Antisecretory Factor-Mediated Inhibition of Cell Volume Dynamics Produces Antitumor Activity in Glioblastoma

Abstract

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AF's ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 777-90. ©2018 AACR.

Conflict of interest statement

Conflicting interests: The authors have no financial or competing interest. Dr. Persson is a co-author on patent WO2005030246 A1 entitled “Novel use of antisecretory factor”.

©2018 American Association for Cancer Research.

Figures

Fig. 1. AF increases MRI-based ADC levels and lowers IFP in GBM xenografts

(A–B) MRI-based measurements of ADC levels following acute nasal injections of AF peptide or chronic induction of AF protein (SPC diet) showed increased ADC levels in GBM43 xenografts versus control. (C) Setup for telemetry-based recordings of IFP in GBM xenografts. (D–E) Parallel measurements of tumor growth (bioluminescence) and IFP (mmHg) in GBM43 xenografts. (F) Immunoreactivity for AF protein ten days following administration of SPC diet to GBM43 xenografts showing high expression in tumor cells (dotted line; tumor border) but not in CD31+endothelial cells, GFAP+ reactive astrocytes, or CD45+ immune cells. Scale bar: top panel 50 μm, bottom panel 10 μm. (G) IFP measurements in GBM43 xenografts. SPC diet lowered IFP levels versus control. (H) Nasal administration of AF peptide effectively reduced IFP levels for 10-30 minutes. *P < 0.05, **P < 0.01.

Fig. 2. AF treatment increases doxorubicin uptake in GBM xenografts

(A) Mice xenografted with GBM43 cells were given a combination of doxorubicin (Dox) and SPC diet or nasal AF. (B–D) Doxorubicin (red) and DAPI (blue) in tumors 20 min after tail-vein injection of doxorubicin. Scale bar: 40 μm. (E) Quantification of fluorescence (red) shows increased uptake of doxorubicin in tumor tissues of mice treated with SPC diet or nasal AF (n=3, **P < 0.01, one-way ANOVA). Kaplan-Meier (n=6 for control, n=5 for AF, and n=9 for SPC, **P < 0.01, ***P < 0.001; one-way ANOVA for control v/s AF and SPC, respectively) curve showing survival benefit following combination treatments with doxorubicin and SPC diet or nasal AF.

Fig. 3. AF treatment reduces tumor growth in GBM models

(A) Parallel IFP and bioluminescence measurements showed that treatment with SPC diet reduced IFP levels and tumor growth compared to control GBM43 xenografts. (B–C) SPC treatment reduced proliferation (Ki67) and induced apoptosis (cleaved caspase 3) in GBM43 xenografts. Scale bar: 30 μm. (n=3, *P < 0.05, Student’s t test) (D) Kaplan-Meier curves showing increased survival in GBM43 xenografts treated with SPC diet (n=7 for control and n=12 for SPC, **P < 0.01; Log-rank Mantel-Cox test) or Salovum (n=7 for control and n=5 for Salovum, *P < 0.05; Log-rank Mantel-Cox test) compared to control. SPC diet and Salovum also increased survival in EGFRvIII-expressing GBM6 xenografts (n=8 for control, n=13 for SPC, and n=10 for Salovum *P < 0.05, **P < 0.01; one-way ANOVA). (E) Immunocompetent GBM model based on established neurospheres from the subventricular zone of Ink4a/Arf null FVBn mice that were lentivirally transduced to express human EGFRvIII, mCherry, and LUC. Mouse GBM cells were allografted into adult FVBn mice and administered SPC diet on day 6. (F–G) SPC treatment resulted in AF induction in tumor cells (dotted line; tumor border) and induction of apoptosis (cleaved caspase 3). Scale bar: top panel 50 μm, bottom panel 20 μm. (n=3, **P < 0.01, Student’s t test). Kaplan-Meier (n=5 for control and n=5 for SPC, **P < 0.01; Log-rank Mantel-Cox test) curve showing increased survival following SPC treatment in GBM43 xenografts.

Fig. 4. AF prevents restoration of cell volume and chloride permeability under hyperosmolar conditions

(A–F) Phalloidin (F-actin, green) and DAPI (nuclei, blue) stainings show significantly reduced cellular and nuclear volumes after SPC treatment of mice xenografted with GBM43 cells. (C) Orthogonal view show that Phalloidin (arrows) is expressed around DAPI+ nuclei. (D) Imaris software was used to demonstrate a correlation between approximate values for cell (Phalloidin, F-actin) and nuclear (DAPI) volumes in GBM cells. Scale bar: 20 μm. (n=3, *P < 0.05, Student’s t test). (G–H) SPC treatment reduced immunoreactivity of phosphorylated NKCC1 (pNKCC1) in GBM43 tumors. (I) AF peptide (5 μg/ml) and bumetanide (BMT, 10μM) reduced fluorescence of excited calcein-AM dye when GBM43 cells were transferred to hypertonic conditions (432 mOsm). Scale bar: 10 μm. (J) Cell shrinkage was induced with hypertonic exposure in both BMT and AF peptide treated cell with no regulatory volume increase response recorded within 20 minutes of hypertonic exposure. (K) Quantification of regulatory volume increase data. (n=3, *P < 0.05, one-way ANOVA) (L) Time-lapse recordings of intracellular chloride concentrations in GBM14 cells in the presence of BMT or AF peptide. (M) Quantifications following adjustment for pH changes showing changes in [Cl−] following incubation with BMT or AF peptide versus control (median values of 30, 39 and 39 time-lapse experiments for BMT, AF and control, respectively).

Fig. 5. NKCC1 is necessary for AF-mediated compression-induced proliferation in GBM tumorspheres

(A) NKCC1 is expressed in human GMB43 cells at the cell membrane and in the cytoplasm. Scale bar: 10μm. (B) NKCC1 protein is expressed in four human GMB tumorsphere cultures. (C) AF treatment has no proliferative effects on GBM43 cells grown under atmospheric pressure. (D) Human GBM tumorspheres were grown under static compression (30%) for 48 hours in HA-gels. (E) Tumorsphere size was measured following 48 h compression (30%) for human GBM tumorspheres grown in 3D HA-gels. (n=3, **P < 0.01, ***P < 0.001, Student’s t test). (F) AF and NKCC1 knockdown reversed compression-induced expansion of tumorsphere size. Scale bar: 100 μm (insert 7 μm). (G) Knockdown of NKCC1 by shRNA in GBM43 cells. (H) shRNA-mediated knockdown of NKCC1 and the NKCC1 inhibitor bumetanide (BMT) restored compression-induced expansion of GBM tumorspheres. No further effects of AF or BMT were found in shNKCC1 cells. (I) AF peptide reverses the protein levels of pH3 and pAKT in cells subjected to static pressure (30%) for 48 hours. (n=3, ***P < 0.001, one-way ANOVA).

Fig. 6. Combined AF and temozolomide treatment blocks regrowth in GBM xenografts

(A) Bioluminescence data of mice treated with control diet, SPC, TMZ (5 mg/kg, 2 consecutive days) alone, or in combination. (B) TMZ alone or in combination with SPC diet effectively reduced tumor growth at early stages of tumor development. (C–D) Bioluminescence showing regrowth of tumors following TMZ treatment, but not after a combination of TMZ and SPC diet. (E) Kaplan-Meier (n=9 for control, n=8 for SPC, n=9 for TMZ and n=7 for SPC + TMZ, **P < 0.01; Log-rank Mantel-Cox test) curve displays regrowth after ~50 days post-TMZ treatment. Tumors in mice treated with a combination of SPC and TMZ never displayed regrowth.