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Abstract
The study of the development of anti-cancer drugs and preclinical toxicity tests has until today encountered a major problem identified as lack of a reliable in vitro-tumor model able to mimic in vivo conditions. These models provide a clear basis for understanding tumor-development processes and assists in the selection of agents from various chemicals to test the efficacy of drugs on cancer cells or tumor tissues. There are two important issues for an ideal cancer culture model. These include tumor-like cell aggregates, and the in vivo-like culture microenvironments. To address these two problems, an in vitro perfused alginate based on a three-dimensional cancer model and a 3D culture of cancer cells and related toxicity tests of the cancer drugs were done. The cells in perfusion culture showed higher proliferation rates and significantly, higher cell viabilities after a 6-day culture compared to statically cultured cells, especially for the cells in the 3D culture. MTs formed from these cancer models showed significant tumor-like morphological characteristics, a denser and highly stable structure, a higher cell viability, and varied drug response rates compared with spheroids.
Studies on the toxicity of drugs considered in this thesis relied on a systematic approach based on bi-directional cell viability studies, micro tumor formations, and drug responses on cells in different models of cancer in static and perfusion culture environments. The toxicity of paclitaxel and cisplatin drugs were compared between cells on monolayer spheroids, MTs, and real human tumor tissues with observations showing that the cells in 2D culture had the lowest cell viabilities while human tumors showed the lowest drug responses. Besides that, the response of drug to MTs was significant on human tumors, when the values of spheroids are relatively closer to the cells in 2D culture. When compared, the toxicity of the drugs on cells in static and perfusion culture with the cells in different models showed significantly different drug responses except for the cells on the monolayer. In addition to that, the difference between static and perfusion culture have shown varied behaviors on cells in different models with different action mechanisms on anti-cancer agents. The perfusion culture provides a steady, homogenous, and physiological tumor microenvironment for the growth of cells and thus affects the response of the drug and resistance rates. In conclusion, in vitro perfused 3D cancer model, developed in this thesis, proved valuable for cancer cell culture and related anti-cancer drug tests.
I could like to thank the following individuals, without their support, this work could not have been completed. Professor Zhanfeng Cui for his academic, administrative, intellectual, and moral support, Dr. Paul Ananda Raju who provided the initial foundational knowledge to conduct this work, Dr. Richard for providing the cell lines; Dr. Yasser for his fluomicroscope training and technical supports. Further, thanks to Dr. Cathy and Dr. Shengda Zhou for their administrative support and thoughtful advice, Dr. UdayTirlapur for the knowledge on the multi-photon microscope. On the other hand, my thanks go to Dr. zhaohui Li for her academic advice and bioassay advice, Dr. Xia Xu for her training session on Tissue Flex bioreactor making and setup; Mr Pierre-Alexis Mouthuy for his support and discussion of collagen scaffold; Dr. FengGu for his laboratory/human tissue facilities, assistance with the human cancer tissue work. On the other hand, my thanks go to Miss Kathy for advice on statistical analysis. All the other members of the Institute of the Biomedical Engineering group for scientific discussions, Tianjin Medical University Cancer Institute and Hospital for the supply of human tumor tissue samples, and all my friends and family members (especially mom and dad) for their moral support.
Introduction
Background
Despite significant advances in both radiotherapy and chemotherapy in the treatment of cancer over the last 200 years, cancer, one of the most dangerous diseases still remains one of the leading causes of deaths in the world today (Jemal et al., 2005). A statistical analysis by Thurston (2006) has shown that nearly one in three of the population in the world suffer from cancer while one in four die from cancer. In addition to that, Thurston (2006) and Thurston (2006) have shown that cancer is increasingly becoming prevalent and the chances of getting completely cured have declined significantly.
Different techniques are available for the treatment of cancer today. The technique often applied on a patient depends on the type of cancer and the progress or stage of the disease in the patient. Combinations of chemotherapy, surgery, and radiotherapy have shown significant improvements in the treatment of cancer when compared with the single treatment approach. Chemotherapy is applied before surgery to shrink tumor tissues making their removal easier. In addition, applying chemotherapy after surgery destroys and clears the remaining cancer cells completely. The importance of applying chemotherapy is to provide a chance for the development of anti-cancer drugs. However, currently, a major limitation in the development of the drug is limited knowledge on tumor model that could provide the selection criteria for agents from various chemicals to test the anti-cancer drugs for their efficiency in destroying cancer cells or tissues before sanctioning clinical usage of the drugs.
According toHorning et al., (2008), earlier studies and tests were at first animal-based in vivo models while current models are mostly 2D cell line based on in vitro models. However, these models were ineffective. Researchers took nearly 100 years to develop a reliable model for testing the drugs. The animal model presents similar drug responses due to the in vivo environment but the difference between animals, usually the mice and human body cannot be ignored. Cells based on the monolayer assay have become the main techniques in many stages of anti-tumor drug tests especially in the first step of studying the discovery of the anticancer drug. That is due to the ease of providing enough samples for large amount chemicals to test from the monolayer structure. However, when applying the model to test the efficiency of a drug, the discrepancy between the monolayer structure and the in vivo situation is significant. The three-dimensional culture is believed to mimic the 3D structure, in vivo-like tumor characteristics, which spans the gap of the monolayer culture and the animal test model.
The 3D structure affects the growth of cells and genes thus affecting the drug response as well. It has been proved that the IC50 values of anti-cancer drugs are significantly higher in 3D models than in 2D culture models (Horning et al., 2008). An in vitro 3D tumor model that could accurately show the toxicity assay of anticancer agents is apparently required for in vitro cancer cell cultures and in the research for anticancer drug development. However, in the study of 3D cancer cell culture, it is a challenge to design a scaffold, which could process the desired tissue properties (Horning et al., 2008). Current scaffolds have more or less limitations not only tumor biology but also have technical limitations such as time-changing environments, are time-consuming, and costly. This study took into consideration biomedical, technical, and economic needs when developing the alginate-based perfusion in vitro3D culture model, which employed the 3D cancer cell culture using related drug tests techniques.
Scope of Thesis and Thesis Outline
There are many techniques to carrying out 3D culture. These include scaffolds based 3D models with the most popular being the spheroids model based on culturing onto an agar-coated surface. Depending on the culture approaches and biomaterials used for 3D culture, test cells present different growth status and characters and all of these will have an effect on the response of drugs to the agents during the test. Thus, the main purpose of this study is to build a reliable model to better present in vivo tumor characteristics and provide accurate data as a baseline for clinical treatments.
In order to improve the accuracy of tests the toxicity of anti-cancer drugs, two main methods are used to improve both tumor tissues and the tests environment. Improving tumor tissue implies building a 3D micro tumor tissue, which has similar characteristics to the real tumor tissues from the human body by culturing cells in 3D scaffold. This could reduce the differences between real tumor tissue and the samples, to be used in drug tests to provide more reliable toxicity test results. In order to achieve this purpose, DLD1 and NCI/ADR cells were cultured in the developed alginate based in vitro3D culture model to form 3D micro tumors that have the 3D structure and similar characteristics to real solid tumor tissues. The MTs were compared with the currently most used 3D model (spheroids) and real human tissues from breast cancer and colon cancer patients to study the discrepancy between these models. Secondly, the perfusion culture was added into the alginate based 3D culture model in a stable physiological environment for tumor formation and drug tests procedures. Not only can the perfusion system improve the tests environment, which is similar to the internal human environment, but also it can support MTs formation and improve cell viability by keeping perfusing nutrients in which it is acting as blood vessels in tumor tissues active. Therefore, owing to these two improvements, the alginate based perfusion 3D culture model developed in this thesis is believed to improve the accuracy of anti-cancer toxicity of the drugs tests. The results and concepts in the proceeding chapters will be of value to researchers in the development of tumor models for anti-cancer drug tests.
Chapter 2 discusses the historical developments of the cancer disease, chemotherapy, and types of anti-cancer drug test models. Chapter 3 focuses on the basic toxicity study of cisplatin and paclitaxel to DLD1 and NCI/ADR cell lines cultured on a monolayer. In addition to that, the concentrations of each of the three drugs selected according to the drug dose responses will be conducted to study cisplatin and paclitaxel. Besides this, observations on the toxicity of alamar blue were done during the cytotoxicity study with the MTT assay selected for further toxicity of the drugs measurements. The next chapter is the basic study of the research with the following structure: Chapter 4 is devoted to adaptation of bioreactors into the monolayer cell culture, perfused cell culture, and toxicity of the drugs assay. On the other hand, in order to apply the three dimensional culture, the structure of bioreactor was improved as compared to the old version developed by Z.F.Cui and X.Xu in our lab (Cui et al., 2007). As a comparison of static and perfusion culture, study cells were cultured based on the method of monolayers. The proliferation and viability of cells were measured during the culture periods and the toxicity of the drugs responses were compared for the two drugs. The study in this chapter provided the basis for developing 3D perfusion culture model in the next part of the work.
In Chapter 5, the alginate based in vitro three-dimensional perfusion cancer model was developed. This model was employed to DLD1 and NCI/ADR 3D cell cultures and toxicity of the drugs study of cisplatin and paclitaxel. This part of the study focuses on three types of comparisons. First, comparisons of the drug responses of cisplatin and paclitaxel to DLD1 and NCI/ADR cells were done between 2D and 3D culture. Secondly, the characteristics of MTs formed in alginate beads were compared and drug responses to spheroids from the agar-coated surface were examined. Finally, the growth of cells status, MTs formation, and drug responds are examined when cultured under static and perfused conditions. Based on these comparisons, the 3D perfused cancer culture model developed in this thesis was proved as an improvement where cells in this model were presented with characteristics of the tumor-like structure. Thus, the model can provide data on the in vivo-like microenvironment for 3D cancer cell culture, relative toxicity of the drugs, and chemo-resistance in the study. Chapter 6 is a report on the study of the toxicity of drugs on the human tumor tissues, breast cancer tissue, and colorectal cancer tissues. This study compares the results with MTs, spheroids, and monolayer culture. A conclusion and suggestions for future works to understand in detail the complexity of in vitro tumor models, the studies are discussed in Chapter 7.
Literature Review
Cancer Disease and Chemotherapy
What Is Cancer
Nearly one third of the global population suffer from different types of cancer today with a prevalence rate showing significant appreciation. Studies by Thurston (2006) in the United Kingdom as an example showed the percentage of deaths related to cancer as only 10% of the entire deaths in the 1900s. New statistic show that about 160,000 people die from cancer every year, which is approximately 25% of all annually, registered deaths in the U.K. In addition to that, a statistical analysis by Thurston (2006) has shown that nearly one in three of the population in the world suffer from cancer while one in four die from cancer. On the other hand, Thurston (2006) has shown that cancer is increasingly becoming more prevalent and the chance of getting completely cured has diminished significantly. Even if the lumpectomy is successful, chemotherapist treatments remain dangerous on the normal tissues during and after the treatment period when applied concurrently with drugs to kill the tumor cells. This might leads to renal failure or damage to the immune system damage resulting from toxic reactions. One optimistic view is that while the patient has successfully undergone all treatments and seems to attain a cancer free cells status, we still are not sure whether the cancer will attack the patient the following years. Since cancer is a gene-related disease, it is a hidden problem mainly due to the occurrence of unexpected changes of the gene structure, a situation not to ignore, giving us a big therapeutic challenge(Kamb, 2005).
Cancer is a complex disease since caused by uncontrolled growth of cells in any part of the human body, introducing a lump of solid mass of cells known as a tumor. This is different from normal tissues with a growth rate of new cells and the death of old cells balanced. A disruption of the balance characterizes cells attacked by cancer. This disruption results from uncontrolled growth of cells or the loss of the cells ability to undergo suicide by a process called Apoptosis .
Cancer could originate in any organ and could spread to other parts if it is a malignant tumor. Scientists use a variety of technical names to distinguish different types of carcinomas, sarcomas, lymphomas, and leukemia (Weinberg, June 2006). Carcinoma is the most common type of cancer, arising from the cells that cover external and internal surfaces of the body such as the Lung, the breast, and colon. Sarcomas are cancers caused by cells found in the supporting tissues of the body such as bone, cartilage, fat, connective tissue, and muscle. Lymphomas are cancers that results from the lymph nodes and tissues of the bodys immune system. Leukemia is a class of cancer of immature blood cells that grow in the bone marrow and tends to accumulate in large numbers in the bloodstream.
Previously, people regarded cancer as a potentially dangerous disease due to cancer cells ability to spread to any part of the body, making surgical intervention impossible. Cancer is capable of spreading by two mechanisms: invasion and metastasis (Cavalli, 2009). Invasion refers to the direct migration and penetration of cancer cells into neighboring tissues. Metastasis refers to the ability of cancer cells to penetrate into lymphatic and blood vessels, circulating through the bloodstream to invade normally functioning tissues in other parts of the body. The tissue, which spreads this way, are called malignant tumors.
Cancer is perceived as a disease that attacks its victim with no apparent medical reason. While scientists have not known actual causes of cancer, a number of causes have been identified in scientific circles (Danaei et al., 2005). According to Doll and Peto, (1981), besides the intrinsic factors such as genetic heredity, diet, tobacco, and hormonal problems, scientific studies point to key extrinsic factors contributing to cancer developments as triggered by changes in a cells genes, chemicals, radiation, and viruses or bacteria (Kuper et al., 2000). Chemical and radiation induced damages on genes, virus induced damages, and alterations introduced into genes and hereditary transmitted alterations in genes makes a person more susceptible to cancer attacks. Genes are inherited instructions that reside within a persons chromosomes. Each gene instructs a cell on ways to build a specific product, in most cases, a particular kind of protein. Gene alterations or mutations in various ways forms part of the mechanism causing cancer.
One group of genes leading to the development of cancer are damaged genes called oncogenes (Paramythiotis et al., 2003). By producing abnormal versions or quantities of cellular growth-control proteins, oncogenes cause a cells growth-signaling pathway to become hyperactive. The second in the group of genes strongly correlated to the development of cancer are the tumor suppressor genes. Tumor suppressor genes, such as the p53, according to Greenblatt et al., (1994), are normal genes with an absence that has the susceptibility of causing cancer. In other words, if a pair of tumor suppressor genes are either lost from a cell or inactivated through mutations, the functional absence allows the development of cancer. Individuals who inherit genes have an increased risk of developing cancer since they are born with a defective copy of a tumor suppressor gene. On the other hand, genes come in pairs (one inherited from each parent). An inherited defect in one copy will not lead to cancer, but if the second copy undergoes mutation, the person may develop cancer since there are no longer any functional copies of the gene.
A third type of genes implicated in cancer is the DNA repair gene. DNA repair genes code the proteins whose normal function is to correct errors that arise when cells duplicate their DNA prior to cell division. Mutations in DNA repair genes can lead to a failure in repair, which in turn allows subsequent mutations to accumulate (Edelmann et al., 1997).
The treatment of cancer is often a multipronged approach depending on the type of cancer and the stage or progress it has made into patient. Currently, the main approaches of cancer treatments are surgery, radiotherapy, and chemotherapy. Early diagnosis can lead to higher chances of cure and a reduced risk of the formation of secondary tumors when chemotherapy and radiotherapy are systematically applied.
Surgery is the first line of treatment used to fight cancer but is only effective when tumors are small and well defined. Even in the early stages, a patient needs chemotherapy and radiotherapy treatments after undergoing surgery to eliminate any left out cancer cells. Typically, chemotherapy and radiotherapy are complimentary before surgery to reduce the tumor and make surgery easier to remove any remaining cancer cells. However, when a tumor has spread to other organs of the body, it is difficult to remove all the tumor tissues and keep the organs functional.
On the other hand, radiotherapy uses X-ray or radiopharmaceuticals, as sources of ³-rays, which damage DNA and kill cells. As X-ray cannot only damage tumor tissue, radiation is directed towards the local tumor from a focused beam to protect healthy tissues. However, stray radiation exposes healthy tissues to the destructive effects of the side effect caused by the radiation. However, the latest technology consists of multiple beams configured to focus high-energy intensities on tumor tissues while exposing surrounding tissues to lower radiation energies. However, cancer cells are hypoxic compared to normal tissues, hence less sensitive to irradiation. Thus, oxygen or radio sensitizing drugs applied prior to radiotherapy sensitize tumor cells to get better results.
Chemotherapy cancer treatments rely on low-molecular-weight drugs to control the growth of cancer cell by killing tumor cells. Chemotherapy could assist surgery and radiotherapy to control the growth of tumor by killing cancer cells left behind after surgery or cells in the metastases status (Herskovic et al., 1992). Nevertheless, anti-tumor drugs are toxic to both cancer cells and healthy cells inducing side effects such as bone marrow suppression, GI tract lesions, hair loss, nausea, and the rapid development of clinical resistance (Gottesman, 2002). It is important to understand the development of chemotherapy as explained in the next section.
According to Talalay et al., (1988), to prevent cancer, the first step is to reduce the chances of exposure to particular kinds of carcinogens and virus with the greatest cancer hazards. Thus, people are advised to pay daily attention by reducing the use of tobacco products and avoiding excessive exposure to sunlight. In addition to that, limiting uptake of alcohol, tobacco, fats, and calories are other recommended measures (Armstrong and Kricker, 2001; Xu et al., 1992).
History of cancer
The Greek physician Hippocrates 460370 B.C., regarded as the father of medicine, was the first to coin the word Cancer . He described tumors as carcinos and carcinoma also referred to as crab in Greek (2010). At the time, Hippocrates thought that the finger-like spreading tumor was similar to the shape of a crab. Later, in 28-50 B.C.,Celsus, a Roman physician, used the term Cancer instead. The term cancer also referred to which also referred to the crab in Latin. Another Roman physician Galen named tumor as oncos , in Greek, meaning swelling. He believed cancer was curable by cutting or cauterizing the infection at an early stage. Although this term is not well accepted as cancer , oncology is used instead to describe a specialized area of cancer research (Houten, 2009).
According to historical records, the earliest evidence recorded about cancer was by the ancient Egyptians, dating back to 1500 B.C. According to a copy found in an ancient Egyptian textbook on trauma, 8 cases of surgery were documented on tumors that happened on breasts. They did the palliative treatment with a tool called a fire drill, the only recorded cancer treatment. The ancient Egyptians thought God caused cancer. However, recent research at Manchesters KNH Centre for Biomedical Egyptology of Egypt mummies has shown cancer was a rarity in ancient Egypt. The conclusion of this study is that cancer is a manmade disease and might be cause by environmental factors (Manchester, 2010).
Hippocratess theory proposed at the time that the human body was composed of four fluids, the blood, phlegm, yellow, and black bile. He argued that the cause of cancer was an excess of black bile. It was until the beginning of the Renaissance period, as early as the 15th century, when the Italians started to get a better understanding of the human body. At that time, scientists showed some important theories during that period, laying the foundation for the study of cancer. These included the understanding of blood circulation, the idea that the removal of a tumor was not possible if it had invaded the surround tissues without anesthesia. In 19th century, Rudolf Virchow, also known as the finder of cellular pathology made a big step in the cancer research owing to the use of the microscope. The microscope not only enabled a better understanding but also helped doctors to better diagnose the problem. The 20th century saw progress in many relevant area of research in cancer. Researchers had spread their research into cancer stem cells, Angiogenesis, Tumor Suppressor Genes, Tumor Microenvironment, Oncogene, Targeted Cancer Therapy, and any others similar areas. Better understandings of the characteristics and behavior of cancer cells lead doctors to apply systematic treatment methods to kill these cells, which spread through the body after surgery. In addition to that, after long time fighting with cancer, today doctors are able to give patients better diagnosis with multiple modern treatments. That is achievable by removing the primary tumors by combining radiation therapy with chemotherapy.
History of Chemotherapy and Anticancer Drug Development
Prior to the 20th century, the treatment of cancer was mainly limited to surgery and radiotherapy. However, due to micro metastases, the rate of cure was limited to approximately 33%. The use of Chemotherapy to fight cancer started as early as the 1900s. At that time, the German chemist, Paul Ehrlich, the father of chemotherapy emphasized on the technique by using chemicals to treat the disease. He was also the first person to employ the animal model to test the potentiality of anticancer chemicals. It was the first case introduced using drugs in the treatment of cancer in the human breast infected with cancer. The evidence showed that combining chemotherapy with surgery and radiotherapy might cure advances in cancer and make the drug attain optimal effects (Urschel et al., 2002, Steel & Peckham, 1979). The importance of chemotherapy created the opportunity for the development of anti-cancer drugs. The following picture lists the major events of cancer chemotherapy and related drug developments.
The two major limitations in drug developments using the tumor models were the inability to mimic accepter agents and the supply of facilities to test these drugs in any clinical organization. The first animal model used for drug tests was a rabbit model, used by Paul Ehrlich to test arsenicals to treat syphilis. However, early models for cancer drug tests were mostly tumor models in mice. In 1910s, George Clowes of Roswell Park Memorial Institute (RPMI) in Buffalo, New York, in the Roswell Park Memorial Institute made a major breakthrough with the first transplantable tumor model developed in rodents (DeVita and Chu, 2008).
Based on Paul Ehrlichs research, early research scientists focused their studies for decades on different cancer models to test cancer drugs. The early models based on mice included the Sarcomas 37 model, usually used to test an array of compounds discovered by Murray Shear in 1937, the S180 model, and some other models also based on murine. Among the large amounts of different tumor models, Leukemia 1210 (L1210), murine leukemia induced by a carcinogen was considered as the most versatile animal tumor screening system and was adopted by the NCI as its primary screen (Grever et al., 1992).
It was in 1975 when scientists wanted to improve their prediction activities on anticancer drugs in humans that the L1210 murine model was replaced with Xenograft models. Here, human cancer was transplanted into an immune-deficient mice (Mattern et al., 1988).This new screen method can test a drug in vivo using similar tissues. However, due to the high cost and long pre-work periods, the numbers of drugs tested reduced significantly from 40,000 per year to 10,000. Similar researches on xenograft models and drug tests were conducted at the time (Dass et al., 2007, Needham et al., 2000).
Ying Zhang et al., described a systematic method to inhibit the growth of tumors in vivo culture of anti angiogenicendostatin-secreting Chinese hamster ovary (CHO) cells in microcapsules as small as 200¼m in diameter (Zhang et al., 2007). The microencapsulation-based in vivo culture method offers a safe, highly efficient, and low-cost anti-angiogenesis approach to tumor therapy. The implantation of microcapsules of small sized and highly concentrated peritoneal cavity has the potential for use in the treatment of solid tumors.
J. K. Peterson and P. J. Houghton (Peterson & Houghton, 2004) reviewed the value and limitations of xenograft models by comparing data from animals and the human body. Results showed a significant difference between them, i.e. the in vitro serum protein binding showed 8082% irofulven binding in mice and 5152% binding in man, when systematically exposed to irofulven across the species.
These findings helped researchers understand the variance between preclinical tests with concrete clinical activity results. The agents had antitumor activities on tumors in mice, but were shown to fail when tested in man. These disappointments led to reasonable doubts about the true value of xenograft rodent tumor models by accurately identifying agents with significant clinical utility (Voskoglou-Nomikos et al., 2003). As discussed above, perhaps the basic problem was the difference between man and animals.
By 1990, developments in drug testing methods saw new developments in screen system based on human cancer cell lines become the main cancer tests and treatment methods. This improvement was due to the development of culture solutions first developed by the English physiologist Sydney Ringer. After nearly 30 years of study, Ross Granville Harrison published the results of his experiment and the tissue culture technique used in the study. In later years, cell culture techniques advances became the main methods to support research in biology, virology, and oncology. Thus, culturing cells on monolayers in the laboratory exposure of drugs became new and significantly utilized techniques for tests the toxicity of cancer fighting drugs.
Anticancer drug
Overview of mechanisms of action of chemotherapeutic agents
The anticancer drugs use different active mechanisms to control or interfere with the growth of tumor cells. Anti-cancer drugs fall into six groups based on the different target mechanisms. These mechanisms include, antimetabolites, DNA-Interactive Agents, anti-tubulin agents, signal transduction inhibitors, hormonal agents, and biological response modifiers, anti-angiogenic agents and so on. The following table lists typical anti-cancer drugs of each group and the specific action mechanisms for each category (Table 2.1).