MR spectroscopy (MRS) also can potentially differentiate low-grade versus high-grade gliomas in the brain. All gliomas have an increased choline peak and a reduced N-acetyl aspartate peak (NAA) which are markers of membrane turnover and neuronal cell death respectively. Levels of lipid and lactate are markers of necrosis and hypoxia respectively and are decidedly elevated in high-grade compared to low-grade gliomas (McBride et al., 1995, Nafe et al., 2003, Negendank et al., 1996). Creatine (Cr), which serves as a marker of energy metabolism, is decreased in brain tumors (Meyerand et al., 1999, Moller-Hartmann et al., 2002), however, this reduction does not appear to correlate with tumor grade by itself (Moller-Hartmann et al., 2002). Using the choline/Cr ratio may be more effective, however, as low-grade gliomas tend to have a lower ratio of choline/Cr (McBride et al., 1995, Murphy et al., 2002, Sijens & Oudkerk, 2002), as well as an increase in NAA/Cr ratio (Law et al., 2003, McKnight et al., 2002, Murphy et al., 2002, Nafe et al., 2003, Negendank et al., 1996). MRS is a promising technique in differentiating low- from high-grade gliomas with sensitivity between 73% and 92% and specificity between 63% and 100% (Astrakas et al., 2004, Fayed & Modrego, 2005, Law et al., 2003, Nafe et al., 2003, Setzer et al., 2007). MRS may also be capable of identifying regions that have undergone malignant transformation within a given tumor that may not be identifiable by other imaging techniques although one such study attempting to detect malignant transformation within low-grade glioma yielded a specificity of only 57.1% (Alimenti et al., 2007).

Positron emission tomography (PET) imaging has been employed to examine gliomas in the brain by measuring the metabolic activity of tissue. Fluorinated glucose analogue 2-[18F] fluoro-2-deoxy-D-glucose (FDG), which is administered to patients intravascularly, has high sensitivity for identifying areas of increased tumor metabolism and has been used as an index to predict tumor aggressiveness. While low-grade gliomas tend to have the same or even lower uptake of FDG than normal brain matter, high-grade gliomas demonstrate increased uptake of FDG on PET imaging (Derlon et al., 1997, Tamura et al., 1998), Studies have shown that it is possible to differentiate low- from high-grade gliomas with a sensitivity of 94% and specificity of 77% using a tumor-to-white-matter ratio of greater than 1.5 and tumor-to-grey-matter ratio of greater than 0.6 (Delbeke et al., 1995).

Bone Marrow-Derived Cells Support Malignant Transformation of Low-Grade Glioma 211

(vasculogenesis). These factors mobilize bone marrow precursor cells which then travel to the site of ischemia via the bloodstream. It is theorized that these cells facilitate vasculogenesis by breaking down existing structures and creating an environment that

Tumors are capable of adopting this machinery to increase growth and invasiveness by activating the angiogenic switch (Bergers & Benjamin, 2003, Rafii & Lyden, 2008). During early tumor development, neoplastic cells rely on existing blood flow and grow in a slow linear fashion (Mandonnet et al., 2003). Once the switch is initiated and neovascularization brings more oxygen and nutrients, tumor cells grow at a much faster rate and tumor size increases significantly (Rees et al., 2009). This initial process is thought to occur mostly by angiogenesis (Kioi, 2010). The release of proteases and proangiogenic factors causes pericytes to detach from existing vessels creating a defect in the extracellular matrix in the environment surrounding the vessel wall (Bergers & Benjamin, 2003). Endothelial cells proliferate locally and sprout outward into the tumor bed creating newly formed blood vessels feeding the tumor. While angiogenesis is an important factor in the angiogenic switch, vasculogenesis and the contribution of BMDC play a critical role as well. For example, when recruitment of BMDC is impaired in an animal model of lymphoma and lung carcinoma, tumor angiogenesis and growth is significantly decreased (Lyden et al., 2001) suggesting that BMDCs contribute significantly to neovascularization and growth in

In metastatic disease, the contribution of BMDC has been well described (Wels et al., 2008). Endothelial (EPCs) and hematopoietic precursor cells (HPC), mesenchymal stem cells (MSC), myeloid-derived suppressor cells (MDSCs), Tie-2 expressing monocytes (TEM) and tumor associated macrophages (TAM) all are mobilized from the bone marrow to future metastatic sites prior to tumor formation. It should be noted that these primitive cells are prominent during embryology and that a significant population of these cells is not present under normal conditions. While the exact role of each cell type has yet to be fully elucidated, their basic function is to break down normal structures and promote vasculogenesis and tumor invasiveness. The net result is a tumor friendly environment capable of sustaining tumor growth. This has been demonstrated experimentally in a murine model of metastatic disease by implanting m-cherry labeled melanoma cells into the flank of mice with GFPlabeled bone marrow and examining the lungs of these animals over time (Kaplan et al., 2005). It was observed that the first cells to arrive in future metastases were not tumor cells, but actual BMDC. This suggested, at least in metastatic disease, that the environment in future metastatic sites is primed by cells from the bone marrow before tumors can begin to grow in these distant areas (Rafii & Lyden, 2008). This also supports the hypothesis by Stephen Paget over 100 years ago that the tumor microenvironment may play as important a

In the brain, the role of BMDC has only recently garnered attention. One of the basic histological differences between low-grade and high-grade gliomas is a lack of neovascularization. Thus, activation of the angiogenic switch is a key element in the transformation of low-grade to high-grade glioma. Two elements are likely to contribute to this process. Genetic changes in tumor cells that occur during progression of disease activate pro-angiogenic factors. This has been observed in human tumor samples whereby genes involved in angiogenesis are upregulated in glioblastoma as compared to low grade astrocytoma (Godard et al., 2003). Kioi et al. also showed in their animal model that release of soluble factors by tumor cells or cells within the tumor microenvironment including

promotes new blood vessel growth.

role as the tumor cells themselves.

solid tumors.

In addition to FDG, other tracers have been utilized in attempts to further characterize these tumors such as carbon 11 and fluorine 18 (18F)-labeled amino acid (Isselbacher, 1972). Methionine PET appears to have a higher sensitivity than FDG PET in detecting low-grade versus high-grade gliomas (Derlon et al., 1997, Giammarile et al., 2004, Ogawa et al., 1993). In particular, methionine PET exhibits a heightened sensitivity in detecting radiation necrosis from recurrent tumors, as inflammatory cells in radiation necrosis have little uptake of methionine (Thiel et al., 2000). Perhaps the most promising technique for diagnosing lowgrade gliomas is 18F-FDOPA PET imaging. 18F-FDOPA PET is more accurate that FDG PET and has been shown to be highly predictive in determining tumor grade on initial diagnosis and may help differentiate tumor necrosis from recurrence (Chen et al., 2006, Fueger et al., Tripathi et al., 2009).

While there currently is no one imaging modality capable of definitively determining lowgrade from high-grade tumors on its own, advanced imaging technology continues to develop and complement standard MRI. As we come to understand the behavior and variability of these tumors, advance imaging techniques provide exciting new possibilities for more precise treatments. Given the variability within a given tumor, advanced imaging techniques may allow for more precise targets for biopsy, vigilant monitoring of malignant transformation, and improved prognostic power in the management of low- and high-grade gliomas.

#### **8. The Role of bone-marrow derived cells in malignant transformation**

The vast majority of brain tumor research, molecular profiling, histological characteristics, diagnostic imaging modalities and treatment targets have focused on the actual tumor cells themselves. As mentioned earlier, one of the key events in the transition from the low-grade to the high-grade state is the angiogenic switch. It is theorized that in the low-grade state, tumor growth may be limited, at least in part, by a lack of blood supply. In this state, the tumor is only capable of a steady-state or linear growth (Mandonnet et al., 2003). Once the tumor acquires the ability to recruit or form new blood vessels, exponential growth occurs (Rees et al., 2009) resulting in rapid clinical decline. While there is considerable evidence that tumor cells undergo continued molecular changes that increase their malignant potential, these changes also allow these cells to initiate the angiogenic switch. It must also be noted that while recent evidence suggests that tumor cells may be capable of directly forming new blood vessels (Ricci-Vitiani et al., Wang et al.), a considerable body of evidence suggests that tumor cells do not do this completely on their own. While the exact details of this process still remain to be fully elucidated, tumor cells acquire the ability to transition the local tumor niche to an environment capable of rapid blood vessel formation. A variety of growth factors, signaling pathways, and indigenous populations of cells is hypothesized to participate in this process. If this theory proves to be correct, this population of cells forms an additional therapeutic target that may be as important as the tumor cells themselves. As current therapies directed at neoplastic cells are limited in part to their toxicity, elucidating other potential treatment pathways may further benefit patient outcome.

Neovascularization is a normal process in tissues and the brain during ischemia. In low oxygen states, cells release signals such as VEGF, PDGF, PlGF and HIF-1 that recruit from local existing vessels within the tissue itself (angiogenesis.) In addition, this can activate distant processes that facilitate neovascularization and may even form *de novo* blood vessels

In addition to FDG, other tracers have been utilized in attempts to further characterize these tumors such as carbon 11 and fluorine 18 (18F)-labeled amino acid (Isselbacher, 1972). Methionine PET appears to have a higher sensitivity than FDG PET in detecting low-grade versus high-grade gliomas (Derlon et al., 1997, Giammarile et al., 2004, Ogawa et al., 1993). In particular, methionine PET exhibits a heightened sensitivity in detecting radiation necrosis from recurrent tumors, as inflammatory cells in radiation necrosis have little uptake of methionine (Thiel et al., 2000). Perhaps the most promising technique for diagnosing lowgrade gliomas is 18F-FDOPA PET imaging. 18F-FDOPA PET is more accurate that FDG PET and has been shown to be highly predictive in determining tumor grade on initial diagnosis and may help differentiate tumor necrosis from recurrence (Chen et al., 2006, Fueger et al.,

While there currently is no one imaging modality capable of definitively determining lowgrade from high-grade tumors on its own, advanced imaging technology continues to develop and complement standard MRI. As we come to understand the behavior and variability of these tumors, advance imaging techniques provide exciting new possibilities for more precise treatments. Given the variability within a given tumor, advanced imaging techniques may allow for more precise targets for biopsy, vigilant monitoring of malignant transformation, and improved prognostic power in the management of low- and high-grade

**8. The Role of bone-marrow derived cells in malignant transformation**

The vast majority of brain tumor research, molecular profiling, histological characteristics, diagnostic imaging modalities and treatment targets have focused on the actual tumor cells themselves. As mentioned earlier, one of the key events in the transition from the low-grade to the high-grade state is the angiogenic switch. It is theorized that in the low-grade state, tumor growth may be limited, at least in part, by a lack of blood supply. In this state, the tumor is only capable of a steady-state or linear growth (Mandonnet et al., 2003). Once the tumor acquires the ability to recruit or form new blood vessels, exponential growth occurs (Rees et al., 2009) resulting in rapid clinical decline. While there is considerable evidence that tumor cells undergo continued molecular changes that increase their malignant potential, these changes also allow these cells to initiate the angiogenic switch. It must also be noted that while recent evidence suggests that tumor cells may be capable of directly forming new blood vessels (Ricci-Vitiani et al., Wang et al.), a considerable body of evidence suggests that tumor cells do not do this completely on their own. While the exact details of this process still remain to be fully elucidated, tumor cells acquire the ability to transition the local tumor niche to an environment capable of rapid blood vessel formation. A variety of growth factors, signaling pathways, and indigenous populations of cells is hypothesized to participate in this process. If this theory proves to be correct, this population of cells forms an additional therapeutic target that may be as important as the tumor cells themselves. As current therapies directed at neoplastic cells are limited in part to their toxicity, elucidating other potential treatment pathways may further benefit patient

Neovascularization is a normal process in tissues and the brain during ischemia. In low oxygen states, cells release signals such as VEGF, PDGF, PlGF and HIF-1 that recruit from local existing vessels within the tissue itself (angiogenesis.) In addition, this can activate distant processes that facilitate neovascularization and may even form *de novo* blood vessels

Tripathi et al., 2009).

gliomas.

outcome.

(vasculogenesis). These factors mobilize bone marrow precursor cells which then travel to the site of ischemia via the bloodstream. It is theorized that these cells facilitate vasculogenesis by breaking down existing structures and creating an environment that promotes new blood vessel growth.

Tumors are capable of adopting this machinery to increase growth and invasiveness by activating the angiogenic switch (Bergers & Benjamin, 2003, Rafii & Lyden, 2008). During early tumor development, neoplastic cells rely on existing blood flow and grow in a slow linear fashion (Mandonnet et al., 2003). Once the switch is initiated and neovascularization brings more oxygen and nutrients, tumor cells grow at a much faster rate and tumor size increases significantly (Rees et al., 2009). This initial process is thought to occur mostly by angiogenesis (Kioi, 2010). The release of proteases and proangiogenic factors causes pericytes to detach from existing vessels creating a defect in the extracellular matrix in the environment surrounding the vessel wall (Bergers & Benjamin, 2003). Endothelial cells proliferate locally and sprout outward into the tumor bed creating newly formed blood vessels feeding the tumor. While angiogenesis is an important factor in the angiogenic switch, vasculogenesis and the contribution of BMDC play a critical role as well. For example, when recruitment of BMDC is impaired in an animal model of lymphoma and lung carcinoma, tumor angiogenesis and growth is significantly decreased (Lyden et al., 2001) suggesting that BMDCs contribute significantly to neovascularization and growth in solid tumors.

In metastatic disease, the contribution of BMDC has been well described (Wels et al., 2008). Endothelial (EPCs) and hematopoietic precursor cells (HPC), mesenchymal stem cells (MSC), myeloid-derived suppressor cells (MDSCs), Tie-2 expressing monocytes (TEM) and tumor associated macrophages (TAM) all are mobilized from the bone marrow to future metastatic sites prior to tumor formation. It should be noted that these primitive cells are prominent during embryology and that a significant population of these cells is not present under normal conditions. While the exact role of each cell type has yet to be fully elucidated, their basic function is to break down normal structures and promote vasculogenesis and tumor invasiveness. The net result is a tumor friendly environment capable of sustaining tumor growth. This has been demonstrated experimentally in a murine model of metastatic disease by implanting m-cherry labeled melanoma cells into the flank of mice with GFPlabeled bone marrow and examining the lungs of these animals over time (Kaplan et al., 2005). It was observed that the first cells to arrive in future metastases were not tumor cells, but actual BMDC. This suggested, at least in metastatic disease, that the environment in future metastatic sites is primed by cells from the bone marrow before tumors can begin to grow in these distant areas (Rafii & Lyden, 2008). This also supports the hypothesis by Stephen Paget over 100 years ago that the tumor microenvironment may play as important a role as the tumor cells themselves.

In the brain, the role of BMDC has only recently garnered attention. One of the basic histological differences between low-grade and high-grade gliomas is a lack of neovascularization. Thus, activation of the angiogenic switch is a key element in the transformation of low-grade to high-grade glioma. Two elements are likely to contribute to this process. Genetic changes in tumor cells that occur during progression of disease activate pro-angiogenic factors. This has been observed in human tumor samples whereby genes involved in angiogenesis are upregulated in glioblastoma as compared to low grade astrocytoma (Godard et al., 2003). Kioi et al. also showed in their animal model that release of soluble factors by tumor cells or cells within the tumor microenvironment including

Bone Marrow-Derived Cells Support Malignant Transformation of Low-Grade Glioma 213

Mobilization of BMDC in peripheral blood samples has similarly been observed in patients with astrocytomas. Circulating CD133+ and VEGFR2+ EPC were measured in patients with different grade gliomas. This population of cells was significantly elevated in brain tumor patients versus controls, correlated with tumor grade, and predicted survival. In one patient, this population also predicted recurrence prior to detection by serial radiographic study. Currently, patients are followed with serial imaging in order to diagnose recurrence or malignant progression. While advances in imaging technology show promise in earlier more accurate diagnosis, the critical event has already occurred and prognosis worsens considerably. Therefore, the identification of a potential surrogate biomarker that measures tumor angiogenicity and aggressiveness may potentially serve as an index for ongoing

As histological and molecular differences between low-grade and high-grade gliomas are further defined and it becomes apparent that tumors cannot be loosely classified, specific treatments based on the particular characteristics of each individual tumor can potentially be designed. In addition, the presence of particular populations of BMDC in these tumors may also provide additional information on tumor behavior and serve as an additional treatment target along with tumor cells themselves. It has already been shown that the presence of BMDC in the blood correlates with tumor grade and initial animal studies suggest that BMDC are present in high-grade tumors only (Greenfield et al., 2009). In addition, TAM have been associated with poorer prognosis in metastatic lesions and other solid tumors (Wels et al., 2008). Thus histological stains aimed at identifying this population of cells may provide more accurate diagnosis and prognosis. Likewise, the molecular markers of this particular population of cells may offer an even more specific therapeutic target. Based on data collected in glioma patients, EPC can be identified by cell surface markers including CD133 and VEGFR2. Knocking down this population with specifically designed drug therapies has the potential for preventing recurrence by decreasing migration of these cells and reducing vasculogenesis within the tumor bed. Finally, one can also envision a role for advanced imaging technologies for improved diagnosis and treatment. For instance, PET has been used to specifically measure VEGF that has been labeled with copper in an orthotropic mouse model of GBM (Cai et al., 2006). If one could identify and label molecular targets that are specific to individual tumors subtypes and sensitive to new imaging techniques, this provides exciting non-invasive possibilities for tumor specific

In summary, one of the primary factors predicting outcome in patients with low-grade glioma is malignant progression to high-grade tumor and it is evident that the angiogenic switch is an important event in this process. Initial management often entails surgical resection while adjuvant therapy for low-grade gliomas remains a controversial topic. Tumor grade is determined by histological analysis of tumor specimens, but the molecular fingerprint of these tumors is now being analyzed more thoroughly and holds promise for more exciting targeted treatment options. In addition, distinct, but as yet undefined, populations of cells are recruited to the tumor site and participate in neovascularization and promote tumor growth and invasiveness. Therefore, this population may represent an important therapeutic target in combating these tumors. Since survival is directly correlated with tumor grade, preventing tumor progression is imperative. While BMDC certainly are

treatment effectiveness or recurrence.

identification and treatment for each individual patient.

**9. Conclusions**

VEGF, FGF and EGF stimulates local angiogenesis (Kioi, 2010). Secondly, hypoxia is an additional critical event in triggering the switch (Kioi, 2010). As tumor size grows and metabolic demand exceeds local perfusion, hypoxic conditions occur. Release of hypoxia inducible factor-1 (HIF-1α) by tumor cells or cells within the hypoxic tumor environment, combined with stromal cell-derived factor-1 (SDF-1) and CXCR-4 receptor activation, mobilizes BMDCs to the tumor site and promotes vasculogenesis in gliomas (Du et al., 2008, Greenfield et al., 2010, Kioi, 2010).

In an attempt to further understand these processes in gliomas, Du et al. utilized an orthotopic model of GBM in mice to demonstrate recruitment of BMDC in gliomas (Du et al., 2008). Based on their results they theorize that hypoxia and the subsequent release of HIF-1α is the key event in tumor progression. Elevation of VEGF, and subsequent SDF-1 release and CXCR-4 receptor activation, mobilizes BMDC and recruits EPC and myeloid cells to the tumor. The net effect tips the balance to a pro-angiogenic state and neovascularization within the tumor bed. Kioi et al also further theorized that radiation treatment may exacerbate the vasculogenesis process and boost eventual tumor recurrence observed in current treatment regimens (Greenfield et al., 2010, Kioi, 2010). The endothelialmesenchymal transition and MSC have also been described in metastatic disease (Singh & Settleman). MSC exist within the brain and mobilize to the tumor site as well (Hata et al., Kang et al.). The exact roles of these particular BMDC remain elusive and require more study before they are fully delineated.

In our laboratory, we have begun to investigate the correlation of BMDC mobilization and tumor grade in gliomas (unpublished data.) We used a PDGF-driven mouse model of GBM within which tumors develop slowly from low-grade to high-grade. Low-grade tumors have a clear absence of neovascularization and BMDC are not present within these lesions. In high-grade tumors, however, we have observed a profound increase in larger, irregularly shaped, hemorrhagic vessels and a significant population of BMDC exists that is not observed in low grade tumors. In addition, these cells are located near newly forming blood vessels in the perivascular niche. We have also observed that BMDC are mobilized in the bone marrow and are elevated in the peripheral blood of tumor bearing animals versus controls. In addition, a significant difference in this population of cells in the blood exists between low-grade and high-grade animals. While much work is yet to be done before this process is fully elucidated, it appears that the presence of BMDC correlates with tumor grade and the process of neovascularization. Thus, BMDC have a potential role in the angiogenic switch as tumors progress from low-grade to high-grade tumors.

Fig. 2. Bone marrow-derived cells in human glioma. A, Immunofluorescence in grade II astrocytoma shows normal blood vessels (red, VE Cadherin) and a paucity of CD11b+ myeloid suppressor cells (Green). B, GBM shows abnormal vessel formation and an influx of CD11b+ cells (unpublished data.)

Mobilization of BMDC in peripheral blood samples has similarly been observed in patients with astrocytomas. Circulating CD133+ and VEGFR2+ EPC were measured in patients with different grade gliomas. This population of cells was significantly elevated in brain tumor patients versus controls, correlated with tumor grade, and predicted survival. In one patient, this population also predicted recurrence prior to detection by serial radiographic study. Currently, patients are followed with serial imaging in order to diagnose recurrence or malignant progression. While advances in imaging technology show promise in earlier more accurate diagnosis, the critical event has already occurred and prognosis worsens considerably. Therefore, the identification of a potential surrogate biomarker that measures tumor angiogenicity and aggressiveness may potentially serve as an index for ongoing treatment effectiveness or recurrence.

As histological and molecular differences between low-grade and high-grade gliomas are further defined and it becomes apparent that tumors cannot be loosely classified, specific treatments based on the particular characteristics of each individual tumor can potentially be designed. In addition, the presence of particular populations of BMDC in these tumors may also provide additional information on tumor behavior and serve as an additional treatment target along with tumor cells themselves. It has already been shown that the presence of BMDC in the blood correlates with tumor grade and initial animal studies suggest that BMDC are present in high-grade tumors only (Greenfield et al., 2009). In addition, TAM have been associated with poorer prognosis in metastatic lesions and other solid tumors (Wels et al., 2008). Thus histological stains aimed at identifying this population of cells may provide more accurate diagnosis and prognosis. Likewise, the molecular markers of this particular population of cells may offer an even more specific therapeutic target. Based on data collected in glioma patients, EPC can be identified by cell surface markers including CD133 and VEGFR2. Knocking down this population with specifically designed drug therapies has the potential for preventing recurrence by decreasing migration of these cells and reducing vasculogenesis within the tumor bed. Finally, one can also envision a role for advanced imaging technologies for improved diagnosis and treatment. For instance, PET has been used to specifically measure VEGF that has been labeled with copper in an orthotropic mouse model of GBM (Cai et al., 2006). If one could identify and label molecular targets that are specific to individual tumors subtypes and sensitive to new imaging techniques, this provides exciting non-invasive possibilities for tumor specific identification and treatment for each individual patient.

#### **9. Conclusions**

212 Glioma – Exploring Its Biology and Practical Relevance

VEGF, FGF and EGF stimulates local angiogenesis (Kioi, 2010). Secondly, hypoxia is an additional critical event in triggering the switch (Kioi, 2010). As tumor size grows and metabolic demand exceeds local perfusion, hypoxic conditions occur. Release of hypoxia inducible factor-1 (HIF-1α) by tumor cells or cells within the hypoxic tumor environment, combined with stromal cell-derived factor-1 (SDF-1) and CXCR-4 receptor activation, mobilizes BMDCs to the tumor site and promotes vasculogenesis in gliomas (Du et al., 2008,

In an attempt to further understand these processes in gliomas, Du et al. utilized an orthotopic model of GBM in mice to demonstrate recruitment of BMDC in gliomas (Du et al., 2008). Based on their results they theorize that hypoxia and the subsequent release of HIF-1α is the key event in tumor progression. Elevation of VEGF, and subsequent SDF-1 release and CXCR-4 receptor activation, mobilizes BMDC and recruits EPC and myeloid cells to the tumor. The net effect tips the balance to a pro-angiogenic state and neovascularization within the tumor bed. Kioi et al also further theorized that radiation treatment may exacerbate the vasculogenesis process and boost eventual tumor recurrence observed in current treatment regimens (Greenfield et al., 2010, Kioi, 2010). The endothelialmesenchymal transition and MSC have also been described in metastatic disease (Singh & Settleman). MSC exist within the brain and mobilize to the tumor site as well (Hata et al., Kang et al.). The exact roles of these particular BMDC remain elusive and require more

In our laboratory, we have begun to investigate the correlation of BMDC mobilization and tumor grade in gliomas (unpublished data.) We used a PDGF-driven mouse model of GBM within which tumors develop slowly from low-grade to high-grade. Low-grade tumors have a clear absence of neovascularization and BMDC are not present within these lesions. In high-grade tumors, however, we have observed a profound increase in larger, irregularly shaped, hemorrhagic vessels and a significant population of BMDC exists that is not observed in low grade tumors. In addition, these cells are located near newly forming blood vessels in the perivascular niche. We have also observed that BMDC are mobilized in the bone marrow and are elevated in the peripheral blood of tumor bearing animals versus controls. In addition, a significant difference in this population of cells in the blood exists between low-grade and high-grade animals. While much work is yet to be done before this process is fully elucidated, it appears that the presence of BMDC correlates with tumor grade and the process of neovascularization. Thus, BMDC have a potential role in the

angiogenic switch as tumors progress from low-grade to high-grade tumors.

Fig. 2. Bone marrow-derived cells in human glioma. A, Immunofluorescence in grade II astrocytoma shows normal blood vessels (red, VE Cadherin) and a paucity of CD11b+ myeloid suppressor cells (Green). B, GBM shows abnormal vessel formation and an influx of

Greenfield et al., 2010, Kioi, 2010).

study before they are fully delineated.

CD11b+ cells (unpublished data.)

In summary, one of the primary factors predicting outcome in patients with low-grade glioma is malignant progression to high-grade tumor and it is evident that the angiogenic switch is an important event in this process. Initial management often entails surgical resection while adjuvant therapy for low-grade gliomas remains a controversial topic. Tumor grade is determined by histological analysis of tumor specimens, but the molecular fingerprint of these tumors is now being analyzed more thoroughly and holds promise for more exciting targeted treatment options. In addition, distinct, but as yet undefined, populations of cells are recruited to the tumor site and participate in neovascularization and promote tumor growth and invasiveness. Therefore, this population may represent an important therapeutic target in combating these tumors. Since survival is directly correlated with tumor grade, preventing tumor progression is imperative. While BMDC certainly are

Bone Marrow-Derived Cells Support Malignant Transformation of Low-Grade Glioma 215

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not the only factor in progression of disease and neovascularization, blocking recruitment of these cells to tumors has been shown to reduce growth in animal models of other tumor types. Therefore, a greater understanding of this process may define a role for targeting this population of cells. In addition, BMDC exist in the periphery, and make for an easier therapeutic target than tumor cells within the blood brain barrier. Lastly, current management of tumor recurrence relies on serial imaging studies. Therefore, an effective and accurate biomarker capable of predicting progression of disease may allow for earlier detection and better treatment outcomes. This makes the case for monitoring BMDC in the periphery in addition to therapy aimed at this population of cells as potential adjuvant therapy in glioma.

#### **10. References**

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**Glioma Progression**

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*2.5. E*ff*ecst of PADI2 Knockout on the Protein and mRNA Expression of Adhesion-Related Genes*

In the U937 cells and the differentiated macrophages, we found that the protein expression levels of phospho-FAK, FAK, phospho-paxillin, paxillin, and PAK1 were all decreased in the PADI2 knockout group compared with in the control group (Figures 5 and 6). For the gene expression of the adhesion-related proteins, we found that there were no statistically significant differences in the mRNA expression of *FAK*, *paxillin*, or *PAK1* between the PADI2 knockout group and the control group in the U937 cells and the differentiated macrophages.

**Figure 5.** Effects of PADI2 knockout in the protein and mRNA expression of focal adhesion kinase (*FAK*), *paxillin*, and *PAK1* in the U937 cells: (**A**) comparison of the relative protein expression levels of FAK and phospho-FAK in the PADI2 knockout group and the control group; (**B**) comparison of the relative protein expression levels of paxillin and phospho-paxillin in the PADI2 knockout group and the control group; (**C**) comparison of the relative protein expression levels of PAK1 in the PADI2 knockout group and the control group; and (**D**) Representative images showing the relative protein expression levels of FAK, paxillin, and PAK1 in the PADI2 knockout group and the control group; (**E**) the relative mRNA expression levels of *FAK*, *paxillin*, and *PAK1* in the U937 cells. In the U937 cells, the protein expression levels of the cell adhesion-related proteins, including FAK, phospho-FAK, paxillin, phospho-paxillin, and PAK1, were performed by Western blotting in the PADI2 knockout group and the control group. The relative protein expression was defined as the ratio of specific protein/hsp90 band intensity.

**Figure 6.** Effects of PADI2 knockout in the protein and mRNA expression of *FAK, paxillin*, and *PAK1* in the differentiated macrophages: (**A**) comparison of the relative protein expression levels of FAK and phospho-FAK in the PADI2 knockout group and the control group; (**B**) comparison of the relative protein expression levels of paxillin and phospho-paxillin in the PADI2 knockout group and the control group; (**C**) comparison of the relative protein expression levels of PAK1 in the PADI2 knockout group and the control group; and (**D**) Representative images showing the relative protein expression levels of FAK, paxillin, and PAK1 in the PADI2 knockout group and the control group; (**E**) the relative mRNA expression levels of *FAK*, *paxillin*, and *PAK1*. In differentiated macrophages, the protein expression levels of the cell adhesion-related proteins including FAK, phospho-FAK, paxillin, phospho-paxillin, and PAK1 were performed by Western blotting in the PADI2 knockout group and the control group. The relative protein expression was defined as the ratio of a specific protein/hsp90 band intensity.

#### **3. Discussion**

Vossenaar et al. [14], Hojo-Nakashima et al. [15], and our previous study [7] have shown that PADI2 protein levels increased during the macrophage differentiation, resulting in increasing protein citrullination. In the current study, we demonstrated that PADI2 is essential for macrophage proinflammatory cytokine secretion, cell adhesion, and apoptosis using CRISPR/Cas9-mediated knockout of PADI2. As for the target proteins of PADI2, we found the histone H3 citrullination did not change in the U937 cells, the differentiated macrophages, or the macrophages stimulated with LPS in the PADI2 knockout group compared with in the control group. Darrah et al. reported that histone H3 was prone to be citrullinated by PADI4, which might explain our finding [16]. Different antibodies used in diffident studies might detect different epitopes, which could affect the results of histone H3 citrullination. However, we demonstrated that the protein level of citrullinated NF-κB p65 was decreased in the PADI2 knockout group compared with in the controls. Sun et al. showed that the citrullination of NF-κB p65 could enhance *IL-1*β and *TNF-*α expression [11]. Our result also showed that the expression levels of *IL-1*β and *TNF-*α were indeed decreased in the U937 cells after PADI2 knockout. The differentiation of macrophage and further stimulation with LPS could decrease the *IL-1*β and *IL-6* expression in the PADI2 knockout group compared with in the controls. Most importantly, we found that the IL-1β, IL-6, and TNF-α concentrations in the culture soup were dramatically decreased in the PADI2 knockout group compared with in the controls. In addition, Sun et al. showed citrullinated NF-κB p65 was mediated by PADI4 using HeLa cells and neutrophils, which expressed high levels of PADI4 compared with those of monocytes and lymphocytes [6]. In the current study, we used U937 cells, a representative cell line for human monocytes. We demonstrated that PADI2 was also required to citrullinated NF-κB p65 in macrophages upon LPS stimulation. Lee et al. showed that PADI2 could interact with an inhibitor of nuclear factor kappa-B kinase subunit gamma (IKKγ) to suppress NF-κB activity using a murine cell line [17]. However, our results suggested that NF-κB activity decreased in PADI2 knockout U937 cells from a decreased expression of proinflammatory cytokines. Different cell lines used in different studies might explain these variations. Mishra et al. demonstrated that PADI2 and PADI4 activity in macrophages were required for inflammasome assembly and IL-1β release in a murine model [18], which is consistent with our findings. Since proinflammatory cytokines including IL-1β, IL-6, and TNF-α play a critical role in the immunopathogenesis of RA [19,20] and increased PADIs activities have been documented in patients with RA [21], targeting PADIs would be a novel strategy for RA treatment.

We found that PADI2 knockout significantly decreased the apoptotic rates in the U937 cells, the differentiated macrophages, and the macrophages stimulated with LPS. Liu et al. demonstrated the overexpression of PADI4-induced cell apoptosis in human leukemia (HL)-60 cells and human acute T leukemia Jurkat cells [22]. These findings suggested that both PADI2 and PADI4 could promote apoptosis. For the detail mechanism of cell apoptosis, Liu et al. found that PADI4 induced apoptosis mainly through cell cycle arrest and a mitochondria-mediated pathway [22]. Our study showed that PADI2 could activate caspase-2 and -9, leading to the activation of capases-3. Further studies are needed to clarify their molecular mechanisms.

We noted that the inhibition of PADI2 impaired the protein expression of phospho-FAK, FAK, phospho-paxillin, paxillin, and PAK1, resulting in impaired cell adhesion ability in macrophages. We could not detect the phosphorylation of PAK1, which was also not detected in macrophages in a previous study [23]. PAK1 belongs to one of the members of the PAKs family, which plays an important role in cell motility [24], and the phosphorylation of PAK1 is critical for cell migration instead of adhesion [25]. We noted that the protein levels, but not mRNA expression levels of FAK, paxillin, and PAK1, were decreased in the PADI2 knockout group compared with in the controls. We speculated that the citrullination of proteins might accelerate their degradation by changing their binding affinity to proteasome [7,26] or altered protein structure [27], and further studies are needed.

In conclusion, our study showed that PADI2 is essential for the multiple functions of macrophages in enhancing inflammatory cytokines production through the citrullination of NF-κB p65, promoting cell apoptosis with capase-2, -3, and -9 activation, and facilitating cell adhesion via FAK, paxillin, and PAK1.Targeting PADI2 could be a novel strategy for controlling inflammation triggers by macrophages.

#### **4. Material and Methods**

### *4.1. Purification of Anticitrullinated Protein Antibodies (ACPA) from Pooled ACPA(*+*) Sera in Patients with RA*

The study protocol was approved by the institutional review board of Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (No. B10902001, 1 April 2020). The study was performed in accordance with the Declaration of Helsinki. Serum samples from ACPA-positive RA patients with high concentration of ACPAs (>340 IU/mL) and aged 20 years and above, which fulfilled the

2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria [28], were detected using an ELISA kit (Pharmacia Diagnostics AB, Uppsala, Sweden), collected and pooled. ACPAs were purified according to the method described previously [29]. In brief, the pooled sera containing high concentration of ACPAs from patients with RA were purified by affinity chromatography using an ÄKTA purifier 10 (GE Healthcare, Little Chalfont, UK) with UV detection at 280 nm for the collection of the desired fractions.


**7. Discussion**

Recent reports in the public media [51,52] have argued that phonics (word recognition) instruction is a necessity for reading success and that current phonics instruction in American schools is inadequate. We agree that proficiency in word recognition accuracy is critical to reading success and that intentional and systematic instruction in phonics is required in grades kindergarten through two. However, mere accuracy in word recognition is insufficient for proficient reading. Given our understanding of the interplay between and cognitive requirements for word recognition and comprehension, the true goal for word recognition instruction should be automaticity in word decoding. For decoding skills to become automatic, fluency instruction that includes regular opportunities for repeated and assisted reading is recommended. Interestingly, neither of the previously mentioned public media articles advocating phonics instruction included reading fluency as a significant part of the recommended instruction or automatic word recognition as the ultimate foundational reading goal.

In Chall's [29] stages of reading development, the foundational reading focus in Stage 1 is decoding and occurs in grades one and two. At Stage 2 the focus of foundational reading moves to fluency and occurs in grades two and three. While we agree with and support the trajectory of Chall's model of word recognition accuracy, we argue that there is no reason why fluency cannot also be developed simultaneously in grades one and two. Perhaps Chall's fluency stage of development may be more appropriately implemented earlier in students' school careers (e.g., grades one–two) and lead to an acceleration in students' reading development.

The results of the present study offer some preliminary insight into challenging the oft-stated suggestion that phonics instruction alone is sufficient for students to achieve mastery of foundational reading. The study also questions the notion that fluency instruction should be delayed until after students have achieved full proficiency in word recognition accuracy (Stage 1). The results of our study suggest that intentional, multi-component fluency instruction along with phonics and word work may provide an additional boost to students' reading achievement greater than phonics alone. Moreover, our results suggest that phonics and fluency instruction can, and perhaps should, occur simultaneously as early as grade one.

Fluency's place in the history of American reading instruction could be viewed as one of benign neglect. Although oral reading recitation was a major component of reading instruction more than a century ago, it was ignored in the later half of the 20th century to the point that by 1983 Allington [2] referred to fluency as the neglected reading goal. Even though fluency did gain some resurgence with its identification as a critical component of effective reading instruction based on the National Reading Panel's [18] evidence-based assessment of the scientific research literature, fluency has continued to be viewed more as a tangential component of the overall literacy curriculum. An analysis of Reading First [25], a U.S. reading initiative in the first decade of the 21st century, whose goal was for all third

grade students to be reading at grade level, found that less than five minutes per day was devoted to fluency instruction, even in schools where there was a commitment to provide instruction in the essential instructional components of reading, including fluency, identified by the National Reading Panel. Reading First failed to achieve the goal of all children reading at grade level, and we are left to wonder if a portion of those disappointing results were due to the fact that fluency was not su fficiently emphasized in instruction.

The results of the present study provide a tentative suggestion that intentional and multi-component fluency instruction, along with phonics, may be an appropriate part of a foundational first-grade reading curriculum. Indeed, recognizing the stagnant growth in reading achievement in the United States, and given the present tentative results in favor of the students receiving fluency instruction, integrating fluency into the first-grade reading curriculum has the possibility of making a significant and substantial impact on improving students' literacy outcomes. The implementation of the FDL required no more than 20 min per day, a time period that could easily be integrated into existing first-grade reading foundational skills instruction.

At a minimum, this study suggests that larger-scale and more in-depth and controlled research into the role of fluency instruction, and in particular the Fluency Development Lesson, on improving reading outcomes in first grade students may be worthwhile. If we are interested in raising reading achievement in the elementary grades, fluency cannot be neglected.