Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment

Background

Three-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular structures and migration of cells in the brain of mice, however, with limited imaging depth. To enable comprehensive analysis of GBM and the brain microenvironment, in-depth 3D imaging methods are needed. Here, we employed methods for optical tissue clearing prior to 3D microscopy to visualize the brain microvasculature and routes of invasion of GBM cells.

Methods

We present a workflow for ex vivo imaging of optically cleared brain tumor tissues and subsequent computational modeling. This workflow was used for quantification of the microvasculature in relation to nuclear or cellular density in healthy mouse brain tissues and in human orthotopic, infiltrative GBM8 and E98 glioblastoma models.

Results

Ex vivo cleared mouse brain tissues had a >10-fold imaging depth as compared to intravital imaging of mouse brain in vivo. Imaging of optically cleared brain tissue allowed quantification of the 3D microvascular characteristics in healthy mouse brains and in tissues with diffuse, infiltrative growing GBM8 brain tumors. Detailed 3D visualization revealed the organization of tumor cells relative to the vasculature, in both gray matter and white matter regions, and patterns of multicellular GBM networks collectively invading the brain parenchyma.

Conclusions

Optical tissue clearing opens new avenues for combined quantitative and 3D microscopic analysis of the topographical relationship between GBM cells and their microenvironment.

     

Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells

Neural stem cells (NSCs) are considered to be the cell of origin of glioblastoma multiforme (GBM). However, the genetic alterations that transform NSCs into glioma-initiating cells remain elusive. Using a unique transposon mutagenesis strategy that mutagenizes NSCs in culture, followed by additional rounds of mutagenesis to generate tumors in vivo, we have identified genes and signaling pathways that can transform NSCs into glioma-initiating cells. Mobilization of Sleeping Beauty transposons in NSCs induced the immortalization of astroglial-like cells, which were then able to generate tumors with characteristics of the mesenchymal subtype of GBM on transplantation, consistent with a potential astroglial origin for mesenchymal GBM. Sequence analysis of transposon insertion sites from tumors and immortalized cells identified more than 200 frequently mutated genes, including human GBM-associated genes, such as Met and Nf1, and made it possible to discriminate between genes that function during astroglial immortalization vs. later stages of tumor development. We also functionally validated five GBM candidate genes using a previously undescribed high-throughput method. Finally, we show that even clonally related tumors derived from the same immortalized line have acquired distinct combinations of genetic alterations during tumor development, suggesting that tumor formation in this model system involves competition among genetically variant cells, which is similar to the Darwinian evolutionary processes now thought to generate many human cancers. This mutagenesis strategy is faster and simpler than conventional transposon screens and can potentially be applied to any tissue stem/progenitor cells that can be grown and differentiated in vitro.Glioblastoma multiforme (GBM) is the most common form of malignant brain cancer in adults. Patients with GBM have a uniformly poor prognosis, with a mean survival of 1 y (1). Thus, advances on all fronts, both basic and applied, are needed to combat this deadly disease better. Recent studies have provided evidence for self-renewing, stem-like cells within human gliomas (2). These glioma-initiating cells constitute a small minority of neoplastic cells within a tumor and are defined operationally by their ability to seed new tumors (3). To target these rare glioma-initiating cells, a better understanding of the molecular mechanisms that regulate their formation is essential.Considerable progress has been made in understanding the mutations responsible for GBM. The Cancer Genome Atlas network has cataloged the recurrent genomic abnormalities in GBM by genome-wide DNA copy number events and sequence-based mutation detection for 601 genes (4). Gene expression-based molecular classification has also defined four subtypes of GBM termed proneural, neural, classical, and mesenchymal (5). Proneural GBM is enriched for the oligodendrocyte gene signature, whereas the classical group is associated with the astrocytic signature. The neural class is enriched for genes differentially expressed by neurons, whereas the mesenchymal class is associated with the cultured astroglial signature (5). Several recurrent mutations, such as PDGFRA, IDH1, EGFR, and NF1, also correlate with these GBM subtypes, providing additional support for their existence. Numerous other, often rare, mutations have also been identified in GBM. Although these datasets are valuable for understanding the molecular pathogenesis of GBM, it is still difficult to distinguish between mutations that contributed to tumor initiation and those acquired later during tumor progression.The cell of origin (COO) of GBM is still controversial. Neural stem cells (NSCs) are good candidates because the adult brain has very few proliferating cells capable of accumulating the numerous mutations required for gliomagenesis. NSCs are also more susceptible to malignant transformation than differentiated cells in the adult brain (6, 7). However, the genetic pathways that can transform NSCs into glioma-initiating cells still remain elusive. Transposon-based mutagenesis provides an unbiased, high-throughput method for identifying genes important for GBM (8). Here, we describe a unique two-step insertional mutagenesis strategy that makes it possible to identify genes and signaling pathways that are able to transform a NSC into a cancer-initiating cell for the mesenchymal subtype of GBM. In this two-step approach, NSCs are first mutagenized in vitro and the mutagenized cells are then transplanted into immunocompromised mice for subsequent tumor development following additional rounds of transposon-based mutagenesis. This makes it possible to discriminate between the genetic changes that occur early in tumor initiation and those required for tumor progression. In addition to identifying several previously undescribed GBM candidate cancer genes, our studies suggest that transposon-induced tumors mimic the evolutionary processes now thought to generate many human cancers, in which tumors have a branched cellular and genetic architecture reminiscent of Darwin’s iconic evolutionary tree.     

A predictive microfluidic model of human glioblastoma to assess trafficking of blood–brain barrier-penetrant nanoparticles

The blood–brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood–brain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood–brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood–brain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.

High-grade gliomas are the most common primary malignant brain tumors in adults (1). These include grade IV astrocytomas, commonly known as glioblastoma multiforme (GBM), which account for more than 50% of all primary brain cancers and have dismal prognoses, with a 5-y survival rate of less than 5% (2). Due to their infiltrative growth into the healthy brain tissue, surgery often fails to eradicate all tumor cells (3). While chemotherapy and radiation modestly improve median survival (4), most patients ultimately succumb to their tumors. This is primarily due to the presence of a highly selective and regulated endothelium between blood and brain parenchyma known as the blood–brain barrier (BBB) (5), which limits the entry of therapeutics into the brain tissue where tumors are located. The BBB, characterized by a unique cellular architecture of endothelial cells (ECs), pericytes (PCs), and astrocytes (ACs) (6, 7), displays up-regulated expression of junctional proteins and reduced paracellular and transcellular transports compared to other endothelia (8). While this barrier protects the brain from toxins and pathogens, it also severely restricts the transport of many therapeutics, as evidenced by the low cerebrospinal fluid (CSF)-to-plasma ratio of most chemotherapeutic agents (9). There is thus an important need to develop new delivery strategies to cross the BBB and target tumors, enabling sufficient drug exposure (10).Despite rigorous research efforts to develop effective therapies for high-grade gliomas, the majority of trialed therapeutics have failed to improve outcomes in the clinic, even though the agents in question are effective against tumor cells in preclinical models (11). This highlights the inability of current preclinical models to accurately predict the performance of therapeutics in human patients. To address these limitations, we developed an in vitro microfluidic model of vascularized GBM tumors embedded in a realistic human BBB vasculature. This BBB-GBM platform features brain microvascular networks (MVNs) in close contact with a GBM spheroid, recapitulating the infiltrative properties of gliomas observed in the clinic (12) and those of the brain tumor vasculature, with low permeability, small vessel diameter, and increased expression of relevant junctional and receptor proteins (7). This platform is well suited for quantifying vascular permeability of therapeutics and simultaneously investigating modes of transport across the BBB and into GBM tumor cells.There is strong rationale for developing therapeutic nanoparticles (NPs) for GBM and other brain tumors, as they can be used to deliver a diverse range of therapeutic agents and, with appropriate functionalization, can be designed to exploit active transport mechanisms across the BBB (13, 14). Liposomal NPs have been employed in the oncology clinic to improve drug half-life and decrease systemic toxicity (15), but, to date, no nanomedicines have been approved for therapeutic indications in brain tumors. We hypothesize that a realistic BBB-GBM model composed entirely of human cells can accelerate preclinical development of therapeutic NPs. Using our BBB-GBM model, we investigated the trafficking of layer-by-layer NPs (LbL-NPs) and ultimately designed a GBM-targeted NP. The LbL approach leverages electrostatic assembly to generate modular NP libraries with highly controlled architecture. We have used LbL-NPs to deliver a range of therapeutic cargos in preclinical tumor models (16, 17) and have recently demonstrated that liposomes functionalized with BBB-penetrating ligands improved drug delivery across the BBB to GBM tumors (18). Consistent with clinical data (19), we observed that the low-density lipoprotein receptor-related protein 1 (LRP1) was up-regulated in the vasculature near GBM spheroids in the BBB-GBM model and leveraged this information to design and iteratively test a library of NPs. We show that the incorporation of angiopep-2 (AP2) peptide moieties on the surface of LbL-NPs leads to increased BBB permeability near GBM tumors through LRP1-mediated transcytosis. With intravital imaging, we compared the vascular permeabilities of dextran and LbL-NPs in the BBB-GBM platform to those in mouse brain capillaries and validated the predictive potential of our in vitro model. Finally, we show the capability of the BBB-GBM platform to screen therapeutic NPs and predict in vivo efficacy, demonstrating improved efficacy of cisplatin (CDDP) when encapsulated in GBM-targeting LbL-NPs both in vitro and in vivo.     

BRG1 expression is increased in human glioma and controls glioma cell proliferation, migration and invasion in vitro

Purpose

The purposes of our study were to elucidate the role of BRG1 in the development of human glioma and to determine the effect of BRG1 on glioma cell growth, migration and invasion.

Methods

Using tissue microarray and immunohistochemistry, we evaluated BRG1 staining in 190 glioma tissues, 8 normal brain tissues and 8 tumor adjacent normal brain tissues. We studied glioma cell proliferative ability with reduced BRG1 expression by siRNA using CCK-8 cell proliferation assay and cell cycle analysis. We studied the role of BRG1 in glioma cell migration and invasion by cell migration assay and matrigel invasion assay. We performed western blot to detect cyclin D1, cyclin B1 and MMP-2 protein expression. We also detected MMP-2 enzyme activity by gelatin zymography.

Results

Our results showed that BRG1 expression was increased in benign tumor and malignant tumor compared with tumor adjacent normal brain tissue (P?Conclusions Our data indicated that BRG1 expression is significantly increased in human glioma and it may be involved in the process of glioma cell proliferation, migration and invasion.     

In vivo chemical exchange saturation transfer imaging allows early detection of a therapeutic response in glioblastoma

Glioblastoma multiforme (GBM), which account for more than 50% of all gliomas, is among the deadliest of all human cancers. Given the dismal prognosis of GBM, it would be advantageous to identify early biomarkers of a response to therapy to avoid continuing ineffective treatments and to initiate other therapeutic strategies. The present in vivo longitudinal study in an orthotopic mouse model demonstrates quantitative assessment of early treatment response during short-term chemotherapy with temozolomide (TMZ) by amide proton transfer (APT) imaging. In a GBM line, only one course of TMZ (3 d exposure and 4 d rest) at a dose of 80 mg/kg resulted in substantial reduction in APT signal compared with untreated control animals, in which the APT signal continued to increase. Although there were no detectable differences in tumor volume, cell density, or apoptosis rate between groups, levels of Ki67 (index of cell proliferation) were substantially reduced in treated tumors. In another TMZ-resistant GBM line, the APT signal and levels of Ki67 increased despite the same course of TMZ treatment. As metabolite changes are known to occur early in the time course of chemotherapy and precede morphologic changes, these results suggest that the APT signal in glioma may be a useful functional biomarker of treatment response or degree of tumor progression. Thus, APT imaging may serve as a sensitive biomarker of early treatment response and could potentially replace invasive biopsies to provide a definitive diagnosis. This would have a major impact on the clinical management of patients with glioma.Glioblastoma multiformes (GBMs) account for more than 50% of all gliomas, and are among the deadliest of all human cancers. Approximately 10,000 patients in the United States are diagnosed each year with GBM, and the median survival time remains at 15 mo despite recent advances in surgery, radiation therapy, and chemotherapy (1). During the past two decades, our knowledge of the aberrant molecular mechanisms that underlie gliomas has increased extensively (2); however, advancement in the treatment of glioma has lagged far behind that in other cancers, except for significant progress made by the introduction of temozolomide (TMZ) (3, 4). TMZ, an oral alkylating chemotherapeutic agent, disturbs DNA replication and induces apoptosis of cancer cells, and subsequently arrests progression of the tumor. For these reasons, TMZ has been widely adopted as standard-of-care treatment for GBM (1, 5). Although TMZ can significantly prolong survival on average, only 45% of patients with newly diagnosed GBM gain substantial benefits from TMZ (6). Furthermore, even in cases in which the tumor responds well to initial treatment and appears to have disappeared on follow-up, recurrence is common and fatal in the middle of treatment or 1–2 y after completion of the treatment in most cases (7, 8).The prognosis and management is vastly different depending on whether one observes tumor progression or treatment effects. Thus, it is critical to decide whether the treatment should be continued or switched to avoid continuing ineffective treatments. Currently, management decisions in all phases of diagnosis, treatment and follow-up rely on MRI (9–11). Generally, clinicians make a decision based on interpretation of signal changes in T2-weighted (T2W) or fluid-attenuated inversion recovery and gadolinium enhancement on T1-weighted (T1W) imaging from one time point to another, typically covering several months. In addition, patients are taken back to the operating room for a repeat craniotomy and biopsy to make the confirmative diagnosis, as the currently available imaging methods often do not suffice to make the final decision. Thus, overall, there is an urgent need for the development of novel imaging techniques that can differentiate between continued progression vs. a positive response to therapy at these critical decision points as early and accurate as possible.Chemical exchange transfer (CEST) has drawn considerable attention as a novel mechanism of MRI contrast. This method provides more detailed physiological and functional information than conventional MRI and has emerged in the field of molecular imaging (12, 13). CEST contrast is achieved by applying a presaturation pulse at the resonance frequency of a slow–intermediate exchanging proton site (−NH, −OH, or metal-bound water molecule) of endogenous or exogenous agents. The resulting saturated or partially saturated spin is transferred to bulk water via chemical exchange. Consequently, specific molecular information is obtained indirectly through the MRI signal of tissue water. The net effect of CEST is to reduce the bulk water signal intensity detected in an imaging experiment, thereby providing negative contrast (14).Amide proton transfer (APT) imaging is one subset of CEST imaging that refers specifically to chemical exchange between protons of free tissue water (bulk water) and amide groups (−NH) of endogenous mobile proteins and peptides. It has been reported that such exchangeable protons are more abundant in tumor tissues than in healthy tissues (15). When applied to rats implanted with 9L gliosarcoma tumors in brain, APT imaging was able to distinguish between pathology-confirmed regions of tumor vs. tissue edema, whereas standard T1W, T2W, and fluid-attenuated inversion recovery imaging or diffusion-weighted imaging could not. Other previous reports demonstrated that the APT signal increased by 3–4% in tumor compared with peritumoral brain tissue in an experimental rat glial tumor at 4.7 T (16) and human brain tumor at 3 T (17). We recently demonstrated that APT imaging can distinguish histopathological World Health Organization grade of diffuse gliomas in patients (18). The mean APT signal increased with tumor grade (grade II, 2.1 ± 0.4%; III, 3.2 ± 0.9%; and IV, 4.1 ± 1.0%) and clearly discriminated between low-grade (i.e., grade II) and high-grade (i.e., grades III and IV) gliomas. Moreover, Zhou et al. clearly demonstrated in rat models that the method could distinguish tumor from radiation necrosis and further that the APT signal decreased in the tumor after radiation therapy (19). These previous studies indicate that APT imaging may be a more sensitive and specific biomarker for characterization of tumor grade or therapeutic response to radiation in brain tumors than other more conventional MRI methods.Here, we demonstrate quantitative assessment of early treatment response in short-term chemotherapy with TMZ in vivo by APT imaging of brain gliomas. A validated noninvasive biomarker of progression or therapeutic response of glioblastoma in patients could potentially reduce the need for craniotomy and biopsy during and after chemotherapy in these patients. Among the imaging methods that potentially can be used for this purpose, APT imaging appears to be a most promising method for early detection of a therapeutic response.     

Multispecific targeting of glioblastoma with tumor microenvironment-responsive multifunctional engineered NK cells

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking, have rendered glioblastoma (GBM) highly resistant to therapy. To address these obstacles, here we describe a unique, sophisticated combinatorial platform for GBM: a cooperative multifunctional immunotherapy based on genetically engineered human natural killer (NK) cells bearing multiple antitumor functions including local tumor responsiveness that addresses key drivers of GBM resistance to therapy: antigen escape, immunometabolic reprogramming of immune responses, and poor immune cell homing. We engineered dual-specific chimeric antigen receptor (CAR) NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site–specific activity in the tissue, and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising NK cell–based combinatorial strategy that can target multiple clinically recognized mechanisms of GBM progression simultaneously.

Glioblastoma (GBM) is the most common and deadliest malignant type of primary brain tumor (1). GBM patients are poorly responsive to traditional treatments, resulting in a grim prognosis that has only modestly improved over the past several decades, motivating the hunt for new treatment approaches (2). So far, chimeric antigen receptor (CAR)-engineered natural killer (NK) cells targeting single GBM antigens—EGFR, EGFRvIII, or ErbB2/HER2—have been limited to the use of NK cell lines, and the overall response rates have been disappointingly low and inconsistent (3–5). These responses appear to mirror the clinical hurdles of single antigen-targeted CAR-T therapies for GBM (6–9). CAR-T cells, administered to target single GBM antigens via intracavitary, intraventricular, or intravenous routes, have so far resulted in inconclusive durable responses (8).Preclinical and patient data have pointed to the heterogeneity of the GBM tumor microenvironment (TME) as a uniquely complex obstacle to overcome ( 10). This is reflected in immunotherapies tested so far having struggled to improve GBM patient overall survival (OS) in phase III clinical trials (11, 12). GBM induces localized lymphopenia to drive disease progression and resist treatment (13). In addition, the tumor’s heterogeneity is broad, with each of the known GBM subtypes—classical, mesenchymal, neural, and proneural—displaying diverse genetic and epigenetic signatures associated with distinct and variable cell plasticities (14). Not surprisingly, the outgrowth of antigen escape variants has been recorded clinically with most GBM-associated antigens to date, resulting in immune evasion and resistance to treatment (7, 15). And though strategies including dual antigen-targeting or programmable tumor-sensing CARs—so far primarily in the context of adoptive T cell therapy—have been evaluated preclinically to combat such evasion, GBM employs mechanisms beyond antigen escape to avoid targeting. Treatment evasion by GBM is fueled by a heavily immunosuppressive, hypoxic TME, which provides a niche unfavorable to NK cell effector function (16). A subset of GBM cells, glioma stem-like cells (GSCs), contribute to treatment resistance and are poorly recapitulated by conventional GBM model cell lines, including U87MG (17). Metabolic and functional pathways, moreover, converge to fuel the tumor’s invasiveness by driving exhaustion of immune cells (18). For instance, immunometabolic dysregulation of NK cell function in GBM is driven in part by the activity of ecto-5′-nucleotidase (CD73). CD73 is a hypoxic ectoenzyme that we and others have found to be associated with a negative prognosis and has emerged as an attractive clinical target (19, 20). In addition, we have previously shown that the CD73-driven accumulation of extracellular adenosine (ADO) leads to significant purinergic signaling–mediated impairment of NK cell activity (21, 22).Although they are among the most abundant lymphocytes found within the GBM TME, NK cells are still present in insufficient amounts in these tumors and exhibit a highly dysfunctional phenotype (23, 24). The need to recapitulate NK cell function lost to multiple complex mechanisms not only presents a significant challenge to traditional CAR-NK therapy but requires a greater presence specifically within the tumor tissue to mount meaningful clinical responses.Here, we describe an example of a multifunctional, engineered human NK cell–based therapy for glioblastoma developed around the programmed targeting of three clinically recognized pathways of GBM progression: antigen escape, immunometabolic suppression, and poor intratumoral NK cell presence. We achieved dual antigen recognition by modifying NK cells with multi-CARs to target disialoganglioside (GD2) and ligands to NK group 2D (NKG2D), which are widely expressed on human GBM (25, 26). Within the same NK cells, we engineered the concomitant local release of an antibody fragment that impairs immunosuppressive purinergic signaling by blocking the activity of CD73 via the cleavage of a tumor-specific linker. This cleavage is dependent on the activity of proteases that are up-regulated in the tumor microenvironment (27). Such local release is able to avoid systemic toxicities owing to its tumor-specific activation that occurs independently of CAR-based signaling.We report the homing of such multifunctional NK cells was enhanced when administered in conjunction with autophagy inhibitors in patient-derived GBM xenografts. Disabling autophagy further revealed a sophisticated and complex reorganization of anti-GBM immunological responses which could contribute to enhanced CAR-NK effector function. The clinical efficacy of adding an autophagy inhibitor to GBM therapy has shown that such treatments are clinically safe and well tolerated (28). We reveal a nuanced and potentially important role for autophagy inhibitors in adoptive human NK therapy.These studies aim to expand the repertoire of GBM-targeting NK cell–based immunotherapy and describe a first example of addressing, simultaneously, the challenge of tumor antigen heterogeneity, an immunosuppressive TME, and insufficient intratumoral trafficking of NK cells.     

Hepatic arterial infusion but not systemic application of cetuximab in combination with oxaliplatin significantly reduces growth of CC531 colorectal rat liver metastases

Purpose

Systemic chemotherapy still represents the gold standard in the treatment of irresectable colorectal liver metastases. Modern anticancer agents like the monoclonal antibody cetuximab have improved the outcome of patients in clinical studies. As hepatic arterial infusion (HAI) is capable to potentially increase the anticancer effect of cytostatics, we herein studied whether HAI of cetuximab (CE) as a single agent or in combination with oxaliplatin (OX) exerts increased anticancer effects compared to the systemic application (SYS) of the drugs.

Methods

WAG/Rij rats were randomized to eight groups and underwent 10 days after subcapsular hepatic tumor implantation either HAI or SYS of CE, OX, or the combination of both agents (CE + OX). Saline-treated animals served as controls. Tumor volume was measured at days 10 and 13 using three-dimensional ultrasound. On day 13, liver and tumor tissue was sampled for histological and immunohistochemical analysis.

Results

In controls, the tumor volume significantly increased from day 10 to 13. Application of OX alone via HAI or SYS did not inhibit tumor growth compared to controls. SYS of CE or CE + OX did also not reduce tumor growth. In contrast, HAI of CE and CE + OX significantly inhibited tumor growth. HAI of CE significantly reduced tumor vascularization as measured by the number of platelet endothelial cell adhesion molecule-1-positive cells and significantly increased the number of apoptotic tumor cells as measured by the cellular caspase-3 expression.

Conclusion

HAI of CE and CE + OX reduces tumor growth of colorectal rat liver metastases involving the inhibition of angiogenesis and induction of tumor cell apoptosis.     

Targeting CD146 with a 64Cu-labeled antibody enables in vivo immunoPET imaging of high-grade gliomas

Given the highly heterogeneous character of brain malignancies and the associated implication for its proper diagnosis and treatment, finding biomarkers that better characterize this disease from a molecular standpoint is imperative. In this study, we evaluated CD146 as a potential molecular target for diagnosis and targeted therapy of glioblastoma multiforme (GBM), the most common and lethal brain malignancy. YY146, an anti-CD146 monoclonal antibody, was generated and radiolabeled for noninvasive positron-emission tomography (PET) imaging of orthotopic GBM models. ⁶⁴Cu-labeled YY146 preferentially accumulated in the tumors of mice bearing U87MG xenografts, which allowed the acquisition of high-contrast PET images of small tumor nodules (∼2 mm). Additionally, we found that tumor uptake correlated with the levels of CD146 expression in a highly specific manner. We also explored the potential therapeutic effects of YY146 on the cancer stem cell (CSC) and epithelial-to-mesenchymal (EMT) properties of U87MG cells, demonstrating that YY146 can mitigate those aggressive phenotypes. Using YY146 as the primary antibody, we performed histological studies of World Health Organization (WHO) grades I through IV primary gliomas. The positive correlation found between CD146-positive staining and high tumor grade (χ² = 9.028; P = 0.029) concurred with the GBM data available in The Cancer Genome Atlas (TCGA) and validated the clinical value of YY146. In addition, we demonstrate that YY146 can be used to detect CD146 in various cancer cell lines and human resected tumor tissues of multiple other tumor types (gastric, ovarian, liver, and lung), indicating a broad applicability of YY146 in solid tumors.About 23,000 new cases of brain and central nervous system tumors are expected to be diagnosed in 2015 in the United States alone (1). More importantly, 15,320 patients will likely die of brain cancer by the end of the year, the majority of them due to malignant tumors types (1, 2). Glioblastoma multiforme (GBM) is the most common brain malignancy, accounting for more than 45% of all primary malignant brain tumors. Incidence rates of GBM increase with age, peaking at ages between 75 and 84; as a result, the number of glioblastoma cases is expected to increase in the United States due to population aging (3). Amid the significant efforts devoted to find effective therapeutic strategies for the treatment of GBM, it remains an incurable disease with a dismal 5-y survival rate of only 5%.Recent understanding of the complex molecular mechanisms underlying GBM’s pathogenesis has revealed the considerable heterogeneity inherent to the disease and has led to the emergence of several promising, patient-tailored therapies (3, 4). However, these therapies benefit only a specific subset of patients and almost invariably need the implementation of combinatorial regimes that simultaneously target several tumor-associated pathways to avoid tumor recurrence and rapid development of resistance. Therefore, it is critical to find new relevant GBM molecular signatures that allow for better patient stratification into specific molecular subtypes and the design of effective targeted therapeutic agents. The creation of The Cancer Genome Atlas (TCGA), and with it the availability of invaluable cancer genome data, has been instrumental in creating the opportunity for researchers to explore the genomic profile of several malignancies and identify new targets that might allow the emergence of novel diagnostics and therapeutic paradigms. GBM was the first malignancy incorporated to TCGA for which extensive genomic and matched phenotypical and clinical data are available.We identified CD146 as a promising diagnosis and therapeutic target for GBM. Subsequent analysis of the TCGA data revealed a statistically significant correlation between the expression of CD146 and decreased disease-free survival and overall survival in glioblastoma patients (Fig. S1). Thus, we devoted our efforts to validate CD146 as a target for noninvasive diagnosis and stratification of GBMs and to evaluate its potential as a therapeutic target. CD146, also known as MCAM, Mel-CAM, MUC18, or S-endo1, was first identified as a tumor progression and metastasis marker in malignant melanomas (5, 6). The major roles of CD146 have been associated with intercellular and cell-matrix adhesion. However, its involvement in several other processes, including development, cell migration, signal transduction, stem cell differentiation, immune response, angiogenesis, and, more recently, induction of epithelial-mesenchymal transition (EMT), has also been documented (7, 8). Despite the copious body of data describing the expression of CD146 in a myriad of cancers, noninvasive in vivo molecular imaging of CD146 expression has remained unexplored.Open in a separate windowFig. S1. CD146 clinical relevance in glioblastoma multiforme patients. Clinical data were obtained from TCGA. (A) The table summarizes the demographics of the analyzed patient cohort. A Kaplan–Meier plot showing a significant difference in (B) disease-free survival (DFS) and (C) overall survival between CD146(+) and CD146(−) GBM patients. P values were determined by the log-rank test. Molecular imaging techniques such as positron emission tomography (PET) and fluorescence imaging are becoming indispensable tools to study tumor biology in a clinical setting (9). ImmunoPET, which combines the excellent sensitivity and quantification capabilities of PET with monoclonal antibodies’ (mAbs’) exquisite binding affinity and specificity for their cognate antigen, is one of the most valuable techniques (10, 11). In this study, we used an improved method to produce YY146, an mAb against human CD146, which we implemented as an immunoPET agent for noninvasive in vivo imaging of CD146 expression in an orthotopic GBM mouse model. We further investigated how CD146 expression associates with several stem cell-like and mesenchymal cell traits in tumor cells and determined the ability of YY146 to actuate preferentially on cell subpopulations presenting these aggressive phenotypes. Finally, histological analysis of different WHO grade human brain tumor tissue samples confirmed the clinical relevance of CD146 for diagnosis and stratification of high-grade glioma patients and suggested its feasibility as a target for YY146-based targeted therapies (e.g., YY146 alone or in combination with other drugs, radioimmunotherapy, antibody-drug conjugates, etc.).     

Stage-specific embryonic antigen-4 as a potential therapeutic target in glioblastoma multiforme and other cancers

Glioblastoma multiforme (GBM), the grade IV astrocytoma, is the most common and aggressive brain tumor in adults. Despite advances in medical management, the survival rate of GBM patients remains poor, suggesting that identification of GBM-specific targets for therapeutic development is urgently needed. Analysis of several glycan antigens on GBM cell lines revealed that eight of 11 GBM cell lines are positive for stage-specific embryonic antigen-4 (SSEA-4), and immunohistochemical staining confirmed that 38/55 (69%) of human GBM specimens, but not normal brain tissue, were SSEA-4⁺ and correlated with high-grade astrocytoma. In addition, an SSEA-4–specific mAb was found to induce complement-dependent cytotoxicity against SSEA-4^hi GBM cell lines in vitro and suppressed GBM tumor growth in mice. Because SSEA-4 is expressed on GBM and many other types of cancers, but not on normal cells, it could be a target for development of therapeutic antibodies and vaccines.Glioblastoma multiforme (GBM), accounting for 60–70% of malignant gliomas, is the most aggressive form of glioma and the most common primary brain tumor in adults (1). Despite treatment, including surgery, and chemo- or radiotherapy, the prognosis for GBM patients is poor, with a median survival rate of 14–15 mo (2). GBM is notoriously resistant to most anticancer drugs and is extremely infiltrative, hampering complete surgical resection; therefore most patients develop tumor recurrence or progression even after multiple therapies. Because of the high mortality, new therapeutic approaches, such as immunotherapy and gene therapy, have been proposed for the treatment of GBM (3).Altered glycosylation is a feature of cancer cells, and several glycan structures are well-known tumor markers (4, 5). These aberrant changes include the overall increase in the branching of N-linked glycans (6) and sialic acid content (7) and the overexpression of certain glycan epitopes, such as sialyl Lewis x (sLe^x), sialyl Tn (sTn), Lewis y (Le^y), fucosyl Gb5 (Globo H), and polysialic acid (8–10). Many tumors also exhibit increased expression of certain glycolipids, especially the gangliosides, glycosphingolipids (GSLs) with sialic acid(s) attached to the glycan chain. Gangliosides normally are observed in neural systems and are elevated in tumors, particularly the complex gangliosides associated with malignancy (11).It has been reported that human glioma biopsies show elevation of monosialylated GM3 and GM2 and their disialylated derivatives GD3 and GD2 (12–14). The increase of GD3 was most significant, but the correlation between GD3 and malignancy remains obscure (15, 16). In addition, the lacto-series gangliosides 3′-isoLM1 and 3′,6′-isoLD1 are reported to be major gangliosides in human gliomas (16–18). Because some of these glioma-associated gangliosides are rarely expressed or even are absent in normal tissues (19), they are suitable for targeted therapy (20). Hence, discovering novel glioma-associated GSLs would provide new targets for development of new therapies against gliomas.The GSLs of globo-series feature a Galα1–4Gal linkage to lactosylceramides, and this linkage is catalyzed by the lactosylceramide 4-alpha-galactosyltransferase, encoded by the A4GALT gene. Although globotriosylceramide (Gb3Cer) and globoside (Gb4Cer) constitute the basis of the P-blood group system (21), galactosyl globoside (Gb5Cer) and sialyl galactosyl globoside (sialyl Gb5Cer, SGG, MSGG), also known as “stage-specific embryonic antigen-3” (SSEA-3) and “stage-specific embryonic antigen-4” (SSEA-4) (22), respectively, are cell-surface markers widely used to define human embryonic stem cells (hESCs). Globo-series GSLs also have been observed in tumors: Globo H is overexpressed in many epithelial cancers [e.g., ovarian, gastric, prostate, lung, breast, and pancreatic cancers (23)]; SSEA-3, SSEA-4, and Globo H are expressed not only on breast cancer cells but also on breast cancer stem cells (24, 25). Moreover, high-level expression of SSEA-4 and disialosyl galactosyl globoside (disialosyl Gb5Cer) is observed in renal cell carcinoma (26), but whether globo-series GSLs are expressed in GBM is not known.In the present study, we examined the expression levels of globo-series GSLs and several tumor-associated glycans in GBM cell lines by flow cytometry. The result showed that SSEA-4, a ganglioside rarely found in normal brain tissues, was highly expressed on GBM cells and GBM specimens, as confirmed by high-performance TLC (HPTLC) immunostaining and MS. We found that anti–SSEA-4 mAb (MC813-70) could induce complement-dependent cytotoxicity in vitro and inhibit the growth of GBM in nude mice. SSEA-4 is displayed on many other types of cancers and therefore can be a target for the development of therapeutic antibodies and vaccines against SSEA-4⁺ cancers.     

Brain tumor invasion model system using organotypic brain-slice culture as an alternative to in vivo model

PURPOSE: The primary cause of local recurrence and therapeutic failure in the treatment of malignant gliomas is the invasion of tumor cells into the surrounding normal brain. While it is known that malignant gliomas infiltrate diffusely into regions of normal brain, it is frequently very difficult to unequivocally identify the solitary invading glioma cell in histopathological preparations, or in experimental glioma models. We have developed an experimental invasion assay system, which allows us to track the solitary invasive glioma cell, using human brain tissue obtained from routine craniotomies for seizures or trauma. METHODS: This tissue is cut into 1-mm thick slices and cultured in the upper chamber of Transwell culture dishes on top of a 0.4- micro m pore size polyester membrane, which is fed on medium provided in the lower chamber. Glioma cells are stably transfected with vectors containing a green fluorescent protein (GFP) cDNA. Stable, high-level expression GFP transfectants were selected by direct visualization under fluorescence microscope. In addition, various tumor spheroids are stained with vital dye, DiI, to track the invading cells. GFP-expressing glioma cells or stained spheroids were then implanted on the center of the brain slice, and the degree of brain tumor invasion into the brain tissue was evaluated at different time points by optical sectioning using a confocal microscope. RESULTS: We observed that GFP-expressing glioma cells or stained spheroids could be readily tracked and followed with this model system. Individual tumor cells that exhibited green or red fluorescence could be identified and their migration path through the brain slices unequivocally followed. CONCLUSION: This experimental invasion system may be of considerable utility in studying the process of brain tumor invasion and in evaluating its invasiveness in individual brain tumor because it not only provides a better representation of extracellular matrix molecules normally encountered by invading glioma cells, but also provides the fluorescent tag applied to the tumor cells.     

Highly penetrative,drug-loaded nanocarriers improve treatment of glioblastoma

Current therapy for glioblastoma multiforme is insufficient, with nearly universal recurrence. Available drug therapies are unsuccessful because they fail to penetrate through the region of the brain containing tumor cells and they fail to kill the cells most responsible for tumor development and therapy resistance, brain cancer stem cells (BCSCs). To address these challenges, we combined two major advances in technology: (i) brain-penetrating polymeric nanoparticles that can be loaded with drugs and are optimized for intracranial convection-enhanced delivery and (ii) repurposed compounds, previously used in Food and Drug Administration-approved products, which were identified through library screening to target BCSCs. Using fluorescence imaging and positron emission tomography, we demonstrate that brain-penetrating nanoparticles can be delivered to large intracranial volumes in both rats and pigs. We identified several agents (from Food and Drug Administration-approved products) that potently inhibit proliferation and self-renewal of BCSCs. When loaded into brain-penetrating nanoparticles and administered by convection-enhanced delivery, one of these agents, dithiazanine iodide, significantly increased survival in rats bearing BCSC-derived xenografts. This unique approach to controlled delivery in the brain should have a significant impact on treatment of glioblastoma multiforme and suggests previously undescribed routes for drug and gene delivery to treat other diseases of the central nervous system.Of the ∼40,000 people diagnosed with primary brain tumors in the United States each year, an estimated 15,000 have glioblastoma multiforme (GBM), a World Health Organization grade IV malignant glioma (1). Despite considerable research efforts, the prognosis for GBM remains poor: median survival with standard-of-care therapy (surgery, systemic chemotherapy with temozolomide, and radiation) is 14.6 mo (2) and 5-y survival is 9.8% (3), with the vast majority of GBMs recurring within 2 cm of the original tumor focus (4). Histopathologically, GBM is characterized by its infiltrative nature and cellular heterogeneity, leading to a number of challenges that must be overcome by any presumptive therapy.The blood–brain barrier (BBB) is a major obstacle to treating GBM (5). It is estimated that over 98% of small-molecule drugs and ∼100% of large-molecule drugs or genes do not cross the BBB (6). Delivery of chemotherapeutics to the brain can be potentially achieved by using nanocarriers engineered for receptor-mediated transport across the BBB (7, 8), but the percentage of i.v. administered particles that enter the brain is low. It is not yet clear whether sufficient quantities of drug can be delivered by systemically administered nanoparticles to make this a useful method for treating tumors in the human brain. An alternate approach is to bypass the BBB: Clinical trials have demonstrated that the BBB can be bypassed with direct, locoregional delivery of therapeutic agents. For example, local implantation of a drug-loaded biodegradable polymer wafer (presently marketed as Gliadel), which slowly releases carmustine over a prolonged period, is a safe method for treating GBM. However, use of the Gliadel wafer results in only modest improvements in patient survival, typically 2 mo (9, 10). In prior work we showed that these wafers produce high interstitial drug concentrations in the tissue near the implant, but—because drugs move from the implant into the tissue by diffusion—penetration into tissue is limited to ∼1 mm, which could limit their efficacy (11, 12).We hypothesize that treatment of GBM can be improved by attention to three challenges: (i) enhancing the depth of penetration of locally delivered therapeutic agents, (ii) providing for long-term release of active agents, and (iii) delivering agents that are known to be effective against the cells that are most important in tumor recurrence. The first challenge can be addressed by convection-enhanced delivery (CED), in which agents are infused into the brain under a positive pressure gradient, creating bulk fluid movement in the brain interstitium (13). Recent clinical trials show that CED is safe and feasible (14–16), but CED alone is not sufficient to improve GBM treatment. For example, CED of a targeted toxin in aqueous suspension failed to show survival advantages over Gliadel wafers (14, 17). Although CED of drugs in solution results in increased penetration, most drugs have short half-lives in the brain and, as a result, they disappear soon after the infusion stops (17, 18). Loading of agents into nanocarriers—such as liposomes, micelles, dendrimers, or nanoparticles—can protect them from clearance. Significant progress has been made in CED of liposomes to the brain (19), although it is not clear that liposomes offer the advantage of long-term release. By contrast, CED of polymeric nanoparticles, such as nanoparticles made of poly(lactide-coglycolide) (PLGA), offers the possibility of controlled agent release. However, CED of PLGA nanoparticles, which are typically 100–200 nm in diameter, has been limited by the failure of particles to move by convection through the brain interstitial spaces (20–23), which are 38–64 nm in normal brain (24) and 7–100 nm in regions with tumor (25). Therefore, to overcome the first and second challenges, it is necessary to synthesize polymer nanocarriers that are much smaller than conventional particles and still capable of efficient drug loading and controlled release. We report here reliable methods for making PLGA nanoparticles with these characteristics.Drug developers have long been frustrated by the BBB, which severely limits the types of agents that can be tested for activity in the brain. We reasoned that creation of safe, versatile, brain-penetrating nanocarriers should enable direct testing of novel agents that address the complexity of GBM biology. For example, cells isolated from distinct regions of a given GBM bear grossly different expression signatures but seem to arise from a common progenitor (26): A small subpopulation of these progenitors drives tumor progression, promotes angiogenesis, and influences tumor cell migration (27–30). These cells have features of primitive neural stem cells and are called brain cancer stem cells (BCSCs) (29, 31–37). BCSCs, many of which are marked by CD133 (PROM1), are resistant to conventional drugs (28, 38), including carboplatin, cisplatin, paclitaxel, doxorubicin, vincristine, methotrexate, and temozolomide (39–42), as well as radiotherapy (29). These observations suggest that agents that affect BCSCs are more likely to lead to a cure for GBM (28, 38, 43, 44). Therefore, to illustrate the translational potential of brain-penetrating nanoparticles, we conducted a screen of ∼2,000 compounds that were previously used in Food and Drug Administration (FDA)-approved products for their ability to inhibit patient-derived BCSCs, encapsulated the best agents to emerge from the screen into brain-penetrating PLGA nanoparticles, and administered these nanocarriers by CED in a BCSC-derived xenograft model of GBM.     

Glioblastoma cellular architectures are predicted through the characterization of two-cell interactions

To understand how pairwise cellular interactions influence cellular architectures, we measured the levels of functional proteins associated with EGF receptor (EGFR) signaling in pairs of U87EGFR variant III oncogene receptor cells (U87EGFRvIII) at varying cell separations. Using a thermodynamics-derived approach we analyzed the cell-separation dependence of the signaling stability, and identified that the stable steady state of EGFR signaling exists when two U87EGFRvIII cells are separated by 80–100 μm. This distance range was verified as the characteristic intercellular separation within bulk cell cultures. EGFR protein network signaling coordination for the U87EGFRvIII system was lowest at the stable state and most similar to isolated cell signaling. Measurements of cultures of less tumorigenic U87PTEN cells were then used to correctly predict that stable EGFR signaling occurs for those cells at smaller cell–cell separations. The intimate relationship between functional protein levels and cellular architectures explains the scattered nature of U87EGFRvIII cells relative to U87PTEN cells in glioblastoma multiforme tumors.Pathological analysis of tumor tissues is typically led by the analyses of cellular architectures within those tumors. Relationships between those architectures and molecular biomarkers of disease are often poorly understood. We seek to establish such a relationship, starting from physical principles. We take as an example glioblastoma multiforme (GBM) cancer cells that express the EGF receptor (EGFR) variant III oncogene receptor (EGFRvIII). Although these cells enhance tumorigenicity, invasion, and other hallmarks of cancer (1, 2), they comprise only a subpopulation of the cancer cells within an EGFRvIII+ tumor, and their distribution is diffuse (1, 3, 4). To help understand this diffuse cellular architecture, we developed an experimental–theoretical methodology based on analysis of EGFR signaling in two interacting cells. In many physical systems—from planets to atomic solids—the interactions of an element of that system with its surroundings can be understood within the context of two-body interactions. This broad observation inspired our experimental approach, which was to measure EGFR-associated signaling activity in statistically significant numbers of two EGFRvIII+ GBM cells, as a function of intercellular separation. Our theoretical approach was similarly inspired: it assumed that the resultant two-cell data sets could be interpreted using thermodynamic-like considerations.Our approach allows a determination of the stability of a phosphoprotein signaling network in two interacting cells, and demonstrates how that stability dictates the cell–cell distance distribution in a bulk culture. Using this concept we determined the most probable intercellular separation distance range within cell populations, and the deviations thereof. The available literature suggests our conclusions can be extended to bulk tumors (1).EGFR signaling plays an important role in motility and promoting tumor growth within EGFRvIII+ GBM tumors (2, 5–8). We thus hypothesized that a detailed examination of the EGFR signaling pathway, within two GBM cells at different separations, would allow a determination of a distance range that exhibited the most stable EGFR signaling. This approach assumes that cell–cell separations with the most stable EGFR signaling will appear with a higher frequency within a bulk population.Our experimental/theoretical analysis combines measurements of functional proteins, such as phosphorylated kinases, within the EGFR signaling pathway in isolated pairs of GBM cells, at varying cell separations, with surprisal analysis (9–11). Here we use surprisal analysis to determine the most balanced state of the two cells at different distance ranges. We thereby identified a steady-state separation distance between two U87EGFRvIII cells of 80–100 μm. The steady-state separation of two cells was found to correspond to the most probable distance range determined through microscopy measurements of the radial distribution function (RDF) of those same cells in bulk culture. The RDF represents the measured distributions of cell locations with respect to each other. We then turned this approach around, and used measurements of the RDF from a bulk culture of the less tumorigenic U87PTEN cells [model GBM cells expressing wild-type EGFR and the tumor suppressor phosphatase and tensin homolog (PTEN)] to identify the most probable cell–cell separation distance. Thereby we predict that the most stable cell–cell pairwise signaling in U87PTEN cells occurs at smaller cell–cell separations. Those predictions were then shown to be consistent with two-cell, functional proteomics assays.Our results may help explain the scattered distribution of EGFRvIII cells and less infiltrative nature of U87PTEN cells; furthermore, they point to an intimate relationship between cellular signaling activity, distance dependent cell–cell interactions, and cell culture architectures. The methodology demonstrated here shows how a thermodynamic-like approach, coupled with quantitative functional protein measurements, can provide information about the stability of a cellular system. This approach should be broadly applicable.