Photochemical Strategies to Eliminate Cancer Cells

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Judith Jing Wen Wong

Photochemical Strategies to Eliminate Cancer Cells

Thesis submitted for the degree of Philosophiae Doctor

Department of Radiation Biology

Institute for Cancer Research - The Norwegian Radium Hospital, Oslo University Hospital

Section of Pharmaceutics and Social Pharmacy Department of Pharmacy

Faculty of Mathematics and Natural Sciences

2021

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© Judith Jing Wen Wong, 2021

Series of dissertations submitted to the

Faculty of Mathematics and Natural Sciences, University of Oslo No. 2460

ISSN 1501-7710

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Photo cover: Øystein Horgmo-UiO

Print production: Reprosentralen, University of Oslo.

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For mom - my best cheerleader

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Preface

This thesis is submitted in partial fulfillment of the requirements for the degree ofPhilosophiae Doctor at the University of Oslo and presents the results of the work conducted under the supervision of Dr. Pål Kristian Selbo and Professor Kristian Berg at Berg’s research group at the Institute for Cancer Research - The Norwegian Radium Hospital, Oslo University Hospital. The financial support by the South-Eastern Norway Regional Health Authority (Helse Sør-Øst), and the Norwegian Radium Hospital Research Foundation (RadForsk) is gratefully acknowledged.

The thesis is a collection of four papers, where the common theme to them is the use of the clinical relevant photosensitizer TPCS2a which is specifically developed for the intracellular drug delivery technology photochemical internalization (PCI) for use in cancer therapy. The papers are preceded by an introductory chapter that relates them to each other and provides background information and motivation for the work.

Acknowledgements

I want to thank all the people who made it possible to fulfill this thesis. I feel incredibly privileged to have worked with such exciting technology together with the foremost experts in the PCI/PDT field. First and foremost, I wish to express my deepest gratitude to my principal supervisor Dr. Pål Kristian Selbo, for allowing me to embark on this research journey and believing in me. Your patient guidance, positive attitude, enthusiasm, and insightful feedback have made this possible. Thank you for sharing all your knowledge and ideas with me.

My appreciation also goes to my co-supervisor, Professor Kristian Berg, whose unparalleled knowledge has been essential in this project. Special thanks to Dr. Anette Weyergang, my supervisor during my master’s project, who first introduced me to the PCI/PDT field. Her enthusiasm and pragmatic approach to research is inspiring and has shaped me as a researcher. Thank you all for always keeping your office door open for questions and discussions.

I want to thank all the co-authors for their excellent contributions to this thesis’s papers: Maria Brandal Berstad, Ane Sofie Fremstedal, Sebastian Patzke, Vigdis Sørensen, Qian Peng, and Susanne Lorenz.

Furthermore, I also wish to thank former and current colleagues at the PCI

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Preface

research group and the Department of Radiation Biology for an inspiring and fun working environment. I want to express my gratitude to the lab oracles Ane S. and Ane L. for sharing their best tips and tricks in the lab. A special thanks to the PCI members Monika, Victoria, Anne Grete, and Mary for good times in and outside the lab.

Last but not least, I am grateful to my family for their continuous support and encouragement throughout these years. Thank you for always being proud of me. I would never have come this far without you. My final thanks go to my partner Vu. You have been solid as a rock and wrap me in your hilarious chillness when I’m stressed. For that, I will always be grateful.

Judith Jing Wen Wong Oslo, August 2021

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List of Papers

Paper I

Wong J.J.W., Berstad M.B., Fremstedal A.S.V., Berg K., Patzke S., Sørensen V., Peng Q., Selbo P.K., and Weyergang A. “Photochemically-induced release of lysosomal sequestered sunitinib: obstacles for therapeutic efficacy”. In: Cancers.

Vol. 12, Issue 2 (2020), Article no. 417. DOI: 10.3390/cancers12020417.

Paper II

Wong J.J.W. and Selbo P.K. “High aldehyde dehydrogenase activity does not protect colon cancer cells against TPCS_2a-sensitized photokilling”. In Photochemical and Photobiological Sciences. Vol. 19, Issue 3 (2020), P.p. 308-312.

DOI: 10.1039/C9PP00453J.

Paper III

Wong J.J.W., Lorenz S. and Selbo P.K. “ATRA improves the anti-tumor effect of photodynamic therapy”. Manuscript 2021.

Paper IV

Wong J.J.W.and Selbo P.K. “Light-controlled elimination of PD-L1+ cells”.

Preprint inbioRxiv (2021). DOI: 10.1101/2021.08.13.456199.

Co-authored work not included in the thesis Paper V

Lund K., Olsen C.E., Wong J.J.W., Olsen P.A., Solberg N.T., Høgset A., Krauss S., and Selbo P.K. “5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin”. In: Journal of Experimental & Clinical Cancer Research, Vol. 36 (2017), Article no. 187. DOI: 10.1186/s13046-017-0662-6.

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List of abbreviations

ALDH Aldehyde dehydrogenase

AlPcS2a Aluminium phthalocyanine disulfonate AML Acute myeloid leukemia

ATF3 Activating transcription factor 3 ATRA All-trans retinoic acid

APL Acute promyelocytic leukemia

CSC Cancer stem cell

CTLA-4 Cytotoxic T-lymphocyte associated protein-4 DAMP Damage-associated molecular patterns

DC Dendritic cell

DEG Differentially expressed gene DMSO Dimethyl sulfoxide

EGR1 Early growth factor 1

EC Endothelial cell

EMA European Medicine Agency

EPR Enhanced permeability and retention FACS Fluorescence-activated cell sorting FDA Food and Drug Administration GSEA Gene-set enrichment analysis

HSP Heat shock protein

IC Internal conversion

ICI Immune checkpoint inhibitor ICT Immune checkpoint treatment IGFBP6 Insuline-like growth factor 6

IF Immunofluorescence

IT Immunotoxin

IPA Ingenuity Pathway Analysis ISC Intersystem crossing

LCN2 Lipocalin 2

LDL Low-densitiy lipoprotein MDR Multi-drug resistance

MHC Major histocompatibility complex

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List of abbreviations

MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide REG4 Regenerating islet-derived protein 4

rGel Recombinant gelonin

RA Retinoic acid

RAR Retinoic acid receptor RCC Renal cell carcinoma

RIP Ribosome-inactivating protein RNA-seq RNA sequencing

ROS Reactive oxygen species

RT Radiotherapy

RT-qPCR Quantitative reverse transcription polymerase chain reaction PD-1 Programmed death protein 1

PD-L1 Programmed death ligand 1

PDT Photodynamic therapy

PCI Photohemical internalization

PpIX Protoporhyrin IX

PS Photosensitizer

scFv Single-chain variable fragment

TME Tumor microenvironment

TKI Tyrosine kinase inhibitor

TUNEL Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling TPPS2a Disulfonated tetraphenyl porphine

TPCS2a Disulfonated tetraphenyl chlorin VEGF Vascular endothelial growth factor

VEGFR Vascular endothelial growth factor receptor

1O2 Singlet oxygen

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Chapter 1 Aims of the project

Drug resistance is a major obstacle in cancer therapy. There is, therefore, a high need for strategies that bypass therapy resistance and improve cancer patient survival. The overall aim of this thesis is the use of PCI-based strategies to overcome mechanisms of drug resistance in cancer therapy. Following are the sub-aims of the thesis (broken down as paper I-IV):

• Paper I: To in vitro and in vivo investigate the use of sunitinib in combination with PCI, and its significance in sunitinib-resistant cancer cells.

• Paper II: To in vitro study the impact of the cancer stem cell (CSC) marker aldehyde dehydrogenase (ALDH) on photodynamic therapy (PDT) using the PCI-photosensitizer TPCS2a.

• Paper III: To in vitro and in vivo investigate the efficacy of pre- conditioning cells to all-trans retinoic acid (ATRA) followed by TPCS2a- PDT in carcinomas.

• Paper IV:Toin vitroassess PCI of anti-PD-L1-saporin, an immunotoxin (IT) intended for targeting and elimination of immunosuppressive stromal

and tumor cells in the tumor microenvironment.

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Chapter 2 Introduction

2.1 Cancer

A healthy human body is made up of trillions of cells that, over a lifetime, grow and divide as needed. We pay a price for having cells that can renew and repair themselves. The intracellular control mechanisms in each cell are delicately adjusted, and if this goes astray, it can lead to a catastrophic disruption of the body’s structure, such as cancer. Cancers arise from violations of the basic rules of cell behavior. During the last decades, our understanding of cancer biology has increased dramatically. Despite this, cancer continues to be the leading cause of death worldwide [1]. Moreover, based on estimates by the International Agency for Research on Cancer, the global cancer burden is expected to be 28.4 million new cancer cases in 2040, a 47 % increase (19.3 million cases) from 2020 [1].

Cancer is a heterogeneous disease and refers to all types of malignancies that can reside in almost every body part. Broadly, cancer can be divided into two main categories; hematologic and solid tumors, of which the latter is the focus of this thesis. Two heritable properties define cancer cells; firstly, they and their progeny proliferate in defiance of the normal constraints. Secondly, they invade and colonize tissues usually reserved for other cells. It is the combination of these traits that makes cancer lethal.

Although the term cancer refers to a broad range of malignancies, some unique traits exist, as reviewed by Hanahan and Weinberg [2], which include;

sustained proliferative signaling, resisting cell death, enabling replicative immor- tality, inducing angiogenesis, activating invasion and metastasis, deregulated metabolism, and the ability to avoid immune destruction. These hallmarks are acquired during the course of multistep tumorigenesis, of which genomic instability is considered as an enabling characteristic, and inherent to the majority of human cancer cells [2]. Understanding the hallmarks of cancer has provided insight into therapeutic targets, which has aided in the development of targeted therapeutics such as tyrosine kinase inhibitors and checkpoint inhibitors. Currently, surgery, radiation therapy, immunotherapy, chemotherapy, and radiotherapy (RT) are the cornerstones in cancer treatment.

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2. Introduction

2.1.1 Cancer stem cells (CSCs)

Despite great progress in cancer drug development, in many cases, drug resistance occurs, giving rise to inevitable relapses in patients [3]. In recent years, increasing evidence has indicated that a small population of cancer cells across many solid tumors have stem-like properties, can give rise to heterogeneity in a tumor, and are intrinsically resistant to conventional cancer therapies. These cells are called cancer stem cells (CSCs) (also known as tumor-initiating cells) due to their self- renewal and differentiation capacity and tumorigenic properties [4]. Due to this unique biology which mimics normal stem cells, CSCs are suggested to drive and maintain tumor development [5–7]. While a universal CSC marker does not exist, markers such as in particular CD133, CD44, epithelial cell adhesion molecule (EpCAM), and aldehyde dehydrogenase (ALDH), and the side-population assay (Hoechst 33342 exclusion) have been commonly used to isolate CSCs [4, 8].

Although these markers have been associated with CSCs, isolated cells has to be validated in vitro and in vivo using limiting-dilution assays, of which in vivo serial-transplantation is considered the gold standard [4, 8]. CSC has been suggested to be responsible for the tumor recurrence after therapy as they are intrinsically resistant to conventional therapy. There is, therefore, a need to develop new cancer treatment strategies that improve tumor response and disease control.

In this work, the impact of the CSC marker ALDH on disulfonated tetraphenyl chlorin (TPCS2a)-based photodynamic therapy (PDT) was studied (paper II).

Furthermore, the anti-cancer efficacy of the photosensitizer (PS) TPCS2a was evaluated in combination with approved (sunitinib and all-trans retinoic acid) or experimental (anti-PD-L1-saporin) therapeutics (paper I, III, and IV). The anti-cancer therapeutics relevant to this work is described in more detail below.

2.1.2 Sunitinib - a tyrosine kinase inhibitor (TKI)

One of the hallmarks of cancer is angiogenesis, which is the formation of new blood vessels from preexisting ones [2]. Both pro- and anti-angiogenic factors tightly regulate this process. In tumors, this balance is shifted toward pro- angiogenic factors to sustain angiogenesis. The newly formed tumor vasculature supplies oxygen, nutrients, and growth factors necessary to support the rapid tumor growth. The observations between cancer and angiogenesis were first described in the late 1960s [9, 10]. Later, it was observed that the solid tumor growth relies on this process, and it was proposed by Folkman that inhibiting angiogenesis is a feasible anti-tumor strategy [11, 12]. The discovery of several angiogenic factors made inhibiting angiogenesis possible [13].

Several angiogenic factors are important, of which the vascular endothelial growth (VEGF) plays a crucial role in angiogenesis. The discovery of VEGF-A led to the development and approval of bevacizumab (Avastin), a humanized anti-VEGF-A monoclonal antibody, as part of combination therapy for the treatment of metastatic colorectal cancers [13]. Bevacizumab binds to VEGF-A 4

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Cancer

and prevents it from binding to vascular endothelial growth factor receptor 2 (VEGFR-2), which inhibits the signaling pathways involved in the endothelial cells (ECs) proliferation and thereby tumor angiogenesis [13]. Since the approval of bevacizumab in 2004, other strategies aimed to inhibit angiogenesis has been developed, such as small-molecule tyrosine kinase inhibitors (TKIs).

Unlike antibodies, which targets are restricted to the cell surface, TKIs are designed to be small and lipophilic enough to cross the plasma membrane passively. Intracellularly, TKIs inhibit corresponding kinases from phosphorylating tyrosine residues of their substrates resulting in a subsequent block of activation of downstream signaling pathways. To date, more than 50 small TKIs are approved by the US Food and Drug Administration (FDA) [14]. VEGFR is the primary target for many of these TKIs; however, there are also so-called multi-targeted TKIs designed to target several kinases such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and VEGFR. Blocking multiple signaling pathways is thought to be more effective than blocking a single pathway [15].

Figure 2.1: Chemical structure of sunitinib, logP = 5.2, pKa = 8.95. Adapted from Drugbank Online [16] and created with Chemdraw.

Sunitinib (Sutent, Pfizer) was among the first approved multi-targeted kinase inhibitors and is formulated as oral capsules [16, 17]. Sunitinib has affinity to VEGFR-1 and -2), PDGFR-alpha and -beta), stem cell factor receptor (KIT), FMS-like tyrosine kinase-3 (FLT3), glial cell-line derived neurotrophic factor receptor (RET), and the receptor of macrophage-colony stimulating factor (CSF1R) [16, 17]. Several kinases involved in angiogenesis are inhibited by sunitinib; however, this drug also has apoptotic and antiproliferative effects on tumor cells [18]. Sunitinib was first approved for gastrointestinal stromal tumors (GISTs) and advanced renal cell carcinoma (RCC). Later, sunitinib has also been approved for the treatment of unresectable or metastatic pancreatic neuroendocrine tumors. In addition, more recently, sunitinib is also indicated as adjuvant treatment in patients at high risk of recurrent RCC [17].

The physicochemical properties of sunitinib (figure 2.1.2) make it increas- ingly protonated at decreasing pH [16]. Due to the lipophilic nature and size of sunitinib, it can passively diffuse through the plasma membrane and the intra-

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2. Introduction

Nucleus Cytosol pH ~ 7.3

Lysosome pH ~ 5

Hydrophobic weak-base drug

Low drug concentration Low drug concentration

-H+

-H+ -H+

High drug concentration

Figure 2.2: Passive lysosomal sequestration of small, hydrophobic weak-base drugs impacts their intracellular distribution and reduces its target interaction. Hydrophobic weak-base drugs passively enter the cell and the lysosomes. Protonation of, e.g., sunitinib occurs in the acidic lysosomes and leads to accumulation in these organelles, impacting its intracellular drug distribution. Figure adapted from Zhitomirsky and Assaraf [19] and created with Biorender.

cellular vesicle membrane at physiological pH. However, the relatively low pH in the endosomes and lysosomes, compared to the cytosol, results in protonated sunitinib and sequestration. Lysosomal sequestration affects the accessibility of sunitinib to the target, thus reduce the cytotoxic effect. Lysosomal sequestration is known to passively trap lipophilic weak-base drugs and can mediate drug resistance [19], which has been reported as a drug resistance mechanism for sunitinib [20, 21]. Thus, sunitinib is a proper candidate for endosomal escape methods and was therefore evaluated in combination with the intracellular drug delivery technology PCI in paper I.

2.1.3 PD-L1 - a target for immunotherapy

Immune checkpoint inhibitors (ICIs) block the immune checkpoints, regulators of the immune system, cytotoxic T-lymphocyte associated protein-4 (CTLA-4), programmed death-1 (PD-1), or programmed death-ligand 1 (PD-L1) using monoclonal antibodies. ICIs represent a group of cancer immunotherapy to treat malignancies by leveraging the cytotoxic potential of the immune system [22, 23].

Tumor cells employ multiple defense strategies to evade detection by the immune system and down-regulate T cell responses. Up-regulation of immune checkpoint proteins and ligands is a common strategy, which takes advantage of the immune mechanism for self-tolerance and prevention of collateral tissue 6

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Cancer

MHC

PD-L1 TCR

Immune attack

PD-1

Cancer cell

T cell inhibitory signals blocked

Tumor cell Activated T cell

Figure 2.3: Blockade of PD-1/PD-L1 in tumor immunotherapy. Monoclonal antibodies against PD-L1 or PD-1 block the PD-1/PD-L1 interaction and facilitates T cell activation. Adapted from Ribas [23] and created with BioRender.com

damage. PD-L1 is commonly up-regulated on the surface of tumor cells in the tumor microenvironment (TME). However, recent discoveries suggest that the PD-L1 expression on malignant cells is not a prerequisite for ICT. Kleinovinket al. demonstrated that PD-L1 expression is also found on myeloid-infiltrating cells in the TME. Both tumor cells and immune cells are therefore involved in the mechanism of PD-L1 blockade [24].

In 2011, ipilimumab became the first FDA-approved ICI targeting CTLA- 4. A decade later, six other ICIs have been approved, including targeting the PD-1/PD-L1 axis with indications across 19 different types of cancer [25].

Although immune checkpoint therapy (ICT) provides clinical responses and can improve the overall survival in patients, only a subset of patients benefit from such treatment [26]. Thus, significant challenges remain, including identifying ra- tional therapeutic combinations. As PD-L1 has been shown to be overexpressed on CSCs [27–29] and to be taken up by endocytosis [30], cytotoxic targeting by PCI of an immunotoxin against PD-L1 was investigated in paper IV.

2.1.4 Retinoids in cancer therapy

The therapeutic potential of retinoids is based on their key role in the regulation of differentiation, apoptosis, and growth (figure 2.4). ATRA, which was used in this thesis, is the major active metabolite of vitamin A. ATRA plays an essential role in many biological processes, including embryonic development,

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2. Introduction

maintenance of the immune system, and vision. These biological effects are a result of ligand-inducible transcription factors. Nuclear retinoic acid receptors (RARs) are ligand-inducible transcription factors that function as heterodimers with retinoid X receptors (RXRs) that bind to the regulatory regions of different target genes and control their transcription. ATRA is a pan-RAR agonist that binds and activates RARα, RARβ and RARγ[31]. Transcriptional activation by RARs regulates apoptosis, induces differentiation, and cell cycle arrest [32], which are traits that make ATRA and other retinoids attractive in cancer therapy.

However, ATRA can also bind and activate peroxisome proliferator–activated receptor β/δ (PPARβ/δ) which induces pro-survival genes as opposed to RAR activated growth inhibition [33].

Currently, ATRA (also known as tretinoin) is successfully used in combination with arsenic trioxide (ATO) in the induction therapy of acute promyelocytic leukemia (APL), a specific subtype of acute myeloid leukemia (AML). ATRA acts as a differentiating agent and drives the maturation of primitive promyelocytes derived from leukemic clones, in part because of ATRA’s ability to induce differentiation, and arrest proliferation [31, 34]. This successful drug combination (ATRA+ATO) has improved the outcome in APL patients - from highly fatal to being the most curable form of AML in adults [31, 34].

Interestingly, pharmacological doses of 13-cis-RA (isotretinoin) can induce differentiation for some stages of human neuroblastoma and is currently used as part of the treatment regimen in high-risk neuroblastoma patients [35]. Retinoids are one of the few drugs known to drive differentiation and are therefore also relevant in regards to CSCs [36]. Due to the clinical success of using ATRA in APL patients, several clinical studies have attempted to use ATRA to treat solid tumors. However, no breakthrough has been achieved so far. Several factors contribute to the limited clinical translation and lack of potency. Retinoids, including ATRA, have low aqueous solubility andex vivothey are chemically unstable and readily oxidized when exposed to heat, UV light, or oxygen. The physicochemical properties makes RA formulation challenging. Currently, ATRA is formulated as oral capsules [37]. In humans, ATRA has a very short half-life which is estimated to∼45 minutes [38]. Further complicating matters, ATRA induces its metabolism, resulting in decreased plasma concentration during continuous dosing [38]. Moreover, as ATRA is involved in many biological effects, using ATRA pharmacologically leads to off-target effects. Retinoids are associated with adverse effects such as toxicity, including liver and lipid alterations, and bone and connective tissue damage, which substantially limits the long-term systemic administration [39].

In addition to the physicochemical properties and pharmacokinetics, the lack of ATRA potency in solid tumors is also partly due to dysregulated retinoid metabolism. In APL patients, it is estimated that at least 98 % of these patients carry the t(15;17)(q22;q21) chromosomal translocation resulting in the PML-RARαfusion protein, which has been identified as the main driver 8

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Cancer

Co-activator complex

CRABP ATRA

Retinoid uptake 1

Retinal ALDH 2

Nucleus Cytosol

RAR RXR

3 Activation of transcription

Differentiation Apoptosis Growth inhibition

4

Figure 2.4: Simplified overview of the classical retinoic acid signaling pathway. (1) Various metabolic forms of retinoids are taken up from the blood circulation. (2) In a series of enzymatic steps, both reversible or irreversible, converts metabolic forms of retinoids to retinal. Retinal is then irreversibly metabolized by aldehyde dehydrogenase (ALDH) to retinoic acid, including ATRA. (3) ATRA is transported to the nucleus by the cellular retinoid binding-protein CRABP and binds to the RAR-RXR dimer to induce the expression of target genes. (4) The effect of ATRA is related to differentiation, apoptosis, and growth inhibition. Adapted from Connollyet al. [36] and Chlapeket al.

[40]. Figure created with BioRender.com

of APL [39, 41]. The PML-RARαfusion protein has increased affinity to the co-repressors and does not respond to physiological ATRA levels. This represses target genes and thus prevents differentiation of promyelocytes. Pharmacological levels of ATRA are therefore necessary to drive terminal differentiation of promyelocytes [41]. However, in solid tumors, multiple oncogene pathways are involved and are considerably more complex than in leukemia, e.g., APL. There is evidence suggesting that solid tumors develop intrinsic resistance to retinoids during carcinogenesis and have aberrant retinoid signaling [40, 42]. For instance, in solid tumors, RARβ expression is frequently lost, or the RAR promoter is epigenetically silenced, resulting in ATRA-resistant cancer cells [36, 40, 42].

Thus, successful cancer therapy with ATRA in solid tumors will likely require improved drug delivery system strategies and combination therapy. ATRA in combination with TPCS2a-based PDT was therefore investigated in paper III.

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2. Introduction

2.2 Photodynamic therapy (PDT)

2.2.1 PDT in oncology

PDT is a well-established treatment modality used clinically for the treatment of several neoplasms, including non-melanoma skin cancer, esophageal cancer, non-small-cell lung cancer (NSCLC), bladder cancer, cervical cancer, head and neck cancer, breast cancer, pancreatic cancer or prostate cancer [43, 44].

Moreover, PDT has also been used in different types of non-malignant diseases in dermatology (e.g. actinic keratosis) and ophthalmology (e.g. age-related macular degeneration, AMD) [45, 46]. Three components are essential to achieve the PDT effect: a photosensitizer (PS), light at appropriate wavelength and dose, and molecular oxygen. The PS is administered, either systemically or topically, prior to illumination of the region of interest. Various approaches can be employed to deliver light to the site of interest for PDT; superficial, intracavitary, endoscopic, or interstitial by inserting one or more laser fibers via needles/catheter into the malignancy [47, 48]. The efficacy of PDT derives from a light-induced activation of a photosensitizer which converts molecular oxygen, via energy transfer, into reactive oxygen species (ROS), of which singlet oxygen (¹O2) is the most common. PDT-induced ROS interact with biomolecules in the surroundings and inflict damage on vital organelles of the cell resulting in cell death [45].

2.2.2 Intracellular PDT targets

Specifically, unsaturated fatty acids, cholesterol, amino acids (tryptophan, histidine, cysteine, tyrosine and methionine), and guanine are susceptible to oxidation by ¹O2 [49–52]. However, the extent of cellular damage and the outcome is dependent on several factors, such as the uptake, the subcellular localization of the PS, and the amount of ROS generated. The lifetime ¹O2

and the diffusion length is estimated as < 0.05 µs and < 0.02µm, respectively [53]. Thus, only biomolecules near the PS are oxidized by ¹O₂. Depending on the molecular structure of the PS, PS can target cellular compartments such as mitochondria, lysosomes, endoplasmic reticulum (ER), Golgi, or plasma membrane [52]. As very few PS accumulates in the nucleus, the mutagenic potential of PDT is therefore low [54].

2.2.3 The selectivity of PDT

PDT offers several advantages over conventional cancer therapies such as surgery, chemotherapy, and radiotherapy; PDT is minimal-invasive, has low systemic toxicity, and is site-specific. PDT can also be repeated as many times as necessary and does not have accumulated toxicity as opposed to conventional chemotherapy. Moreover, the PS remains pharmacological in- active until light exposure in the presence of oxygen. As the PDT effect is dependent on light, the penetration depth of the light is an important limiting factor. The light penetration is limited by optical scattering within the tumor 10

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Photodynamic therapy (PDT)

and by endogenous chromophores, including melanin, hemoglobin, and water [45].

Most of the PSs passively accumulate in the tumor compared to the normal tissue. This tumor selectivity leads to restricted damage in the tumor and the surrounding tissue upon light activation, while the effect on normal tissue is minimal.The selective tumor accumulation or retention of PSs is not entirely understood; however, several tumor properties possibly contribute to this selectiveness. One possible explanation behind the preferential accumulation or retention of PS is the enhanced permeability and retention (EPR) effect [55].

Most PSs are lipophilic and bind to serum proteins, e.g., albumin or low-density lipoprotein (LDL). As PS binds to serum proteins, it behaves as a macromolecule when injected into the bloodstream. It can pass through the large endothelial fenestrations in the tumor endothelial vasculature and accumulate in the cancer tissues due to poor lymphatic drainage [55].

Another possible explanation is the binding of PS to LDL. It is proposed that tumor tissues display elevated LDL receptor expression on their plasma membranes compared to normal tissues [55]. The LDLs are efficient carriers of lipophilic and amphiphilic PSs, forming an LDL-PS complex that binds to the LDL receptors on the tumor cell surface and is subsequently taken up, resulting in cancer-specific accumulation of the PSs [56–58]. It has also been proposed that tumor-associated macrophages efficiently take up PS and contribute to the PS retention at the tumor site [59].

2.2.4 Photochemical reactions

As highlighted above, an essential component of PDT is the PS, a molecule that absorbs photons and transfers this energy to another molecule. Upon light exposure of appropriate wavelength, the PS is excited to an unstable and transient singlet state. An excited molecule cannot persist in this state indefinitely, and thus several competing physical processes can occur, leading to energy dissipation and deactivating the excited states [45, 60, 61]. The energy dissipation from the excited singlet state may be via internal conversion (IC), a nonradiative transition, or photon emission (fluorescence), resulting in a return to the ground state [45, 60, 61]. As many PS preferentially accumulate in tumors or neoplastic tissues, photodynamic diagnosis takes advantage of the fluorescent properties from PSs to detect tumor tissue(s) [62]. In addition, it is also used in fluorescence-guided surgery [63].

Alternatively, intersystem crossing (ISC) may occur from the excited singlet state to a metastable triplet state. This state is favored as it is of lower energy than the singlet state and is relatively long-lived (microseconds) compared to the singlet state (nanoseconds), allowing sufficient time for the PS to interact with other molecules. As for the singlet state, energy dissociation may also occur here via IC or photon emission (phosphorescence), leading to the excited triplet state’s deactivation. The excited PS may at triplet states undergo type I or

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2. Introduction

type II photochemical reactions [45, 60, 61]. Type I reaction involves electron transfer or hydrogen abstraction between the excited PS and a substrate, which leads to the formation of free radicals (e.g., the superoxide anion radical (O^−.₂ )) that further react with oxygen resulting in the generation of other ROS.

Energy

S 0

S 1

Absorption Fluorescence

Internal conversion

ISC

Type I Electron transfer T 1

Phosphorescence

Internal conversion

Type II Energy transfer

3 O 2

ROS Oxidation reactions PS*

PS*

PS

1 O 2

Figure 2.5: Simplified Jablonski diagram illustrating the activation of a photosensitizer (PS) leading photochemical reactions and thereby oxidation reactions. After activation by light absorption, the PS is excited (PS^∗) from its ground state (S0) to the excited singlet state (S1). The PS^∗ can return to S0 by emitting fluorescence or internal conversion. Alternatively, the (PS^∗) can, by intersystem crossing (ISC), convert to the excited triplet state (T1). From here, the (PS^∗) can return to S0 by emitting phosphorescence, internal conversion, or react through type I or II reactions which subsequently lead to oxidation reactions that inflict damage on biomolecules. Figure adapted from Abrahamse and Hamblin [60], and created with BioRender.com In a type II reaction, the excited PS transfers its energy to molecular oxygen which generates ¹O2 and ground state PS [60]. With porphyrin-based PSs such as TPCS2a/fimaporfin, and other structurally related compounds, type II reactions are considered the most important. If the PS is not photobleached, the ground state PS can be regenerated. Thus, further cycles of excitation and generation of ¹O2 can take place if light and a sufficient amount of molecular oxygen are present [64].

Both type I and type II reactions can co-occur; however, the ratio between these reactions is influenced by several factors, including tumor oxygen tension and substrate concentrations, type of PS, and the binding affinity between the PS and the substrate [65]. The ROS generated from these reactions can induce cell toxicity by inflicting damage on biomolecules such as proteins, unsaturated lipids, and nucleic acids.

2.2.5 The mechanism of action of PDT

Three distinct biological mechanisms contribute to the PDT-induced anti-tumor efficacy; direct tumor cell death, vascular shutdown, and induction of anti- tumor immune response. The relative contribution of these mechanisms and the extent of tumor damage is dependent on several factors such as type of PS, its extracellular and intracellular localization, PS dose, the total light dose, the 12

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fluence rate, and oxygen tension in the tumor tissue. However, a combination of all three mechanisms is necessary to achieve long-term tumor control [45].

Vascular shutdown Tumor cell death Light

Inflammation ROS

1 O 2

Dendritic cells Neutrophil

Figure 2.6: PDT’s mechanism of action on a tumor. Three distinct mechanisms can occur as a result from the¹O2 and ROS generated by light-activated PS; vascular shutdown, tumor cell death and induction of the immune system. Adapted from Castanoet al. [66] and created with BioRender.com

Direct cytotoxic effects

Although primary ROS are short-lived, PDT also induces prolonged oxidative stress in PDT-treated cells. The post-PDT oxidative stress stems from peroxidized reaction products such as lipids and proteins that have a longer lifetime and can have detrimental effects on the cell. In case of excessive damage or stress, cell death occurs. Depending on which intracellular substrates are most affected by ROS and where the PS is localized, cell death is mediated via necrosis, apoptosis, or autophagy [67, 68].

Apoptosis is characterized by cytoplasmic shrinkage, chromatin condensation, nuclear fragmentation, and plasma membrane blebbing [69]. Mitochondria- localizing PS are rapid inducers of apoptosis due to mitochondrial damage, resulting in cytochrome c release [68, 70]. Moreover, both ER- and mitochondrial damage results in destruction of anti-apoptotic proteins, Bcl2 and Bcl-xL, without damaging the pro-apoptotic protein Bax [71–73]. The death receptors can also induce apoptosis on the plasma membrane if the PS is localized there.

Damage to the plasma membrane can result in multimerization of death receptors such as Fas. Fas signaling results in cleavage of pro-caspase 8, thus triggering the apoptotic program [68].

Autophagy is a process that involves the uptake of cytoplasmic material, of endogenous or exogenous origin, into a double membrane vesicle called autophagosomes which fuse with lysosomes. Once fused with lysosomes, the content is subsequently degraded and recycled [74]. Autophagy plays a vital role in promoting survival following stress, such as nutrient limitation by recycling

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2. Introduction

essential cellular components. In the context of PDT, autophagy is a common response post-PDT [75]. Autophagy can mediate cell death [68, 76] or play a cytoprotective role [45, 77] depending on the type of PS, cell type, and PDT dose. Low-dose PDT and photodamage to the ER and/or mitochondria have been associated with autophagy as a pro-survival mechanism, which is thought to be initiated in an attempt to recycle injured organelle. Interestingly, lysosomal/endosomal-localizing PSs are suggested to impair the autophagic process post-PDT due to the essential role of lysosomes in autophagy [45, 77].

Although autophagy is described as a pro-survival or pro-death mechanism post-PDT, autophagy can also promote resistance to PpIX-mediated PDT as demonstrated by Wei et al. in CD133⁺ CSCs [78]. Interestingly, it has also been demonstrated that photochemical damage of lysosomes (after PDT using the PCI PS TPC2a/fimaporfin) and subsequent inhibition of autophagic flux results in enhanced cytotoxic effects of 5-FU in 5-FU-resistant pancreatic cell lines [79].

The third major death mode associated with PDT is necrosis. Necrosis has no distinctive morphotypes as apoptosis and autophagy [69]. Necrotic cells swell and disrupt the plasma membrane, which results in the release of intracellular components. This cell death occurs as a result of severe stress, e.g., in strongly photosensitized cells [67].

Vascular damage

Tumor cells are dependent on oxygen and nutrients supplied by the blood vessels to stay viable and proliferate. Accumulation of the PS in vasculature and light activation results in vascular damage. Particularly endothelial cells and the vascular basement membrane are considered sensitive sites [67]. A general hypothesis of the vascular effects of PDT is the initiation of thrombogenesis in the vessel lumen. Thus occlusion and vascular shutdown, which deprives the tumor cells of both oxygen and nutrients [67]. Based on this mechanism, the European Medicines Agency (EMA) recently approved the PS Tookad (padeliporfin) for the treatment of low-risk prostate cancer. Padeliporfin is retained in the vasculature of the prostate, which is illuminated immediately after intravenous injection of the PS [80, 81]. Interestingly, photochemical-induced vasculature damage by PDT led to approval of the PS Visudyne (verteporfin) for treatment of age- related macular degeneration. This condition causes vision loss due to abnormal blood vessel growth [43].

Induction of immune responses

PDT can induce inflammatory responses and tumor-specific adaptive immune reactions [68]. Anti-tumor immunity response can be triggered by a series of events post-light exposure, where an acute inflammatory response is considered to be an important step [45, 82]. PDT-induced tumor tissue damage leads to rapid upregulation of cytokines (e.g., IL-6) and an immediate influx of neutrophils and 14

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later followed by other immune cells to the targeted site [68]. Several reports have documented local and systemic neutrophilia upon PDT, which is suggested as necessary for an effective anti-tumor response and enhanced immunity [76, 82–

85]. Necrotic cell death leads to exposure or release of intracellular components known as damage-associated molecular patterns (DAMPs) [68, 76]. Dendritic cells (DCs) are professional antigen-presenting cells that recognize DAMPs and activate the adaptive immune system by presenting antigens on their surface through major histocompatibility complexes (MHCs) to T cells [68, 76]. Mature DCs interact with T-cells through binding between MHC-complexes I or II, of which CD8⁺T cell activation through MHC I plays an essential role in facilitating PDT-mediated anti-tumor immunity and contribute to the long-term tumor control [66, 86].

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2. Introduction

2.3 Photochemical internalization (PCI)

Photochemical internalization (PCI) is light-facilitated intracellular drug delivery technology based on photodynamic therapy (PDT). The principle shares many of the fundamental characteristics of PDT. However, the main aim of using PCI is to release drugs from the endosomes or lysosomes into the cytosol. Several drugs are entrapped in these compartments due to their physicochemical properties and/or size. PCI may be used to potentiate the effect of drugs that are limited by endosomal and lysosomal entrapment. For most therapeutic agents, the primary goal is not only to reach inside the cells but also to interact with specific cytoplasmic compartments or nuclear targets. Most small-molecule drugs can enter the cytoplasm directly by diffusion. However, for macromolecules and particulate drug carriers, the uptake process is complex. These molecules can enter the cells by endocytosis and are then entrapped in endosomes or lysosomes.

To achieve cytoplasmic delivery, the entrapped drug needs to be relatively stable in the endosomes/lysosomes and be able to escape [87]. Thus, PCI has the potential to reduce or eliminate the adverse events of many chemotherapeutics by lowering the doses without reducing the efficacy.

2.3.1 The therapeutic potential of PCI

PCI utilizes an amphiphilic photosensitizer, particularly, the clinical relevant PS fimaporfin/TPCS_2a, that accumulates in the membrane of endocytic vesicles by adsorptive endocytosis. The excess photosensitizer on the plasma membrane will over time dissociate which leads to localization on the membrane of endosomes and lysosomes. Upon light exposure, the photosensitizer is activated and subsequently rupture the endocytic vesicles caused by ROS formation, and release its content into the cytosol. The internalized drug can then interact with its target in the cytosol. As described in the PDT section, in addition to¹O2formation which in PCI permeabilizes the endocytic membranes, lipid peroxidation chain reactions appear to be important [51, 88]. Previous studies have indicated that lipid peroxidation chain reactions are important for the rupture and relocalization of the vesicle content This may take place for more than 20 minutes post-light.Without light exposure, the content in these vesicles are degraded by lysosomal enzymes.

PCI was initially developed as a drug delivery method for macromolecules in cancer cells, which cannot cross the plasma membrane and is taken up by endocytosis. Since the first PCI publication in 1999 by Berg et al. [89], the PCI technology has been evaluated in a wide range of macromolecules and small molecules in several models. This includes peptides/proteins (ribosome- inactivating proteins (RIPs) type I, RIP type I-based immunotoxins (IT), vaccine antigens), nucleic acids (RNA and DNA), and small molecule-drugs (doxorubicin, sunitinib, and gemcitabine) [44, 90, 91]. The use of PCI is not limited to drug delivery to cancer cells, but has also been demonstrated to enhance the MHC I-presentation of peptide-based vaccine antigens in antigen-presenting cells 16

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Photochemical internalization (PCI)

(APCs) [92, 93], and to enhance the cytosolic release of antibiotic gentamicin and increases its efficacy against S. epidermidis andS. auerus infection [94].

This underlines the versatility and the potential of the PCI technology.

Drug

"Light after" PCI

Target 1

2

3

Light

Photosensitizer

"Light first" PCI

1

2

3

4

5

Target Light

Figure 2.7: Schematic illustration of the two PCI protocols. In the "light after" PCI protocol, the drug is incubated with an amphiphilic PS. Without light exposure, the enclosed content is hydrolyzed by lysosomal enzymes. Over time, the drug is localized in endocytic vesicles with the PS on the vesicle membrane. Light-activation of the PS leads to disruption of the membrane and subsequent cytosolic release of the content allowing drug-target interaction. In the "light first" PCI protocol, the PS is activated prior to drug administration. Drug-containing vesicles fuse with photochemically-ruptured vesicles, which results in drug release. Adapted from Dietze et al. [95] and created with BioRender.com

2.3.2 The PCI treatment sequence

Two different protocols can be used for PCI. The standard and first protocol that was developed is the so-called "light after" protocol, where the light is delivered after drug incubation. Later, it was discovered that the drug incubation can also take place after light exposure, and an effect could be achieved up to 6-8 hours after light exposure [96]. This protocol is called the "light first" protocol meaning that photodamage of endosomal/lysosomal membranes is applied before drug exposure. The proposed mechanism behind this "light first" protocol is the fusion of photodamaged/ruptured vesicles and intact vesicles carrying the drug leading to endo-/lysosomal release of the drug [96]. The "light first" protocol seems to work best for receptor-independent drugs as the photochemical processes may disrupt phosphorylation of the cytosolic tail of the receptor, which may block receptor internalization [97–100].

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2. Introduction

2.3.3 PCI photosensitizers (PS)

PS for PCI should be amphiphilic in order to enter the cells by adsorptive endocytosis and localize specifically on the endosomal and lysosomal membranes. The amphiphilic property makes it possible for the PS to reside on the membrane without fully crossing it [91, 101, 102]. The hydrophobic part of the PS associates with the plasma membrane, whereas the hydrophilic part faces the extracellular space. Several PSs have been evaluated for the PCI method, where the most commonly used are AlPcS2a (aluminium phthalocyanine disulfonate), TPPS2a (tetraphenyl porphyrin disulfonate) and TPCS2a

(tetraphenyl chlorin disulfonate, also known as fimaporfin). TPCS2a is currently the most used PCI-PS and under clinical evaluations [44, 89, 101, 103, 104].

These PSs contain two sulfonate-groups, typically with low pK a values and remain ionized throughout relevant physiological pH ranges, which are localized structurally close providing sufficient hydrophilicity [105, 106]. In the first PCI reports, TPPS2a and AlPcS2a were used [89, 104]. However, TPPS2a absorbs light mostly in the blue region which makes itsin vivoapplication limited due to poor tissue light penetration depth [45]. AlPcS2a, on the other hand, absorbs light around 670 nm and has therefore been used for preclinical PCI studies.

Although AlPcS_2a is useful for preclinical studies, the PS is not optimal in a clinical setting due to a large number of isomers. The isomers can lead to a large batch-to-batch variation. Moreover, the biological effects of these isomers may also vary upon light exposure. Finally, AlPcS2ais not patent protected. In order

Figure 2.8: The chemical structure of one of three isomers of TPCS2a. Adapted from Berget al. [105] and created with ChemDraw.

to resolve these challenges related to the production and the spectral properties, TPCS2a was developed for clinical use (figure 2.8)[106]. TPCS2a is synthesized from TPPS2a by reduction from porphyrin to chlorin [105, 106] which leads to formation of three possible isomers. Reduction of one of the double-bound systems in the aromatic ring system results in increased light absorption in the red region, which is necessary to achieve sufficient tissue light penetration and 18

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activation of the PS [105, 106]. Similar to other PSs, TPCS2a also has enhanced retention in the tumor compared to most normal tissues [105].

Both red and blue light can be used for TPCS2a activation as the PS absorbs light in both regions. Forin vitro studies, blue light is adequate whereas red light is often necessary for TPCS2a-activation in e.g. tumors. However, blue light is sufficient for PCI-enhanced vaccination in the skin [92, 93]. TPCS2a

is commercially known as fimaporfin and is PCI Biotech’s (Oslo, Norway) proprietary drug. The clinical formulation made for TPCS_2ais named Amphinex containing Tween80, mannitol and Tris-buffer at pH 8.5 [107]. Currently, fimaporfin is at the clinical development stage (reviewed in section 2.3.5).

2.3.4 PCI-based circumvention of resistance in cancer cells Drug and therapy resistance remains one of the main challenges in cancer treatment, which exist across all types of cancer and modes of treatment, including newer drugs that are approved in recent years, such as TKIs and immunotherapy.

The mechanisms contributing to resistance are complex and multifactorial.

Drug resistance can be orchestrated through different mechanisms, such as in multidrug resistance (MDR).

MDR is defined as resistance to multiple drugs that are not necessary structurally related. The ATP-binding cassette (ABC) transporters have been associated with MDR. Interestingly, previous studies have indicated that TPCS2a is not a substrate to ABCG2 nor P-glycoprotein (ABCB1) [108]. In contrast, pheophor- bide a (PhA), chlorin e6, and the photosensitizing compound protoporphyrin IX (PpIX) have been identified as ABCG2 substrates [108–110]. The PgP substrates are a wide variety of structurally diverse compounds; however, they share some common features. Most Pgp substrates are mainly hydrophobic, weakly amphiphilic, and ranges between 0.3-2 kDa [111, 112].

PCI of type I RIP toxins

The use of hydrophilic macromolecular drugs in combination with PCI may circumvent resistance mediated by these transporters. PCI of type I RIPs toxins, specifically gelonin and saporin (∼30 kDa [113]), have been extensively studied [90, 107]. These ribosome-inactivating proteins are potent when located in the cytosol where as few as 1-10 RIP toxin molecules are thought to be sufficient to kill a cell [114]. Gelonin and saporin are mainly taken up by endocytosis and lack an escape mechanism from the endo/lysosomal compartments. This has limited the clinical translation. However, in combination with PCI, gelonin and saporin are highly effective. PCI of gelonin was found to be very efficient in the PDT- and radiotherapy-resistant sarcoma cell line MESA-SA/Dx5in vitro [115]. Interestingly, MESA-SA/Dx5 was susceptible to PCI despite increased expression of ROS-scavengers [115].

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2. Introduction

A targeted moiety linked to type I RIPs provides enhanced specificity in combination with PCI, given that the receptor is internalized. Indeed, the EGFR-targeting gelonin-based toxin, rGel/EGF, was highly effective in the cetuximab-resistant head and neck squamous cell carcinoma (HNSCC) cell line SCC-040. The efficacy of this cell line was comparable to the cetuximab-sensitive HNSCC cell line SCC-026 [116]. Similarly, the HER2-expressing ovarian cancer cell line SKOV-3 was found relatively resistant to trastuzumab and the HER2- targeting toxin, MH3-B1/rGel. Despite this, SKOV-3 was responsive to PCI of MH3-B1/rGel [99]. The use of PCI of targeted toxin is not limited to cancer cells resistant to targeted therapies, as exemplified here. Previously, Lund et al. demonstrated that 5-FU-resistant sub-clones derived from the Panc03.27 pancreatic cancer cells overexpress endoglin/CD105. Targeting this receptor was highly effective in killing the CD105 5-FU-resistant clones [79].

In several proof-of-concept studies, PCI of CSC-targeting immunotoxins are highly specific and effective at killing cancer cells and CSCs [117]. PCI of a CD133-targeting immunotoxin (AC133-saporin), was indicated as a promising strategy to eliminate CD133 expressing cancer cells. AC133-saporin was highly efficient to kill CD133 high-expressing WiDr colorectal cancer cells that are resistant to PDT, chemo-, and radiotherapy [118]. This strategy was also confirmed in vivo of which PCI of AC133-saporin gave a significant tumor growth delay in WiDr xenografts compared to the controls [118]. The use of PCI-based CSC-targeting strategy is extensively reviewed by Selbo et al., PPS 2015 [117].

PCI-induced release of lysosomal sequestered doxorubicin

A number of anti-cancer therapeutics are ABCG2 substrates, including, doxorubicin, daunorubicin, mitoxantrone, topotecan, irinotecan and its metabolite SN-38, methotrexate, and TKIs (erlotinib, imatinib, gefitinib) [117]. Resis- tance to the chemotherapeutic drug doxorubicin has been associated with Pgp-expression; however, altered intracellular doxorubicin distribution has also been identified as a resistance mechanism [119]. Weak basic hydrophilic drugs, including doxorubicin, has been shown to accumulate in lysosomes that are acidic [19]. Doxorubicin directly inhibits topoisomerase II which prevents the double helix DNA from being resealed during DNA replication [19]. Lysosomal sequestration of doxorubicin limits the amount of doxorubicin accessible to enter the target site, the nucleus.

Lou et al. demonstrated that PCI could reverse doxorubicin resistance in the MCF-7/ADR MDR breast cancer cells [120]. The doxorubicin sensitivity in the resistant cells was found to be comparable to the doxorubicin-sensitive parental MCF-7 cells when PCI was applied [120]. The use of other strategies to overcome the physical barrier of endo/lysosomal entrapment of doxorubicin has also been demonstrated using chloroquine and omeprazole. These strategies are, however, not cancer-specific [121]. PCI, on the other hand, is cancer-specific due 20

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to the enhanced tumor-retention of the PCI PS and light-controlled exposure.

PCI is ,therefore, a promising strategy to overcome resistance in cancer cells that sequester anti-cancer drugs in endo/lysosomes.

2.3.5 The clinical application of PCI

The first PCI clinical phase I study was evaluated in patients with various advanced solid tumors (NCT00993512) [122]. In this clinical study, TPCS2a

was administered in escalating dose in combination with the chemotherapeutic drug bleomycin to patients. The combination was found to be safe and tolerable;

moreover, promising anti-tumor activity was observed in patients receiving TPCS_2a doses of 0.25 mg/kg and above [122]. Currently, TPCS_2a-PCI of gemcitabine followed by gemcitabine/cisplatin chemotherapy in patients with inoperable cholangiocarcinoma (bile duct cancer) is under clinical evaluation.

Based on positive data from a phase I dose-escalation study to assess the safety of TPCS2a-induced PCI of gemcitabine in these patients (NCT01900158), a pivotal phase-II trial is currently ongoing and recruiting patients worldwide (NCT04099888) [44, 123].

Recently, a phase I clinical study in healthy volunteers (NCT02947854) demonstrated that PCI-enhanced vaccination combined with an adjuvant is safe and enhances both the cellular and the humoral immune responses to an HPV peptide-based vaccine and to the keyhole limpet hemocyanin (KLH) protein, which was used as a vaccine control [124].

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Chapter 3 Summaries of papers and

manuscripts

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3. Summaries of papers and manuscripts

Paper I

This study aimed to explore the impact of PCI of sunitinib in both parental and sunitinib-resistant HT-29 colon cancer cells. Sunitinib is a TKI that blocks the protein signal transduction by inhibiting the ATP-binding pocket of multiple intracellular kinases. TKIs, including sunitinib, are small and lipophilic enough to passively cross cellular membranes and interact with their targets. Sunitinib can also passively cross the vesicle membranes. However, sunitinib is a weak base and is therefore subjected to increased protonation at decreasing pH in acidic lysosomes where it is protonated and sequestered due to its charge. This impacts sunitinib’s intracellular distribution and reduces its target interaction and hence, drug efficacy. Several studies have indicated that sunitinib accumulates in acidic vesicles due to its weak-basic properties, and which is a possible resistance mechanism.

In this study, PCI of sunitinib was found to enhance the in vitro cytotoxicity of HT-29 and CT26.WT cells, however, the treatment sequence is crucial.

Sunitinib incubation after light exposure was most efficient as sunitinib is susceptible to photodamage due to its close proximity to the photosensitizer. In sunitinib-resistant HT-29 cells, no enhanced toxicity was observed, indicating that lysosomal sequestration of sunitinib is not the main resistance mechanism in our model.

PCI of sunitinib was further evaluated in two animal models. A modest tumor growth delay was found in the HT-29 xenograft-bearing athymic nude mice. Surprisingly, the survival in an immunocompetent model, CT26.WT- bearing mice were poor compared to sunitinib and PDT monotherapy despite enhanced necrosis compared to the other treatment groups. At the endpoint, the tumors were evaluated for the influx of CD3 positive cells, a marker for T-cells. The number of CD3 positive cells in the PCI group was comparable to the non-treated tumors, whereas increased tumor influx of CD3 positive cells were found in the sunitinib and PDT monotherapy groups. This observation indicates an antagonistic immune response by PCI of sunitinib and may explain the overall failed response.

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Paper II

This work was undertaken to evaluate the potential role of the cancer stem cell (CSCs) marker aldehyde dehydrogenase (ALDH) on PDT sensitivity using the PCI photosensitizer TPCS2a. ALDH is a group of enzymes with detoxifying properties. Overexpression of ALDH has been associated with CSC biology, therapy resistance and poor prognosis in cancer patients. In this work, several cancer cell lines, both human and murine, were screened for ALDH activity using the ALDEFLUOR assay. Among all the cell lines, HT-29 cells were shown to have heterogeneous ALDH activity and therefore selected for further analysis. By using the ALDEFLUOR assay, the HT-29 cells were sorted using flow cytometry into three populations: ALDH^bright, ALDH^dim, and unsorted cells, and subjected to different treatments; 5-FU, oxaliplatin, radiotherapy, and TPCS2a PDT. Colony formation assay revealed that HT-29 ALDH^bright cells had significantly higher survival after RT at 4 Gy compared to ALDH^dim and unsorted cells. No significant differences between the different populations were found after 5-FU, oxaliplatin, and TPCS2aPDT treatment. In conclusion, our study supports the concept of using PCI for cancer stem cell-targeted therapeutics as high ALDH activity does not cause resistance to TPCS2a-based PDT as opposed to RT.

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3. Summaries of papers and manuscripts

Paper III

The overall aim of this work was to study the effect of all-trans retinoic acid (ATRA), the major metabolite of vitamin A, in combination with TPCS2a-based PDT. ATRA is successfully used in the induction therapy of acute promyelocytic leukemia (APL). ATRA acts as a differentiating agent and drives the maturation of primitive promyelocytes derived from leukemic clones, in part because of ATRA’s ability to induce differentiation and arrest proliferation. Several clinical studies have attempted to use ATRA to treat solid tumors, however, no breakthrough has been achieved so far. We hypothesized that low-dose ATRA combined with PDT (ATRA+PDT), is a suitable combination to improve the treatment outcomes in solid tumors.

The cytotoxicity of ATRA+PDT, was evaluated in five different cancer cell lines; two human breast cancer cell lines (MDA-MB-231 and SKBR3), and three colon cancer cell lines (HCT116, HT-29 and MC-38), of which the latter is murine. Enhanced cytotoxicity was found in all cell lines, except HCT116.

ATRA+PDT blocked cell proliferation, induced inhibition of cell viability and colony-forming ability. Moreover, the TUNEL assay indicated increased apoptosis in ATRA+PDT-treated HT-29 cells compared to monotherapy. RNA-seq was used to identify differentially expressed genes (DEGs) and pathways important for the survival or death after ATRA+PDT compared to PDT. Pathway analysis by Ingenuity Pathway Analysis (IPA) indicated activation of apoptosis pathways by ATRA+PDT. Furthermore, the unfolded protein response (UPR) was also identified as significantly activated post-ATRA+PDT.

To verify thein vitroanti-tumor effects, ATRA+PDT was evaluated in HT-29 xenografts. Significant initial tumor delay was observed during the first 20 days, including complete responses in 2/5 animals. In conclusion, ATRA+PDT represents a promising novel combination strategy for the treatment of solid tumors.

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Paper IV

This work explores the potential of PCI of a programmed death ligand-1 (PD- L1)-targeting immunotoxin. PD-L1 is a transmembrane molecule critical in the regulation of cell-mediated immune response through interaction with the receptor programmed death protein (PD-1). PD-L1 overexpression has been observed in a number of carcinomas and in immunosuppressive stromal cells in the TME and provides an escape mechanism from immune surveillance. Blocking antibodies, checkpoint inhibitors against PD-1 or PD-L1 have revolutionized cancer immunotherapy. However, only a fraction of patients has durable antitumor responses. Thus, there is a high need for novel strategies to improve the therapeutic success in tumors with an immunosuppressive TME with PD-L1- expressing cells. Here, we explored a PD-L1-targeting approach combined with PCI to enhance the cytosolic release of the toxin saporin conjugated to a PD-L1 antibody (anti-PD-L1-saporin). We screened a panel of human cancer cell lines of which the triple-negative breast cancer cell line MDA-MB-231 had the highest PD-L1 expression. Treatment of anti-human-PD-L1-saporin in MDA-MB-231 alone reduced the viability compared to the non-targeting saporin. Interestingly, PCI of anti-PD-L1-saporin was found to be highly effective in the pico- to nanomolar range. In contrast, no enhanced toxicity was found in the triple- negative breast cancer cell line (MDA-MB-453) with low PD-L1 expression. This work lays the foundation for developing a recombinant PD-L1-targeting toxin suitable for evaluation in combination with PCI in syngeneic mouse models.

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Chapter 4 Experimental considerations

4.1 Cancer cell lines

Cell lines are an integral part of biological research and provide great insight into the biological processes and their response to treatment. Immortalized cell lines offer a cost-effective method with standardized cultivation methods provided by vendors. Moreover, immortalized cell lines are a convenient and stable platform. All cell lines used in this work, except MC-38, were acquired from American Type Tissue Collection (ATTC). The MC-38 cell line was obtained from Kerafast. All culture media was supplemented with antibiotics (penicillin and streptomycin) to prevent bacterial infections. The cells were mycoplasma negative throughout all experiments.

The cells were not cultured for more than 25 passages to ensure a repro- ducible experimental setting. For tumor grafts in animal models, all passages were kept under 15. One exception is in paper I, where continuous sunitinib exposure over 25 passages was necessary to generate sunitinib-resistant HT-29 cells. In that study, the non-treated HT-29 cells were cultivated along sunitinib- resistant cells to account for potential genotypic and phenotypic variations over an extended time. All experiments with cells were initiated when the confluence was around 70-80 %.

Both murine and human adherent cancer cell lines were used in this work.

In paper I, the human colon carcinoma cell line HT-29 was selected based on the work by Gotinket al. [20], who first demonstrated lysosomal sequestration of sunitinib. The murine colon carcinoma cell line CT26.WT was also included in paper I to serve as a model in syngeneic BALB/c mice (immunocompetent).

The HT-29 cell line was also included in paper II, together with five pancreatic carcinoma (Panc 03.27-derived) cell lines [79], CT26.WT, and the murine mammary carcinoma 4T1 cell line. All these cell lines were included in paper II to evaluate the aldehyde dehydrogenase (ALDH) activity, a CSC marker. The HT-29 displayed heterogenous ALDH expression and was selected to evaluate the impact of ALDH expression on TPCS_2a-PDT response. The HT-29 cells also express high levels of CD133; another important CSC marker [125]. High CD133 expression has previously been associated with TPCS_2a-PDT resistance [126].

In paper III, we selected the cell lines based on their ATRA sensitivity as we explored the use of ATRA in combination with TPCS2a-PDT (ATRA+PDT).

Five cell lines were included in this paper; the ATRA-resistant human colon carcinoma HCT116 cells and the human breast carcinoma MDA-MB-231 cells,

Photochemical Strategies to Eliminate Cancer Cells

Judith Jing Wen Wong