CHARACTERIZATION OF BREAST CANCER WITH MANGANESE-ENHANCED MAGNETIC RESONANCE IMAGING. Joris Igor Nofiele Tchouala

Transcription

1 CHARACTERIZATION OF BREAST CANCER WITH MANGANESE-ENHANCED MAGNETIC RESONANCE IMAGING by Joris Igor Nofiele Tchouala A thesis submitted to the Department of Medical Biophysics in partial fulfillment of the requirements for the degree of Master of Science in Medical Biophysics at the University of Toronto c Copyright by Joris Igor Nofiele Tchouala (2014)

2 ABSTRACT CHARACTERIZATION OF BREAST CANCER WITH MANGANESE-ENHANCED MAGNETIC RESONANCE IMAGING Joris Igor Nofiele Tchouala Master of Science Department of Medical Biophysics University of Toronto 2014 Highly metastatic cancer cells are more likely to escape and form metastases, and only minimal improvements in treatment can be achieved. Despite metastases being the primary cause of cancer-related mortality, they often proceed unnoticed. Current imaging modalities rely solely on the morphological features of the tumor for characterization, rather than cellular differences. Our goal is to develop an MR cellular imaging capability for characterizing the potential of breast cancer cells to metastasize and enable early cancer detection using manganese. Experiments on breast cell lines demonstrated that aggressive cancer cells significantly enhanced on T 1 -weighted MR images as a result of a higher uptake and retention of manganese. These results suggest that differences in uptake of manganese can help the detection and characterization ii

3 of breast cancers. The proposed technique can also be useful for other cancers, and could bring a critically needed dimension to cancer imaging. iii

4 ACKNOWLEDGMENTS I wish to thank the following people: The Czarnota Lab at the Sunnybrook Health Science center for providing me with the initial training, facility, and subsequent technical help without which this work could not have been achieved. The Cheng Lab at the Hospital for Sick Children, for their continuous support in the timely completion of this work. All of the imaging technicians who provided me with training on the MR scanners. My fellow MBP colleagues and classmates. iv

5 Contents Table of Contents List of Tables List of Figures v vii viii 1 Introduction The normal breast anatomy The parenchyma The stroma and connective tissue Breast cancer Statistics Diagnosis Characterization Metastasis Aggressiveness Imaging of breast cancer Mammography Ultrasound Magnetic resonance imaging Models of breast cancer In vitro model In vivo model Outline In vitro characterization of breast cancer cells Rationale Experimental procedures Cell cultures Cell proliferation Cell viability and labeling with manganese Quantification of intracellular manganese content Immunological staining v

6 vi CONTENTS Colorimetric assessment MR imaging and data analysis Results Cell culture and viability Solutions and pellet imaging Manganese quantification Cellular CaSR histology and quantification Discussion Conclusions Future directions Preamble Experimental procedures Tumor innoculation Manganese administration Rat imaging Observations and Discussion Manganese-enhanced contrast Tumor localization Conclusions Bibliography 42 Index 46

7 List of Tables 2.1 Cell viability ICP-AES measurement of intracellular manganese following 1 hour treatment with 0 to 1.0 mm of manganese chloride Colorimetric assessment of the calcium-sensing receptor vii

8 List of Figures 1.1 Sagittal view of the breast Growth cycle of adherent cells Growth curves T 1 -weighted image of cell samples R 1 relaxation rate of cell samples R 1 relaxation rate of cell samples 1, 24 and 72 hours after incubation with manganese Immunohistochemistry staining of surface CaSR for each cell line in culture, growing exponentially MRI of rats prior and 24 hours after manganese administration viii

9 Chapter 1 Introduction 1.1 The normal breast anatomy The breast is an important organ of the integumentary system. It is supported by the ribs and the pectoral muscles of the chest wall (Fig. 1.1). The breast is composed of the skin, the subcutaneous tissue and the breast tissue [1]. Breast tissue is made of the parenchyma (composed of the lobes and ducts) and the stroma The parenchyma The parenchyma is typically made of 15 to 20 lobes, each containing several lobules [1] responsible for milk production. The lobes are arranged in a radial fashion, converging via milk transporting ducts to the nipple. The lobules themselves contain 10 to 100 alveoli lined with milk-producing cells. Several lobules (20 to 40) are drained by a single duct, a hollow conduit which carries milk to the nipple via approximately 10 major collecting ducts. All of these structures are supported by the stroma and connective tissue. 1

10 2 Chapter 1 Introduction Figure 1.1 The breast is supported by the pectoralis and intercostal muscles, which are connected to the ribs. The internal mammary artery supplies the breast. [1]

11 3 Chapter 1 Introduction The stroma and connective tissue The stroma and connective tissue are composed of fat, ligaments, local vasculature, lymphatic vessels, nerves and regional lymph nodes. Their purpose is the delivery of nutrients to mammary glands as well as their support. The breast receives blood supply from two main arteries: the internal mammary artery and the lateral thoracic artery [1]. Drainage of the blood from the breast back to the heart is achieved by the internal thoracic vein, the axillary vein and intercostal veins. Lymphatic vessels are part of the lymphatic organ system, whose role is mainly the defense against infection and disease as well as transport of interstitial tissue fluid or lymph back to the bloodstream [2]. Lymphatic vessels carry lymph to the lymph nodes, a storage of white blood cells and an important point of defense for infections. Usually, lymph from the breast drains to nearby lymph nodes such as the axillary lymph nodes. However, lymph may drain via different routes in the presence of abnormalities such as cancer. 1.2 Breast cancer Cancer in general occurs when a cell escapes the normal proliferation cycle and begins to proliferate uncontrollably. When the parent cell is from an epithelial tissue, the malignant growth is called carcinoma. Furthermore, if the cancer originates in a gland, it is refered to as adenocarcinoma. Thus, breast adenocarcinoma is a cancer that originates in the epithelial layer of the mammary glands. The vast majority of breast cancers are of this type, as they most often occur in the milk ducts and the breast lobules.

12 4 Chapter 1 Introduction Statistics In Canada, it is estimated that 1 in 9 women will develop breast cancer over the course of her lifetime. This disease represents 15% of all cancer-related deaths in Canada [3]. It is expected that 2012 statistics will report an estimates 22,700 new cases of breast cancer and 5,100 deaths from it [4]. On a worldwide scale, it is estimated that in 2008, breast cancer caused 482,485 deaths [5]). Breast cancer also remained the highest occuring form of cancer among women in the world Diagnosis Diagnosis of breast cancer usually begins with palpation, when a patient or physician can feel a solid mass within their breast. First, the patient is sent for a mammography exam, which is imaging of the breast with x-rays. This technique is commonly used to screen patients and to confirm the presence of breast cancer. It is almost always complemented with MRI and sometimes ultrasound, to confirm diagnosis. Once imaging confirms the presence of a suspicious mass in the breast, a biopsy or invasive removal of a piece of tissue from the cancer is performed in order to characterize the cancer and decide on the best course of treatment. In summary, imaging is typically used to confirm the presence of a suspicious enhancing mass and determine its morphological features, and biopsy is used to visualize cells removed from the diseased tissue and perform an histology-based characterization Characterization Whereas mammography purely relies on the presence of calcium deposits to detect cancer, MRI provides a much better tool for the characterization of breast cancer. The morphological appearance of cancer on an MR image is used for characterization.

13 5 Chapter 1 Introduction For instance, a spiculated shape is commonly perceived as more aggressive than a well defined round mass. Furthermore, because cancerous masses have vasculature, MR contrast agents can be used in combination with imaging to assess vasculature and aid the staging process. In addition to imaging, a biopsy provides useful information about cells composing the cancer. The general appearance of the cells, their arrangement, their location and other parameters are evaluated by a pathologist on a microscope to determine how likely the cancer cells are to have escaped the primary site of origin of the cancer. Biopsy is also performed on regional lymph nodes to determine if cancer has already spread. This information is further used to determine how aggressive the cancer is. Furthermore, hormone receptors expression by tumor cells is determined to decide on the best treatment. The hormones of interest are estrogen (ER), progesterone (PR) and the human epidermal growth factor receptor 2 (Her2/Neu). Tumor cells that possess receptors for estrogen and progesterone are called hormone-dependent cell lines. Tumors composed of such cells can usually be targeted with hormonal therapy and thus have a more favorable prognosis. Furthermore, for estrogen-receptive tumors, a commonly used test to evaluate the potential benefit of adding chemotherapy to hormone therapy is Oncotype DX. This test uses cancer tissue from the biopsy to analyze 21 genes, from which a recurrence score that aids treatment planning is calculated. Tumor cells that do not express any of these three receptors are known as triple negative cells, and they generally present a much greater difficulty to treat as they cannot be targeted with therapies. Such cells are usually fast growing and present a poorer prognosis than tumors composed of hormone-receptive cells. Furthermore, due to the impossibility to target them with specific treatment, they remain under-treated, as the effects of chemotherapy are limited by its toxicity to nearby healthy tissue. Overall, all of these parameters provide useful information on the cancer, and poten-

14 6 Chapter 1 Introduction tially effective treatments when the tumor is found at an early stage. Late stage and aggressive tumors however, present several difficulties affecting their management Metastasis As a tumor develops, several physiological events happen to sustain its growth, among which is the formation of new blood vessels. Typically, a solid tumor cannot grow beyond 1 to 2 mm in size without intratumoral blood vessels supplying nutrients to cancer cells [1]. As a tumor grows larger, its cells secrete growth factors that result in the formation of new branches from existing nearby blood vessels. The tumor vasculature formed is leaky, inefficient and erratic [6]. Such characteristics allow cells from aggressive tumors to escape, through the vasculature, to other parts of the body and form metastases, or malignant growths in a different location from the primary site of the tumor. Furthermore, local lymphatic vessels drain back to the vasculature, providing another route for cancer cells to enter vasculature and form distant metastases. Cancers with detectable distant metastases at the time of diagnosis are labelled as end stage cancer and have a very poor survival rate, which is commensurate with their aggressiveness. The diagnosis of cancer at its early stage is therefore of critical importance Aggressiveness Several criteria are used clinically to determine breast cancer aggressiveness [7]. First, the size of the tumor at the time of diagnosis is evaluated. The larger the tumor the more aggressive the cancer is. This characteristic can also be linked to the rate of tumor cells proliferation, as the volume of the tumor partially depends on the number of cancer cells actively dividing. Next, the grade of the tumor upon diagnosis

15 7 Chapter 1 Introduction is examined to determine its aggressiveness. Tumor grades range from 1 to 3, and are determined by the tumor s growth pattern and overall appearance. Grade 3 tumors are considered more aggressive than grade 1 tumors, and typically spread to other parts of the body. Additionally, as mentioned before, the hormone receptor status of the tumor cells can also give an indication about the tumor s aggressiveness. Triple negative tumors are typically more aggressive than estrogen, progesterone and Her2/Neu positive tumors. Furthermore, triple negative tumors are more likely to spread, to recur after treatment, and generally have a lower 5-year survival rate [8]. 1.3 Imaging of breast cancer Mammography Mammography is an x-ray based imaging modality widely used for screening breast cancer. Mammography primarily relies on the presence of calcifications, which appear bright on a mammogram. These calicum deposits usually indicate the presence of cancer or other disease. For an x-ray image of the breast to be acquired, the breast is compressed to reduce overlap of tissue and decrease the scatter of photons. This however, represents a major source of discomfort to patients. Furthermore, ultrasound and MRI are usually required to complement mammographic findings, as calcifications can also be due to other non pathologic sources. Also, despite its widely spread use and relatively low cost, mammography still makes use of ionizing radiation, which is harmful to normal tissue.

16 8 Chapter 1 Introduction Ultrasound Ultrasound is an inexpensive, non-ionizing imaging modality used in breast cancer diagnosis. Ultrasound is often used in the area of cancer imaging to differentiate cysts from solid masses, which appear similar on a mammogram. Furthermore, because of the high acoustic reflection of needles, ultrasound is also used to guide biopsy needles. In addition, this imaging modality is portable and readily available, which makes it ideal for several applications. The resolution of ultrasound systems however, are less than adequate for more complex analyses. Depth of penetration is also a barrier for the use of ultrasound to locate deep seated cancers. Operator skill as well is critical for ultrasound imaging to be useful. Overall, ultrasound is currently limited in terms of its usefulness in the area of breast cancer imaging. However, new ultrasound-based functional imaging techniques such as elastography might be more useful Magnetic resonance imaging Magnetic Resonance Imaging is an imaging technology that emerged in the 1970s as a powerful new method that relies on imaging the hydrogen atom. Atoms that possess an odd number of protons and/or neutrons possess a property called spin, which gives rise to a magnetic moment. The hydrogen atom, due to its large abundance in the body, is an excellent source of signal in magnetic resonance imaging. Magnetic resonance physics In the absence of an external magnetic field, protons are arranged in a random fashion, giving rise to a net magnetization of zero. When placed within an MR scanner and therefore subject to an external magnetic field B 0 created by the scanner s magnet, the spins align with B 0 in either a parallel or an antiparallel fashion. The spins aligned in

17 9 Chapter 1 Introduction parallel with B 0 are in a lower energy configuration, and therefore are more abundant than spins aligned antiparallel to B 0. This results in a net magnetization that is aligned with B 0. Furthermore, by the classical explanation of MR, the spins rotate at a particular frequency, related to the external field by the following expression: ω = γ B 0, (1.1) where γ is the gyromagnetic ratio of the hydrogen atom at a particular B 0. The spin system described above can be perturbed using a radiofrequency wave at the Larmor frequency, which alters the magnetization alignment. When the excitation RF pulse is turned off, the system will return to equilibrium due to the presence of B 0, and at a rate that is dependent on the particular tissue containing the spins, a process known as relaxation. MR pulse sequences can further be used to tune this magnetization recovery and obtain several useful parameters characteristic of different tissues. T1-weighted imaging The T1 relaxation time is a parameter that can be inferred from T1-weighted MR images. T1 relaxation depends on the exchange of energy between the spins and the surrounding medium. This medium generally contains water, and thus hydrogen atoms, in three states: free or unbound water, structured or partially bound water, and bound water. The rotation, translation and vibration of water molecules in these three states, known as tumbling, determines the speed of recovery of the net magnetization vector, and therefore affect T1. In addition, various T1 contrast agents exist that can alter the T1 relaxation time.

18 10 Chapter 1 Introduction T1 contrast agents Given that T1 relaxation depends on energy exchange with the surrounding lattice, chemical compounds possessing special magnetic properties can be used to accelerate the energy exchange which in turn reduces the T1 relaxation time and creates a better contrast. The main elements composing contrast agents have unpaired valence electrons exhibiting strong resonance phenomena, which accelerate T1 relaxation. Gadolinium and manganese for instance, have 7 and 5 valence electrons respectively, which in the presence of B 0 possess a magnetic field of their own that interacts with the hydrogen field, thereby accelerating relaxation. The effectiveness of MR contrast agents is termed relaxivity. It is typically measured in water, and is expressed in mm 1 sec 1. For T1 contrast agents, the contrast agent relaxivity is represented as r 1. r 1 is related to the relaxation time by the following expression: 1 T 1 = 1 T 0 + r 1 [CA], (1.2) where [CA] is the concentration of the contrast agent, T 1 is the T1 relaxation time of the solution containing the contrast agent and T 0 is the T1 relaxation time of the solution without contrast agent. This equation can also be represented by R 1 = R 0 + r 1 [CA], (1.3) where we define R as the relaxation rate, or the inverse of the relaxation time. T1-weighted imaging of breast cancer Current clinical imaging of cancer utilizes gadolinium-based intravascular contrast agents, which are used in three major ways:

19 11 Chapter 1 Introduction (i) For morphological assessment, since the size and shape of the suspect mass is correlated to tumor invasiveness. (ii) For vascular assessment, given that tumor vasculature provides a good indication of the potential effectiveness of systemic cancer drugs. (iii) For staging. The pharmacokinetics of the contrast agent assist in the staging of the tumor. However, the current techniques listed above are not capable of differentiating metastatic cancer cells from non-metastatic ones. Furthermore, these techniques cannot detect and characterize tumors during their early development. Gadolinium-based contrast agents currently used in clinic, are inconclusive in determining cancer aggressiveness, as the morphological features of aggressive tumors can sometimes be the same as non-aggressive tumors. Manganese-based contrast agents, such as the FDA-approved Telascan, are chelated manganese-ii-containing compounds used primarily for imaging liver lesions. These compounds have not been used either to assess cancer aggressiveness, which is understandable, given that they are chelated so as to be taken up by liver cells. For these compounds, MR contrast is generated by the Mn 2+ ion bound to other structures. The manganese chloride contrast agent however (MnCl 2 ), dissociates when placed in an aqueous solution, releasing Mn 2+ ions, which are then able to enter cells through ion channels. Because Mn 2+ can provide MR contrast from within cells, it provides a different type of contrast as compared to Gadolinium. Gadolinium is an intravascular and extracellular contrast agent, but it does not enter actual cells and therefore cannot provide intracellular contrast.

20 12 Chapter 1 Introduction 1.4 Models of breast cancer The study of human cancers often requires the use of models that simulate natural cancer growth. These models are necessary to test new imaging techniques, new treatments and various other analyses that cannot be performed clinically. In vitro and in vivo models are commonly used for the purpose of studying cancers In vitro model The in vitro model of cancer makes use of cancer cell cultures. A cell culture refers to the extraction of cells from an animal and their subsequent growth in an adequate environment. Human cancer cell lines currently used were extracted from cancerous tissue after surgical removal from patients with cancer. These cell lines typically retain their malignant properties when cultured in the appropriate conditions, thereby providing a useful model to study cancer cells themselves. One of the largest entities that distributes cancer cell lines is the American Tissue Culture Collection (ATCC), which stores many cell lines that were adapted in the past. In vitro evaluation of cancer cells aggressiveness has not been clearly done. Instead, particular genes are usually identified in vitro, that correlate with in vivo phenotypes of aggressiveness. Nevertheless, it is reasonable that aggressiveness of cancer cells in vitro can be associated with speed of proliferation, patterns of colony formation, general appearance of the cells and their nuclei, etc. The fastest proliferating cell lines can partially be expected to grow fast in vivo as well. Also, while observing cells in culture, more aggressive cells can be identified by their ability to form colonies from individual cells. In other words, if one aggressive cell was to be plated alone in a dish with culture medium, it would very likely form a colony and reach confluency, whereas a less aggressive cell would probably decay and die. Additionally, in a culture dish, a large

21 13 Chapter 1 Introduction portion of aggressive cells typically appear more round, as they are always actively dividing. Less aggressive cell lines on the other hand, depending on their stage of cell cycle, will only appear round when dividing. One final measure of in vitro agressiveness is the metabolic rate of cells. Through observation of cells in culture, it is visible that more aggressive cells reduce the ph of their culture medium more rapidly than the same number of less aggressive cells, which is indicated by a change in color of the medium from red to yellow. This change in ph is commonly attributed to the lactate being excreted by cancer cells as part of the glycolysis process [9]. Although in vitro models of cancer are useful to study actual cancer cells and for proof of principle experiments, they do not represent the much more complex in vivo environment In vivo model A living biological entity represents a complex system in which many biological pathways affecting each other happen simultaneously. In pre-clinical models of cancer studies, animals are typically used as a medium to simulate a human environment. In the case of breast cancer, animals with a compromised immune system are used to generate tumors in mice or rats. Contrast-enhanced imaging can subsequently be done along with histological analysis. Tumor aggressiveness in vivo is linked, as discussed before, to the general appearance of cancer cells, the speed of growth of the tumor, the receptor status of the tumor, etc. Current MRI-based identification of aggressive tumors currently relies solely on the morphological features of the tumor, rather than on cancer cells themselves.

22 14 Chapter 1 Introduction 1.5 Outline The aim of this thesis project is the early detection of breast cancer through noninvasive characterization of tumor cells using manganese-enhanced magnetic resonance imaging. In essence, we aim to detect cancer cells before the tumor becomes large, and regardless of the presence of tumor vasculature. Furthermore, the hypothesis tested is that metastatic breast cancer cells take up more manganese than less metastatic ones, leading to a difference in their enhancement of MR signal. Additionally, we predict a differential time profile release of intracellular manganese by cells of different aggressiveness. To achieve these aims, four breast cancer and one epithelial cell lines are utilized to demonstrate a differential uptake of manganese. Then, one cancer cell line is injected in vivo in rat model to test the validity of the same concept.

23 Chapter 2 In vitro characterization of breast cancer cells Note: This chapter has been submitted as a manuscript for publication in contrast media & molecular imaging. 2.1 Rationale Current MR contrast agents used for the imaging of cancer rely on the presence of tumor blood vessels. However, before a well vascularized mass is detected, aggressive cancer cells have already had a chance to spread to other parts of the body, even if metastases are not yet detectable. Breast cancer specifically, is the most common malignancy among women in the world [10]. Some improvements in cancer management have slightly reduced the death rate due to breast cancer, but this disease remains the second cause of mortality among cancers in women [10]. This mortality rate is attributed in large part to the inability to detect and characterize metastatic cancer cells early on, before actual metastases appear. Once metastasis is diagnosed, a long 15

24 16 Chapter 2 In vitro characterization of breast cancer cells term improvement is rarely attainable, which is why metastasis is the primary cause of cancer-induced mortality [11]. It is therefore of prime importance to detect such aggressive cancer cells at the earliest time possible, such that the treatment course can be adjusted in time. As stated in the first chapter, current imaging techniques for breast cancer (ultrasound, mammography and MRI) rely only on the gross morphological appearance of the tumor in question for characterization, and does not characterize actual tumor cells. Biopsy, which is also used to perform tumor characterization, can only sample select very limited regions of the tumor, and relies mainly on receptor status of cancer cells for characterization of aggressiveness. There is currently no imaging modality capable of characterizing the entire tumor non-invasively. Furthermore, such a modality would be of even better use if it did not rely on tumor vasculature, but rather on actual differences between aggressive, less aggressive and normal cells. Thus, the early detection of such cells would need to rely on their activity at the intracellular level. Cancer aggressiveness is associated with an altered control of cell proliferation, differentiation, migration and apoptosis, which are among the processes regulated by the calcium ion, Ca 2+ [12 14]. Intracellular levels of calcium ions have also been shown to increase during cancer proliferation [12] and migration [15, 16]. Such ions, because of their charge, primarily utilize ion channels to enter cells. In particular, the calcium sensing receptor (CaSR) has recently been investigated as a potential entry point for calcium ions. CaSR is present in small amounts in cells of the breast [17] and of the bone [18], and is believed to promote metastatic progression of breast cancer [19], prostate cancer [20] and other cancers [21]. The manganese ion Mn 2+, can be used as a calcium analogue that can probe Ca 2+ activity within the cell, given their similarity in size and charge [22]. Mn 2+, as

25 17 Chapter 2 In vitro characterization of breast cancer cells mentioned in chapter 1, also possesses magnetic properties that enable it to provide positive MR contrast (i.e. bright signal). Manganese has not typically been used for the imaging of cancer. The few reports that exist note a difference in manganese uptake between normal and malignant cells [23 26]. Using breast tumor bearing mice, one investigator showed that different regions of the tumor enhanced following intraveneous injection of manganese, and that these regions correlated with increased levels of CaSR expression [27]. However, to the best of our knowledge, manganese uptake by cancer cells of different aggressiveness has not been explored. The characterization of breast cancer cells of different aggressiveness is therefore undertaken to test the prediction that aggressive cancer cells take up and retain manganese ions in a differential manner as compared to less aggressive cancer cells. 2.2 Experimental procedures Cell cultures Four breast cancer cell lines of different aggressiveness were selected, as well as one non cancerous epithelial breast cell line. All of these cell lines were adherent cell lines, i.e. cells that can attach to the flask they re grown in. The two most aggressive cell lines selected were 231/LM2-4 (hereafter refered to as LM2) and MDA-MB-231 (hereafter refered to as MDA). The MDA cell line is a well characterized epithelial breast cancer cell line, which was derived from a pleural effusion metastatic site in a patient with breast cancer [30]. MDA is a triple negative cell line, which can form tumors in mice. The LM2 cell line on the other hand, was especially selected for its higher likelihood of forming metastases [28]. To obtain the LM2 cell line, MDA cells were injected in the mammary fat pad of mice. The primary tumor formed was then resected and the animal was kept alive to allow metastases to form. Several weeks

26 18 Chapter 2 In vitro characterization of breast cancer cells later, cells were extracted from the actual metastatic sites, adapted to culture and injected again in mice. The primary tumor was removed again, and cells collected from the metastatic site of this second round of injection were adapted to culture and labelled 231/LM2-4 [28]. The LM2 cell line therefore, was selected from the cells in the MDA population of cells that were able to form metastases. For this reason, the LM2 cell line is considered more aggressive than the MDA cell line, in addition to its triple negative status and its more aggressive phenotype observed in cell culture. The other two cancerous cell lines, MCF-7 (hereafter refered to as MCF7 [31]) and ZR-75-1 (hereafter refered to as ZR75 [29]) were also derived from metastatic sites. However, they are estrogen and progesterone positive, which makes them, as discussed above, less invasive than LM2 and MDA. MCF7 is an epithelial cell line derived from a pleural effusion metastatic site, whereas ZR75 is a metastatic epithelial cell line derived from malignant ascites. The fifth cell line used, MCF10A, was an epithelial, non-cancerous cell line derived from a patient with a fibrocystic disease. Due to its storage conditions, this cell line developed a particular sensitivity to calcium. Furthermore, its growth medium included various growth factors and other substances not used for the other cell lines, as such cell lines can only growth in their optimal growth medium. The base medium used for MCF10A was MEGM supplemented with 2 ml BPE, 0.5 ml hegf, 0.5 ml insulin, 0.5 ml hydrocortisone and 100 ng/ml cholera toxin. These components were obtained as a kit, and combined together into the medium as per ATCC instructions. The four cancer cell lines were grown in 1640-RPMI growth medium supplemented with 10% fetal bovine serum and 0.5% penicilin streptomycin. All 5 cells lines were kept in an incubator at 37 degrees Celsius with 5% CO 2 until harvesting. To detach cells from flasks, 80% to 90% confluent flasks were washed with PBS and exposed to 0.05% trypsin EDTA. To create samples, cells were centrifuged

27 19 Chapter 2 In vitro characterization of breast cancer cells Figure 2.1 Cell number as a function of the number of days following subculture. The cycle of growth of cells includes a lag phase, a log phase and a plateau phase. Important parameters, such as doubling time, can be derived from such a curve for a particular cell line [33]. at 440g for 10 minutes, while the temperature was kept at 4 degrees Celsius Cell proliferation Cell proliferation is one measure of in vitro cancer aggressiveness, therefore the 5 cell lines were partly characterized by finding their doubling times. When cells are first plated after trypsinization, they undergo a latent or lag phase during which they do not grow, but instead recover from trypsinization and repair their cytoskeleton (Fig. 2.1). Following this, cells enter the log phase, during which they proliferate exponentially. Finally, as the flask becomes confluent, cells enter the plateau or stationary phase, where cells proliferate very slowly. The doubling time of a cell line can be obtained from data points of cells growing in the log phase, as derived from the growth curve [33]. Doubling time of cell lines in this study were obtained by plating 10 5 cells in a several dishes, and counting the number of cells from each dish each day.

28 20 Chapter 2 In vitro characterization of breast cancer cells Cell viability and labeling with manganese Given that the Mn 2+ is an intracellular contrast agent, cells were exposed to different concentrations of aqueous manganese chloride for one hour to allow intake. The manganese chloride molecule breaks up and liberates ions once in water. The molecular weight of the actual product was g/mol, and its formula: MnCl 2 4H 2 O. To prepare the treatment medium, specific amounts of the crystal were added to complete growth medium up to concentrations of 0.05 mm, 0.1 mm, 0.2 mm, 0.5 mm and 1.0 mm. This medium was subsequently filtered using a 0.22µm pore size filter, and added to the flasks containing exponentially growing cells. The flasks were then returned to the incubator, and one hour after, the medium was discarded, and the flasks were rinsed twice with PBS to further remove lingering non intracellular manganese ions. Initial tests were performed to assess the viability of the cells after the one hour exposure using trypan blue. Trypan blue is a chemical compound which can enter cells if their membrane is damaged, but cannot enter cells with an intact membrane. Thus, small amounts of treated cells were exposed to trypan blue for 5 min, after which stained and non stained cells were counted on the microscope with a hemocytometer. The ratio was then used to determine the percentage of live and dead cells, as measured by their membrane integrity. This test was performed up to 3 days following exposure. Treated cells were rinsed and given fresh non treated medium for 3 days, and viability was done again to evaluate potential late toxic effects caused by exposure to manganese. In addition to the trypan blue test, overall cell morphology was evaluated using microscopy after exposure to manganese chloride.

29 21 Chapter 2 In vitro characterization of breast cancer cells Quantification of intracellular manganese content In order to chemically measure and quantify intracellular manganese, treated cells were lysed and manganese quantified using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES). First, cells were treated as described above (Section 2.2.3), and after treatment, cells were collected, counted and placed in 1M nitric acid for 30 min at 70 degrees Celsius. Next, the mixture was resuspended in double distilled water, and the solution was filtered using a 0.22µm pore size filter. After this procedure, an acidic solution containing manganese previously in cells was obtained. The ICP-AES technique consists in combusting the sample using argon gas, which evaporates water and excites the manganese ions. Upon cooling, the ions release photons at a wavelength of 293 nm, which can be detected and correlated with the concentration of manganese in the sample Immunological staining To visualize membrane ions channels, specimens are typically stained using immunohistochemistry, which essentially consists in exposing antigens to antibodies bound to fluorophores. In this study, the calcium-sensing receptor ion channel on the cell membrane was stained and quantified using immunohistochemistry and a colorimetric assessment respectively. To do this, cells growing in the log phase on a circular glass cover slip were fixed with 4% paraformaldehyde for 10 min. Cells were then washed with a non-perforating solution, i.e. PBS not containing triton X. The primary anti- CaSR antibody was then added to cells for 1 hour at room temperature, followed by incubation with a secondary fluorophore-bound antibody at room temperature and for 1 hour. The parameters of antibody exposure were optimized for this procedure, as recommended by the antibody suppliers.

30 22 Chapter 2 In vitro characterization of breast cancer cells Colorimetric assessment To add a quantitative correlate to this histology staining, a colorimetric assessment was performed [34]. First, cells were grown in multiwell plates, such that the procedure could be carried out at the same time for all of the cell lines. Once the cells were growing exponentially, medium was removed and the cells were fixed as decribed above (Section 2.2.5) and subsequently incubated with 3% bovine serum albumin for 1 hour at 4 degrees Celsius. Then without washing, cells were incubated with a 1:2000 dilution of the primary anti-casr antibody for 1 hour at room temperature. Cells were washed and incubated with a 1:800 dilution of secondary antibody for 1 hour at room temperature. Following more washing, cells were incubated with 1 ml OPD for 2 min, and then with 0.6 ml of 3N hydrochloric acid for 10 min to stop the reaction. OPD is a substance that reacts with the fluorophores bound to the secondary antibody and gives the solution a color whose optical absorbance can be measured and correlated with the actual quantity of secondary antibody present on the cells. Antibody still bound to the cells after all the washing are directly related to the amount of CaSR present on the cells surface, hence providing a quantitative measure. The optical absorbance was measured using a spectrophotometer at a wavelength of 492 nm. The mean value from three independent measurements was normalized to the untreated cells and number of cells MR imaging and data analysis Prior to data acquisition with cells, the relaxivity of manganese chloride was measured by preparing samples containing various concentrations of manganese chloride and imaging them as described below. The concentrations used were 0, 0.05, 0.1, 0.2, 0.5, 1.0 and 1.5 mm in water, PBS and complete cell medium. Treated and untreated cells

31 23 Chapter 2 In vitro characterization of breast cancer cells were centrifuged at 440 g for 10 min in borosilicate glass, and the tubes were placed in a block of ultem resin with drilled holes corresponding to the size of the glass tubes. Ultem resin was used to minimize air-to-sample interface-induced artifacts. MR imaging was performed immediately on a 3 Tesla scanncer using a 32-channel receive-only head coil. Quantitative T 1 relaxation times were measured using a 2D inversion-recovery turbo spin echo sequence, with inversion times of 50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 and 2500 ms, a repetition time of 3000 ms, and echo time of 18.5 ms, a turbo spin echo factor of 4, a 60 mm field of view, a 3 mm slice thickness, and a 0.5 by 0.5 mm in-plane resolution. High resolution T 1 -weighted images were also acquired using a spin echo sequence and a repetition time of 100 ms, an echo time of 14.1 ms, a 60 mm field of view, a 3 mm slice thickness, a 0.5 by 0.5 mm in-plane resolution and 8 signal averages. MR data was analyzed using in-house software Matlab software. T 1 times were calculated on a pixel-by-pixel basis in each cell pellet. In other words, for each inversion time during signal acquisition, one image was acquired, and a particular pixel in each of the images was used to calculate the T 1 time for that pixel. At each pixel, the signal intensity versus inversion time curve was fitted to the function A 1 2e T I T 1 + e T R T 1, (2.1) where A and T 1 are free parameters, TI is the inversion time and TR is the repetition time. Relaxation rates were calculated by taking the inverse of T 1 and reported as mean values and standard deviations.

32 24 Chapter 2 In vitro characterization of breast cancer cells Figure 2.2 Logarithmic scale of the number of cells as a function of the days after plating. 5 measurements were obtained to generate each data point, and the counting was stopped once the dish containing the cells reached confluency and the cells entered the plateau growth phase. 2.3 Results Cell culture and viability Following treatment of cells with 1.0 mm of manganese chloride, viability was assessed immediately after treatment, and at 24 hours and 72 hours after treatment. None of the cancer cell lines exhibited a significant decrease in cell viability. However, the basal MCF10A cell line showed a slight decrease in viability, to a minimum of 80% viability 72 hours after treatment Solutions and pellet imaging Relaxivity measurements, as described above (Section 2.2.7), were performed with manganese chloride dissolved in PBS, complete growth medium and double distilled

33 25 Chapter 2 In vitro characterization of breast cancer cells Table 2.1 Fraction of live cells in a population of cell treated with 1 mm MnCl 2 for 1 hour. 5 measurements were made, and dead and live cell were counted to generate numbers. Figure 2.3 T 1 -weighted images of cell pellets labelled with concentrations of manganese ranging from 0 mm to 1 mm. rows from top to bottom: LM2, MDA, MCF7 and ZR75 cell lines. Columns from left to right: 0 mm, 0.05 mm, 0.1 mm, 0.2 mm, 0.5 mm and 1.0 mm. Scale bar = 6mm

34 26 Chapter 2 In vitro characterization of breast cancer cells Figure 2.4 R 1 relaxation rate of cell pellets. R 1 was calculated from images obtained with an inversion recovery sequence as previously described (Section 2.2.7). Each color bar represents one cell line and its R 1 at each concentration of manganese. Figure 2.5 R 1 relaxation rate of cell pellets 1, 24 and 72 hours after incubation with manganese. Following treatment, cells were maintained in normal, untreated growth medium and harvested at different time points for imaging.

35 27 Chapter 2 In vitro characterization of breast cancer cells water. The graph of relaxation rates versus manganese concentration was expected to yield a series of points which could be fitted to a straight line. However, in complete growth medium, this was not the case. Similiarly, measurements made in PBS did also not fit a straight line, though the deviation was less apparent than with growth medium containing bovine serum and other components. In water however, the measurements were successfully made and the relaxivity of Mn 2+ was measured to be 7.4mM 1 s 1. With cells, MR imaging was done immediately after the 1 hour incubation time and at 24 hours and 72 hours post labeling. Figure 2.3 shows images of the 4 cancer cell lines labelled with increasing concentrations of Manganese. A significantly brighter signal is clearly evident in LM2 and MDA cell pellets (top two rows respectively) as the concentration of manganese increases from 0 mm to 1 mm, as opposed to MCF7 and ZR75 cell pellets (bottom two rows respectively). The longitudinal relaxation rate R 1 was measured for these cell pellets. Figure 2.4 summarizes these measurements. The most aggressive cell lines, LM2 and MDA showed a significantly greater increase in intracellular Mn 2+ concentration, which is particularly evident at the 0.5 mm and 1.0 mm concentrations. Given that only intracellular manganese ions can cause T 1 shortening, the change in T 1 observed can be attributed to the manganese ions sequestrated into the cells, and R 1 can be calculated and related to the quantity of these ions taken up by cells. Thus, these results suggest that there is a large differential uptake of Mn between aggressive and less aggressive cell lines. The large increase in intracellular manganese observed in aggressive cell lines did not remain over 24 hours and 72 hours following treatment. Figure 2.5 summarizes T 1 values measured at later times. At the 24 hour time, R 1 for MDA and LM2 had already dropped back to pre-treatment levels, whereas ZR75 and MCF10A retained intracellular manganese. Manganese levels for MCF7 cells also dropped after 24 hours,

36 28 Chapter 2 In vitro characterization of breast cancer cells Table 2.2 Inductively Coupled Plasma Atomic Emission Spectroscopy measurement of intracellular manganese following 1 hour treatment with 0, 0.05, 0.1, 0.2, 0.5 and 1.0 mm of manganese chloride, in femtograms. Measurements were made by first quantifying intracellular level of manganese in untreated cells. Then, the mass of manganese was found and normalized to the number of cells. Therefore, this table summarizes the increase of manganese per cell in femtograms (10 15 g) before and after treatment. though this decrease was not as high as with LM2 and MDA Manganese quantification Manganese content was also measured chemically immediately after cells were incubated with different concentrations of manganese. Table 2.2 summarizes these results. Generally, an increase in intracellular Mn 2+ was observed for all cell lines. MDA cells had the highest increase among all cell lines, over 200-fold increase at 1.0 mm. The less aggressive cell lines exhibited much lower levels of manganese as expected. Of note was the manganese content in LM2 cells, which was unexpectedly lower compared to MDA.

37 29 Chapter 2 In vitro characterization of breast cancer cells Figure 2.6 Immunohistochemistry staining of surface CaSR for each cell line in culture, growing exponentially. Cells growing exponentially on cover slips were fixed and stained without perforating the cell membrane, such that only surface CaSR would be stained. The brown color represents a qualitative measurement of surface CaSR. Cells Nuclei are stained dark blue for better visualization. scale bar = 100µm Table 2.3 Colorimetric assessment of the calcium-sensing receptor. A quantitative measure of surface CaSR was obtained by performing a colorimetric assay as described earlier (Section 2.2.6). Three independent measurements were made and the relative optical absorbance per cell in arbitrary units is reported for each cell lines Cellular CaSR histology and quantification Histology showed a low presence of surface CaSR (Figure 2.6) for LM2, MDA and MCF10A cell lines. MCF7 had a moderate staining, and ZR75 had a high amount of CaSR. These measurements were positively correlated with the colorimetric assessment (Table 2.3). 2.4 Discussion Clinical screening and diagnosis of breast cancer relies on finding manifestations of obvious morphological changes, such as formation of a sizeable tumor mass or an

38 30 Chapter 2 In vitro characterization of breast cancer cells abnormal blood supply. Unfortunately, at such a stage, malignant transformation at the cellular level may have already occurred, potentially leading to the formation of metastases. To better manage breast cancer, we need to identify cancer cells at their earliest appearance and be able to determine their metastatic potential in order to personalize treatment. themselves is limited to biopsy. Currently, the ability to characterize cancer cells This study proposes a novel, MR imaging-based method to determine the metastatic potential of breast cancer cells non invasively. Four breast cancer cell lines from very aggressive to less aggressive were investigated (LM2, MDA, MCF7 and ZR75) for their ability to take up manganese, an MR contrast agent. MR imaging showed that aggressive cell lines appeared significantly brighter than less aggressive cell lines due to a significantly larger increase in relaxation rate R 1. Quantification of intracellular Mn 2+ content with ICP-AES attributed the R 1 increase to a higher accumulation of manganese or a more uniform distribution. These results strongly support the potential of Mn-enhanced MR imaging for non-invasive characterization of metastatic potential to achieve very early breast cancer detection. The observations made in this study were mostly consistent with our hypothesis. If manganese is indeed a calcium analogue to assess altered calcium signaling recently identified in cancer progression and metastasis, then MR imaging should be capable of distinguishing cancer cells by virtue of differences in cell uptake of manganese. Cells with greater metastatic potential would accumulate more manganese, which would translate to a brighter signal on MRI with standard signal acquisition techniques. These predictions agree with our MR imaging and ICP-AES results for all cell lines. One discrepancy however, was observed with the LM2 cell line, as the treated cell pellet showed a high increase in R 1, but the ICP-AES measurement was lower than expected. This discrepancy cannot be explained by the errors in Mn 2+ quantification that may have risen from incomplete dissolution of cellular structures. The lower

39 31 Chapter 2 In vitro characterization of breast cancer cells amount of intracellular Mn 2+ observed for LM2 most likely arises from a more uniform intracellular distribution of Mn 2+ as compared to the other cell lines. For this most aggressive cancer cell line, it is possible that the ability to compartmentalize metals is reduced, thus leading to a greater accumulation in the cytosol. Uniform distribution of manganese ions throughout the cell would allow water proximity to Mn 2+ and therefore increase the T 1 effects of the smaller number of Mn 2+ present. A further analysis into the compartmentalization of manganese ions intracellularly in breast cancer cells of different metastatic potential was also performed. Figure 2.4 shows that at 0.5 mm and 1.0 mm, MDA and LM2 had a significantly greater amount of manganese than the other cell lines, but this difference was minimal at lower incubation concentrations. This suggests that between 0.1 mm and 0.2 mm, the intracellular concentration of manganese begins to diverge. One explanation is that in aggressive cells, accumulation of manganese ions into the mitochondria is impaired, leading to the accumulation of free manganese ions in the cytosol and thereby a higher increase in R 1. This is a plausible explanation, given that manganese ions can enter cells through calcium channels, and since high intracellular levels of certain ions, including calcium, initiates their transport into the mitochondria, a process known as uniporter [35]. In other words, more aggressive cancer cells may have an increased tolerance to high cytosolic concentrations of manganese and therefore a higher threshold for the activation of mitochondrial accumulation pathways. In this instance, it is possible that MCF7, ZR75 and MCF10A cells continuously accumulate manganese ions in the mitochondria, whereas LM2 and MDA do not. The mechanism of Mn 2+ uptake by the cells was also investigated. We tested the hypothesis that the CaSR was the primary route of entry of manganese ions into the cells, based on previous reports suggesting that manganese ions enter cells through the CaSR [27]. However, our results do not support this claim. Histology

40 32 Chapter 2 In vitro characterization of breast cancer cells of surface CaSR (Fig. 2.6) revealed that the most aggressive cell lines and the basal cell line had the lowest amount to CaSR, while the less aggressive ZR75 cell line had a much higher amount. Quantitative assessment of these surface receptor agreed with histology findings. Our results suggest that the CaSR is not the primary route of entry of manganese ions into cells. A few of many possibilities include T-type channels [12] and non-voltage gated calcium channels [36]. To understand the mechanisms of manganese uptake in breast cancer cells, further mediators need to be identified and systematically tested. In addition to manganese uptake, we also investigated retention up to three days after cell labeling. LM2 and MDA returned to basal levels of intracellular manganese by 24 hours following treatment. On the other hand, the less aggressive ZR75 cell line and the non-tumorigenic MCF10A cell line both showed sustained levels of manganese even after 72 hours post treatment. This observations reveals that aggressive cancer cells take up manganese more readily, but also release it more quickly. The reasons are unclear, although it is expected that if Mn 2+ is found in the cytosol (as presumed to be the case in aggressive cancer cells), then diffusion back into the extracellular environment could occur quite readily. Although we do not yet understand why differences in retention exist, this difference may serve as a potential metric to more specifically assess metastatic potential of cancer cells. A practical consideration of the proposed technique is whether or not intracellular accumulation of manganese is toxic. It is known that a high intracellular level of Mn 2+ or Ca 2+ can induce cell death through membrane and subsequent mitochondrial damage [37]. However, in the sub-millimolar concentration range used in this study, we did not observe cytotoxicity up to 7 days post cell labeling at the maximum concentration of 1.0 mm tested. The decrease in viability with MCF10A could be attributed to the fact that the cell line was acquired and cultured in low Ca 2+ concen-

41 33 Chapter 2 In vitro characterization of breast cancer cells tration medium for a long time. It is possible that after a long exposure to calcium, changes in cell morphology occurred, which may be responsible for an overall slightly lower viability. Future studies will need to establish the proposed Mn-enhanced MR imaging technique in vivo in animal models of breast tumors and to ensure low toxicity. Fundamentally, we want to prove that this method can identify aggressive cancer cells in the early stages of their appearance and that their identification accurately predicts later onset of metastasis. We also need to establish the optimal protocol for contrast administration, dosing and imaging time-points. Secondly, it would be useful to understand why a differential uptake and release of manganese exists, so that MR imaging can be optimized for more sensitive early detection of breast cancer. In order to achieve this, we would need to determine the fate of manganese ions once they enter cells, as well as the point of entry of these ions into the cell. These studies would provide a better understanding of the biology that MR imaging had indicated to be different in this study. 2.5 Conclusions This study proposes a novel, potentially transformative MR imaging-based capability to characterize breast cancer cell metastatic potential to achieve very early breast cancer detection. It reaps the advantages of MR imaging (non-irradiative, deep tissue penetration, high spatial resolution) for a new cellular imaging method to detect and identify aggressive cancer cells based on altered uptake of manganese ion. It goes beyond traditional approaches, bringing a completely new perspective to cancer imaging, and would be valuable for screening, diagnosis, and treatment monitoring. By enabling early non-invasive identification of breast cancer cells with high metastatic

42 34 Chapter 2 In vitro characterization of breast cancer cells potential, the proposed MR imaging technique may greatly improve cancer patient management and survival, as it would enable early intervention and selection of a treatment most effective for the particular cancer phenotype. Following further improvements in the imaging technique, this research could have tremendous impact and broad implications, providing the basis for investigating many other cancers.

43 Chapter 3 Future directions 3.1 Preamble In the previous chapter, five cell lines were used to show that the aggressiveness of breast cancer cells could potentially be characterized based on their uptake of manganese and subsequent imaging with MRI. As a first step to future work in vivo, one of the breast cancer cell lines was injected orthotopically into rats, from which tumors were formed and imaged before and after administration of the same manganese compound to the rats. The experiment was designed to test whether the manganese ions would indeed enter cancer cells in vivo in sufficient quantities such that it would be detectable by MRI, and to test whether contrast would be improved. 35

44 36 Chapter 3 Future directions 3.2 Experimental procedures Tumor innoculation Successful innoculations LM2 cells were grown as described above (Section 2.2.1) until they reached 80% confluency. Then, cells were harvested, counted, and resuspended in PBS at a concentration of cells/ml. From this solution, 250µL ( cells) were injected into the mammary fat pad of nude (hairless) female immunodeficient rats from Harlan Laboratories. The rats were 6 to 7 weeks old at the time of injection, and weighing approximately 160 g each. For the injection, the rats were anaesthesized with 1.5% isoflurane gas, and a 25G needle was used to minimize trauma to the cells. The injection was carried out with ultrasound guidance, using a 10 MHz pediatric cardiac probe (10 FPA from GE). The ultrasound machine used was a 1992 GE VingMed system equipped with array transducers. Knowing the acoustic scatter given off by the mammary fat pad and following the strong signal reflection given off by the needle, cells were carefully injected into the mammary fat pad of rats, such that based on a visual observation, the entire solution was contained within the fat pad. Following injection, tumors were visible and imaged beginning at 6 days after injection. Unsuccessful innoculations Prior to successful innoculation of the previously described tumors, several unsuccessful attempts were made. First, ultrasound guidance proved difficult to use because the visualization of the needle reflection depended on the alignment of the array transducer with the needle, and this was not easily achieved with a single operator. Therefore, it is possible that for these injection attempts, cells were injected intramuscularly rather than orthotopically, thereby reducing the probability of tu-

45 37 Chapter 3 Future directions mor formation. Next, some injections were done subcutaneously into the flank of these rats, but although this model works well with immunodeficient mice, it proved more difficult to do with rats. There are theories that propose that rats acquire immunodeficiency over time, which if true, would definitely agree with our poor tumor formation outcome. An additional technique tried involved the mixture of a solution of cells with matrigel for injection. Matrigel is a viscous substance that solidifies at temperatures above 10 degrees Celsius and forms an extracellular matrix, which supports initial cell attachment and provides nutrients for cancer cells newly injected into animals. It supposedly also supports the formation of tumor vasculature, and allows cells to stay in one place after injection. For the matrigel injections, a cell solution containing 25% matrigel (v/v) was prepared and kept on ice until injection. Rats were anaesthesized as described in the previous section, injected into the flank, and maintained under anaesthesia for 10 min to allow matrigel to solidify completely. Note that for all of these injections, the total number of cells injected was always per rat, and the volume of injected solution was approximately the same. Although the rats injected as described in this section are labelled as unsuccessful, some of these tumors grew, albeit much more slowly than the successful orthotopic model Manganese administration Manganese chloride was dissolved into sterile saline to a concentration of 25 mm, and filtered using a 0.22µm filter. The dose administered in this study was chosen with regards to doses previously used with mice [27]. A dose of 0.05 mmol/kg was administered per rat, which is equivalent to 9.9 mg/kg. Therefore, following pre-contrast scans, 0.05 mmol/kg manganese chloride in saline was administered subcutaneously in the neck of rats, and the animals were imaged again after 24 hours.

46 38 Chapter 3 Future directions Rat imaging Rats were maintained anaesthesized with 1.5% isoflurane gas delivered through inhouse set up. Rats were imaged in a 8-channel wrist coil on the same 3T scanner as cell pellets were. A T 2 -weighted turbo spin echo sequence was used to localize the tumor and a high resolution T 1 -weighed spin echo sequence with fat saturation was used to assess manganese contrast. The sequences were set up as described above (Section 2.2.7). The rats laid prone in the coil, and coronal slices were acquired so as to cover the entire tumor, and representative slices were selected and shown below. 3.3 Observations and Discussion Manganese-enhanced contrast For the two tumors shown, a rim-like enhancement was observed 24 hours after injection of manganese chloride (Figure 3.1). Furthermore, we noticed that the tumor had sharper boundaries, suggesting that cells at the rim of the tumor had a higher uptake of manganese than cells in the center of the tumor. This observation makes sense, as the blood vessels typically nourish the periphery of the tumor better than the core, thereby providing peripheral cells a higher and longer exposure to circulating intracellular manganese. Furthermore, the necrotic core observed with one of the tumors enhanced, based on a qualitative assessment, following manganese administration. This observation suggests, given that tumor blood vessels do not reach necrotic cores, that manganese passively flows into the tumor and gets taken up by cells around the core of the necrosis. If this proves to be true, then manganese could potentially be used to visualize live cells at the center of an area of necrosis. It was also observed that the quantity of manganese chloride administered was

47 39 Chapter 3 Future directions Figure 3.1 MR images of rats 1 and 2 prior and 24 hours after manganese administration. T 2 -weighted images were obtained for tumor localization. The tumor of each of the two rats is circled in yellow. T 1 -weighted spin echo with fat saturation images of rats were acquired before and after subcutaneous injection of 0.05 mmol/kg of manganese chloride. On the T 2 -weighted image of rat 2, a necrotic core (dark region) can be seen. scale bar = 5cm.