Convective forces increase CXCR4-dependent glioblastoma cell invasion in GL261 murine model

Abstract

Glioblastoma is the most common and malignant form of brain cancer. Its invasive nature limits treatment efficacy and promotes inevitable recurrence. Previous in vitro studies showed that interstitial fluid flow, a factor characteristically increased in cancer, increases glioma cell invasion through CXCR4-CXCL12 signaling. It is currently unknown if these effects translate in vivo. We used the therapeutic technique of convection enhanced delivery (CED) to test if convective flow alters glioma invasion in a syngeneic GL261 mouse model of glioblastoma. The GL261 cell line was flow responsive in vitro, dependent upon CXCR4 and CXCL12. Additionally, transplanting GL261 intracranially increased the populations of CXCR4⁺ and double positive cells versus 3D culture. We showed that inducing convective flow within implanted tumors indeed increased invasion over untreated controls, and administering the CXCR4 antagonist AMD3100 (5 mg/kg) effectively eliminated this response. These data confirm that glioma invasion is stimulated by convective flow in vivo and depends on CXCR4 signaling. We also showed that expression of CXCR4 and CXCL12 is increased in patients having received standard therapy, when CED might be elected. Hence, targeting flow-stimulated invasion may prove beneficial as a second line of therapy, particularly in patients chosen to receive treatment by convection enhanced delivery.

Figure 1

Interstitial flow increases GL261 invasion in a CXCR4-CXCL12 dependent manner. (A) Schematic representation of tissue culture insert setup for static and flow experimental conditions. (B) Percent invasion of GL261 in static and flow conditions with and without addition of 10 µM AMD3100 (n = 5, *p < 0.05). (C) Percent GL261 invasion in static and flow conditions with and without addition of 100 nM CXCL12 (n = 4, *p < 0.05). Bars show standard error.

Figure 2

Population-level expression of CXCR4 and CXCL12 in GL261 depends on growth conditions. Flow cytometry was used to determine the percent of CXCR4⁺, CXCL12⁺, and double positive GL261 in 2D, 3D, and in vivo environments. Representative plots gated on live glioma cells are shown in the left column for (A) CXCR4⁺, (B) CXCL12⁺, and (C) double positive populations. Correlating quantifications are shown on the right. *p < 0.05, ****p < 0.0001. Bars show standard error.

Figure 3

Interstitial flow increases murine glioma cell invasion in vivo in a CXCR4-dependent manner. (A) Schematic of intratumoral convection enhanced delivery. (B) Experimental timeline. (C–J) Representative fluorescence images of in vivo glioma invasion for (C,D) untreated controls, (E,F) CED alone group, (G,H) AMD alone group, and (I–J) + CED/ + AMD group. Top: Full brain slice scans with nuclei labeled with DAPI (blue), with tumor defined by white dotted line. Scale bar = 1 mm. Bottom: GFP-labeled GL261 tumor cells at the border location depicted above (red boxes). Scale bar = 100 µm. (K) Quantification of tumor cells beyond the tumor border averaged per mouse from five locations in three sections through tumors. Bars show standard error.

Figure 4

Treatment of GL261 with AMD3100 decreases convection-driven increases in pCXCR4. Representative fluorescence images at GL261 tumor (T) borders of GFP-GL261 (green) and pCXCR4 (magenta) in (A) untreated animals, (B) animals receiving only CED, and (C) animals dosed with AMD3100 for two days prior to CED. Scale bars = 100 μm.

Figure 5

Immunoreactivity for CXCR4 and CXCL12 is increased in patients who received therapy. (A) Representative fluorescence image of a resected tumor from a patient prior to therapy stained for CXCR4 (red) and its ligand CXCL12 (cyan). (B) Close up of white boxed area for CXCR4, and (C) Close up for CXCL12. (D) Representative fluorescence image of a resected tumor from a patient after standard of care therapy stained for CXCR4 (red) and its ligand CXCL12 (cyan). (E) Close up of white boxed area for CXCR4, and (F) Close up for CXCL12. The entire tissue sample is outlined with a white dashed line. Scale bars are 7 mm for A/D and 200 μm for B/C/E/F. (G) Quantification of fluorescence intensity from high magnification images (B/C/E/F) at five random locations throughout the tumor sample. *p < 0.05 and **p < 0.01.