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4.20.4.1.1 Tissue invasion and metastasis

One of the six essential alterations in cell physiology of tumour cells is the capability of tissue invasion and metastasis. To escape the limiting nutrients and space available around the primary tumour, tumour cells colonize distant tissue in the body. Proteases play a major role in facilitating tumour cells to invade adjacent tissue and metastasize to distant sites. Many tumours have elevated levels of proteolytic enzymes (Keppler et al., 1996; Kim et al., 1998; Koblinski et al., 2000). Proteases can be classified by their mechanisms of action: serine, cysteine, threonine, aspartate, glutamic acid proteases, and metalloproteases (Yang et al., 2009a). The main focus of interest in targeting proteases for NIR fluorescence imaging has been in cathepsins, metalloproteases, and the urokinase plasminogen activator. Cysteine cathepsins are involved in protein degradation. They are a member of the family of papain-like cysteine proteases and were linked to cancer as long as 30 years ago (Sloane et al., 1981). An elevated cathepsin expression has been associated with poor prognosis and progression in a number of cancers (Turk et al., 2012). Moreover, high serum cathepsins have been correlated with metastases (Turk et al., 2012). Cathepsin expression is elevated in cancer cells as well as in macrophages and other host cells that are located within the tumour microenvironment (Mohamed and Sloane, 2006; Vasiljeva et al., 2006). Next to the well-known extracellular function of promoting migration and invasion of

cancer cells, cathepsins play a pro- and antitumorigenic intracellular role in tumour progression (Turk et al., 2012). Eleven human cathepsins are known (i.e., cathepsins B, C, F, h, K, L, O, S, V, X, and W) of which especially cathepsins B, D, and S are important because of the high expression in malignancies. Therefore, these particular cathepsins have been used as targets in optical imaging studies. Weissleder et al. (1999) were the first to image protease activity using the so-called ‘smart probes’ (Figure 9).

Figure 9 (a) Schematic diagram of probe activation.

The initial proximity of the fluoro- chrome molecules to each other results in signal quenching. (b) NIRF image (top) and bright light image (bottom) of nonactivated probe (left) and activated probe (right). Note the difference in signal intensity between enzyme-activated and unactivated probe. (c) Chemical structure of repeating graft copolymer segment indicating quenching of fluorophore Cy and enzymatic degradation site (green arrow). Reproduced from Weissleder R, Tung CH, Mahmood U, and Bogdanov A Jr. (1999) In vivo imaging of tumours with protease-activated near- infrared fluorescent probes. Nature Biotechnology 17(4): 375–378.

(Further information about ‘smart probes’ can also be found in Part I, Chapter 4.05 and Part III, Chapter 4.15) The smart probes are based on multiple self-quenching (i.e., inactivated) fluorophores that are located close to each other. Until the probe is dequenched by the protease of interest, excited fluorophores will absorb energy from each other that would otherwise result in the emission of a photon (Kobayashi et al., 2010). Weissleder et al. (1999) coupled the fluorophore Cy5.5 to a synthetic graft copolymer consisting of poly-L-lysine sterically protected by multiple methoxypolyethylene glycol (MPEG) side chains. Selective in vivo accumulation of probe in a breast and small cell lung tumour was effectuated by the EPR effect (Fang et al., 2011). Intratumoral dequenching (i.e., activation) of the fluorophores resulted in a 12-fold increase in NIR fluorescent signal that allowed for the detection of tumours with submillimetre- sized diameters. The substrate-based probe was dequenched by a series of cysteine/serine proteases, including cathepsins B, H, and L. Shortly after the finding of the smart probe, Bremer et al. (2002) reported the use of aforementioned probe to differentiate the cathepsin B expression levels of breast cancer cell lines. Considering the presentation of both human and murine cathepsin B in this study, Bremer et al. (2005) conducted a second study in which a spontaneous breast tumour was used. Again, breast tumours could be detected. Many resembling probes, targeting different cathepsins and built around a reporter substrate, have been developed (Abd- Elgaliel and Tung, 2010; Cruz-Monserrate et al., 2011; Tung et al., 1999). Thereby, tumour-

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4.20.4 Tumour Detection Using Organic Fluorescent Probes

Specific targeting of the primary tumour and metastases by fluorescent agents to intraoperatively identify the tumour margin is expected to be of great importance in future surgical oncology. Different targeting strategies can be used to visualize tumours and subsequently identify their margins for radical resection. Preclinically, many targeting moieties have been explored in the past decennium. Nonetheless, cancer targeting with high sensitivity and specificity remains challenging, mostly because of a great extent of intra- and intertumoral heterogeneity (Gerlinger et al., 2012; Marusyk et al., 2012). Hence, the quest for a universal tumour-specific target remains to be ongoing. In 2000, Hanahan and Weinberg (2000) reported six essential alterations in cell physiology that collectively dictate malignant growth, namely, tissue invasion and metastasis, sustained angiogenesis, limitless replicative potential, self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, and evasion of programmed cell death (Keereweer et al., 2011a). In the following, the preclinical and clinical work of NIR fluorescence tumour imaging will be discussed, based on specific targeting of each of these hallmarks of cancer (see also Part I, Chapter 4.05).

4.20.4.1 Tumour Imaging: Preclinical Studies

4.20.4.1.1 Tissue invasion and metastasis

One of the six essential alterations in cell physiology of tumour cells is the capability of tissue invasion and metastasis. To escape the limiting nutrients and space available around the primary tumour, tumour cells colonize distant tissue in the body. Proteases play a major role in facilitating tumour cells to invade adjacent tissue and metastasize to distant sites. Many tumours have elevated levels of proteolytic enzymes (Keppler et al., 1996; Kim et al., 1998; Koblinski et al., 2000). Proteases can be classified by their mechanisms of action: serine, cysteine, threonine, aspartate, glutamic acid proteases, and metalloproteases (Yang et al., 2009a). The main focus of interest in targeting proteases for NIR fluorescence imaging has been in cathepsins, metalloproteases, and the urokinase plasminogen activator. Cysteine cathepsins are involved in protein degradation. They are a member of the family of papain-like cysteine proteases and were linked to cancer as long as 30 years ago (Sloane et al., 1981). An elevated cathepsin expression has been associated with poor prognosis and progression in a number of cancers (Turk et al., 2012). Moreover, high serum cathepsins have been correlated with metastases (Turk et al., 2012). Cathepsin expression is elevated in cancer cells as well as in macrophages and other host cells that are located within the tumour microenvironment (Mohamed and Sloane, 2006; Vasiljeva et al., 2006). Next to the well-known extracellular function of promoting migration and invasion of

cancer cells, cathepsins play a pro- and antitumorigenic intracellular role in tumour progression (Turk et al., 2012). Eleven human cathepsins are known (i.e., cathepsins B, C, F, h, K, L, O, S, V, X, and W) of which especially cathepsins B, D, and S are important because of the high expression in malignancies. Therefore, these particular cathepsins have been used as targets in optical imaging studies. Weissleder et al. (1999) were the first to image protease activity using the so-called ‘smart probes’ (Figure 9).

Figure 9 (a) Schematic diagram of probe activation.

The initial proximity of the fluoro- chrome molecules to each other results in signal quenching. (b) NIRF image (top) and bright light image (bottom) of nonactivated probe (left) and activated probe (right). Note the difference in signal intensity between enzyme-activated and unactivated probe. (c) Chemical structure of repeating graft copolymer segment indicating quenching of fluorophore Cy and enzymatic degradation site (green arrow). Reproduced from Weissleder R, Tung CH, Mahmood U, and Bogdanov A Jr. (1999) In vivo imaging of tumours with protease-activated near- infrared fluorescent probes. Nature Biotechnology 17(4): 375–378.

(Further information about ‘smart probes’ can also be found in Part I, Chapter 4.05 and Part III, Chapter 4.15) The smart probes are based on multiple self-quenching (i.e., inactivated) fluorophores that are located close to each other. Until the probe is dequenched by the protease of interest, excited fluorophores will absorb energy from each other that would otherwise result in the emission of a photon (Kobayashi et al., 2010). Weissleder et al. (1999) coupled the fluorophore Cy5.5 to a synthetic graft copolymer consisting of poly-L-lysine sterically protected by multiple methoxypolyethylene glycol (MPEG) side chains. Selective in vivo accumulation of probe in a breast and small cell lung tumour was effectuated by the EPR effect (Fang et al., 2011). Intratumoral dequenching (i.e., activation) of the fluorophores resulted in a 12-fold increase in NIR fluorescent signal that allowed for the detection of tumours with submillimetre- sized diameters. The substrate-based probe was dequenched by a series of cysteine/serine proteases, including cathepsins B, H, and L. Shortly after the finding of the smart probe, Bremer et al. (2002) reported the use of aforementioned probe to differentiate the cathepsin B expression levels of breast cancer cell lines. Considering the presentation of both human and murine cathepsin B in this study, Bremer et al. (2005) conducted a second study in which a spontaneous breast tumour was used. Again, breast tumours could be detected. Many resembling probes, targeting different cathepsins and built around a reporter substrate, have been developed (Abd- Elgaliel and Tung, 2010; Cruz-Monserrate et al., 2011; Tung et al., 1999). Thereby, tumour-

specific imaging in pancreatic cancer, pancreatic intraepithelial neoplasia, peripheral lung cancer, breast cancer, oral squamous cell carcinoma, and lymph node metastases have been reported using the cathepsins B, H, L, and S sensitive and commercially available probe ProSense680 or ProSense780 (PerkinElmer, Waltham, Massachusetts) (Eser et al., 2011; Figueiredo et al., 2006; Keereweer et al., 2011b, 2012a; von Burstin et al., 2008; Xie et al., 2012). Mieog et al. (2011b) determined the accuracy of real-time NIR fluorescence imaging in obtaining tumour-free resection margins by using ProSense680 and ProSense780 (Figure 10).

Figure 10; (a) In vivo activation of ProSense750 by syngeneic rat model of primary breast cancer: a typical example of

a spectral unmixed image of an EMR86 tumour-bearing female WAG/Rij rat, acquired 24 h after intravenous administration of 10 nmol ProSense750. Shown is the separation of the autofluorescence signal (pseudocolored green) and the ProSense750 signal (pseudocolored red; IVIS spectrum). (b) Emission curve plot of the spectrally unmixed fluorescence signals from (a) demonstrates matching of the tumour signal (red line) with the predefined ProSense750 emission curve (blue line), confirming the localization of activated ProSense750 at the tumours. (c) In a dose- dependent and time-dependent experiment, nine tumor bearing rats (N¼35 tumours) were randomized to three ProSense750 dose groups and imaged 24 h (gray bars) and 48 h (open bars) after intravenous administration of ProSense750 using the IVIS Spectrum. Bars represent mean_SEM. (d) Scatter plot of fluorescence intensity and tumour volume of the 10 nmol dose group imaged 24 h after intravenous administration of ProSense750 (R¼0.934, P<0.0001, N¼12 tumours from three rats). Reproduced from Mieog JS, Hutteman M, van der Vorst JR, et al. (2011) Image-guided tumour resection using real-time near-infrared fluorescence in a syngeneic rat model of primary breast cancer. Breast Cancer Research and Treatment 128(3): 679–689.

Complete resection was attained in 17 out of 17 tumours with minimal excision of healthy tissue. Next to cathepsins, matrix metalloproteinases (MMPs) are important matrix-degrading proteinases involved in organ morphogenesis, wound healing, and embryonic development. Similar to the proteases mentioned above, MMPs are overexpressed in many tumours. Over 20 MMPs are known of which predominantly MMP-2, MMP-9, and MMP-7 are involved in the progression of cancer. Their complex role in carcinogenesis includes the stimulation of cancer cell growth, angiogenesis, migration, invasion, and metastasis. Like cathepsins, MMPs are overexpressed in cancer cells as well as tumour stromal cells (Egeblad and Werb, 2002; Hua et al., 2011; Overall and Lopez-Otin, 2002). The expression of MMPs correlates with advanced tumour stage, increased invasion, metastasis, and poor survival (Egeblad and Werb, 2002).

By modifying the aforementioned cathepsin-sensing probe, Bremer et al. (2001a) were the first to image MMP activity using NIR fluorescence imaging in vivo. Although cleavable by other MMPs, the peptide substrate was primarily cleaved by MMP-2. The probe allowed identification ofMMP-2-positive tumours and the assessment ofMMP-2 activity after treatment with the potent MMP inhibitor prinomastat (Bremer et al., 2001a,b). Next to imaging of nonmembrane-bound MMPs, Zhu et al. (2011a,b) explored the imaging of membrane-type MMPs (MT-MMP). Membrane type-1 matrix metalloproteinase (MMP-14), one of the six MT-MMPs, is highly expressed in different cancers (Zhu et al., 2011a). A nonsubstrate MT1-MMP-binding peptide was conjugated to the NIR-fluorescent dye Cy5.5 and targeted the high MT1-MMP that expressed MDA-MB-435 xenografts. In the line of research with the commercially available probes targeting cathepsins, a substrate-based MMP targeting probe, MMPSense680 (PerkinElmer, Waltham, Massachusetts) was developed and used in NIR fluorescence imaging of oncology (Keereweer et al., 2011b, 2012a; Xie et al., 2012). In healthy tissue, the urokinase-type plasminogen activator receptor (uPAR) is moderately expressed in variable tissues. Increased expression is related to extensive tissue remodelling through the regulation of extracellular proteolysis in healthy tissues, as well as in many pathological conditions including cancer, inflammation, and infections (Blasi and Sidenius, 2010). The nonproteolytic functions consist of the promotion of cell proliferation, spreading, migration, and invasion (Blasi and Sidenius, 2010). Although a variety of studies reported the use of radioactive or iron oxide labeled uPA-based peptides, reports on targeting uPAR by using a targeting moiety conjugated to a fluorophore are relatively rare (Li et al., 2008; Liu et al., 2009; Yang et al., 2009b,c). Law et al. (2004) described the development of a selective uPA activatable NIR fluorescence imaging probe in vitro. A copolymer of L-lysine and MPEG was used as a backbone for an uPA selective substrate and conjugated to Cy5.5 or Cy7. Upon addition of recombinant human uPA to the probe, a significant amplification of fluorescence was observed. No amplification was observed using the negative control probe or when uPA inhibitors were added. The same probe was used to assess the validation in animal models of human colon adenocarcinoma and fibrosarcoma (Hsiao et al.,

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specific imaging in pancreatic cancer, pancreatic intraepithelial neoplasia, peripheral lung cancer, breast cancer, oral squamous cell carcinoma, and lymph node metastases have been reported using the cathepsins B, H, L, and S sensitive and commercially available probe ProSense680 or ProSense780 (PerkinElmer, Waltham, Massachusetts) (Eser et al., 2011; Figueiredo et al., 2006; Keereweer et al., 2011b, 2012a; von Burstin et al., 2008; Xie et al., 2012). Mieog et al. (2011b) determined the accuracy of real-time NIR fluorescence imaging in obtaining tumour-free resection margins by using ProSense680 and ProSense780 (Figure 10).

Figure 10; (a) In vivo activation of ProSense750 by syngeneic rat model of primary breast cancer: a typical example of

a spectral unmixed image of an EMR86 tumour-bearing female WAG/Rij rat, acquired 24 h after intravenous administration of 10 nmol ProSense750. Shown is the separation of the autofluorescence signal (pseudocolored green) and the ProSense750 signal (pseudocolored red; IVIS spectrum). (b) Emission curve plot of the spectrally unmixed fluorescence signals from (a) demonstrates matching of the tumour signal (red line) with the predefined ProSense750 emission curve (blue line), confirming the localization of activated ProSense750 at the tumours. (c) In a dose- dependent and time-dependent experiment, nine tumor bearing rats (N¼35 tumours) were randomized to three ProSense750 dose groups and imaged 24 h (gray bars) and 48 h (open bars) after intravenous administration of ProSense750 using the IVIS Spectrum. Bars represent mean_SEM. (d) Scatter plot of fluorescence intensity and tumour volume of the 10 nmol dose group imaged 24 h after intravenous administration of ProSense750 (R¼0.934, P<0.0001, N¼12 tumours from three rats). Reproduced from Mieog JS, Hutteman M, van der Vorst JR, et al. (2011) Image-guided tumour resection using real-time near-infrared fluorescence in a syngeneic rat model of primary breast cancer. Breast Cancer Research and Treatment 128(3): 679–689.

Complete resection was attained in 17 out of 17 tumours with minimal excision of healthy tissue. Next to cathepsins, matrix metalloproteinases (MMPs) are important matrix-degrading proteinases involved in organ morphogenesis, wound healing, and embryonic development. Similar to the proteases mentioned above, MMPs are overexpressed in many tumours. Over 20 MMPs are known of which predominantly MMP-2, MMP-9, and MMP-7 are involved in the progression of cancer. Their complex role in carcinogenesis includes the stimulation of cancer cell growth, angiogenesis, migration, invasion, and metastasis. Like cathepsins, MMPs are overexpressed in cancer cells as well as tumour stromal cells (Egeblad and Werb, 2002; Hua et al., 2011; Overall and Lopez-Otin, 2002). The expression of MMPs correlates with advanced tumour stage, increased invasion, metastasis, and poor survival (Egeblad and Werb, 2002).

By modifying the aforementioned cathepsin-sensing probe, Bremer et al. (2001a) were the first to image MMP activity using NIR fluorescence imaging in vivo. Although cleavable by other MMPs, the peptide substrate was primarily cleaved by MMP-2. The probe allowed identification ofMMP-2-positive tumours and the assessment ofMMP-2 activity after treatment with the potent MMP inhibitor prinomastat (Bremer et al., 2001a,b). Next to imaging of nonmembrane-bound MMPs, Zhu et al. (2011a,b) explored the imaging of membrane-type MMPs (MT-MMP). Membrane type-1 matrix metalloproteinase (MMP-14), one of the six MT-MMPs, is highly expressed in different cancers (Zhu et al., 2011a). A nonsubstrate MT1-MMP-binding peptide was conjugated to the NIR-fluorescent dye Cy5.5 and targeted the high MT1-MMP that expressed MDA-MB-435 xenografts. In the line of research with the commercially available probes targeting cathepsins, a substrate-based MMP targeting probe, MMPSense680 (PerkinElmer, Waltham, Massachusetts) was developed and used in NIR fluorescence imaging of oncology (Keereweer et al., 2011b, 2012a; Xie et al., 2012). In healthy tissue, the urokinase-type plasminogen activator receptor (uPAR) is moderately expressed in variable tissues. Increased expression is related to extensive tissue remodelling through the regulation of extracellular proteolysis in healthy tissues, as well as in many pathological conditions including cancer, inflammation, and infections (Blasi and Sidenius, 2010). The nonproteolytic functions consist of the promotion of cell proliferation, spreading, migration, and invasion (Blasi and Sidenius, 2010). Although a variety of studies reported the use of radioactive or iron oxide labeled uPA-based peptides, reports on targeting uPAR by using a targeting moiety conjugated to a fluorophore are relatively rare (Li et al., 2008; Liu et al., 2009; Yang et al., 2009b,c). Law et al. (2004) described the development of a selective uPA activatable NIR fluorescence imaging probe in vitro. A copolymer of L-lysine and MPEG was used as a backbone for an uPA selective substrate and conjugated to Cy5.5 or Cy7. Upon addition of recombinant human uPA to the probe, a significant amplification of fluorescence was observed. No amplification was observed using the negative control probe or when uPA inhibitors were added. The same probe was used to assess the validation in animal models of human colon adenocarcinoma and fibrosarcoma (Hsiao et al.,

2006). A threefold higher signal intensity was reported, correlating with tumour-associated uPA activity. Dullin et al. (2009) published the use of a nonactivatable uPAR antibody conjugated to Cy5.5 to visualize breast carcinoma. In contrast to the nonspecific antibody that was used as a control, clear visualization of the breast tumour was observed. Next to the substrate-based probes described above, fluorescent activity-based probes (ABPs) have been reported (Blum et al., 2005, 2007). In contrast to the substrate-based probes, ABPs label target proteases through covalent binding (Figure 11). One of the major advantages of using a substrate as a reporter in ‘smart probes’ is that a single active protease can process many substrates resulting in the amplification of fluorescent signal over time. However, such substrates do usually not target a specific single protease, making it difficult to determine which particular protease is being imaged (Blum et al., 2009). ABPs allow direct biochemical analysis of targets after in vivo imaging and enable a direct link to the imaging data. Moreover, due to small molecules, ABPs have shorter half-lives and shorter elimination times, resulting in higher contrast images. ABPs targeting cathepsins B, L, S, and X have been synthesized and were reported to successfully delineate tumours in vivo (Blum et al., 2007; Paulick and Bogyo, 2011; Tedelind et al., 2011; Verdoes et al.,