EVALUATION OF PROPHYLACTIC ANTINEOPLASTIC ACTIVITY OF PLACENTAL LYSATE IN HEPATOCELLULAR
CARCINOMA IN EXPERIMENTAL ANIMAL MODEL
A DISSERTATION SUBMITTED TO THE TAMIL NADU DR.
M.G.R. MEDICAL UNIVERSITY IN PARTIAL FULFILMENT OF THE REGULATIONS FOR THE AWARD OF M.D. DEGREE IN PHARMACOLOGY (BRANCH VI) EXAMINATION TO BE HELD IN
APRIL, 2017
DEPARTMENT OF PHARMACOLOGY AND CLINICAL PHARMACOLOGY
CHRISTIAN MEDICAL COLLEGE
VELLORE: 632 002, TAMIL NADU, INDIA
CERTIFICATE
This is to certify that this dissertation entitled “Evaluation of Prophylactic Antineoplastic Activity of Placental Lysate in Hepatocellular Carcinoma in Experimental Animal Model” submitted by Dr. Saibal Das, in partial fulfillment of university regulations for the award of M.D. Pharmacology (Branch VI) degree examination of The Tamil Nadu Dr. M.G.R. Medical University, Chennai to be held in April, 2017 is a bona fide original work done under my direct guidance and supervision and completed to my utmost satisfaction.
Date:
Place: Vellore
Dr. Margaret Shanthi F.X.
Guide, Professor, Department of Pharmacology and Clinical Pharmacology,
Christian Medical College, Vellore
CERTIFICATE
This is to certify that this dissertation entitled “Evaluation of Prophylactic Antineoplastic Activity of Placental Lysate in Hepatocellular Carcinoma in Experimental Animal Model” is a bona fide original work of Dr. Saibal Das under the guidance of Dr. Margaret Shanthi F.X., Professor, Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore and Dr. Sathya Subramani, Professor and Head, Department of Physiology, Christian Medical College, Vellore towards partial fulfillment of university regulations for the award of M.D. Pharmacology (Branch VI) degree examination of The Tamil Nadu Dr. M.G.R.
Medical University, Chennai to be held in April, 2017.
Date:
Place: Vellore
Dr. Binu S. Mathew Professor and Ag. Head, Department of Pharmacology and Clinical Pharmacology,
Christian Medical College, Vellore
This is to certify that this dissertation entitled “Evaluation of Prophylactic Antineoplastic Activity of Placental Lysate in Hepatocellular Carcinoma in Experimental Animal Model” is a bona fide original work of Dr. Saibal Das under the guidance of Dr. Margaret Shanthi F.X., Professor, Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore and Dr. Sathya Subramani, Professor and Head, Department of Physiology, Christian Medical College, Vellore towards partial fulfillment of university regulations for the award of M.D. Pharmacology (Branch VI) degree examination of The Tamil Nadu Dr. M.G.R.
Medical University, Chennai to be held in April, 2017.
Date:
Place: Vellore
Dr. Alfred Job Daniel Principal, Christian Medical College, Vellore
DECLARATION
I, Dr. Saibal Das, do hereby declare that this dissertation entitled “Evaluation of Prophylactic Antineoplastic Activity of Placental Lysate in Hepatocellular Carcinoma in Experimental Animal Model” has been done by me under the direct guidance of Dr. Margaret Shanthi F.X., Professor, Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore and Dr. Sathya Subramani, Professor and Head, Department of Physiology, Christian Medical College, Vellore in partial fulfillment of university regulations for the award of M.D. degree in Pharmacology (Branch VI). I have not submitted this dissertation in any part or full to any other university or towards any other degree.
Date:
Place: Vellore
Dr. Saibal Das
First and foremost I thank the Almighty God for the completion of this research work and His overall support in my life. He enabled my guide, co-investigators and me with necessary intellect and wisdom in carrying forward the project which may help in steering the progress of future research in this subject.
I would like to thank my parents Mr. Chandan Kumar Das and Mrs. Ruma Das for their constant guidance, love and support at all times.
I express my sincere gratitude to my guide Dr. Margaret Shanthi F.X., Professor, Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore and co-guide Dr. Sathya Subramani, Professor and Head, Department of Physiology, Christian Medical College, Vellore for their genuine guidance in this project. Their able supervision helped to plan, conduct and overcome every troubles and obstacles faced while doing this work.
I whole heartedly thank Dr. Kalpana Ernest, Professor and Head (retired), Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore for the endless support she has provided in carrying out this research work. So also I would like to thank Dr. Binu S. Mathew, Professor and Ag. Head, Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore for her constant motivation and support.
Besides, I would like to express my heartiest gratitude to Mr. Soosai Manickam A.,
Vellore, who were all instrumental in conducting the work at the bench side.
I would like to specially thank Dr. Anne George Cherian, Assistant Professor, Department of Community Health and Development Hospital, Christian Medical College, Vellore for providing the placenta sample used in the experiment, Dr.
Deepak V. Francis, Assistant Professor and Dr. J. Suganthy, Professor, Department of Anatomy, Christian Medical College, Vellore for assisting us in the histopathological examinations. I am also indebted to Dr. Joe Varghese, Department of Biochemistry, Christian Medical College, Vellore for helping with the protein assay. I also thank the Center for Stem Cell Research, Christian Medical College, Vellore for using the fluorescent microscope and imaging facility.
I would like to thank my other esteemed faculties from the Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore viz.
Dr. Manoj G. Tyagi, Dr. Jacob Peedicayil, Dr. Denise H. Fleming, Dr. Ratna Prabha Gupta and Dr. Sumith K. Mathew for their insights and inspirations. Especially I like to acknowledge the valuable inputs of Dr. Jacob Peedicayil while writing the thesis.
I would like to thank my seniors, colleagues and friends from the Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore viz.
Dr. Rohit Kodagali, Dr. Aswathy Rachel Thomas, Dr. Sumalya Sen, Dr. Jaya Ranjalkar, Dr. Deepasree Sukumaran, Dr. Jayanta Kumar Dey, Dr. Jeana Jacob, Dr.
Niranjan Prabhu S. S. and Dr. Premila Wilfred for their constant support, intelligent
I would like to thank Mrs. Anita J., Senior Technician, Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore, Mrs. Sudha J., Secretary and Mr. Anbarasu E. and Mr. Karunakaran L., Attendant, Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore for their timely help and assistance.
Also I would like to thank the mother who cheerfully consented to donate the placenta after her delivery in the Community Health and Development Hospital, Christian Medical College, Vellore to facilitate this work.
Finally, I would like to thank Ms. Preeti Barnwal, Junior Research Fellow, Department of Medical Elementology and Toxicology Faculty of Science Jamia Hamdard (Hamdard University), New Delhi for the overall support and assistance in the write-up.
Last but not the least, I would most gratefully acknowledge the Institutional Review Board and Institutional Animal Ethics Committee, Christian Medical College, Vellore for giving approval to the project and providing intra-mural fluid research grant, without which the work would not have been completed.
Abbreviation Expansion AFP Alpha fetoprotein BCA Bicinchoninic acid DEN Diethylnitrosamine HBV Hepatitis B virus
HCC Hepatocellular carcinoma HCV Hepatitis C virus
HLA Human leukocyte antigen i.p. Intraperitoneal
IPT Immunoplacental therapy
MHC Major histocompatibility complex
MP Membrane proteins
PBS Phosphate buffer saline
PP Placenta proteins
TAA Tumor associated antigen
VEGF Vascular endothelial growth factor
Table no. Table legend Page no.
1 Various tumor antigens common to trophoblast and
tumor 23
2
Body weights (g) at the time of death than before administering DEN in the three groups of mice (n=6 in each group)
47 3 Survival (days) of Group 1 and 2 animals after
administration of DEN (n=6 in each group) 49
Figure no. Figure legend Page no.
1 The structure of placenta by cross sectional view 16 2 Structure of a villous tree connecting the maternal surface
(basal plate) and the fetal surface (chorionic plate) 18 3 Standard curve for BCA protein assay by
spectrophotometry (540 nm) 38
4
Box-plot showing the changes in the body weights at the time of death than before administering DEN in the three groups of mice.
48 5 Kaplan-Meier survival analysis in three groups of animals
after administration of DEN. 49
6a, 6c, 6e Histopathological features of liver from a representative
Group 1 mouse showing features of HCC (H & E, 10X). 51-53 6b, 6d, 6f Histopathological features of liver from a representative
Group 1 mouse showing features of HCC (H & E, 40X). 52-54 7a, 7c, 7e Histopathological features of liver from a representative
Group 2 mouse showing features of HCC (H & E, 10X). 54-56 7b, 7d, 7f Histopathological features of liver from a representative
Group 2 mouse showing features of HCC (H & E, 40X). 55-57 8a, 8c, 8e Histopathological features of liver from a representative
Group 3 mouse showing features of HCC (H & E, 10X). 57-59 8b, 8d, 8f Histopathological features of liver from a representative
Group 3 mouse showing features of HCC (H & E, 40X). 58-60 9a Histopathological features of kidney from a representative
Group 1 mouse showing no abnormalities (H & E, 40X). 60 9b Histopathological features of kidney from a representative
Group 2 mouse showing no abnormalities (H & E, 40X). 61 9c Histopathological features of kidney from a representative
Group 3 mouse showing no abnormalities (H & E, 40X). 61 10a, 10b AFP expression in liver from a representative Group 1
mouse suggesting HCC (Fluorescent microscopy, 20X). 62-63 11a, 11b AFP expression in liver from a representative Group 2
mouse suggesting HCC (Fluorescent microscopy, 20X). 63-64 12a, 12b AFP expression in liver from a representative Group 3
mouse suggesting HCC (Fluorescent microscopy, 20X). 64-65
Topic Page no.
Abstract 1
Introduction 4
Hypotheses 10
Aim and objectives 12
Review of literature 13
The placenta 14
Immunoplacental therapy (IPT) 15
Similarity between pregnancy and cancer 22
IPT was not always successful 24
Chemical constitution and preparation of placental extract 24
Hepatocellular carcinoma (HCC) 27
HCC animal models 30
Materials and methods 34
Preparation of placental lysate 35
Estimation of total protein concentration in the extract 37
Experimental animal model 39
Measurement of different parameters 40
Statistical analysis 45
Results 46
Changes in body weight 47
Changes in survival 48
Histopathological findings in liver 50
Histopathological findings in kidney 51
Immunohistochemistry of liver 62
Discussion 66
Conclusion 78
Limitations 80
Future scopes 82
Bibliography 84
Annexures
Institutional Review Board approval letter a Institutional Animal Ethics Committee approval letter f Participant information sheet and consent form g
Abstract
Objectives: Similar to cancer cells, the placenta has immunological and cellular characteristics. This study was conducted to evaluate the prophylactic antineoplastic property of placental lysate in chemical-induced hepatocellular carcinoma in C57BL/6 mice.
Methods: Lysate from a full-term human placenta was prepared following a standard protocol. The total protein concentration of the lysate was assessed.
18 male C57BL/6 mice (3 weeks age, 8-10 g body weight) were used in the experiment and equally divided into 3 groups to receive the following treatments: Group 1: 20 μg placental lysate i.p. weekly for 3 consecutive weeks, followed by diethylnitrosamine (DEN) 75 mg/kg i.p. weekly from the 3rd week for 4 consecutive weeks; Group 2: Normal saline (positive control) i.p. per mouse weekly for 3 consecutive weeks, followed by injection of DEN 75 mg/kg i.p. weekly from the 3rd week for 4 consecutive weeks; Group 3: No intervention (negative control). Changes in body weight and mortality were recorded in all the animals. Histopathological and immunohistochemical examination (alpha fetoprotein expression) of livers were done to check for the development of hepatocellular carcinoma.
Results: There was a significant reduction of body weight in both Group 1 (p=0.027) and 2 (p=0.013) animals at the time of death than after administration of diethylnitrosamine. Significant improvement in survival was
observed in Group 1 animals compared to Group 2 ones (p=0.043). However, hepatocellular carcinomas were detected in both Group 1 and 2 animals.
Conclusion: Although prophylactic administration of placental lysate could not prevent hepatocellular carcinoma, it significantly improved survival in comparison to placebo.
Keywords: Placental lysate, hepatocellular carcinoma (HCC), xenogeneic, antineoplastic activity, diethylnitrosamine (DEN).
Introduction
Pregnancy is one of the models that can be used to study cancer.1 The immunological activities which are involved in trophoblast proliferation resemble that of oncogenesis.1 The placenta show strikingly similarity in features with tumors, including similar antigenic determinants, defective death receptor signaling, surface-expressed tumor-associated antigens, growth factors, angiogenic factors, complement regulatory protein expressions, regulatory T cells, modes of activation of matrix metalloproteases, immune- escape properties, impaired apoptotic processes, immune escape mechanisms, etc.1-4
Experimental models have shown that placental extract has a definite immunostimulating activity both at humoral and cellular levels. Valentin I.
Govallo, a Russian physician and immunologist in the 1970s, based on this observation, developed the concept of developing a prophylactic „universal cancer vaccine‟ by using chorionic villi extract, as a sort of „immunoplacental therapy (IPT)‟. The formulated hypothesis stated that the placental vaccine would induce humoral and cell-mediated immune response against embryo- like antigens as well as the various angiogenic factors which are present in both cancer cells and placenta.2-4 A similar putative mechanism has been published stating that on injecting placental extract, the „IPT‟ might act as a polyvalent vaccine; which will identify the placental cells and tumor associated
antigens (TAA) expressed in placenta as foreign and will target tumor and embryo-like proteins and surface antigens which are involved in complement regulation, apoptosis, angiogenesis and other antiproliferative mechanisms.3 Since the necessary requirement for the co-evolution towards a malignant disease state is an ever-happening process between cancer cell and its tumor microenvironment, the success of a cancer vaccine might be dependent on how efficiently it acts on pre-tumor cells and their microenvironment. It was also thought that a vaccine originating from placental lysate could induce cell- mediated and humoral immune response against the embryo-like antigens as well as the different angiogenic factors which are commonly present in placenta and cancer cells. The net proposal of this vaccination is to induce an immunological state which in turn will result in rejection of cancer stem cells.
At the same time, to define immunogenic and safe cancer antigens that can serve as suitable targets for vaccines, including tumor-specific antigens and proteins over expressed on the tumor, but not on normal tissues, is a major challenge for designing prophylactic cancer vaccines. Presently, only a limited number of such cancer antigens have been found with limited successes.5-7 It is well documented that expression of embryonic antigens to variable extents is found in most solid tumor, and as the similarity of expression of antigen between embryonic tissues and cancer is very much, this provides the potential
to target embryonic components as an effective strategy to prevent the appearance of cancers.8 Induction of protective immune response against cancers in animal models has shown by vaccination with embryonic or stem cell antigens.9-11
Cross-reactive cell-mediated and humoral immune response is stimulated by IPT against TAA and proteins that are involved in carcinogenesis. Also, by triggering important cytokines at vaccination site, the vaccine might result in the balance of cytokine-network and also promote T helper cell-mediated immunity in the tumor microenvironment.
The activities of placental extracts have been tested in many studies like, in already developed carcinoma in human subjects, cancer cell lines, different tumor cell-induced or spontaneous carcinogenesis (in knockout mice) in experimental models. No conclusive study has been conducted to evaluate the antineoplastic activity of placental extract prophylactically in hepatocellular carcinoma (HCC).
The most common primary malignancy of the liver is HCC. Globally HCC is detected in approximately 7.5 lakhs patients in each year and mostly is associated with grave prognosis.12 It is also one of the most lethal cancers and the highest incidence of this carcinoma is found in sub-Saharan Africa and East Asia.13
The single largest risk factor for HCC is cirrhosis of any etiology, followed by hepatitis B (HBV) and hepatitis C (HCV) infection. Susceptibility to develop cirrhosis and HCC is greater in patients with genetic hemochromatosis.12 Incidence of liver cancer also increases in patients with autoimmune hepatitis.
Risk for HCC is also increases in individuals with cirrhosis related to non- alcoholic fatty liver disease and non-alcoholic steatohepatitis. Approximately 2-fold increased incidence of HCC have been shown in individuals with diabetes mellitus.12 Cigarette smoking and alcohol consumption among environmental factors has become more recognized as potential factors in the development of liver cancer.14
After the United States Food and Drug Administration approved the first cancer vaccine, a wide spectrum of other cancer vaccines which target a diverse range of TAA has been tested in different randomized clinical trials. To counteract various immunosuppressive entities, like the combined use of different immune checkpoint inhibitors as vaccines, certain chemotherapeutics, small-molecule targeted therapies and radiation strategies are currently being tried both pre-clinically and clinically.15 For breast, lung, colon, skin, renal, prostate and other cancers therapeutic vaccines are now being actively investigated in clinical trials.16 So, it will be also justified to screen for
prophylactic anti HCC vaccine to be used in high risk individuals predisposed for developing HCC subsequently.
Immunopharmacology is an emergent and important discipline in pharmacology. Precise details about many immunotherapeutic agents are not known regarding their mechanism of actions. However, several agents augmenting the immune system or balancing the normal immune physiology are particularly becoming important in the management of cancer, autoimmune or inflammatory diseases.16
Hypotheses
When placental lysate containing multiple tumor antigens is injected:
1. Immunoplacental therapy (IPT) will function as a multi-epitope polyvalent vaccine thereby recognizing placental cells and antigens as foreign and target tumor and embryo-like surface antigens and proteins involved in apoptosis, complement regulation, angiogenesis and other antiproliferative mechanisms.
2. There will be activation of both humoral (e.g. anti-alpha fetoprotein antibody) and cell-mediated (e.g. cytotoxic T cells) immune systems in the host which will be protective against subsequent tumorgenesis.
Aim and objectives
Aim:
To evaluate the antineoplastic activity of intraperitoneal (i.p.) xenogeneic (human) placental lysate prophylactically in comparison to placebo in chemical-induced HCC in C57BL/6 mice.
Objectives:
To evaluate the following parameters in the HCC-induced mice in the treatment group (receiving prophylactic placental lysate) and placebo group and compare those with the negative control group at the end of the experiment:
1. Survival.
2. Body weight.
3. Carcinogenic changes in liver as noted by histopathology and immunohistochemistry.
Review of literature
The placenta:
The placenta connects the developing fetus to the uterine wall. It attaches to the uterine wall and the umbilical cord develops from the placenta. Nutrition, thermo-regulation, waste elimination and gas exchange of the fetus via the mother's blood supply, fight against internal infection and hormone productions to support pregnancy are provided by the placenta.17
The placenta is a feto-maternal organ having two components: the fetal placenta (chorion frondosum), which embryonically originates from the blastocyst and forms the fetus and the remaining maternal placenta (decidua basalis) which gives rise to the uterus.17, 18
In humans, the approximate size of a placenta is 2-2.5 cm (0.8-1 inch) in thickness and 22 cm (9 inch) in length. It has thick center with thin edges. It weighs around 500 grams (just over 1 lb). Its color is dark crimson or reddish- blue. The fetus is connected to the placenta by the means of an umbilical cord, which is approximately 55-60 cm (22-24 inches) in length and it contains one umbilical vein and two umbilical arteries.17
The human placenta usually has a discoid shape, but between different mammalian species, the size can vary much.17,18 The umbilical cord inserts into the chorionic plate (eccentric attachment). Over the placenta blood vessels branch out and further divide to form a network which is again covered by a
thin layer of cells. The result of this is the formation of villous tree anatomical structures. On the maternal side of the placenta, the villous tree structures are grouped together into lobules called cotyledons.
Figure 1: The structure of placenta by cross sectional view.
(Figure adopted from: Placenta. In Wikipedia, The Free Encyclopedia.
Available at:
https://en.wikipedia.org/w/index.php?title=Placenta&oldid=713667669. Last accessed: 10 April 2016)
As mentioned, the main components of the placenta are villous “trees”. Based on ontogeny, vessel branches, villous structure, vessel-cell type components and histologic features, different types of villi17 have been described:
1. Stem villi: The chorionic plate is connected by stem villi. They have dense fibrous stroma where large and small blood vessels are present.
These villi develop vessels with smooth muscle in the outer covering and central stromal fibrosis within.
2. Immature intermediate villi: These are bulbous, in shape and peripheral in location and are basically immature continuations of the stem villi. These have Hofbauer cells, reticular loose stroma and distinct vessels and discontinuous cytotrophoblast layer.
3. Mature intermediate villi: These types of villi are slender, long, having some peripheral ramifications and are deficit of vessels in the stroma. They mainly give rise to all the terminal villi. The large amount of fetal vascularization and involvement in various surface exchanges make them suitable for feto-maternal exchange.
4. Terminal villi: They have some kind of attachment to the stem villi by intermediate structures. These villi have the shape of grapes and are featured by an extensive vascularization and presence of sinusoids which are highly dilated. However, in a term placenta, terminal villi are
smaller with lesser stroma having a cytotrophoblast layer which is discontinuous and contain around 4-6 fetal capillaries per cross section.
5. Mesenchymal villi: These types of villi are the most primitive villi which are present from early pregnancy. They are characterized by the presence of stroma, two complete trophoblast layers, undistinguished capillaries, single cytotrophoblast layer surrounding the villous core and an outer syncytiotrophoblast layer on the surface.
Figure 2: Structure of a villous tree connecting the maternal surface (basal plate) and the fetal surface (chorionic plate).
(Figure adopted from: Wang Y, Zhao S. Vascular Biology of the Placenta. San Rafael (CA): Morgan & Claypool Life Sciences; 2010. Chapter 3, Structure of the Placenta. Available at: http://www.ncbi.nlm.nih.gov/books/NBK53256/
Last accessed: 10 April 2016)
Immunoplacental therapy (IPT):
In a retrospective way,3 Harandi described a cancer vaccine which can be obtained from the extract of chorionic villi of the human placenta. VI Govallo, a Russian ophthalmologist first introduced this therapy and reported the characteristic similarities between cancer and pregnancy in terms of immunity set-up.
Like cancer cells the placenta has remarkable similar growth mechanisms, antigenic determinants and immune-escape properties. Placental vaccine might act as a multi-epitope vaccine as the body can recognize the placental antigens as foreign and stimulate cross-reactive cell-mediated and humoral immune response against cancer TAA and proteins that aid in complement regulation, angiogenesis and apoptotic resistance. Thus in various ways, IPT may provide a very good strategy in cancer immunotherapy.3,4
Govallo started experiments with placenta in 1975 when he demonstrated the usefulness of IPT in the management of solid tumors. He also considered pregnancy as a model to study cancer, because the fetus with foreign paternal antigens resembles a „tumor‟ and the fetus is not rejected by the immune system of the host mother. But, the fetus causes physiological suppression of immunity in the host mother, whereas a neoplasm or tumor results in definite immunosuppression through similar mechanisms. Govallo expressed that the
aim of cancer vaccination is to induce an artificial immunological state in the patient which might cause rejection of foreign cancer cells. This is very similar to the immune response which causes spontaneous abortion. However, when Govallo made these early observations, the detailed mechanism of tumor escape was not fully understood.19
Since 1970s, IPT was tried as a prophylactic vaccine in healthy individuals to stimulate the immune system to attack pre-tumor cells.4,19,20 Various studies have reported proteins which are common to cancer and placenta. In the last century, Bohn and Winckler21 isolated and characterized more than 30 new proteins from placental extracts and placental membranes and termed them as placenta membrane proteins (MP) and placenta proteins (PP).
Of these, some are specific to the placenta, and the others are detected in other tissues also; e.g. PP12, PP11, PP10, PP5, that were detected in trophoblast, ovarian, breast and gastrointestinal tumors. Raised levels of PP8 and PP7 in serum were found in respiratory, gastrointestinal and hematological malignancies; PP5 was detected in breast, gynecological and lung cancers;
PP11 was demonstrated in breast, ovarian gastric and testicular cancer; PP10, was detected in serous and mucinous cystadenocarcinomas of ovary, adenocarcinoma of breast and endometrium and also in non-malignant tumors of the ovary, uterus and breast.21,22
Previous studies22 have shown a marked diagnostic antigenic feature of a pharmaceutical allogeneic human placenta suspension, for detection of a specific immune response in the form of delayed hypersensitivity in patients having histopathologically diagnosed substratum epithelial hyperplastic neoplasms, that demonstrates IPT as a substance which could be useful in the development of cancer vaccine.2,3,23-25
These reports suggest that certain genes are expressed in placenta, which are also expressed in tumors and so placental tissue-peptides provide specific targets for cytotoxic T lymphocyte recognition of tumors. A T cell with a good affinity for self-peptide has a fair chance to identify the placental homologous peptides derived from cancer cells.2,3,24,25
Placental lysate also has various other activities against angiogenesis. This angiogenesis of placenta has been associated with placental growth factor, a member of vascular endothelial growth factor (VEGF) family of important pro- angiogenic factors.26 Reduced expression of genes involved in angiogenesis such as transforming growth factor-β, VEGF, metalloproteinase-2, integrin, leptin receptor, basic fibroblast growth factor and plasminogen activator inhibitor was observed in the chorionic villi of women having multiple spontaneous abortions.27 IPT could inhibit the expression of different angiogenic genes within the tumor, and mitigate this growth process.
Similarity between pregnancy and cancer:
As already mentioned, the immunological processes of proliferation of trophoblast and oncogenesis mimic each other. Similar characteristics include expression of same surface tumor-associated antigens, participation of the same types of regulatory T cells,18 gene expression of similar compliment regulatory proteins,27 growth factors,28 angiogenic factors,29 and defective death receptor signaling and apoptosis.30 Moreover, tumors31 and placental tissue32 use same immune escape mechanisms causing apoptosis of immune cells in pregnancy as well as cancer.
Years of studies have shown a list of common TAA shared between proliferating trophoblasts and a variety of tumor cells as shown in Table 1.
Targeting these TAA could be a better vaccination strategy, as the body will try to induce antibody responses against these TAAs, thereby prevent cancer development. This has actually paved the way for developing a universal cancer vaccine with the idea that there will be protective antibody response against diverse tumor antigens in the host by injecting the „polyvalent‟
placental lysate.
Table 1: Various tumor antigens common to trophoblast and tumor3
IPT was not always successful:
Tumors Antigens
Melanoma Glypican-3, CTA, survivan
Colon CSA, HPL, CEA, HCG, AFP,FE, PLAP, SP1, TF, 5T4 Ovarian HCG, AFP,PLAP, Bjorkland‟s antigen, CEA, CA-125, CTA
Breast CD46, PLAP, CEA, PLF, CA-125
Lung CEA, HCG, PLAP, Ki-1 (CD30), CTA (SP1, MAGE)
Prostate CTA, PSA, PSCA, HCG, PAGE
Testicular Ki-1 (CD30), PLAP
Gastric PLAP, 5T4
Sarcoma SSX, CTA (XAGE)
Lymphoma Ki-1 (CD30)
HCC AFP, Glypican-3, CTA
(CTA: cancer testis antigen, CEA: carcino embryonic antigen, CSA: colon specific antigen, SPI: serine protease inhibitor, PLAP: placental alkaline phosphatase, HCG: human chorionic gonadotropin, HPL: human placental lactogen, AFP: alpha fetoprotein, TF: Thomsen-Friedenreich, CA-125: cancer antigen-125, PLF: Placental isoferritin, CD: cluster of differentiation, MAGE:
melanoma-associated antigen, SP: specificity protein, PSCA: prostate stem cell
Hypothetically, placental extract, if injected, can induce immunological responses against TAA shared between the placenta and neoplasia and also against functional proteins that the tumors require to maintain its malignant state. But still IPT has not produced desirable results in certain tumors. A number of theories32 have been suggested for that, which includes:
1. Many people with cancer have reduced immunity and so their immune systems are not able to react to the vaccines.
2. Some tumors produce proteins and chemicals that prevent the immune system from attacking them effectively, even when it has been stimulated by the vaccine.
3. Not all tumor cells are the same and some cells may be different to those in the vaccine. Those different cells will be resistant to and unaffected by such vaccine.
Chemical constitution and preparation of placental extract:
Placental extracts can be grouped into two major types: hydroalcoholic extract and aqueous extract. The constituents present in the extract are largely related to the method of the preparation and depend on the solubility of the components in respective solvents used in the extraction process. Thus, an aqueous extract contains molecules which are more polar like small organic
components, peptides/proteins like nucleotides, amino acids polydeoxyribonucleotides, carbohydrates, and trace amounts of protein-bound lipids which are relatively better soluble in a watery medium. Similarly, various other types of lipids are present in the hydroalcoholic extract, which are hydrophobic and less polar. Hydroalcoholic extract of placenta on chemical analysis, showed the presence of carbohydrates, triglycerides, sialic acids, high density lipoproteins, glycosphingolipids, cholesterol, and other amino acids, carotenes, nucleotides, vitamins, low molecular weight proteins/peptides containing hydrophobic amino acid residues that are better soluble in less polar solvent.2,23,33
By employing Filatov‟s procedure modern indigenous aqueous placental extract is prepared.33 Fresh placentas are stored in ice and primarily checked for human immunodeficiency virus antibody and HBV surface antigen. One cold and hot aqueous extraction are performed after incubating minced and dissected placenta at 60C and 900C respectively. This is followed by sterilization of the extract under a saturated steam (pressure 15-lbs/sq. inch at 1200C for 40 min). After the next step involving filtration and addition of 1.5%
(v/v) benzyl alcohol as preservative, ampoules are filled and sterilized again under the said condition for 20 minutes.
In the initial sterilization process, the extended heat treatment specifically completes the precipitation of various macromolecules, consisting of mostly proteins. This is also done to destroy maximum resistant spore producing organisms possible like Clostridium tetani. The terminal sterilization step is to maintain sterility of the products after they are filled and sealed in ampoules.
From 100 mg of fresh placenta, one milliliter of the drug is derived approximately.33
As repeated sterilization is involved in the extract preparation, it is likely that it might contain degraded macromolecules and other small bioorganic compounds like amino acids, peptides, nucleotides, small polynucleotide fragments, small sized polypeptides, etc. But finally, the molecules which are able to withstand the high sterilization pressure i.e. are heat stable will remain biologically active.33
Other methods of preparation of placental lysates have also been mentioned in studies evaluating immunogenic properties of placental lysates in cancer models.34 Placental tissues were washed in sterile phosphate buffered saline containing 5% antibiotic (penicillin streptomycin) mixture and placed on ice for transport. The placental tissues were then homogenized with a tissue homogenizer and subjected to 4 freeze-thaw cycles, alternating from liquid nitrogen to a water bath at 420C. The cell debris was then precipitated by
centrifugation for 45 minutes at 1500 g. The supernatant was collected and sterilized with 0.2 micron filter papers.34
Hepatocellular carcinoma (HCC):
Among the primary liver cancers, HCC is the most common and is one of the leading causes of global cancer-related death. HCC incidence is rising throughout the world, with a specific geographic, age and sex distribution.
Maximum burden of disease (85%) is borne in developing countries.35
The different typical symptoms of HCC are often absent. Rather, patients manifest symptoms due to cirrhosis, which is underlies in up to 80-90% of patients with HCC. As a result, the majority of patients are diagnosed with advanced disease and often precluding potentially curative therapies. This has resulted, in a 5-year overall survival rate of 12% and a median survival of only 6 to 20 months.35
When symptomatic, HCC is associated with different complaints, like right upper abdominal or epigastric pain, weight loss, early satiety and malaise.
Detection of ascites, encephalopathy, jaundice, or variceal bleeding in patients with compensated cirrhosis warns of HCC. Patients may also present with distension and severe abdominal pain, hypotension and sudden drop in hematocrit due to tumor rupture and i.p. bleeding. Urgent embolization of the
bleeding variceal vessel and/or surgical intervention might be required in such cases.35
HCC is also associated with different paraneoplastic syndromes resulting in hypercholesterolemia, severe watery diarrhea, hypoglycemia, erythrocytosis, hypercalcemia and some cutaneous manifestations. Extra-hepatic spread of cancer at the time of presentation is not common, ranging up to 30%. The most common sites of metastasis include adrenal gland, lung, regional lymph node regions (including celiac and porta hepatis lymph node chains) and bone.35 Cirrhosis is the leading risk factor for HCC, with the maximum number of cases contributed by chronic HCV and HBV infection and abuse of alcohol, although non-alcoholic fatty liver disease is coming up as a relevant cause. The other etiologies are viral infections, inherited errors of metabolism, comorbid conditions, environmental toxins and autoimmune disorders. Primary prevention like HBV vaccination has caused a remarkable reduction in HBV- related HCC. Also, initiation of antiviral therapy has lowered the incidence of HCC in patients with chronic HCV or HBV infection.35,36
Too much alcohol intake is a recognized risk factor for HCC, although the threshold duration and dose have not been clearly established.37 Smoking is also a risk factor for HCC and a recent European-population-based study demonstrated that cigarette smoking was responsible for causing nearly 50% of
all HCCs.38 Various community and hospital based studies have also suggested a close correlation between diabetes and obesity and a high incidence of HCC.39
The most common environmental factor associated with HCC is aflatoxin, produced from Aspergillus fungus.40 Over exposure to dietary aflatoxin, in the form of contaminated peanuts, corn and soybeans are frequent in developing countries and may result in HCC.41
Serum alpha fetoprotein (AFP) and ultrasonography are commonly employed methods for screening of HCC.35,36 The other markers of HCC with varying degrees of sensitivity and specificity and useful for both diagnosis and prognosis of the disease are: α-fetoprotein, heat shock proteins 70, Glypican-3, squamous cell carcinoma antigen, α-l-fucosidase, γ-glutamyltransferase, fucosylated GP73, transforming growth factor-β1, VEGF, miRNA-500, miRNA-21, etc.42 Surveillance is recommended for patients having risk factors for HCC.36 The modalities employed for HCC diagnosis depend on the lesion size as well as liver function and include imaging, biopsy and estimation of serum AFP and other biomarkers.35 HBV vaccination, antiviral treatment and surveillance are the measures which can be implemented against HCC.36
Treatment is mainly staging guided. The therapeutic options include surgical resection, local ablation, orthotopic liver transplantation, radioembolization
and transarterial chemoembolization and other targeted molecular therapies.35,36 Apart from vaccination against HBV and HCV virus, there are no other protective vaccines against HCC having other etiologies. So, a vaccine against HCC might be very useful.
HCC animal models:
Animal models of HCC can help in understanding HCC pathogenesis. The experimental mouse is the most suitable model to study cancer due to its small size, easy handling and marked similarities to humans.43 But, all these mouse models do not meet the necessary criteria of the most ideal animal model (genetic, biologic, etiologic and therapeutic aspects).44
The most commonly employed mouse HCC models45,46 are:
1. Hepatocellular carcinogen-induced model: Hepatocarcinogens are of two classes, non-genotoxic (or epigenetic) and genotoxic. The genotoxic carcinogens causes cancer due to formation of DNA adducts, leading to genetic changes in the cell, which can direct normal cells to a pre- neoplastic state. The non-genotoxic carcinogens, however, do not alter the DNA structure; but stimulate the pre-neoplastic cells to convert into a malignant neoplasm by influencing apoptosis, cell proliferation and cell differentiation.
The role of age, sex and genetic status of experimental mice on the probability of developing HCC are important confounders of these models. The most commonly used hepatocarcinogens in these mice models are phenobarbital and diethylnitrosamine (DEN).44 It has been shown that the suitable DEN treatment range for post-weaning C57BL/6 mouse strain was from 75 mg/kg for 4 weeks (total amount: 300 mg/kg) to 50 mg/kg for 8 weeks (total amount: 400 mg/kg) for HCC model.45 The other chemicals which are used in this model are peroxisome proliferators, (like ciprofibrate, fenofibrate, clofibrate), aflatoxin B1, carbon tetrachloride, thioacetamide, choline deficient diet, etc.46
2. Xenograft models: Here, the tumors are induced by injection of human cancer cells, which are obtained from a laboratory culture in immune deficient mice. As host, severe combined immune deficient or athymic (nude) or mice are often used. Tumor xenografts can be established either by inoculation of the tumor cell lines or direct implantation of the biopsy material. Various types of xenograft models can be identified, like ectopic implantation, orthopic implantation, hollow fiber model, etc.46
3. Genetically modified models: These mouse models are engineered to resemble HCC in terms of their molecular and pathophysiological
characteristics.43 These are distinct and peculiar model to evaluate the effects of oncogenes either alone or in combination with other oncogenes or even other carcinogenic agents. These types of models aid in the detailed investigation of carcinogenic pathways (molecular level) and allow the assessment of pathway co-operability and dependency.
Some representative genetic models are: transgenic models expressing viral genes (e.g. HCV), transgenic mice models over-expressing growth factors (e.g. transforming growth factor-α, transforming growth factor- β, epidermal growth factor), transgenic mice over-expressing oncogenes (e.g. Myc protein, β-catenin).46
4. Viral hepatocarcinogenesis: Different types of animal models have been developed in which human liver cells or tissues are transplanted into mice. Then the transplanted hepatocytes can be finally transinfected with HBV or HCV. With the help of these models, different pathways have been postulated that might be involved in HCC formation due to chronic viral hepatitis.45
Again it is thought that with the chronic inflammation of the liver, continuous hepatocyte regeneration happens after cell death. Viral hepatitis might cause several genetic alterations. These models are very much suitable for the evaluation of therapeutic and prophylactic
strategies against hepatitis due to HBV or HCV. However, these are not of much use to study HCV or HBV-associated HCC.45
Materials and methods
The work was started after getting approval of the Institutional Review board and Institutional Animal Ethics Committee, Christian Medical College, Vellore.
1. Preparation of placental lysate:
A) Materials:
i) Phosphate buffered saline (PBS) (1X, pH: 7.4) (Fisher Scientific, Mumbai, India).
ii) Penicillin (10,000 U/ml), streptomycin (10 mg/ml) and amphotericin B (25 µg/ml) solution (Fisher Scientific, Mumbai, India).
iii) Syringe filter (0.2 µm) (Sigma-Aldrich, Bangalore, India).
B) Methods:
i) One fresh, full-term placenta was collected from a woman undergoing delivery by elective Cesarean section in the Community Health and Development Hospital, Christian Medical College, Vellore.
ii) The placenta was transported in ice cold PBS (1X, pH:
7.4) in a sterile container after draining the excess blood.
iii) The placental cotyledons were sectioned with a sterile scissor at 40C temperature within a laminar air flow cabinet containing UV lamp.22
iv) The cotyledons were washed and swirled thoroughly with PBS multiple times to get rid of blood.22
v) A small portion of the tissue was finely minced and placed in a sterile centrifuge bottle containing ice cold PBS (1X, pH: 7.4) twice the tissue volume.
vi) 5% (v/v) streptomycin (10 mg/ml), penicillin (10,000 U/ml) and amphotericin B (25 µg/ml) solution was added to it and mixed.22
vii) The bottle containing the tissue and solution was centrifuged at 2000 g for 1 minute.22,47
viii) The supernatant was discarded.22,47
ix) The tissue at the bottom was separated and placed in another sterile container.22,47
x) The tissue was homogenized thoroughly using a tissue homogenizer maintaining a temperature of 40C.22
xi) The homogenized tissue was subjected to 4 freeze-thaw cycles alternating between water bath 420C and liquid nitrogen (-800C) for further cell lysis.22,48
xii) After homogenization, the cell debris was precipitated by centrifugation for 45 minutes at 1000 g.22
xiii) The top supernatant was collected and was passed through syringe filter (0.2 µm) for sterilization.
xiv) Sterility was ensured at every step.
2. Estimation of total protein concentration in the extract:
A) Materials:
i) PierceTM Bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Mumbai, India).
B) Methods:
i) The BCA protein assay combines the well-known reduction of Cu++ to Cu+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by BCA.49
ii) At first, copper was chelated with protein in an alkaline environment to form a light blue complex.49
iii) Then BCA was then made to react with the reduced (cuprous) cation that was formed in the previous step.49 iv) The intense purple colored reaction product resulted from
the chelation of two molecules of BCA with one cuprous ion.49
v) The water soluble BCA/copper complex exhibited a strong linear absorbance at 540 nm with increasing protein concentrations and was measured by spectrophotometry (Figure 3).49
Figure 3: Standard curve for BCA protein assay by spectrophotometry (540 nm).
3. Experimental animal model:
A) Animals and materials:
i) 18 male C57BL/6 mice (3 weeks age and 8-10 g body weight).45,46
ii) Diethylnitrosamine (DEN) as hepatocellular carcinogen45 (Sigma-Aldrich, Bangalore, India).
B) Experimental protocol:
i) All the animals were housed in standard wire cages with 6 animals per cage. They were maintained at standard laboratory conditions (temperature of 25 ± 20C and ambient humidity of 55-60%) with alternate light and dark cycles (12/12 hours). They were allowed standard pellet diet and water ad libitium. The animals were acclimatized to laboratory conditions for 1 week before the initiation of the experiment. Animal housing, care and conduct of experimental procedures were conducted following Committee for the Purpose of Control and Supervision of Experiments on Animals guidelines.
ii) The animals were divided into 3 groups as follows:
a) Group 1: Injection of 20 μg (0.5 ml after dilution) placental lysate i.p. per mouse weekly for 3 consecutive weeks, followed by injection ofDEN75 mg/kg weekly i.p. from the 3rd week for the next 4 consecutive weeks.
b) Group 2: Injection of 0.5 ml of normal saline (placebo) i.p. per mouse weekly for 3 consecutive weeks, followed by injection of DEN 75 mg/kg weekly i.p. from the 3rd week for the next 4 consecutive weeks.
c) Group 3: No intervention was done (untreated control).
All the animals were observed regularly and mortality was recorded. After the death of each animals (Groups 1 and 2), livers and kidneys were dissected out and preserved in 10% buffered formalin solution.
All the Group 3 animals were euthanized by chloroform inhalation method after the last animal from Group 1 and 2 died.
4. Measurement of different parameters:
A. Body weight of animals:
i) Measured at the beginning of the experiment and then at every week till the end of the study.
B. Survival:
i) All the animals were observed regularly and mortality rate was calculated for each group.
C. Histopathology of liver and kidney:
i) Materials:
a) 10% buffered formalin (Fisher Scientific, Mumbai, India).
b) Xylene (Fisher Scientific, Mumbai, India).
c) Absolute ethanol (Fisher Scientific, Mumbai, India).
d) Hematoxylin and eosin stain (Thermo Fisher Scientific, Mumbai, India).
ii) Methods:
a) Liver tissues were fixed in buffered formalin solution (10%).50
b) Tissue dehydration was done with ascending concentrations of ethanol.50
c) Tissue clearing was performed with xylene.50
d) The tissues were then transferred to molten paraffin for impregnation and embedded in paraffin blocks.50
e) Then the tissues were sectioned with microtome (5 µm each).50
f) Staining was performed with hematoxylin and eosin (H & E) stain.
g) Finally, the tissues were examined under light microscope.
h) As we observed early mortality with some animals after administering DEN, we also examined the kidneys by histopathology to look for DEN-induced toxicities.
D. Immunohistochemistry for AFP expression
42,51,52in liver:
i) Materials:
a) Buffered formalin (Fisher Scientific, Mumbai, India), b) Xylene (Fisher Scientific, Mumbai, India).
c) Absolute ethanol (Fisher Scientific, Mumbai, India).
d) Phosphate buffered saline (PBS) (1X) (Fisher Scientific, Mumbai, India).
e) Sigma DiagnosticsTM Poly-L-lysine solution (Fisher Scientific, Mumbai, India).
f) Triton X-100 (Thermo Fisher Scientific, Mumbai, India).
g) Bovine serum albumin (Thermo Fisher Scientific, Mumbai, India).
h) Primary antibody to AFP, grown in rabbit (Thermo Fisher Scientific, Mumbai, India).
i) Antirabbit Alexa FluorTM 488 tagged secondary antibody(Thermo Fisher Scientific, Mumbai, India).
j) Diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Mumbai, India).
k) Glycerol (Fisher Scientific, Mumbai, India).
ii)
Methods:
a) Liver tissues were fixed in buffered formalin solution (10%).50
b) Tissue dehydration was done with ascending concentrations of ethanol.50
c) Tissue clearing was performed with xylene.50
d) The tissues were then transferred to molten paraffin for impregnation and embedded in paraffin blocks.50
e) Then the tissues were sectioned with microtome (5 µm each).50
f) The liver tissues were transferred to Poly-L-lysine coated glass slides.53
g) The tissues were deparaffinized in xylene.53
h) Then the tissues were hydrated with descending concentrations of ethanol.53
i) The slides were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS (PBST) at room temperature.53
j) Blocking was done with 2% bovine serum albumin.53 k) The primary antibody to AFP, grown in rabbit
(dilution 1:100) was then added and the slides were left overnight.53
l) On the next day, the slides were again washed with PBST.53
m) Antirabbit Alexa FluorTM 488 tagged secondary antibody (dilution 1:100) was added.53
n) The tissues were finally counter stained with DAPI and mounted with 90% glycerol.53
o) Finally, imaging was done under a fluorescence microscope.
5. Statistical analysis:
i) All analysis was done in statistical program „R‟ (3.1.0).
ii) Wilcoxon-signed rank test (non-parametric test) was performed to compare body weight.
iii) A p value <0.05 was considered as statistically significant.
iv) Kaplan-Meier survival analysis was also done.
Results
1. Changes in body weight:
The mean body weights in both Group 1 (p=0.027) and Group 2 (p=0.013) mice were significantly reduced at the time of death compared to before administering DEN. However, the mean body weights of Group 3 mice had no significant reduction at the time of sacrifice compared to before administering DEN (Table 2 and Figure 4).
Table 2: Body weights (g) at the time of death than before administering DEN in the three groups of mice (n=6 in each group)
Group 1 Group 2 Group 3
Before administering
DEN
At time of death
Before administering
DEN
At time of death
Before administering
DEN
At time of sacrifice
20 14 21 20 23 23
23 13 19 13 22 21
22 10 22 NA 23 20
21 14 20 14 20 20
13 13 21 12 21 21
20 NA 21 14 19 22
Figure 4: Box-plot showing the changes in the body weights at the time of death compared to before administering DEN in the three groups of mice.
2. Changes in survival:
There was significant improvement in survival in Group 1 compared to Group 2 (p=0.043) mice after administration of DEN (Table 3 and Figure 5). There was no death in any Group 3 animals before sacrifice.
All the Group 3 animals were sacrificed on day 112 (death of the last animal from Group 1 and 2).
Table 3: Survival (days) of Group 1 and 2 animals after administration of DEN (n=6 in each group)
Figure 5: Kaplan-Meier survival analysis in three groups of animals after administration of DEN.
Group Survival of each animal (days) Group 1 11, 68, 83, 90, 91, 112 Group 2 7, 23, 29, 35, 45, 49
3. Histopathological findings in liver:
a) Group 1: There were features of atypical hepatocytes in solid and occasionally vague tubular pattern. There was increased thickness of hepatic plates. Individual cells were highly pleomorphic with normal to high nuclear: cytoplasm ratios. Nuclei showed dusty chromatin, nuclear clearing, occasional prominent nucleoli and multinucleated giant cells. Cytoplasm was moderate in the cells and contained bile pigment. There was proliferation of endothelial cells surrounding the malignant hepatocytes giving them island like shapes in some areas.
Acinar patterns were seen occasionally. These features were suggestive of HCC (Figures 6a to 6f).
b) Group 2: There were features of atypical hepatocytes surrounding the central vein of the liver in solid and occasionally vague tubular pattern. There was increased thickness of hepatic plates. Individual cells were highly pleomorphic with normal to high nuclear:
cytoplasm ratios. Nuclei showed dusty chromatin, nuclear clearing, occasional grooving, occasional prominent nucleoli and multinucleated giant cells. Cytoplasm was moderate in the cells and contained bile pigment. There was proliferation of endothelial cells surrounding the malignant hepatocytes giving them island like
shapes in some areas. Acinar patterns were seen sometimes. These features were suggestive of HCC (Figures 7a to 7f).
c) Group 3: Normal cellular architecture, hepatic plates, central veins, nuclei and cytoplasm were seen. There was no evidence of malignancy. These features were suggestive of normal liver (Figures 8a to 8f).
4. Histopathological findings of kidney:
a) Groups 1, 2 and 3: All the kidneys were normal in architecture.
Glomeruli, vessels, tubules and interstitium showed normal morphology (Figures 9a to 9c).
Figure 6a: Histopathological features of liver from a representative Group 1
Figure 6b: Histopathological features of liver from a representative Group 1 mouse showing features of HCC (H & E, 40X).
Figure 6c: Histopathological features of liver from a representative Group 1 mouse showing features of HCC (H & E, 10X).
Figure 6d: Histopathological features of liver from a representative Group 1 mouse showing features of HCC (H & E, 40X).
Figure 6e: Histopathological features of liver from a representative Group 1 mouse showing features of HCC (H & E, 10X).
Figure 6f: Histopathological features of liver from a representative Group 1 mouse showing features of HCC (H & E, 40X).
Figure 7a: Histopathological features of liver from a representative Group 2 mouse showing features of HCC (H & E, 10X).
Figure 7b: Histopathological features of liver from a representative Group 2 mouse showing features of HCC (H & E, 40X).
Figure 7c: Histopathological features of liver from a representative Group 2 mouse showing features of HCC (H & E, 10X).
Figure 7d: Histopathological features of liver from a representative Group 2 mouse showing features of HCC (H & E, 40X).
Figure 7e: Histopathological features of liver from a representative Group 2 mouse showing features of HCC (H & E, 10X).
Figure 7f: Histopathological features of liver from a representative Group 2 mouse showing features of HCC (H & E, 40X).
Figure 8a: Histopathological features of liver from a representative Group 3 mouse showing features of normal liver (H & E, 10X).
Figure 8b: Histopathological features of liver from a representative Group 3 mouse showing features of normal liver (H & E, 40X).
Figure 8c: Histopathological features of liver from a representative Group 3 mouse showing features of normal liver (H & E, 10X).
Figure 8d: Histopathological features of liver from a representative Group 3 mouse showing features of normal liver (H & E, 40X).
Figure 8e: Histopathological features of liver from a representative Group 3 mouse showing features of normal liver (H & E, 10X).
Figure 8f: Histopathological features of liver from a representative Group 3 mouse showing features of normal liver (H & E, 40X).
Figure 9a: Histopathological features of kidney from a representative Group 1 mouse showing no abnormalities (H & E, 40X).
Figure 9b: Histopathological features of kidney from a representative Group 2 mouse showing no abnormalities (H & E, 40X).
Figure 9c: Histopathological features of kidney from a representative Group 3 mouse showing no abnormalities (H & E, 40X).
5. Immunohistochemistry of liver:
a) Group 1: Increased AFP expression in the peri-nuclear region of the hepatocytes, suggesting HCC (Figure 10a and 10b).
b) Group 2: Increased AFP expression in the peri-nuclear region of the hepatocytes, suggesting HCC (Figures 11a and 11b).
c) Group 3: No expression of AFP (Figures 12a and 12b).
Figure 10a: AFP expression in liver from a representative Group 1 mouse suggesting HCC (Fluorescent microscopy, 20X).
Figure 10b: AFP expression in liver from a representative Group 1 mouse suggesting HCC (Fluorescent microscopy, 20X).
Figure 11a: AFP expression in liver from a representative Group 2 mouse suggesting HCC (Fluorescent microscopy, 20X).
Figure 11b: AFP expression in liver from a representative Group 2 mouse suggesting HCC (Fluorescent microscopy, 20X).
Figure 12a: AFP expression in liver from a representative Group 3 mouse suggesting normal liver (Fluorescent microscopy, 20X).
