Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

University of Virginia

The invasive nature of glioblastoma (GBM) represents a major clinical challenge contributing to poor outcomes. Invasion of GBM into healthy tissue restricts chemotherapeutic access and complicates surgical resection. Here, we test the hypothesis that an effective anti-invasive agent can "contain" GBM and increase the efficacy of chemotherapy. We report a new anti-invasive small molecule, Imipramine Blue (IB), which inhibits invasion of glioma in vitro when tested against several models. IB inhibits NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-mediated reactive oxygen species generation and alters expression of actin regulatory elements. In vivo, liposomal IB (nano-IB) halts invasion of glioma, leading to a more compact tumor in an aggressively invasive RT2 syngeneic astrocytoma rodent model. When nano-IB therapy was followed by liposomal doxorubicin (nano-DXR) chemotherapy, the combination therapy prolonged survival compared to nano-IB or nano-DXR alone. Our data demonstrate that nano-IB-mediated containment of diffuse glioma enhanced the efficacy of nano-DXR chemotherapy, demonstrating the promise of an anti-invasive compound as an adjuvant treatment for glioma.

Anti-invasive adjuvant therapy with imipramine blue enhances chemotherapeutic efficacy against glioma.

Brain tumor invasion leads to recurrence and resistance to treatment. Glioma cells invade in distinct patterns, possibly determined by microenvironmental cues including chemokines, structural heterogeneity, and fluid flow. We hypothesized that flow originating from pressure differentials between the brain and tumor is active in glioma invasion. Using in vitro models, we show that interstitial flow promotes cell invasion in multiple glioma cell lines. Flow effects were CXCR4-dependent, because they were abrogated by CXCR4 inhibition. Furthermore, CXCR4 was activated in response to flow, which could be responsible for enhanced cell motility. Flow was seen to enhance cell polarization in the flow direction, and this flow-induced polarization could be blocked by CXCR4 inhibition or CXCL12 oversaturation in the matrix. Furthermore, using live imaging techniques in a three-dimensional flow chamber, there were more cells migrating and more cells migrating in the direction of flow. This study shows that interstitial flow is an active regulator of glioma invasion. The new mechanisms of glioma invasion that we identify here-namely, interstitial flow-enhanced motility, activation of CXCR4, and CXCL12-driven autologous chemotaxis-are significant in therapy to prevent or treat brain cancer invasion. Current treatment strategies can lead to edema and altered flow in the brain, and one popular experimental treatment in clinical trials, convection enhanced delivery, involves enhancement of flow in and around the tumor. A better understanding of how interstitial flow at the tumor margin can alter chemokine distributions, cell motility, and directed invasion offers a better understanding of treatment failure. .

Interstitial flow in a 3D microenvironment increases glioma invasion by a CXCR4-dependent mechanism.

As cancer progresses, a dynamic microenvironment develops that creates and responds to cellular and biophysical cues. Increased intratumoral pressure and corresponding increases in interstitial flow from the tumor bulk to the healthy stroma is an observational hallmark of progressing cancers. Until recently, the role of interstitial flow was thought to be mostly passive in the transport and dissemination of cancer cells to metastatic sites. With research spanning the past decade, we have seen that interstitial flow has a promigratory effect on cancer cell invasion in multiple cancer types. This invasion is one mechanism by which cancers can resist therapeutics and recur, but the role of interstitial flow in cancer therapy is limited to the understanding of transport of therapeutics. Here we outline the current understanding of the role of interstitial flow in cancer and the tumor microenvironment through cancer progression and therapy. We also discuss the current role of fluid flow in the treatment of cancer, including drug transport and therapeutic strategies. By stating the current understanding of interstitial flow in cancer progression, we can begin exploring its role in therapeutic failure and treatment resistance.

Interstitial fluid flow in cancer: implications for disease progression and treatment.

Aggressive surgical resection is the primary therapy for glioma. However, aggressive resection may compromise functional healthy brain tissue. Currently, there are no objective cues for surgeons to distinguish healthy tissue from tumor and determine tumor borders; surgeons skillfully rely on subjective means such as tactile feedback. This often results in incomplete resection and recurrence. The objective of the present study was to design, develop, and evaluate, in vitro and in vivo, a nanoencapsulated visible dye for intraoperative, visual delineation of tumor margins in an invasive tumor model. Liposomal nanocarriers containing Evans blue dye (nano-EB) were developed, characterized, and tested for safety in vitro and in vivo. 3RT1RT2A glioma cells were implanted into brains of Fischer 344 rats. Nano-EB or EB solution was injected via tail vein into tumor-bearing animals. To assess tumor staining, tissue samples were analyzed visibly and using fluorescence microscopy. Area, perimeter ratios, and Manders overlap coefficients were calculated to quantify extent of staining. Nano-EB clearly marked tumor margins in the invasive tumor model. Area ratio of nano-EB staining to tumor was 0.89 ± 0.05, perimeter ratio was 0.94 ± 0.04, Manders R was 0.51 ± 0.08, and M1 was 0.97 ± 0.06. Microscopic tumor border inspection under high magnification verified that nano-EB did not stain healthy tissue. Nano-EB clearly aids in distinguishing tumor tissue from healthy tissue in an invasive tumor model, while injection of unencapsulated EB results in false identification of healthy tissue as tumor due to diffusion of dye from the tumor into healthy tissue.

Evans blue nanocarriers visually demarcate margins of invasive gliomas.

Angiogenesis and lymphangiogenesis often occur in response to tissue injury or in the presence of pathology (e.g., cancer), and it is these types of environments in which macrophages are activated and increased in number. Moreover, the blood vascular microcirculation and the lymphatic circulation serve as the conduits for entry and exit for monocyte-derived macrophages in nearly every tissue and organ. Macrophages both affect and are affected by the vessels through which they travel. Therefore, it is not surprising that examination of macrophage behaviors in both angiogenesis and lymphangiogenesis has yielded interesting observations that suggest macrophages may be key regulators of these complex growth and remodeling processes. In this review, we will take a closer look at macrophages through the lens of angiogenesis and lymphangiogenesis, examining how their dynamic behaviors may regulate vessel sprouting and function. We present macrophages as a cellular link that spatially and temporally connects angiogenesis with lymphangiogenesis, in both physiological growth and in pathological adaptations, such as tumorigenesis. As such, attempts to therapeutically target macrophages in order to affect these processes may be particularly effective, and studying macrophages in both settings will accelerate the field's understanding of this important cell type in health and disease.

Macrophages: An Inflammatory Link Between Angiogenesis and Lymphangiogenesis.

Glioblastoma (GBM) prognosis remains dismal due in part to the invasiveness of GBM cells. Interstitial fluid flow (IFF) has been shown to increase invasion of glioma cells in vitro through the CXCR4 receptor interacting with autologous, pericellular gradients of CXCL12 (autologous chemotaxis) or through the CD44 receptor interactions with the extracellular matrix (hyaluronan-mediated mechanotransduction). These mechanisms have not been examined together and thus we hypothesized that both mechanisms contribute to invasion in populations of cancer cells. Therefore, we examined IFF-stimulated CXCR4-, CXCL12-, and CD44-dependent invasion in patient-derived glioblastoma stem cells (GSCs). Using our 3D in vitro assay and correlative in vivo studies we demonstrated GSC lines show increased invasion with flow. This flow-stimulated invasion was reduced by blockade of CXCR4, CXCL12, and/or CD44, revealing that GSC invasion may be mediated simultaneously by both mechanisms. Characterization of CXCR4(+), CXCL12(+), and CD44(+) populations in four GSC lines revealed different percentages of protein positive subpopulations for each line. We developed an agent-based model to identify the contributions of each subpopulation to flow-stimulated invasion and validated the model through comparisons with experimental blocking studies. Clinically relevant radiation therapy increased flow-stimulated invasion in one GSC line. Our agent-based model predicted that IFF-stimulated invasion is driven primarily by CXCR4(+)CXCL12(+) populations, and, indeed our irradiated cells had an increase in this subpopulation. Together, these data indicate that different mechanisms govern the flow response across GSCs, but that within a single patient, there are subpopulations of GSCs that respond to flow via either CD44- or CXCR4-CXCL12 mechanisms.

Interstitial flow differentially increases patient-derived glioblastoma stem cell invasion via CXCR4, CXCL12, and CD44-mediated mechanisms.

Glioblastomas, the most common primary malignant brain tumors, have a distinct tissue microenvironment. Although non-neoplastic cells contribute to glioblastoma progression, very few quantitative studies have shown the effect of tumor microenvironmental influences on patient survival. We examined relationships of the cellular microenvironment, including astrocytes, microglia, oligodendrocytes, and blood vessels, to survival in glioblastoma patients. Using histological staining and quantitative image analyses, we examined the tumor-associated parenchyma of 33 patients and developed statistical models to predict patient outcomes based on the cellular picture of the tumor parenchyma. We found that blood vessel density correlated with poorer prognosis. To examine the role of adjacent parenchymal versus higher tumor cell density bulk parenchymal tissue, we examined the glial components in these highly variable regions. Comparison of bulk and adjacent astrocytes and microglia in tissue yielded the strongest prediction of survival, with high levels of adjacent astrocytes predicted poor prognosis and high levels of microglia correlated with a better prognosis. These results indicate that parenchymal components predict survival in glioblastoma patients and in particular that the balance between reactive glial populations is important for patient prognosis.

Jennifer M. Munson

Biomedical Engineering

University of Virginia

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Jennifer M. Munson

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University of Virginia

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Biomedical Engineering