Photochemical internalization (PCI) of immunotoxins directed against putative cancer stem cell markers in triple-negative breast cancer and malignant melanoma

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Photochemical internalization (PCI) of immunotoxins directed against putative cancer stem cell markers in triple-negative

breast cancer and malignant melanoma

Marius Strømbo Eng

Master’s thesis at the School of Pharmacy

Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO

May 2012

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For the degreeMaster of Pharmacy(45 credits):

Photochemical internalization (PCI) of immunotoxins directed against putative cancer stem cell markers in triple-negative breast cancer and malignant melanoma

Author:

Marius Strømbo Eng

Main supervisor:

Pål Kristian Selbo Co-supervisor:

Anette Weyergang Internal supervisor:

Kristian Berg

Department of Radiation Biology Institute for Cancer Research The Norwegian Radium Hospital OSLO UNIVERSITY HOSPITAL

Department of Pharmacy School of Pharmacy

Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

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Photochemical internalization (PCI) of immunotoxins directed against putative cancer stem cell markers in triple-negative breast cancer and malignant melanoma

Marius Strømbo Eng

http://www.duo.uio.no/

Print: Reprosentralen, University of Oslo, Oslo, Norway

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Abstract

The surface antigens CSPG4 and CD271 have been identified as possible markers for a population of therapy-resistant and tumor-maintaining cancer stem cells within triple-negative breast cancer (TNBC) and malignant melanoma, respectively. Photochemical internalization (PCI) is a novel drug delivery technology developed for high temporal-spatial controlled delivery of drugs that are normally unable to reach their target within cells. Five breast cancer cell lines, in which three were TNBC/CSPG4-expressing, as well as two CSPG4-expressing malignant melanoma cell lines were treated with PCI of the CSPG4-directed immunotoxin 225.28-saporin. In addition, two malignant melanoma cell lines, one CD271-expressing, were treated with PCI of the CD271-directed immunotoxin ME20.4-saporin. PCI enhanced the cytotoxic effect of the immunotoxins in a synergistic manner and was substantially more cytotoxic compared to PCI of streptavidin-saporin in the antigen-positive cell lines. The antigen-negative cell lines had no advantage of the immunotoxins, and in one cell line, the cytotoxic effect by PCI of 225.28-saporin could be blocked with the addition of a 20-fold excess of 225.28-mAbs. This study provides proof-of-principle that TNBC and malignant melanoma can be efficiently and selectively targeted in vitrousing PCI of an immunotoxin specific for CSPG4, and is the first of its kind to demonstrate targeting of CD271 by PCI-induced delivery of ME20.4-saporin. The present work provides an important foundation for future PCI-based therapy targeting CSPG4 and CD271.

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Abbreviations

ALDH Aldehyde dehydrogenase

ALDH^hi Aldehyde dehydrogenase high-expressing bFGF Basic fibroblast growth factor

CD(number) Cluster of differentiation (number)

CFU Colony forming units

CSC Cancer stem cell

CSPG4 Chondroitin sulfate proteoglycan 4 DMSO Dimethyl sulfoxide

EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor

EGFR Epidermal growth factor receptor 1 EMT Epithelial-mesenchymal transition EpCAM Epithelial cell adhesion molecule

ER Estrogen receptor

ESA Epithelial specific antigen (see EpCAM) FACS Fluorescence activated cell sorting

FBS Fetal bovine serum

Fab Fragment antigen binding Fc Fragment crystallizable

Fv Variable fragment

h. Hour(s)

HER2 Human epidermal growth factor receptor 2 HESCm Human embryonic stem cell medium HMEC Human mammary epithelial cells IgG2a Immunoglobulin G, subclass 2a

LAF Laminar flow

mAb Monoclonal antibody

MAPK Mitogen-activated protein kinase

MM Malignant melanoma or MelMet (cell lines)

MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide

MW Molecular weight

NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells

NGF Nerve growth factor

NOD/SCID Non-obese diabetic, severe combined immunodeficiency

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NSG NOD/SCID IL-2Rγ^null

NT No treatment

NTR Neurotrophin receptor

O⁻₂ Superoxide anion

1O₂ Singlet oxygen

PARP Poly (ADP-ribose) polymerase PBS Phosphate buffered saline PCI Photochemical internalization pCR Pathological complete response

PDT Photodynamic therapy

PE Phycoerythrin (fluorochrome) orPseudomonasexotoxin (bacterial toxin)

PR Progesterone receptor

PROCR Endothelial protein C receptor

PS Photosensitizer

RIP Ribosome-inactivating protein ROS Reactive oxygen species scFv Single-chain variable fragment TNBC Triple-negative breast cancer TPCS_2a Disulfonated tetraphenyl chlorin TPPS_2a Disulfonated tetraphenyl porphine VEGF Vascular endothelial growth factor

VEGFR Vascular endothelial growth factor receptor

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Acknowledgements

The work presented here was carried out in the period of august 2011 to may 2012 at the Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, and is the final part of the five-year master’s programme in pharmacy at the School of Pharmacy, University of Oslo. The project has been a part of the Cancer - Stem Cell Innovation Center (SFI-CAST), one of the Centres for Research-based Innovation appointed by the Norwegian Research Council, through a collaboration between PCI Biotech ASA and the Department of Radiation Biology.

Firstly, I would like to extend my gratitude to my main supervisor dr. Pål Kristian Selbo for providing excellent supervision, an interesting project, as well as constructive feedback and encouragement throughout the whole period. I would like to thank my co-supervisor dr. Anette Weyergang for her comments and the introduction to the laboratory procedures, and my internal supervisor at the School of Pharmacy and group/department head Professor Kristian Berg for his input. I am also grateful for the comments from dr. Anders Høgset at PCI Biotech ASA.

I would like to thank Monica Bostad for her suggestions and the introduction to several of the methods. To the whole of the PCI group: I am very grateful to be included in the group, for your encouraging comments and for being available to answer any questions.

Thanks to my fellow master students Cathrine E. Olsen, Svein R. Angel and Marte Jonsson for the valuable scientific and non-scientific discussions.

My gratitudes to dr. Soldano Ferrone at the University of Pittsburgh Cancer Institute for the generous gift of the 225.28 monoclonal antibodies, and Nina Iversen at the Department of Medical Genetics, Oslo University Hospital for providing us with the SUM149 cell line. I would also like to thank Lina Prasmickaite and Gunhild Mælandsmo at the Department of Tumor Biology for their contributions to the project and for providing us with the MelMet cell lines. Thanks to Kotryna Vasiliauskaite for patiently providing MelMet cells prior to each experiment. I would also like to acknowledge the Flow cytometry core facility by Idun D. Rein and Kirsti S. Landsverk, as well as the Confocal microscopy core facility by Ellen Skarpen for their assistance.

Oslo, May 15, 2012

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Chapter 1 Introduction

1.1 Background

Despite of decades of drug development, no efficient, curative therapy exists for late stages of triple-negative breast cancer (TNBC) and malignant melanoma to this date. Current therapy includes highly cytotoxic agents with low specificity and serious adverse effects. The presence of a population of cancer stem cells (see 1.2) within triple-negative breast cancer and malignant melanoma has been hypothesised to explain the resistance of treatment, and may be recognized by the surface antigenschondroitin sulfate proteoglycan 4(CSPG4) (see 1.5) andcluster of differentiation 271(CD271) (see 1.6), respectively. The use of therapeutic monoclonal antibodies (mAbs) (see 1.8.2) has paved the way for a more specific and targeted treatment of cancer and has made it possible to develop antibody-toxin conjugates, immunotoxins(see 1.8.3), specific for the target antigen.

The aim of the present study was to combine the specificity of immunotoxins with the specificity provided by the novel drug delivery method termed photochemical internalization (PCI) (see 1.7.2), and selectively treat a proposed population of cancer stem cells within TNBC and malignant melanoma. It was hypothesised that the combined treatment would lead to loss of clonal capacity of TNBC and malignant melanoma cell linesin vitro.

1.2 Cancer stem cells

Tumors are thought to derive from single cells that have lost their own restraints on cell division and have acquired properties that favor their survival [1]. The development of cancer can be seen as a microevolutionary process wherein mutations occur and accumulate over time [1].

Mutations in genes important for DNA repair and DNA damage control are common, resulting in a genetic instability of malignant cells [1]. Through several rounds of mutation and natural selection, the most well-adapted cells will proliferate, out-live normal cells and will eventually be dominant [1]. Previously, every cell of the tumor population were thought to be biologically

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equivalent and capable of acquiring tumorigenic properties [2] (fig. 1.1A-a). Hence, tumorigenic cells were thought to occur stochastically and be indistinguishable from the tumor bulk population . This is referred to as the stochastic model.

However, when studying tumorigenicity, only a distinct population of individual tumor cells are able to form tumors in immunodeficient mouse models [3]. With the invention of fluorescence activated cell sorting (FACS), it became possible to recognize and sort out these subpopulations harboring tumorigenic properties [2]. This included analysis based on markers such as CD44, CD24, CD133, EpCAM (ESA) and ABCB5 [4, 5]. One of the first attempts to distinguish tumorigenic cells was performed on cells of acute myeloid leukemia [2]. Using flow cytometry, cells were sorted into two distinct populations. The two populations were designated tumorigenic and non-tumorigenic based on the number of cells required to form a tumor when injected into mice. In the smallest population, cells readily formed tumors, while the larger, non- tumorigenic fraction mostly contained differentiated granulocytes or monocytes with a limited ability to proliferatein vivo[2]. A similar study was conducted on human breast cancer cells by Al-Hajjet al.[6] and revealed a small, tumorigenic subpopulation capable of re-generating the heterogeneity found in the primary tumor (described in more detail in 1.3). More recent studies have made similar observations on the existence of a tumorigenic subpopulation in breast cancer [7, 8]. The observation that differentiated and undifferentiated cells had different tumorigenic properties led to the cancer stem cell model, in which cells are organized in a hierarchy (fig.

1.1A-b) of undifferentiated, stem-like cells as well as progenitor cells and differentiated cells [2]. Stem cells may divide to give stem cell progeny, making them self-renewing and capable of expanding their population [2].

Although several surface antigens are associated with tumorigenicity within certain forms of cancer, no universal cancer stem cell markers exist [9, 5], and surface markers are mainly se- lected by their heterogeneity within the tumor population and ability to be efficiently separated using FACS [9]. Hence, cancer stem cells must be identified using functional assays like two- and three-dimensional clonogenicity assays in vitro, by studying expression of genes involved in normal stem cell biology, or more importantly, by tumorigenicity assays in animal models [5].

Cancer stem cells have been suggested to be resistant to traditional chemotherapy [3]. While a recent review emphasizes that therapy resistance is not an universal trait of cancer stem cells [5], several examples exist in the literature: E.g. a rare population of cancer stem cells has been proposed to explain the resistance to tyrosine kinase inhibitorimatinibin certain patients with chronic myelogenous leukemia [5]. Breast cancer stem cells have been shown to exhibit radiation therapy resistance through an overexpression of genes important for glutathione synthesis [10]. Glutathione is an important cellular anti-oxidant and can neutralize the ionizing radiation- induced cellular damage caused by reactive oxygen species (ROS) [10]. Similarly, treatment of locally advanced breast cancer patients with the traditional chemotherapeutic agents doc- etaxel or a combination of doxorubicin and cyclophosphamide, resulted in an enrichment of

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the CD44⁺/CD24⁻ (see 1.3) fraction, increased mammosphere formation in vitro, as well as an increase in engraftment efficiency in immunodeficient mice [11], possibly demonstrating chemotherapy resistance within the cancer stem cell population.

Hence, regrowth of a tumor following chemotherapy may be explained by the survival of therapy-resistant cancer stem cells. Since bulk tumor cells can not proliferate indefinitely, it was proposed that eradication of all cancer stem cells of a tumor would lead to loss of tumor growth and self-maintenance (fig. 1.1B) [3]. However, recent studies have demonstrated that differentiated breast cancer cells may posses the ability to de-differentiate into cancer stem cells [12, 13]. If this occurs in other forms of cancer as well, it demonstrates the need to eradicate not only cancer stem cells, but also the whole bulk of the tumor.

1.3 Triple-negative breast cancer

Breast cancer is grouped according to molecular subtypes; (i) Luminal A and (ii) luminal B, (iii) Human epidermal growth factor receptor 2 (HER2)-overexpressing and (iv) basal-like [14].

Luminal breast cancers are positive for estrogen receptor (ER) and progesterone receptor (PR) [14], and the luminal B subtype is distinguished from luminal A by its overexpression of HER2, and may present with high rates of cell proliferation [14]. HER2-overexpressing breast cancer is ER⁻/PR⁻, but overexpresses HER2. The basal-like subtype is dominated by a triple-negative phenotype (TNBC) (ER⁻/PR⁻/HER2⁻) [15] and 40-80% of all TNBCs are basal-like [15].

TNBC constitutes 10-20% of all breast cancers [16] and is associated by a poor clinical prognosis, including high rates of central nervous system and lung metastases [15]. A fraction (17-58%) of patients have been shown to achieve pathological complete response (pCR) following chemotherapy [15], however, patients who do not achieve pCR have an exceptionally poor outcome [15], and less than 30% women survive for more than five years [16]. Some of the most effective therapies for breast cancer include anti-HER2 mAb trastuzumaband estrogen receptor antagonist tamoxifen[17]. As cells of TNBC do not express the relevant re-

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ceptors, these treatment strategies are not effective [17]. Novel drugs for TNBC include poly (ADP-ribose) polymerase (PARP)-inhibitors, angiogenesis inhibitors, epidermal growth factor receptor (EGFR)-inhibitors and kinase inhibitors [15]. The angiogenesis inhibitorbevacizumab (Avastin®) recently lost its indication for breast cancer due to lack of efficacy [18]. Moreover, Conleyet al. [19] recently demonstrated that tumor hypoxia induced by the angiogenesis inhibitors bevacizumab and sunitinib can increase the population of cancer stem cells in breast cancer xenografts. Tyrosine kinase-inhibitors have only shown moderate responses in phase II trials [15], while PARP inhibitors have been described as promising therapeutic agents [17], however, drug development proceeds slowly and only a minority of drugs reach the clinic [15].

The presence of cancer stem cells has been suggested as a possible explanation for the poor clinical prognosis of TNBC [20]. As breast cancer is a phenotypically diverse disease, Al-Hajj et al. [6] hypothesised that only a small subset of the cells in metastatic breast cancer would display tumorigenic properties. They found tumorigenic breast cancer cells to be highly enriched in the CD44⁺/CD24⁻ subpopulation. Similarly, Fillmoreet al. [7] analysed several cell lines, including triple-negative cell lines SUM149 and MDA-MB-231, and found cells in the CD44⁺/CD24⁻/EpCAM⁺fraction to be tumorigenic, divide slowly and be resistant to chemotherapy. Furthermore, Hwang-Versleus et al. [8] was able to enrich for tumorigenic cells by isolating the endothelial protein C receptor (PROCR)-positive and EpCAM-positive fraction of the MDA-MB-231 cell line. Another proposed marker for breast cancer stem cells is the aldehyde dehydrogenase enzyme (ALDH), shown to correlate with breast cancer aggressiveness [21, 22], and more recently the ALDH^hi/CD44⁺ fraction of MDA-MB-231 cells was associated with chemotherapy resistance [23].

TNBC was recently proposed to be subdivided into the basal-like, mesenchymal-like and luminal androgen receptor subtype [16]. Mesenchymal-like and mesenchymal stem-like cells are shown to have a gene expression profile consistent with increased cell motility and epithelial- mesenchymal transition (EMT) [16]. The normal function of EMT is to assist in tissue generation during embryonic development, as well as during wound healing, facilitating the motility necessary for epithelial cells to move into wound sites [2]. During EMT, epithelial cells lose their epithelial-like morphology and gene expression pattern, and instead assume the transcrip- tional program typical of mesenchymal cells [2]. This includes the loss of E-cadherin, which is a protein important for epithelial cell-to-cell adhesion [2]. These changes in gene expression result in higher motility and invasiveness, which are important features for metastasis [2].

When EMT was induced in non-tumorigenic, normal, immortalized human mammary epithelial cells (HMEC), the resulting mesenchymal cells acquired the CD44⁺/CD24⁻ phenotype [24]. Furthermore, oncogenically transformed cells formed mammospheres, soft agar colonies and tumors more efficiently following EMT [24]. Similar observations have been made by oth- ers as well [25]. This indicates an important role of EMT in the acquisition of stem cell-like properties in breast cancer.

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1.4 Malignant melanoma

Melanocytes are cells derived from neural crest cells and distribute naturally along the base- ment membrane of the epidermis of the skin [26]. A malignant, neoplastic development of melanocytes is referred to as malignant melanoma, and advanced melanoma is notoriously treatment resistant [27]. Early stages of melanoma involve a non-invasive, horizontal growth phase which is curable by surgery in 80% of cases [27]. At later stages, malignant cells enter the invasive, vertical growth phase, which is associated with a declining clinical prognosis [27]. If allowed to metastasise, treatment becomes increasingly difficult and patients with metastasis- ing melanoma are known to have a very poor outcome [28]. The median survival of metastatic melanoma patients is 6 to 9 months [27]. In the advanced stage, brain metastases is the most common, but can also include metastasis to soft tissues such as the lung or the liver [27]. Pri- mary treatment consists of surgical excision with interferon adjuvant treatment for patients with a high risk of relapse [29]. Distant metastases are rarely curable, but progression-free survival may be improved by BRAF-inhibitorvemurafenibor anti-CTLA4-mAbipilimumab[29].

Melanoma aggressiveness has been suggested to be caused by cancer stem cells [30] and possible virulence mechanisms include immune system evasion, multidrug resistance through efflux pumps, EMT and vasculogenic mimicry, a phenomenon in which tumor cells form a vascular network [31, 32]. Early evidence for melanoma stem cells included sorting for markers like CD20, CD133 and ABCB5 [32]. Later, scepticism arose as subsequent studies were unable to distinguish tumorigenic cells based on surface antigens [32]. Moreover, stem cells have been considered to be a rare part of the tumor, while in melanoma more than 20% of random tumor cells were able to form tumors in highly immunocompromised mice [33]. In contrast, both Civenni et al. [34] and Boiko et al. [35] recently reported that tumorigenic subpopulations could be distinguished by the surface marker CD271 (see 1.6). Additionally, Civenniet al.[34]

demonstrated that different methods and definitions of cancer stem cells between scientists, may explain why reports on melanoma stem cells are conflicting.

1.5 Chondroitin sulfate proteoglycan 4

Chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA), melanoma chondroitin sulfate proteoglycan (MCSP) or as the homologue neuron-glial antigen 2 (NG2) in rat, is a trans-membrane chondroitin sulfate proteoglycan [36], either expressed as a 250 kDa glycoprotein or a 450 kDa proteoglycan [37].

CSPG4 was initially detected in melanocytes, endothelial cells and pericytes, but has recently been shown to be expressed in several normal and malignant cells [36], as in melanoma, where it is found in more than 90% benign nevi and melanoma lesions [38]. Although initially found in melanoma, CSPG4 is expressed in several non-melanocytic cancers including astrocytomas, gliomas, neuroblastomas, squamos cell carcinoma of the head and neck, basal-like breast can-

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cer, mesothelioma and pancreatic carcinoma [36]. CSPG4 can activate signaling pathways through the mitogen-activated protein kinase (MAPK) and the focal adhesion kinase (FAK) cascades (fig. 1.2) [37]. Activation of these pathways may subsequently result in tumor progression through increased survival, chemoresistance, invasion, migration, proliferation and EMT [37], and cells expressing CSPG4 may theoretically have a selective advantage over cells not expressing CSPG4 [37]. Additionally, constitutive activation of the ERK pathway is shown to maintain melanoma cells in the undifferentiated state [39].

Wanget al. [40] assessed the expression of CSPG4 in 44 primary TNBC lesions, 4 TNBC cell lines and in tumor cells derived from pleural effusions of 12 different patients. CSPG4 was found expressed in 32 of the 44 (72.7%) primary TNBC lesions, as well as in the TNBC cell lines and cells of the pleural effusions. The protein was detected at a significantly higher frequency in basal-like breast cancer and TNBC, compared to luminal subtypes. Moreover, anti- CSPG4 mAb 225.28 inhibitied tumor growth and migrationin vitro, as well as tumor growth and metastasis in xenografts in immunodeficient mice [40]. Additionally expression of CSPG4 appeared to be associated with the cancer stem cell phenotype CD44⁺/CD24⁻ desribed by Al- Hajjet al.[6].

In melanoma, CSPG4 has been shown to have an important role in cell proliferation and may serve as a receptor for platelet-derived growth factor AA and basic fibroblast growth factor (bFGF) [36]. The role of CSPG4 in cell migration and invasion was shown in the melanoma cell line M14 [36]. Compared to untreated M14 cells, cells transfected with CSPG4 cDNA displayed a fourfold higher migratory ability [36], and similarly, melanoma migration could be inhibited by CSPG4-specific mAbs [36].

1.6 Cluster of differentiation 271

Cluster of differentiation 271 (CD271), also known as nerve growth factor receptor (NGFR), low-affinity nerve growth factor receptor (LNGFR) or p75 neurotrophin receptor (p75^{N T R}) is a 75 kDa transmembrane receptor and a member of the tumor necrosis factor receptor super fam- ily [41]. It is found widely expressed in the developing central and peripheral nervous system [41], in developing tissues of mesenchymal origin [42], as well as mature endothelial cells, fi- broblasts, prostate epithelial cells and immune B cells [42]. It has been found to be a marker for non-neuronal mesenchymal tumors and may act as a tumor suppressor in prostate and bladder carcinoma [42]. In breast cancer, nerve growth factor (NGF), a natural substrate for CD271, stimulates survival through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway [43]. Similarly, NGF is shown to promote survival through NGF/CD271 in malignant melanoma [44] and is more frequently expressed in benign nevi compared to normal melanocytes [45]. More importantly, CD271 may be associated with melanoma metastasis and tumor heterogeneity [34], vasculogenic mimicry [31], and is suggested to select for melanoma cancer stem cells [35, 34].

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Figure 1.2– CSPG4 may activate MAPK or FAK cascades, leading to events important for tumor progression.

Adapted from "CSPG4, a potential therapeutic target, facilitates malignant progression of melanoma" by Price, M. A.et al., 2011, Pigment Cell Melanoma Res, 24, p. 1153. Copyright 2011 by John Wiley & Sons

1.7 Photodynamic therapy and photochemical internalization

1.7.1 Photodynamic therapy (PDT)

To understand the principle of PCI, one must first be familiar with photodynamic therapy (PDT), on which the PCI principle is based. PDT involves the use of three, usually non-toxic, components; a photosensitiser (PS), oxygen and light, resulting in anti-tumor activity through generation of reactive oxygen species (ROS) as singlet oxygen (¹O₂) [46]. A molecule may be excited by light of an appropriate wavelength and return to the ground state, either by emitting a fluorescent photon (fluorescence) or by losing energy through internal conversion, for example by release of thermal energy (fig. 1.3A) [47]. A photosensitizer used in PDT has the additional property to lose part of its energy by transition to the triplet excited state through a process known as intersystem crossing [47]. Relaxation to the ground state from the triplet excited state can occur through emission of a photon, referred to asphosphorescence, by internal conversion or more importantly, by transfer of energy to another molecule [47]. The latter phenomenon is divided into two groups of photosensitazation reactions (fig. 1.3B). Firstly, in the type I reac-

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Figure 1.3–(A)Simplified Jablonski diagram. Relaxation from the triplet excited state may occur through phosphorescence, internal conversion or a photochemical reaction, exemplified by generation of ¹O2. Re- drawn and adapted from "Basic principles of photodynamic therapy" by Macdonald, I.J. and Dougherty, T.J., 2001, J. Porphyrins Phthalocyanines, 5, p. 106. (B)Type I and type II photochemical reactions. Adapted from "Photodynamic therapy for cancer" by Dolmans D.E.et al., 2003, Nat Rev Cancer, 3, p. 383. Copyright 2003 by Nature Publishing Group

tion, the photosensitizer can transfer its energy to an organic substrate, such as a cell membrane, and form an oxidized substrate and a reduced photosensitizer [47]. The reduced photosensitizer may subsequently react with molecular oxygen to form superoxide anions (O⁻₂). Alternatively, the triplet state photosensitizer may react directly with superoxide radicals to produce O⁻₂ [47].

These O⁻₂ go on to form the highly reactive radical oxygen [47]. With the type II reaction, the photosensitizer transfers its energy to ground-state molecular oxygen (³O2) to produce the reactive oxygen species (ROS)¹O₂ [47, 48].

Some of the most common photosensitizers in PDT include porphyrins, chlorins and bacte- riochlorins (fig. 1.4) [47]. These groups of photosensitizers absorb red light, and all porphyrins have a strong band of absorption of blue light around 400 nm [47]. Early commercially availi- ble photosensitizers include a mixture of hematoporphyrin derivatives known as Photofrin®, the chlorin-type photosensitizer meta-tetrahydroxyphenyl chlorin (Foscan®) and the heme- precursor methyl aminolevulinic acid (Metvix®). Amphiphilicity of photosensitizers was reported to be important for the photodynamic acitivity and led to development of amphiphilic compounds such as sulfonated tetraphenylporphines (TPPS) and sulfonated tetraphenylchlo- rines (TPCS) [47].

The radius of action of¹O2 is less than 0.02µm, which can be explained by its short half- life [49]. The cytotoxic effect of PDT on tumors is mediated through three main mechanisms;

(i) Direct effect of ROS on tumor cells, including mitochondrial damage, membrane damage, induction of late-stage apoptosis [46] as well as autophagy [50], which may either promote survival or promote death [50]. (ii) Damage to tumor vasculature by PDT leads to vascular

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Figure 1.4–Upper panel:Porphyrin, chlorin and bacteriochlorin structures. Adapted from "Basic principles of photodynamic therapy" by Macdonald, I.J. and Dougherty, T.J., 2001, J. Porphyrins Phthalocyanines, 5, p.

108.Lower panel:PCI photosensitizers TPPS2aand TPCS2a.

shutdown and inflammation [46] and lastly (iii) PDT has shown to activate an immune response against tumor cells [51, 46]. The direct effect on tumor cells has certain limitationsin vivo, as the level of tumor cell cytotoxicity is negatively correlated with the distance between tumor cells and vasculature, possibly due to reduced photosensitizer distribution and oxygen supply farther away from the vascular supply [48]. Vascular damage is an important mechanism, as it can restrict access to oxygen and nutrients, thereby leading to cell death [46]. In vivo-studies have shown induction of vasoconstriction, thrombus formation and subsequent inhibition of tumor growth [46, 48]. An upregulation of inflammatory cytokines is associated with PDT treatment, and infiltration of lymphocytes, leukocytes and macrophages has been observed in PDT-treated tissues [51]. The immunological effect was demonstrated clinically in a case report by Thonget al. [52]. Following PDT treatment of a tumor on the patient’s right upper limb, they observed a spontaneous remission of untreated tumors on the same limb two months later, as well as on untreated tumors on the patient’s other limb four months later. Biopsies revealed an infiltration of lymphocytes in untreated tumors, supporting the importance of immunological effects in PDT.

An important advantage of PDT is the possibility to specifically activate photosensitizers within tumor tissues. The dose of PDT is proportional to the concentration of the photosensitizer and the radiance of the light source [47]. Compounds like disulfonated tetraphenyl porphine (TPPS_2a) and disulfonated tetraphenyl chlorine (TPCS_2a) have been shown to preferentially accumulate within tumor tissues [53, 54], enhancing tumor specificity.

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1.7.2 Photochemical internalization (PCI)

Based on PDT, PCI (fig. 1.5) is a novel drug delivery method for selective cytosolic delivery of drugs that are sequestered in endo-lysosomal vesicles. The amphiphilic properties of photosensitizers like TPPS2aand TPCS2aallow them to be inserted within cellular membranes, followed by endocytosis [55], resulting in endocytic vesicles containing membranous photosensitizer.

The observation that lysosomal enzymes were released into cytosol upon illumination [56], led to the discovery that PDT disrupted endosomal and lysosomal membranes and could lead to cytosolic release of substances that are normally entrapped in the organelles. Subsequently, PCI was developed as a method for delivery of drugs and genes which would normally have no efficient mechanisms to reach their target within the cell. For example, ribosome-inactivating protein (RIP) toxins like saporin or gelonin have high molecular weights and are unable to pass directly through cellular membranes. These toxins can be internalized through endocytosis, but would normally be degraded within lysosomes, never reaching their target site within cytosol.

However, co-administered with a PCI photosensitizer, the photochemical treatment leads to a disruption of the endocytic membranes and allows the toxins to reach their target [55].

The photosensitizer and the drug of interest are required to localise within the same endocytic compartments. One might worry that the ROS generated would lead to damage of the delivered drug. However, PCI takes advantage of the short range of ROS, which mostly affect the endosomal membranes while avoiding damage to the drug within the compartment [57, 58].

While this is true for hydrophilic drugs, certain highly lipophilic compounds may localise too close to the photosensitizer to allow degradation-free delivery [58].

The PCI technology has been demonstrated in more than 80 different cell lines [58], as well as in a phase I clinical trial [59], and may enhance delivery of protein toxins, cytostatic drugs as well as genes and nanoparticles [58]. PCI has been described as a minimally invasive treatment with close to no systemic, adverse effects [58]. Similar to PDT, frequent adverse effects include photosensitation of the eyes and skin, but may be avoided by avoiding excessive light exposure following treatment [58]. Important limitations of the technology includes a restriction to treatment of local tumors and by the light penetration in the tissue (up to ≈ 1 cm) [58]. However, as previously mentioned, possible distal immunological effects have been observed after PDT and may contribute to systemic anti-tumor effects [51, 52].

1.8 Immunotoxins

1.8.1 Development of immunotoxins

Immunotoxins consist of an antibody-derived targeting moiety conjugated to a toxic compound.

Early immunotoxins were prepared by conjugating whole antibodies to toxins through disulfide bridges [60]. This method often led to low specificity, poor stability and a heterogenous product [60]. With new knowledge that protein toxins are divided into distinct domains, second genera-

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Figure 1.5– Photochemical internalization. The drug is co-administered with the photosensiziter (PS). The PS accumulates in cell membranes and the drug is taken up through endocytosis. ROS are generated during illumination leading to disruption of the endocytic membrane and release of drug into cytosol.

tion immunotoxins were prepared without the cell-binding domain of the toxin [60]. However, the homogeneity and affinity of the immunotoxins was still not optimal and could cause damage to endothelial cells, leading to vascular leakage syndrome [60]. This led to the development of recombinant immunotoxins consisting only of the necessary elements to recognize and kill tumor cells [61].

Important limitations of immunotoxins include low tumor tissue availability due to high in- terstitial pressure and poor blood perfusion, toxicity to circulating leukocytes and endothelial cells due to prolonged exposure [60], generation of neutralizing antibodies against both the antibody and the toxin fraction [62], as well as retroendocytosis and lysosomal degradation [63].

The immunogenic effect of the toxin has been reduced by removing the B-cell recognizing epitopes by recombinant technology [64], as well as chemical conjugation to polyethylene glycol and co-administration of immunosuppressive drugs [60].

1.8.2 Antibodies

Antibodies are an important part of the function of the adaptive immune system, and their antigen specificity make them suitable for targeted treatment. Briefly, an antibody consists of two identical heavy chains (≈50 kDa) and two identical light chains (≈25 kDa), in total about 150 kDa, and can further be divided into a variable region and a constant region [65]. Antigen-

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Figure 1.6– The basic structure of an antibody. Adapted from "Therapeutic antibodies for human diseases at the dawn of the twenty-first century" by Brekke O.H. and Sandlie I., 2003, Nat Rev Drug Discov, 2, p. 53.

binding sites are termed CDR loops and are localized on the variable regions, while the constant part of the antibody interacts with components of the immune system. If antibodies are cleaved by papain, they divide into three fragments; two fragments termed Fab (fragment antigen binding), containing the variable region and parts of the constant region, and one fragment termed Fc (fragment crystallizable). Antibodies are grouped according to their species of origin as well as their immunoglobulin class. Each type of antibody have a distinct structure and biological activity [65], and antibodies recognizing the same antigen may bind to different sites (epitopes) of their target.

1.8.3 Toxic moiety

The toxic component of an immunotoxin is often derived from bacteria or plants. Immuno- toxins used in clinical trials are usually produced by recombinant technology and incorporates bacterial toxins likePseudomonasexotoxin A as they are more easily produced inEscherichia coliand have fewer adverse effects in humans [60]. Another important group of toxins include ribosome-inactivating proteins (RIP) from plants. RIPs are divided into two subgroups: Type I RIPs, e.g. saporinorgelonin, consist of a single polypeptide chain (termed A-chain) and are usually unable to bind to and enter cells by themselves [66]. In addition to the A-chain, type II RIPs, e.g. ricin, include a galactose-binding lectin domain (termed B-chain) which facilitates cellular binding and uptake through interaction of lectin with the cell membrane [66]. RIPs are highly toxic compunds and immunotoxins containing saporin have been shown to inhibit protein synthesis and induce apoptosis [67].

1.8.4 Internalization

While most proteins can be internalized through non-specific fluid-phase pinocytosis, immunotoxins and targeted toxins are internalized specifically by binding to their target cell surface

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receptor. Upon binding, a signaling cascade is activated to induce internalization of the receptor- ligand complex [63]. As the the cell membrane is invaginated and "pinched" off, the result is an endocytic vesicle which incorporates the receptor-ligand complex [63]. The complex may either be recycled to the cell surface or degraded upon vesicle fusion with lysosomes [63]. The effect of a targeted drug is influenced by several factors [63], including affinity of the targeting moiety, affinity of the antigen, antigen density, target cell, rate of endocytosis, route of internalization, release from target, escape from vesicles and the drug concentration. Low immunotoxin cytotoxicity may be explained by an inability of antibody internalization, low binding affinity or poor endocytosis of the antigen [63]. In fact, the rate of internalization is an important factor when predicting toxicity [63], and may vary greatly across different mAbs targeting the same antigen [63].

1.8.5 PCI of immunotoxins and ligand-toxin conjugates

Delivery of immunotoxins by PCI has the potential to induce a triple-targeted effect through accumulation of the photosensitizer in tumor tissues, accumulation of immunotoxin in target cells and the site-specificity of light exposure. Immunotoxins are therefore considered as at- tractive drugs for delivery by PCI and several successful attempts have been made. An early attempt was PCI of the EpCAM-targeting immunotoxin MOC31-gelonin, which demonstrated a synergistic cytotoxicity in cell lines from small cell lung carcinoma, colon adenocarcinoma and ductal breast carcinoma [68]. However, it was later shown by confocal microscopy that MOC31-gelonin is poorly internalized, even follwing 18 h. incubation, and displays poor re- localization to the cytosol following light exposure [69]. Later on, epidermal growth factor receptor 1 (EGFR) was successfully targeted with PCI of cetuximab-saporin, in carcinoma cell lines from colon, prostate and epidermis [70]. More recently, PCI has been applied to target HER2 with trastuzumab-saporin in an epithelial ductal carcinoma cell line (Berstadet al., not published), as well as with two recombinant immunotoxins (Berstad et al., not published and Bull-Hansenet al., not published). The proposed stem cell-marker CD133/1 has been targeted using AC133-saporin in colorectal carcinoma cell lines (Bostad et al., not published). CSPG4 has previously been targeted using PCI of the fusion toxin scFvMEL/rGel in a melanoma cell line, a lobular breast carcinoma cell line and a malignant glioblastoma cell line in vitro[71].

In the same study, PCI of scFvMEL/rGel, by systemic administration of photosensitizer and fusion toxin, induced complete regression of 33% (n=12) well-developed melanoma xenografts in mice [71].

Additionally, PCI has been exploited to deliver ligand-toxin conjugates. As opposed to immunotoxins, ligand-toxin conjugates are not derived from antibodies, but consist of an en- dogenous ligand in conjugation to a toxin. PCI has been applied for EGFR-targeted delivery of epidermal growth factor (EGF)-saporin [72], vascular endothelial growth factor receptor type 1 (VEGFR1) and 2 (VEGFR2)-targeted delivery of vascular endothelial growth factor 121

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(VEGF121)-saporin (Weyerganget al., not published), as well as VEGFR2-targeted delivery of VEGF121/rGelonin [69].

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Chapter 2 Materials and methods

2.1 Cell culture

2.1.1 Cell lines

Mammary adenocarcinoma cell lines MDA-MB-231 (HTB-26™) and MDA-MB-435 (HTB- 129™), and luminal breast cancer cell line MCF-7 (HTB-22™) were all purchased from Amer- ican Type Culture Collection (Manassas, VA, USA). Breast cancer cell line MA11 was provided by the Department of Tumor Biology (Institute for Cancer Research, Oslo University Hospital).

Metastasing melanoma cell lines MelMet1 and MelMet5 were provided from the Department of Tumor Biology prior to each experiment. SUM149 was a gift from the Department of Med- ical Genetics, Oslo University Hospital. All cell lines were routinely tested forMycoplasma.

Selection of cell lines will be discussed further in chapter 4.

2.1.2 Culture medium

MDA-MB-231, MDA-MB-435, MA11, MelMet1 and MelMet 5 were cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) base medium supplied with L-glutamine, 10% fetal bovine serum (FBS) (PAA Laboratories, Pasching, Austria), 100 IU/ml penicillin (Sigma- Aldrich) and 100 µg/ml streptomycin (Sigma-Aldrich). MCF-7 was cultured in MEM with Earle’s salts (PAA Laboratories), supplied with L-glutamine, 10% FBS, 100 IU/ml penicillin/100 µg/ml streptomycin and 10 µg/ml insulin (Sigma-Aldrich). SUM149 was cultured in Ham’s F12 nutrient mixture (Sigma-Aldrich) supplied with L-glutamine, 5% FBS, 100 IU/ml penicillin/100 µg/ml streptomycin, 1 µg/ml hydrocortisone (Sigma-Aldrich) and 5 µg/ml insulin.

Single-use, stock solutions of hydrocortisone were prepared by dissolving 1 mg hydrocortisone powder per 1 ml absolute ethanol (Kemetyl Norge AS, Vestby, Norway) and stored at -20^◦C.

All cell lines were cultured as monolayers in Nunclon™ surface treated tissue culture flasks (NUNC A/S, Thermo Fisher Scientific, Roskilde, Denmark) in incubators at 37^◦C/5% (v/v) CO₂, medium renewal 2-3 times a week.

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Cell line Type Subtype ER/PR/HER2 CSPG4 Plating density

References MDA-MB-231 Mammary

adenocarcinoma

Mesenchymal stem-like

-/-/- + 13000 [7, 16, 40, 73]

MDA-MB-435 Mammary ductal carcinoma

(Basal/

mesenchymal)

-/-/- + 10000 [40, 74, 75, 76]

MCF-7 Mammary

adenocarcinoma

Luminal +/+/- - 8000 [7, 40, 77]

SUM149 Primary

breast cancer

Basal-like -/-/- + 8000 [7, 16, 40, 78]

MA11 Lobular

breast carcinoma

(Luminal) -/-/¹ (+) 8000 [71, 79]

MelMet 1 Metastasing melanoma

N/A N/A + 4000 [80]

MelMet 5 Metastasing melanoma

N/A N/A + 8000 [80]

1. Not described in the literature.

Table 2.1– Overview of cell lines.

2.1.3 Passaging

All mammary cell lines were adherent and were disassociated using a 0.025%-0.5% trypsin/0.53 mM EDTA solution (Sigma-Aldrich). MelMet cell lines were disassociated using a 0.54 mM EDTA solution. EDTA solution was prepared by dissolving EDTA disodium powder (Sigma- Aldrich) in Dulbecco’s PBS (PAA Laboratories). Prior to disassociation, the monolayer was washed once with Dulbecco’s PBS to remove any remaining trypsin-deactivating enzymes from serum. Cells were grown to a maximum of 80-90% confluency and a maximum of 25 passages to avoid changes in growth, morphology and genetics to affect the reproducibility of the results.

To determine cell concentration prior to an experiment, cells were counted manually using a KOVA® Glastic Slide disposable plastic hemocytometer (Hycor Biomedical, Indianapolis, IN, USA).

2.2 PDT and PCI treatment

2.2.1 Photosensitizer and light source

Disulfonated tetraphenyl chlorin (TPCS_2a, Amphinex™, PCI Biotech ASA, Oslo, Norway) is an amphiphilic photosensitizer, previously indicated to localize within endocytic vesicles [54], thereby making it suitable for PCI. It can be excited by blue light at ≈ 400-440 nm, as well as the more therapeutically relevant red light at ≈ 650 nm [54]. Cells were treated using the LumiSource® light source (PCI Biotech ASA). The lamp consists of a bed of four blue light- emitting (peak at about 435 nm [54]) 18 W Osram L 18/67 [81] tubes and is optimized to give a homogeneous spread of light across the lamp bed. Samples were placed directly on the lamp bed and illuminated for the desired amount of time. The irradiance was 10.7 mW/cm², see table 2.2 for conversion.

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Light exposure time (sec.) Light dose (J/cm²)

0 0

30 0.321

60 0.642

90 0.963

120 1.284

150 1.605

180 1.926

210 2.247

240 2.568

270 2.889

300 3.210

Table 2.2– Interconversion from light exposure time to J/cm²with the LumiSource® lamp of irradiance 10.7 mW/cm²

2.2.2 PDT and PCI

All PDT and PCI procedures were performed protected from light (no ceiling or LAF hood illumination). For a typical PDT or PCI experiment, cells were seeded into Nunc 96 MicroWell™

or 6 well plates (NUNC A/S, Thermo Fisher Scientific) and allowed to adhere. Cells were incubated in medium containing TPCS2aover night (≈18 h.), after which they were washed twice with fresh medium and chased for 4 h. to allow the photosensitizer to be internalized and cleared from the cell membrane. Following chase, samples were illuminated on the LumiSource® lamp for the desired amount of time.

PCI treatment was carried out similarly to PDT treatment, with the addition of the desired toxins. For 18 h. incubation, toxins were added to cells in photosensitizer-containing medium prior to the 18 h. incubation period. For assays of blocking immunotoxin binding, antibodies were added just prior to chase, while toxins were added 30-60 minutes into chase. See table 2.3 for a list of treatments and figure 2.1 for a timeline of a PCI experiment.

2.2.3 Toxins and mAbs

Saporin is a 30 kDa ribosome inactivating protein (RIP) [82]. Recombinant streptavidin is a 53 kDa protein with an extremely high affinity (Ka = 1015 M⁻1) [83] for the vitamin biotin.

The biotin-streptavidin interaction is one of the strongest non-covalent biological interaction known. Streptavidin-ZAP (Advanced Targeting Systems, San Diego, CA, USA) is a chemical conjugate of saporin and streptavidin (mw = 128 kDa), and contains in average 2.5 molecules of saporin per streptavidin. Stock solutions were diluted to 200 nM in PBS and stored at -20^◦C or 2-8^◦C for short term (4-6 weeks).

Biotinylated 225.28, hereby described as 225.28-biotin, (mouse anti-CSPG4, IgG2a mAb) was kindly provided by Dr. Soldano Ferrone (University of Pittsburgh Cancer Institute, Pitts- burgh, PA, USA). Antibodies were preserved by sodium azide and stored as small aliquots at

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Table 2.3– Overview of treatments and controls.

Figure 2.1–Timeline of a typical PCI experiment. Cells are seeded out and allowed to adhere, incubated over night with photosensitizer/toxin, washed twice, chased and illuminated.

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-20^◦C or at 2-8^◦C for short term (4-8 weeks) according to the instructions provided by Dr. Fer- rone. CSPG4-targeting immunotoxin 225.28-saporin was prepared by mixing streptavidin-ZAP and 225.28-biotin (molar ratio 1:4) in PBS. The reaction was assumed to be complete within 20 minutes, due to the high affinity of streptavidin to biotin. Solutions of 200 nM 225.28-saporin were stored as small aliquots at -20^◦C or at 2-8^◦C for short term (4-6 weeks).

ME20.4-SAP (Advanced Targeting Systems) is a 210 kDa chemical conjugate of ME20.4, an anti-CD271 mAb, and saporin [84]. Stock solutions were diluted to 200 nM in PBS and stored at -20^◦C or 2-8^◦C for short term (4-6 weeks).

2.3 Viability assays

2.3.1 MTT

Cell viability was measured 48 h. post illumination using the 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide (MTT) assay. For most cells, the mitochondrial activity is proportional to the cell viability [85]. MTT is converted to formazan crystals within active mitochondria. Formazan crystals can be dissolved in an organic solvent and forms a purple solution which can be analysed by a spectrophotometer [85]. The MTT assay is considered suitable to quantify cytotoxicity in short-term cultures [86], but has some important limitations which will be discussed further in chapter 4.

Cells were incubated in fresh medium containing 0.25 mg/ml MTT reagent (Sigma-Aldrich) for 3-5 h. Additionally, eight empty wells were incubated in the MTT solution to serve as a blank control. Following incubation, MTT solution was aspirated and dimethyl sulfoxide (Sigma-Aldrich) was added to each well to dissolve the formazan crystals. The optical density of each well was measured using a Powerwave XS2 microplate spectrophotometer (BioTek, Winooski, VT, USA) at 570 nm. The absorbance was corrected based on the mean absorbance from the eight blank samples. Relative cell viability was calculated using the absorbance of untreated cells.

To determine the optimal cell density per well for the MTT assay, cells were seeded out at varying densities, treated according to the PDT protocol (drug-free) and examined using the MTT assay.

2.3.2 Clonogenic survival

The clonogenic assay measures the ability of a single cell to form clones, proliferate and es- tablish a colony. Compared to the MTT assay, the clonogenic assay gives a better indication of the long-term effects of a cytotoxic agent [86]. In this study, a modified version of [87] was used. Typically, 1000 cells were seeded into a 6-well plate and treated according to the PCI protocol. Medium was replaced 2 times per week. After 9-14 days, colonies were washed once

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in 0.9% saline (Fresenius Kabi, Halden, Norway), fixed by absolute ethanol (Kemetyl), stained by methylene blue and counted by hand. A colony was defined to consist of at least 50 cells as described in [87]. To determine the optimal cell density per well for the clonogenic assay, cells were seeded at varying densities and treated according to the PDT protocol (drug-free).

Colonies were fixed, stained and counted after 10-14 days.

2.3.3 Trypan blue exclusion stain

Trypan blue is a charged molecule and can not pass intact cell membranes, while can be taken up into damaged cells [88]. This makes it possible to exclude non-viable cells when observed under a microscope. The trypan blue exclusion stain was applied as a brief and simple method to count viable cells. A sample of cell suspension was mixed 1:1 with a 0.4% trypan blue solution in 0.81% sodium chloride/0.06% potassium phosphate (Sigma-Aldrich). After 3-5 minutes, unstained cells were counted in a hemocytometer to calculate the concentration of viable cells.

2.3.4 Spheroids

Dontu et al. [89] attempted to grow human mammary epithelial cells (HMEC) in serum- free medium containing B-27®, EGF, bFGF and heparin. Most primary HMECs died under these culture conditions, while a small amount of cells were able to generate floating spherical colonies (mammospheres, "spheroids" or 3D culture). Furthermore they reported that mammospheres are highly enriched in undifferentiated cells and can be formed at clonal densities. Fang et al. [30] demonstrated that similar spheroids formed when malignant melanoma cells were grown in human embryonal stem cell (HESC) medium as described in [90]. Cells isolated from these spheroids were capable of proliferation, differentiation, and self-renewal. Additionally they were shown to possess higher tumorigenicity compared to cells grown as monolayers.

To study the spheroid forming ability of melanoma cell line MelMet 5, a modified version of the HESC medium was used. Briefly, it consisted of 40% of the methyl cellulose based medium Methocult® H4100 (STEMCELL Technologies SARL, Grenoble, France), 38% KnockOut™

DMEM/F12, 20% KnockOut™ Serum Replacer, 0.1 mM non-essential amino acids, 0.1 mM 2-Mercaptoethanol (all Life Technologies), 4 ng/ml bFGF, 2 mM L-glutamine (Sigma-Aldrich), and penicillin-streptomycin (Sigma-Aldrich). Viable cells were suspended in 300 µl medium and transferred to a 3 ml aliquot of HESCm/Methocult®. Using a plastic syringe (BD Medical, Franklin Lakes, NJ, USA) and a 16 G hypodermic needle (BD Medical), 1,1 ml was added to a well of a Costar® ultra low attachment 6 well plate (Corning Life Sciences, Tewksbury, MA, USA). Fresh, non-Methocult® HESCm was added each week. After 2-3 weeks, spheroids were counted using a GelCount™ (Oxford Optronix, Oxford, UK).

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Figure 2.2 – Simplified schematic overview of a fluorescence microscope filter block. A wide range of excitation light hits the excitation filter. Light of the desired wavelength passes and is reflected by the dichroic mirror at a 90 degree angle through the objective and down on the specimen. As molecules are excited, they emit light of a longer wavelength (Stokes shift). The dichroic mirror allows for the emission light to pass through, while filtered out by the emission filter before reaching the camera.

2.4 Fluorescence microscopy

2.4.1 Epifluorescence microscopy

Certain molecules are able to absorb energy of a particular wavelength and emit a portion of the absorbed energy. The difference in emission and absorption wavelengths is known as the Stokes shift, and each fluorescent molecule has a characteristic absorption and emission spec- trum [91]. In a fluorescence microscope, the excitation light is directed towards the specimen and as fluorophores are excited, they emit light of lower energy. The emission light is collected and allows for observation and recording using a digital camera. To deliver and collect light of specific wavelengths, the microscope incorporates optical filters that filter out undesired light [91]. Additionally, a dichroic mirror is employed to keep excitation light from reaching the observer. Its function is to reflect all light below a specific wavelength, while allowing longer waves to pass. Figure 2.2 gives a schematic overview of an optical filter block.

Samples were studied using a Zeiss Axio microscope, an AxioCamMR3 camera, a plan- apochromat 63x oil immersion objective, and images were processed using Zeiss AxioVision software (all Carl Zeiss AG, Oberkochen, Germany). Cells were seeded and treated directly on 0.17 (±0.01) mm thick coverslips (Assistent, Glaswarenfabrik Karl Hecht GmbH & Co KG, Sondheim, Germany) in Nunc 4-well dishes (NUNC A/S, Thermo Fisher Scientific). Prior to examination, cells were washed twice with cold Dulbecco’s PBS with Ca²⁺/Mg²⁺ (Life Tech- nologies, Carlsbad, CA, USA) and coverslips were transferred to a microscopy slide (Menzel- Gläser, Gerhard Menzel GmbH, Braunschweig, Germany). For TPCS2a a 395-440 nm band pass excitation filter, a 470 dichroic mirror and a 610 nm long pass emission filter was used for both photosensitisers. For LysoTracker® Green and Alexa Fluor® 488, a 450-490 nm band

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Figure 2.3– Simplified schematic overview of optical sectioning in a confocal microscope. Excitation light hits a single spot on the specimen. Fluorphores through the whole depth are illuminated and emit light, which travels at different angles towards the detector. A pinhole in placed in front of the detector to prevent out- of-focus light to be detected. Adapted from "Basic Confocal Microscopy" by Smith C. L., 1999, Current Protocols in Cell Biology, 4.5.3. Copyright 1999 by John Wiley & Sons, Inc.

pass excitation filter, a 495 nm dichroic mirror and a 500-550 nm band pass emission filter was used.

2.4.2 Confocal microscopy

In epifluorescence microscopy, cells are illuminated and fluorescence recorded not only from the vertical plane in focus, but the whole depth of the cell. Specimens may therefore appear blurry and out of focus [92]. Confocal microscopes have the ability to collect fluorescence from a single focal plane, dividing cells vertically into optical sections of ≈1 µm. Excitation light is reflected by a dichroic mirror to the specimen through the objective and is focused towards a single spot on the specimen. Since the excitation light passes vertically through the specimen, both fluorophores at the focal point as well as above and below, is excited and emit light. This causes both in-focus and out-of-focus emission to be reflected at different angles towards the detector. To prevent detection of the out-of-focus regions, a pinhole is placed in front of the detector [92]. The main advantages of this design is the achievement of sharper images and the ability to study co-localization within a single focal plane, thereby reducing the chance of artefacts caused by organelles which overlap vertically.

Samples were studied by staff at the Confocal microscopy core facility using a Zeiss LSM 710 confocal microscope and a C-Apochromat 40x water immersion objective (both Carl Zeiss).

A 488 nm laser was used for TPCS_2aand LysoTracker® Green and a 561 nm laser was used for Cy3. Images were processed using ZEN Lite 2011 (Carl Zeiss).

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2.4.3 Co-localization studies

To study TPCS_2a, cells were incubated in 1 µg/ml TPCS_2a in medium for 18 h. Cells were washed twice in drug-free medium and chased for 4-6 h. prior to examination.

Endocytic compartments were stained using LysoTracker® Green (Life Tehcnologies). Cells were incubated for 30-120 minutes in 0.5-5 µM LysoTracker® Green prior to examination.

LysoTracker® probes consist of a fluorophore linked to a weak base which is protonated in the acidic environment of endosomes and lysosomes, while mostly unprotonated at cytosolic pH.

[93]. As only the unprotonated probe has the ability to pass through endosomal membranes, protonation will prevent the molecules from escaping, thereby facilitating accumulation within these compartments.

To study localization of 225.28, biotinylated antibodies were labeled by a streptavidin-Alexa Fluor® 488 conjugate (Life Technologies). A conjugate of 225.28-biotin and Alexa Fluor® 488 was prepared by mixing the two components in PBS and allowed to react for 20 minutes. Cells were incubated with the 225.28-Alexa Fluor® 488 conjugate for 18 h. followed by 4 h. chase, or alternatively for 4 h. without chase prior to examination. A similar procedure was used to study 225.28-biotin bound to streptavidin-Cy3 (Jackson Immunoresearch).

2.5 Flow cytometry

2.5.1 Principle

In a flow cytometer, suspension cells flow in a stream of fluid and is passed through a beam of light. When the light hits a cell, it will be scattered in all directions. Detectors are placed both opposite to the light source (180^◦) and on the side of the flow stream (90^◦). The amount of scatter identified by the 180^◦(forward scatter) detector correlates to the size of the cell, whereas the scatter at 90^◦ (side scatter) can be used to differentiate between the intracellular complexity of the cells [94]. As a large number of cells can be analysed individually, the side and forward scatter can be plotted to give a graphical representation of the different populations of cells present. For example dead and live cells, single cells, doublets of cells or large and small cells.

When fluorophores are introduced on or into cells, they can be excited by the appropriate light and the subsequent emission can be detected for each cell, thereby making it possible to detect different populations of cells based on their fluorescence. The flow cytometer often incorporates a narrow-wavelength laser source and optical filters, as well as dichroic mirrors to control excitation and emission light [95]. As flow cytometry data incorporates observations for a large number of cells, it is suitable as a quantitative method.

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2.5.2 Antigen analysis

CSPG4 and CD271 expression was investigated using flow cytometry. Monolayer cells were harvested using Accutase™ (Sigma-Aldrich), an enzymatic disassociation agent known to be less harmful and leave more intact plasma membrane proteins compared to trypsin, resulting in improved antibody-antigen recognition prior to flow cytometry [96, 97]. Subsequently, cells were suspended to a concentration of 0.5-5 million cells per ml. They were incubated for 1 h. in 1-10µg/ml 225.28-biotin, followed by incubation in 1-10µg/ml Alexa Fluor® 488-streptavidin (Life Technologies) for 30 minutes. To study CD271 expression, 5µl ME20.4-PE pre-labelled antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) was added per 100µl sample and co-incubated with 225.28-biotin. All incubations were performed on ice/refrigerated to prevent receptor internalisation. To remove cell aggregates, cells were filtered into BD Falcon™ tubes (BD Biosciences, Franklin Lakes, NJ, USA) through a 35 µm nylon mesh (BD Biosciences) and immediately analysed by staff at the Flow cytometry core facility on a BD LSR II flow cytometer (BD Biosciences). Dead cells were eliminated based on Hoechst exclusion staining or by side/forward scatter.

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Chapter 3 Results

3.1 Density curves

To determine the optimal plating density in 96 well plates for use with MTT assay, cells were plated at different densities and treated according to the PCI (without drugs or toxins) and MTT protocol. The density should be low enough not to cause cell confluency at the end of the experiment, while maintaining enough cells for detection by the assay. Based on the density curves (fig. 3.1), the optimal density was determined for each cell line (table 2.1).

3.2 CSPG4-targeting in breast cancer

3.2.1 Surface expression of antigen

CSPG4 surface expression was studied using flow cytometry after incubation with 10 µg/ml 225.28-biotin and Alexa Fluor® 488-streptavidin (fig. 3.2). In MCF-7 and MA11, there was little to no observed difference in fluorescence between stained and unstained samples. MDA- MB-435 was strongly stained (mean fluorescence intensity 6905 versus 167 for unstained).

Stained MDA-MB-231 had a four to five-fold higher mean fluorescence intensity and a two- fold increase in fluorescence intensity range compared to the unstained sample (fig. 3.2).

3.2.2 Intracellular localization of TPCS

2a

Using epifluorescence microscopy, a co-localization study (fig. 3.3) was performed to confirm that TPCS2a localized within endocytic vesicles. MDA-MB-231 was incubated in 1 µg/ml TPCS2aover night and chased for 4 h. 0.25-1µM LysoTracker® Green was added 30 minutes prior to examination. A granular red fluorescence was observed from TPCS2a and a granular green fluorescence from LysoTracker® Green. When merged, a yellow signal was observed where the two fluorophores overlapped. Similar results were obtained in MDA-MB-435, MCF- 7 and MA11 (previously published in [98]).

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Figure 3.1– The optical density with the MTT assay was observed at different plating densities for(A) MDA- MB-231, (B) MDA-MB-435, (C) MCF-7 and (D) SUM149. All figures show a representative result from a single experiment, bars represent the empirical standard deviation.

Figure 3.2–MCF-7, MA11, MDA-MB-435 and MDA-MB-231were stained by 225.28-biotin/streptavidin- Alexa Fluor® 488 and analysed by flow cyometry. Red font represents mean fluorescent intensity. (Prelimi- nary data)

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Figure 3.3– Epifluorescence microscopy shows colocalization (yellow) of LysoTracker® Green and TPCS2a

(red) in(A) MDA-MB-231, (B) MA11, (C) MCF-7 and (D) MDA-MB-435.Corresponding DIC micrograph for each cell line is shown for demonstration of viability in monolayers of the cells.

Photochemical internalization (PCI) of immunotoxins directed against putative cancer stem cell markers in triple-negative breast cancer and malignant melanoma

Photochemical internalization (PCI) of immunotoxins directed against putative cancer stem cell markers in triple-negative

breast cancer and malignant melanoma

Marius Strømbo Eng

Master’s thesis at the School of Pharmacy

Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO

May 2012

Photochemical internalization (PCI) of immunotoxins directed against putative cancer stem cell markers in triple-negative breast cancer and malignant melanoma

Author:

Marius Strømbo Eng

Abbreviations

Acknowledgements

Contents

Chapter 1 Introduction

1.1 Background

1.2 Cancer stem cells

1.3 Triple-negative breast cancer

1.4 Malignant melanoma

1.5 Chondroitin sulfate proteoglycan 4

1.6 Cluster of differentiation 271

1.7 Photodynamic therapy and photochemical internalization

1.7.1 Photodynamic therapy (PDT)

1.7.2 Photochemical internalization (PCI)

1.8 Immunotoxins

1.8.1 Development of immunotoxins

1.8.2 Antibodies

1.8.3 Toxic moiety

1.8.4 Internalization

1.8.5 PCI of immunotoxins and ligand-toxin conjugates

Chapter 2

Materials and methods

2.1 Cell culture

2.1.1 Cell lines

2.1.2 Culture medium

2.1.3 Passaging

2.2 PDT and PCI treatment

2.2.1 Photosensitizer and light source

2.2.2 PDT and PCI

2.2.3 Toxins and mAbs

2.3 Viability assays

2.3.1 MTT

2.3.2 Clonogenic survival

2.3.3 Trypan blue exclusion stain

2.3.4 Spheroids

2.4 Fluorescence microscopy

2.4.1 Epifluorescence microscopy

2.4.2 Confocal microscopy

2.4.3 Co-localization studies

2.5 Flow cytometry

2.5.1 Principle

2.5.2 Antigen analysis

Chapter 3 Results

3.1 Density curves

3.2 CSPG4-targeting in breast cancer

3.2.1 Surface expression of antigen

3.2.2 Intracellular localization of TPCS