.

STAT3 knockdown prolongs survival of tumor-transplanted mice. Kaplan–Meier survival curves are shown for B6C3F1 mice transplanted with Tu-2449/control vector (red line) and Tu-2449/shSTAT3 (blue line) glioma cells, respectively. Fifteen animals were included in each group. Mean survival of Tu-2449/control vector transplanted mice was 18 days, whereas it was 30 days for Tu-2449/shSTAT3 transplanted mice (P< .0007). Knockdown of STAT3 resulted in 27% long-term survivors.

Fig. 6.

STAT3 knockdown reduces podoplanin expression and microvilli formation. (A) TaqMan RT-PCR and western blot analysis for podoplanin. Both mRNA and protein expression are dramatically downregulated in Tu-2449/shSTAT3 cells compared with Tu-2449/control vector cells. (B) Scheme of transwell chamber assay with 0.4-µm pore size excluding transmigration of whole cells. (C) Fluorescent wheat germ agglutinin was used to visualize sugar-containing plasma membrane protrusions on control vector and shSTAT3 transduced glioma cells. Note abundant microvilli in control vector cells poked through the narrow pores. Magnification, 60× (upper panel); 4× (lower panel). Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 7.

STAT3 knockdown decreases podoplanin expression in transplanted tumors. Shown is the immunohistochemical analysis of the podoplanin expression in intracerebrally transplanted gliomas from Tu-2449 cells stably transduced with shRNA against STAT3 or control vector. Podoplanin protein is preferentially found at the junction from tumor to normal brain. Note the absence of podoplanin in the brain parenchyma of tumor-free animals. Scale bar, 100 µm.