Compression-induced changes in the transcriptional GBM phenotype. Differentially expressed genes in tumorspheres derived from human GBMs (GBM5, GBM14, GBM34, and GBM43) when comparing compression to other conditions.

Fig. S2. Interstitial fluid pressure (IFP) pressure sensors do not increase reactive gliosis or microglia activation during tumor progression. (A, B) GFAP+ reactive astrocytes (DAB; brown) and (C, D) Iba1+ tumor-associated macrophages (DAB; brown) in progressing GBM43 tumors in the absence or presence of IFP sensor. Nuclei are counterstained with hematoxylin (blue). Scale bar: 40 μm.

Fig. S3. AF treatment increases drug uptake resulting in increased survival of GBM43 xenografted mice. (A, B) SPC diet increased uptake and reduced clearance of tail-vein injected gadolinium in mice xenografted with GBM43 cells. (C) Treatment regimen with SPC diet and/or Erlotinib for uptake and survival studies in GBM43 xenografts. (D) SPC treatment increased Erlotinib levels in GBM43 tumors 60 min following a single administration of Erlotinib. (n=4 for control and SPC, *P < 0.05; Student's t-test). (E) Kaplan-Meier (n=8 for all groups, **P < 0.01; Log-rank Mantel-Cox test or One way ANOVA) curve increased survival following SPC and Erlotinib treatment versus Erlotinib alone.

Fig. S1. AF treatment increases ADC in tumor tissues. MR imaging showing an increase of (A) ADC in xenografted mice treated with nasal AF or SPC-diet with corresponding (B) T2w images.

Fig. S6. AF peptide have no proliferative effect on pNKCC1 expressing human GBM cells under atmospheric pressure. (A) Adherent human GBM cells show immunoreactivity against an antibody recognizing Thr-phosphorylation site on human NKCC1. (B) AF treatment had no proliferative effects on human GBM5 cells grown under atmospheric pressure.

Fig. S7. AF reversed compression-induced transcriptional phenotype of GBM tumorspheres. Both (A-B) smaller and (C-D) larger GBM43 tumorspheres expanded in response to 48h compression. (***P < 0.05, Student's t-test). (E) Principle components analysis of all expressed genes. The first principle component is associated with GBM subtype, while the second principle component is associated with the presence of compression. (F) Heat map of genes differentially expressed when comparing compression to other conditions (see supplementary table S1 for gene list).

Fig. S5. AF prevents restoration of cell volume of GC22 cells under hyperosmolar conditions. (A) AF peptide (5 μg/ml) and bumetanide (BMT, 10 μM) reduced fluorescence of excited calcein-AM dye when GC22 cells were transferred to hypertonic conditions (432 mOsm). Scale bar: 10 μm. (B) Cell shrinkage was induced with hypertonic exposure in both BMT and AF peptide treated cells with no regulatory volume increase recorded within 20 minutes of hypertonic exposure. (C) Quantification of regulatory volume increase data. (n=3, *P < 0.05, one-way ANOVA).

Fig. S4. AF treatment results in no changes in body weight for mice xenografted with GBM43 cells and resulted in complete tumor regression in an EGFRvIII-expressing GBM allograft model. (A, B) SPC or Salovum treatment had no effect on body weight in GBM43 xenografts (n=7 for control, n=10 for SPC, and n=5 for Salovum). (C) SPC treatment resulted in complete tumor regression in 5/6 FVBn mice allografted with EGFRvIII and mCherry-expressing mouse GBM cells (dotted line; tumor border). Scale bar 2 mm.

Native interactions between lysophospholipids (LPs) and their cognate LP receptors are difficult to measure because of lipophilicity and/or the adhesive properties of lipids, which contribute to high levels of nonspecific binding in cell membrane preparations. Here, we report development of a free-solution assay (FSA) where label-free LPs bind to their cognate G protein-coupled receptors (GPCRs), combined with a recently reported compensated interferometric reader (CIR) to quantify native binding interactions between receptors and ligands. As a test case, the binding parameters between lysophosphatidic acid (LPA) receptor 1 (LPA1; one of six cognate LPA GPCRs) and LPA were determined. FSA-CIR detected specific binding through the simultaneous real-time comparison of bound versus unbound species by measuring the change in the solution dipole moment produced by binding-induced conformational and/or hydration changes. FSA-CIR identified KD values for chemically distinct LPA species binding to human LPA1 and required only a few nanograms of protein: 1-oleoyl (18:1; KD = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; KD = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; KD = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; KD = 1.69 ± 0.1 nM) LPA. These KD values compared favorably to those obtained using the previous generation back-scattering interferometry system, a chip-based technique with low-throughput and temperature sensitivity. In conclusion, FSA-CIR offers a new increased-throughput approach to assess quantitatively label-free lipid ligand-receptor binding, including nonactivating antagonist binding, under near-native conditions.

Posthemorrhagic hydrocephalus (PHH) in premature infants is a common neurological disorder treated with invasive neurosurgical interventions. Patients with PHH lack effective therapeutic interventions and suffer chronic comorbidities. Here, we report a murine lysophosphatidic acid (LPA)-induced postnatal PHH model that maps neurodevelopmentally to premature infants, a clinically accessible high-risk population, and demonstrates ventriculomegaly with increased intracranial pressure. Administration of LPA, a blood-borne signaling lipid, acutely disrupted the ependymal cells that generate CSF flow, which was followed by cell death, phagocytosis, and ventricular surface denudation. This mechanism is distinct from a previously reported fetal model that induces PHH through developmental alterations. Analyses of LPA receptor-null mice identified LPA(1) and LPA(3) as key mediators of PHH. Pharmacological blockade of LPA(1) prevented PHH in LPA-injected animals, supporting the medical tractability of LPA receptor antagonists in preventing PHH and negative CNS sequelae in premature infants.

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.

Glioblastoma (GBM) is among the most aggressive cancers. Although high interstitial fluid pressure presents a barrier to drug uptake, how mechanical stresses and osmotic changes impact tumor biology remains unclear. We show that inhibition of the sodium-potassium-chloride co-transporter 1 (NKCC1) hindered proliferation and invasion of GBM cells. Compressive pressures actually promoted proliferation of GBM, an effect antagonized by the NKKC1 inhibitor bumetanide, which penetrates poorly into brain, or antisecretory factor, a protein that readily accumulates in brain, and has been used clinically. Antisecretory factor reduced IFP in patient-derived GBM xenografts. Both antisecretory factor and bumetanide inhibited osmotic adaptation of cultured GBM cells. In vivo, antisecretory factor reduced tumor growth, increased uptake of chemotherapeutics, and promoted temozolomide-sensitivity in therapy-resistant GBM xenografts. Thus, antisecretory factor represents a novel strategy to improve outcome in GBM patients.

Neurogenesis, the generation of new neurons, is deregulated in neural stem cell (NSC)- and progenitor-derived murine models of malignant medulloblastoma and glioma, the most common brain tumors of children and adults, respectively. Molecular characterization of human malignant brain tumors, and in particular brain tumor stem cells (BTSCs), has identified neurodevelopmental transcription factors, microRNAs, and epigenetic factors known to inhibit neuronal and glial differentiation. We are starting to understand how these factors are regulated by the major oncogenic drivers in malignant brain tumors. In this review, we will focus on the molecular switches that block normal neuronal differentiation and induce brain tumor formation. Genetic or pharmacological manipulation of these switches in BTSCs has been shown to restore the ability of tumor cells to differentiate. We will discuss potential brain tumor therapies that will promote differentiation in order to reduce treatment resistance, suppress tumor growth, and prevent recurrence in patients.

BACKGROUND: High interstitial fluid pressure (IFP) represents a barrier for drug uptake in human GBM, the most common primary brain tumor. Fluid accumulation and high cell density compress tumors and promote osmotic swelling of tumor cells. Although studies have clarified the role of tumor vasculature, it remains unclear whether osmotic swelling of cancer cells regulates tumor growth and drug uptake. METHODS: To address this, we used human GBM tumorspheres and xenografts. We developed methodology to measure IFP in GBM xenografts and mechanical compression in 3D-cultures. Fluorescent-based sensors were used to measure cell volume and chloride levels. To reduce IFP and osmotic swelling in GBM cells, we established exogenous or endogenous induction of antisecretory factor (AF), known to be safe in patients, reduces elevated intracranial pressure in rodents, and lowers IFP in subcutaneous solid tumors. RESULTS: Intriguingly, our data demonstrate that elevated pressure drives proliferation of GBM tumorspheres. Transcriptional profiling showed that increased compression regulates genes involved in translation and ion transport. AF peptide completely blocked compression-induced proliferation and transcriptional changes. We found that AF targeted the sodium-potassium-chloride channel (NKCC1), more potently than the NKCC1 inhibitor bumetanide, and prevented restoration of cell volume and chloride permeability under hyperosmotic conditions. AF therapy effectively reduced tumor growth, increased drug uptake, and extended survival in GBM xenografts, effects that were mediated through lowering of IFP, reduced cell volume, and inhibition of NKCC1 activity. CONCLUSIONS: We find that elevated IFP in human GBMs maintains osmotic swelling in tumor cells as a second barrier for drug uptake. Our results further show that elevated pressure is a driver of proliferation, survival, and translational control. AF therapy represents a novel approach to inhibit NKCC1 activity and osmotic swelling in human GBMs, leading to reduced tumor growth and increased drug uptake, ultimately improving the outcome for this disease.

Glioma is the most common primary malignant brain tumor and arises throughout the central nervous system. Recent focus on stem-like glioma cells has implicated neural stem cells (NSCs), a minor precursor population restricted to germinal zones, as a potential source of gliomas. In this review, we focus on the relationship between oligodendrocyte progenitor cells (OPCs), the largest population of cycling glial progenitors in the postnatal brain, and gliomagenesis. OPCs can give rise to gliomas, with signaling pathways associated with NSCs also playing key roles during OPC lineage development. Gliomas can also undergo a switch from progenitor- to stem-like phenotype after therapy, consistent with an OPC-origin even for stem-like gliomas. Future in-depth studies of OPC biology may shed light on the etiology of OPC-derived gliomas and reveal new therapeutic avenues.

BACKGROUND: (blind field). METHODS: We developed methodology to measure IFP in human GBM xenografts and mechanical compression in 3D-cultures. We used a cell counter and calcein-AM-loaded cells to measure cell volume following treatment with temozolomide and/or blockade of NKCC1 activity. Fluorescent MQAE-loaded cells were recorded to measure intracellular chloride levels. To study uptake of chemotherapy following IFP-reduction in GBM xenografts following AF-induction or treatment with bumetanide, we injected doxorubicin and measured fluorescent doxorubicin levels in tissue sections. RESULTS: Elevated compression increased proliferation in GBM cultures and IFP levels rapidly increased during the exponential growth phase in GBM xenografts. In contrast to Avastin that targets the vasculature, SPC diet induced AF expression only in tumor cells. In addition to reducing IFP levels, AF-induction also inhibited proliferation, induced apoptosis, and increased survival in mice xenografted with human GBM cells. SPC diet and intranasal injection of AF increased uptake of doxorubicin in GBM xenografts. In vitro, AF augmented TMZ-induced apoptosis and reduced proliferation at both baseline and increased hydrostatic compression. CONCLUSIONS: In contrast to IFP-reducing therapies targeting the vasculature, treatments that reduced osmotic pressure in GBM cells effectively reduced tumor growth and invasion in vivo. AF and bumetanide increased uptake and cytotoxic response from chemotherapies by inhibiting NKCC1 activity in GBM cells. Our studies suggest that elevated IFP promotes tumor growth, reduces drug uptake, and limits therapy-response in GBM. AF-induction represents an attractive strategy to reduce invasion, inhibit tumor growth, increase drug uptake, and ultimately improve the survival of GBM patients. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

High interstitial fluid pressure (IFP) represents a barrier for drug uptake in human GBM, the most common malignant primary brain tumor in adults. Increased IFP is due to leakiness of blood vessels and reduced drainage of fluid. This accumulation of fluid and high cell density compresses tumor tissue and promotes swelling of tumor cells. Although studies have clarified the role of tumor vasculature, it is still unclear if elevated IFP and swelling of cancer cells regulates tumor growth and drug uptake. Inhibition of NKCC1 (Na-K-Cl cotransporter) activity with bumetanide renders glioma cells unable to restore cell volume following osmotic challenges, blocks invasion in GBM xenografts, and augments temozolomide-mediated apoptosis in vitro. However, concerns about side-effects following bumetanide treatment warrants development of new approaches. Our preliminary data show that induction of antisecretory factor (AF), a regulator of fluid secretion, inhibited phosphorylation of NKCC1 in intracranial xenografts of human primary GBMs. Since AF therapy is safe in patients and lowers IFP in experimental models of solid tumors, we hypothesize that AF induction lowers IFP levels and increases drug uptake in xenografted GBMs. We have established novel methodology to study IFP in GBMs intracranially grafted into mice and the effects of compression in 3D-cultures. Our preliminary data show that elevated IFP and compression promotes tumor growth of GBMs. Our data suggest that AF induction lowers IFP levels by blocking restoration of cell volume and increases uptake of chemotherapy. Surprisingly, we found that AF induction alone reduced tumor growth and extended the survival of transplanted mice. Our work establish a role for IFP and cell swelling as potential therapeutic targets in GBMs and other solid cancers. As a novel pressure-reducing therapy, AF-induction represents an attractive strategy to reduce tumor growth, increase drug uptake, and improve survival in brain tumor patients.