Detailed longitudinal sampling of glioma stem cells in situ reveals Chr7 gain and Chr10 loss as repeated events in primary tumor formation and recurrence.

Intratumoral heterogeneity at the genetic, epigenetic, transcriptomic, and morphologic levels is a commonly observed phenomenon in many aggressive cancer types. Clonal evolution during tumor formation and in response to therapeutic intervention can be predicted utilizing reverse engineering approaches on detailed genomic snapshots of heterogeneous patient tumor samples. In this study, we developed an extensive dataset for a GBM case via the generation of polyclonal and monoclonal glioma stem cell lines from initial diagnosis, and from multiple sections of distant tumor locations of the deceased patient's brain following tumor recurrence. Our analyses revealed the tissue-wide expansion of a new clone in the recurrent tumor and chromosome 7 gain and chromosome 10 loss as repeated genomic events in primary and recurrent disease. Moreover, chromosome 7 gain and chromosome 10 loss produced similar alterations in mRNA expression profiles in primary and recurrent tumors despite possessing other highly heterogeneous and divergent genomic alterations between the tumors. We identified ETV1 and CDK6 as putative candidate genes, and NFKB (complex), IL1B, IL6, Akt and VEGF as potential signaling regulators, as potentially central downstream effectors of chr7 gain and chr10 loss. Finally, the differences caused by the transcriptomic shift following gain of chromosome 7 and loss of chromosome 10 were consistent with those generally seen in GBM samples compared to normal brain in large-scale patient-tumor data sets.

Organoids as a Systems Platform for SCLC Brain Metastasis.

Small Cell Lung Cancer (SCLC) is a highly aggressive, neuroendocrine tumor. Traditional reductionist approaches have proven ineffective to ameliorate the uniformly dismal outcomes for SCLC - survival at 5 years remains less than 5%. A major obstacle to improving treatment is that SCLC tumor cells disseminate early, with a strong propensity for metastasizing to the brain. Accumulating evidence indicates that, contrary to previous textbook knowledge, virtually every SCLC tumor is comprised of multiple subtypes. Important questions persist regarding the role that this intra-tumor subtype heterogeneity may play in supporting the invasive properties of SCLC. A recurrent hypothesis in the field is that subtype interactions and/or transition dynamics are major determinants of SCLC metastatic seeding and progression. Here, we review the advantages of cerebral organoids as an experimentally accessible platform for SCLC brain metastasis, amenable to genetic manipulations, drug perturbations, and assessment of subtype interactions when coupled, e.g., to temporal longitudinal monitoring by high-content imaging or high-throughput omics data generation. We then consider systems approaches that can produce mathematical and computational models useful to generalize lessons learned from ex vivo organoid cultures, and integrate them with in vivo observations. In summary, systems approaches combined with ex vivo SCLC cultures in brain organoids may effectively capture both tumor-tumor and host-tumor interactions that underlie general principles of brain metastasis.

Cytosolic phospholipase A2 as a mediator of disease pathogenesis.

As efficient catalysts, enzymes help maintain a variety of biological and chemical transformations necessary for cellular metabolism and normal physiology. Unfortunately, pathogenic microbes can also exploit enzymatic reactions in an attempt to spread infection. Cytosolic phospholipase A2 (cPLA(2) ) is an enzyme that is responsible for the hydrolysis of membrane phospholipids such as phosphatidylcholine. Following activation, cPLA(2) cleaves phosphatidylcholine to yield free fatty acid and lysophosphatidylcholine. Both of these products and their downstream metabolites initiate a network of signalling cascades that influence cellular viability and inflammation. Recent observations have shown that viral and bacterial agents often target this intricate organization of signalling molecules. This review briefly discusses the role of cPLA(2) in the biological response to disease-causing pathogens and injury, the immunological process and tumour progression.

Cytosolic phospholipase A2: targeting cancer through the tumor vasculature.

PURPOSE: In vascular endothelial cells, low doses of ionizing radiation trigger the immediate activation of cytosolic phospholipase A2 (cPLA2). This event initiates prosurvival signaling that could be responsible for radioresistance of tumor vasculature. Thus, the development of radiosensitizers targeting these survival pathways may enhance tumor response to radiation therapy. Arachidonyltrifluoromethyl Ketone (AACOCF3), a specific cPLA2 inhibitor, was studied as a potential radiosensitizer. EXPERIMENTAL DESIGN: Vascular endothelial cells (3B11 and MPMEC) and lung tumor cells (LLC and H460) were treated with 1 micromol/L AACOCF3 for 30 minutes prior to irradiation. Treatment response was evaluated by clonogenic survival, activation of extracellular signal-regulated kinase 1/2 (ERK1/2), tubule formation, and migration assays. For in vivo experiments, mice with LLC or H460 tumors in the hind limbs were treated for 5 consecutive days with 10 mg/kg AACOCF3 administered daily 30 minutes prior to irradiation. Treatment response was assessed by tumor growth delay, Power Doppler Sonography, and immunohistochemistry. RESULTS: In cell culture experiments, inhibition of cPLA2 with AACOCF3 prevented radiation-induced activation of ERK1/2 and decreased clonogenic survival of irradiated vascular endothelial cells but not the lung tumor cells. Treatment with AACOCF3 also attenuated tubule formation and migration in irradiated vascular endothelial cells. In both tumor mouse models, treatment with AACOCF3 prior to irradiation significantly suppressed tumor growth and decreased overall tumor blood flow and vascularity. Increased apoptosis in both tumor cells and tumor vascular endothelium was determined as a possible mechanism of the observed effect. CONCLUSION: These findings identify cPLA2 as a novel molecular target for tumor sensitization to radiation therapy through the tumor vasculature.

Generating Patient-Derived Gliomas within Cerebral Organoids.

Glioblastoma (GBM) remains a devastating disease with a median survival of less than two years. Current preclinical models are unable to accurately reflect the complexity of human GBM. We recently established a cerebral organoid glioma (GLICO) model to study the invasion and biology of patient-derived glioma stem cells in miniature replicas of the human brain. Through the dissemination of our detailed methodology, we aim to encourage other scientists to further build upon our existing model for studying these destructive tumors. For complete details on the use and execution of this protocol, please refer to Linkous et al. (2019).

Tumor Microenvironment Is Critical for the Maintenance of Cellular States Found in Primary Glioblastomas.

Glioblastoma (GBM), an incurable tumor, remains difficult to model and more importantly to treat due to its genetic/epigenetic heterogeneity and plasticity across cellular states. The ability of current tumor models to recapitulate the cellular states found in primary tumors remains unexplored. To address this issue, we compared single-cell RNA sequencing of tumor cells from 5 patients across four patient-specific glioblastoma stem cell (GSC)-derived model types, including glioma spheres, tumor organoids, glioblastoma cerebral organoids (GLICO), and patient-derived xenografts. We find that GSCs within the GLICO model are enriched for a neural progenitor-like cell subpopulation and recapitulate the cellular states and their plasticity found in the corresponding primary parental tumors. These data demonstrate how the contribution of a neuroanatomically accurate human microenvironment is critical and sufficient for recapitulating the cellular states found in human primary GBMs, a principle that may likely apply to other tumor models. SIGNIFICANCE: It has been unclear how well different patient-derived GBM models are able to recreate the full heterogeneity of primary tumors. Here, we provide a complete transcriptomic characterization of the major model types. We show that the microenvironment is crucial for recapitulating GSC cellular states, highlighting the importance of tumor-host cell interactions.See related commentary by Luo and Weiss, p. 907.This article is highlighted in the In This Issue feature, p. 890.

Modeling Patient-Derived Glioblastoma with Cerebral Organoids.

The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a "normal" human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor microtubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening.

A Core Regulatory Circuit in Glioblastoma Stem Cells Links MAPK Activation to a Transcriptional Program of Neural Stem Cell Identity.

Glioblastoma, the most common primary malignant brain tumor, harbors a small population of tumor initiating cells (glioblastoma stem cells) that have many properties similar to neural stem cells. To investigate common regulatory networks in both neural and glioblastoma stem cells, we subjected both cell types to in-vitro differentiation conditions and measured global gene-expression changes using gene expression microarrays. Analysis of enriched transcription factor DNA-binding sites in the promoters of differentially expressed genes was used to reconstruct regulatory networks involved in differentiation. Computational predictions, which were biochemically validated, show an extensive overlap of regulatory circuitry between cell types including a network centered on the transcription factor KLF4. We further demonstrate that EGR1, a transcription factor previously shown to be downstream of the MAPK pathway, regulates KLF4 expression and that KLF4 in turn transcriptionally activates NOTCH as well as SOX2. These results demonstrate how known genomic alterations in glioma that induce constitutive activation of MAPK are transcriptionally linked to master regulators essential for neural stem cell identify.

Autotaxin Inhibition with PF-8380 Enhances the Radiosensitivity of Human and Murine Glioblastoma Cell Lines.

PURPOSE: Glioblastoma multiforme (GBM) is an aggressive primary brain tumor that is radio-resistant and recurs despite aggressive surgery, chemo, and radiotherapy. Autotaxin (ATX) is over expressed in various cancers including GBM and is implicated in tumor progression, invasion, and angiogenesis. Using the ATX specific inhibitor, PF-8380, we studied ATX as a potential target to enhance radiosensitivity in GBM. METHODS AND MATERIALS: Mouse GL261 and Human U87-MG cells were used as GBM cell models. Clonogenic survival assays and tumor transwell invasion assays were performed using PF-8380 to evaluate role of ATX in survival and invasion. Radiation dependent activation of Akt was analyzed by immunoblotting. Tumor induced angiogenesis was studied using the dorsal skin fold model in GL261. Heterotopic mouse GL261 tumors were used to evaluate the efficacy of PF-8380 as a radiosensitizer. RESULTS: Pre-treatment of GL261 and U87-MG cells with 1 µM PF-8380 followed by 4 Gy irradiation resulted in decreased clonogenic survival, decreased migration (33% in GL261; P = 0.002 and 17.9% in U87-MG; P = 0.012), decreased invasion (35.6% in GL261; P = 0.0037 and 31.8% in U87-MG; P = 0.002), and attenuated radiation-induced Akt phosphorylation. In the tumor window model, inhibition of ATX abrogated radiation induced tumor neovascularization (65%; P = 0.011). In a heterotopic mouse GL261 tumors untreated mice took 11.2 days to reach a tumor volume of 7000 mm(3), however combination of PF-8380 (10 mg/kg) with irradiation (five fractions of 2 Gy) took more than 32 days to reach a tumor volume of 7000 mm(3). CONCLUSION: Inhibition of ATX by PF-8380 led to decreased invasion and enhanced radiosensitization of GBM cells. Radiation-induced activation of Akt was abrogated by inhibition of ATX. Furthermore, inhibition of ATX led to diminished tumor vascularity and delayed tumor growth. These results suggest that inhibition of ATX may ameliorate GBM response to radiotherapy.

Novel radiosensitizing anticancer therapeutics.

In cancer treatment, radiation therapy is second only to surgery in terms of its curative potential. However, radiation-induced tumor cell death is limited by a number of factors, including the adverse response of the tumor microenvironment to radiation treatment and tumor-acquired mechanisms of evasive resistance. Recent attempts to enhance the therapeutic efficiency of ionizing radiation have produced promising results. In this review article, we discuss the development of novel therapeutic strategies for tumor sensitization to radiation therapy. These innovative approaches incorporate the involvement of the immune response and the role of cancer stem cells, as well as direct targeting of signal transduction pathways. Taken together, these concerted efforts demonstrate that the augmentation of radiotherapeutic efficacy results in significantly improved control not only of local disease, but also of metastatic spread and improved overall patient survival.

Novel therapeutic approaches for targeting tumor angiogenesis.

Current attempts to disrupt the complex process of tumor blood vessel formation are predominantly focused on targeting the vascular endothelial growth factor (VEGF)-VEGFR signaling pathway. Although clinically proven to inhibit VEGF and its receptors, these pharmacologic agents are selective, but not specific. Consequently, many of the approved inhibitors also impair other molecular targets leading to increased toxicity. Current efforts to unravel the complexity of tumor angiogenesis have identified several new candidates for antivascular therapy. In this review article, we identify well-established and novel angiogenic molecules and discuss benefits of the therapeutic approaches based on targeting of such factors.

Angiogenesis in glioblastoma multiforme: navigating the maze.

Blood vessel formation is a fundamental process that occurs during both normal and pathologic periods of tissue growth. In aggressive malignancies such as glioblastoma multiforme (GBM), vascularization is often excessive and facilitates tumor progression. In an attempt to maintain tumors in a state of quiescence, multiple anti-angiogenic agents have been developed. Although several angiogenesis inhibitors have produced enhanced clinical benefits in GBM, many of these pharmacologic agents result in transitory initial response phases followed by evasive tumor resistance. Thus, a significant need exists for the discovery of novel and effective anti-angiogenic therapies. The development of new molecular-targeted therapeutic strategies is often complicated by the complexity of angiogenic signal transduction. Due to the labyrinthine nature of these signaling pathways, increased production of other angiogenic factors may compensate for the inhibition of key vascular targets like vascular endothelial growth factor (VEGF). Such compensatory mechanisms facilitate vascularization and allow tumor growth to proceed even in the presence of anti-angiogenic agents. This review presents the challenges of targeting the intricate vascular network of GBM and discusses the clinical implications for recent advancements in targeted anti-angiogenic drug therapy.

Cytosolic phospholipase A2 as a molecular target for the radiosensitization of ovarian cancer.

In ovarian cancer, the molecular targeted chemotherapeutics could increase the efficiency of low-dose radiotherapy while decreasing injury to adjusted organs. In irradiated A2780 human ovarian carcinoma cells, cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF(3) prevented activation of pro-survival Akt signaling and enhanced cell death. The potential molecular mechanisms of this effect could involve signaling through lysophosphatidic acid receptors. In the heterotopic A2780 tumor model using nude mice, cPLA(2) inhibition significantly delayed tumor growth compared to treatment with radiation or vehicle alone. These results identify cPLA(2) as a molecular target to enhance the therapeutic ratio of radiation in ovarian cancer.

Autotaxin and LPA receptors represent potential molecular targets for the radiosensitization of murine glioma through effects on tumor vasculature.

Despite wide margins and high dose irradiation, unresectable malignant glioma (MG) is less responsive to radiation and is uniformly fatal. We previously found that cytosolic phospholipase A2 (cPLA(2)) is a molecular target for radiosensitizing cancer through the vascular endothelium. Autotaxin (ATX) and lysophosphatidic acid (LPA) receptors are downstream from cPLA(2) and highly expressed in MG. Using the ATX and LPA receptor inhibitor, α-bromomethylene phosphonate LPA (BrP-LPA), we studied ATX and LPA receptors as potential molecular targets for the radiosensitization of tumor vasculature in MG. Treatment of Human Umbilical Endothelial cells (HUVEC) and mouse brain microvascular cells bEND.3 with 5 µmol/L BrP-LPA and 3 Gy irradiation showed decreased clonogenic survival, tubule formation, and migration. Exogenous addition of LPA showed radioprotection that was abrogated in the presence of BrP-LPA. In co-culture experiments using bEND.3 and mouse GL-261 glioma cells, treatment with BrP-LPA reduced Akt phosphorylation in both irradiated cell lines and decreased survival and migration of irradiated GL-261 cells. Using siRNA to knock down LPA receptors LPA1, LPA2 or LPA3 in HUVEC, we demonstrated that knockdown of LPA2 but neither LPA1 nor LPA3 led to increased viability and proliferation. However, knockdown of LPA1 and LPA3 but not LPA2 resulted in complete abrogation of tubule formation implying that LPA1 and LPA3 on endothelial cells are likely targets of BrP-LPA radiosensitizing effect. Using heterotopic tumor models of GL-261, mice treated with BrP-LPA and irradiation showed a tumor growth delay of 6.8 days compared to mice treated with irradiation alone indicating that inhibition of ATX and LPA receptors may significantly improve malignant glioma response to radiation therapy. These findings identify ATX and LPA receptors as molecular targets for the development of radiosensitizers for MG.

Cytosolic phospholipase A2 and lysophospholipids in tumor angiogenesis.

BACKGROUND: Lung cancer and glioblastoma multiforme are highly angiogenic and, despite advances in treatment, remain resistant to therapy. Cytosolic phospholipase A2 (cPLA(2)) activation contributes to treatment resistance through transduction of prosurvival signals. We investigated cPLA(2) as a novel molecular target for antiangiogenesis therapy. METHODS: Glioblastoma (GL261) and Lewis lung carcinoma (LLC) heterotopic tumor models were used to study the effects of cPLA(2) expression on tumor growth and vascularity in C57/BL6 mice wild type for (cPLA(2)α(+/+)) or deficient in (cPLA(2)α(-/-)) cPLA(2)α, the predominant isoform in endothelium (n = 6-7 mice per group). The effect of inhibiting cPLA(2) activity on GL261 and LLC tumor growth was studied in mice treated with the chemical cPLA(2) inhibitor 4-[2-[5-chloro-1-(diphenylmethyl)-2-methyl-1H-indol-3-yl]-ethoxy]benzoic acid (CDIBA). Endothelial cell proliferation and function were evaluated by Ki-67 immunofluorescence and migration assays in primary cultures of murine pulmonary microvascular endothelial cells (MPMEC) isolated from cPLA(2)α(+/+) and cPLA(2)α(-/-) mice. Proliferation, invasive migration, and tubule formation were assayed in mouse vascular endothelial 3B-11 cells treated with CDIBA. Effects of lysophosphatidylcholine, arachidonic acid, and lysophosphatidic acid (lipid mediators of tumorigenesis and angiogenesis) on proliferation and migration were examined in 3B-11 cells and cPLA(2)α(-/-) MPMEC. All statistical tests were two-sided. RESULTS: GL261 tumor progression proceeded normally in cPLA(2)α(+/+) mice, whereas no GL261 tumors formed in cPLA(2)α(-/-) mice. In the LLC tumor model, spontaneous tumor regression was observed in 50% of cPLA(2)α(-/-) mice. Immunohistochemical examination of the remaining tumors from cPLA(2)α(-/-) mice revealed attenuated vascularity (P ≤ .001) compared with tumors from cPLA(2)α(+/+) mice. Inhibition of cPLA(2) activity by CDIBA resulted in a delay in tumor growth (eg, LLC model: average number of days to reach tumor volume of 700 mm(3), CDIBA vs vehicle: 16.8 vs 11.8, difference = 5, 95% confidence interval = 3.6 to 6.4, P = .04) and a decrease in tumor size (eg, GL261 model: mean volume on day 21, CDIBA vs vehicle: 40.1 vs 247.4 mm(3), difference = 207.3 mm(3), 95% confidence interval = 20.9 to 293.7 mm(3), P = .021). cPLA(2) deficiency statistically significantly reduced MPMEC proliferation and invasive migration (P = .002 and P = .004, respectively). Compared with untreated cells, cPLA(2)α(-/-) MPMEC treated with lysophosphatidylcholine and lysophosphatidic acid displayed increased cell proliferation (P = .011) and invasive migration (P < .001). CONCLUSIONS: In these mouse models of brain and lung cancer, cPLA(2) and lysophospholipids have key regulatory roles in tumor angiogenesis. cPLA(2) inhibition may be a novel effective antiangiogenic therapy.