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Characterising the impact of immune dysfunction
and cancer on the gastrointestinal tract in mice
Student Name:
Student Number:
Supervisors:
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Acknowledgement
My sincere appreciation goes to A/Prof. Elisa Hill-Yardin, Dr. Gayathri Balasuriya and Dr
Ashwini Chand who were very cooperative and kind to me throughout this project. It has been a
great experience learning from your wealth of knowledge. You all find time to share and impact
your knowledge despite all uncertainties in the COVID-19 pandemic. Also, my profound
gratitude goes to Mitra Mohsenipour who help with laboratory training and coordination of all
experimental works.
I will like to thank Dr. Sara Baratchi, Dr.Sonia La Vita, Dr.April Kartikasari and Dr. Faith Kwa
for their valuable contributions, feedbacks and guidance during writing this thesis.
To my dear friends Ali Alrsheed, Oluwaseun Akande and their families who have supported me
with encouragements and motivations. I really appreciate you.
Lastly, to my loving wife, children and brother (Mohammed) for their support and understanding
through the challenging process of my studies and the completing my thesis. I say thank you.
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Table of Contents
Acknowledgement……………………………………………………………………………………………………………………. 2
List of Figures …………………………………………………………………………………………………………………………. 6
List of Tables…………………………………………………………………………………………………………………………… 7
Abbreviations………………………………………………………………………………………………………………………….. 8
Abstract ………………………………………………………………………………………………………………………………….. 9
Chapter One: Introduction …………………………………………………………………………………………………….. 10
1.1 Background ………………………………………………………………………………………………………………….. 10
1.2 The Enteric Neuroimmune System …………………………………………………………………………………. 11
1.2.1 The Enteric Nervous System ……………………………………………………………………………… 11
1.2.2 Neuroimmune interactions………………………………………………………………………………… 13
1.3 Impact of immune dysfunction and cancer on the Gastrointestinal Tract …………………………. 15
1.3.1 Cancer and immune dysfunction ……………………………………………………………………….. 15
1.3.2 Histopathological alterations of the GI tract in cancer and symbiosis…………………… 17
1.4 Alterations to the immune system through the introduction of mammary tumour metastasis
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1.4.1 Immune cells at the primary tumour site influence metastatic behaviour of cancer
cells 19
1.4.2 The immune interaction with the gut nervous system ………………………………………….. 20
1.5 Research Questions……………………………………………………………………………………………………….. 22
1.6 The rationale of the study………………………………………………………………………………………………. 22
1.7 Hypothesis…………………………………………………………………………………………………………………….. 23
1.8 Research Project Aims…………………………………………………………………………………………………… 23
Chapter Two: Materials and Methods…………………………………………………………………………………….. 24
2.1 Animal model …………………………………………………………………………………………………………………… 24
2.1.1 Anatomical evaluation …………………………………………………………………………………………….. 24
2.2 Histopathological Studies……………………………………………………………………………………………….. 24
2.2.1 Cryo-preservation …………………………………………………………………………………………….. 24
2.2.2 Cryosectioning………………………………………………………………………………………………….. 25
2.2.3 Haematoxylin and Eosin Staining ………………………………………………………………………. 25
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2.2.4 Imaging (Brightfield microscopy) ………………………………………………………………………. 25
2.3 Immunohistochemistry for muscularis macrophages and neuronal processes……………………. 26
2.3.1 Tissue preparation and microdissection ……………………………………………………………… 26
2.3.2 Primary and secondary antibody……………………………………………………………………….. 26
2.3.3 Imaging (florescent microscopy) ………………………………………………………………………… 27
2.4 Image Analysis………………………………………………………………………………………………………………. 27
2.5 Statistical analysis …………………………………………………………………………………………………………. 27
2.6 Systematic review methodology ……………………………………………………………………………………… 28
2.6.1 Study design……………………………………………………………………………………………………… 28
2.6.2 Search Terms……………………………………………………………………………………………………. 28
2.6.3 Inclusion and Exclusion Criteria………………………………………………………………………… 28
2.6.4 Data Collection and Analysis……………………………………………………………………………… 28
3.1 Anatomical measures for gut tissues in non-tumour and mammary tumour-bearing mice…. 29
3.1.1 Body weight ……………………………………………………………………………………………………… 29
3.1.2 Colon length……………………………………………………………………………………………………… 29
3.1.3 Small intestinal length……………………………………………………………………………………….. 30
3.1.4 Caecal weight……………………………………………………………………………………………………. 31
3.1.5 Number Peyer’s of patches………………………………………………………………………………… 32
3.1.6 Number of caecal patches………………………………………………………………………………….. 33
3.1.7 Number of faecal pellets and their dimensions within the dissected colon……………… 34
3.2 Gastrointestinal histopathology in mammary tumour-bearing mice …………………………………. 36
3.2.1 Villus height in proximal colon…………………………………………………………………………….. 36
3.2.2 Villus width in proximal colon……………………………………………………………………………… 36
3.2.3 Crypt depth in control and mammary tumour-bearing mice proximal colon………………… 36
3.3 Immunofluorescence staining of caecal tissue in mammary tumour-bearing mice……………… 36
3.3.1 Optimization of immunostaining…………………………………………………………………………… 36
3.3.2 Positive and Negative immunofluorescence controls ……………………………………………….. 36
3.3.3 Detection of Iba1, Hu and Tuj-1 in cross sections of control and mammary tumour-bearing
mice caecal tissue……………………………………………………………………………………………………………… 36
3.3.4 Quantification of Iba1 immunoreactivity in caecal tissue of control and mammary tumourbearing mice…………………………………………………………………………………………………………………….. 36
3.4 Systematic Review Results……………………………………………………………………………………………… 36
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3.4.1 Study Design and Location of the Studies …………………………………………………………… 38
3.4.2 Study Participants…………………………………………………………………………………………….. 38
3.4.3 Measurement of Impact…………………………………………………………………………………….. 39
3.4.4 Impact of Immune Dysfunction and Cancer on the Gastrointestinal Tract …………… 39
Chapter 4: Discussion…………………………………………………………………………………………………………….. 40
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List of Figures
Figure 1. Illustration of the anatomy of the ENS……………………………………………………………….. 12
Figure 2. Neuron-macrophage crosstalk in the GI tract………………………………………………………. 14
Figure 3. The mucosa associated lymphoid tissue (MALT)………………………………………………… 16
Figure 4. Components of the GI system. ………………………………………………………………………….. 18
Figure 5. The mucosal immune system. …………………………………………………………………………… 20
Figure 6. Mucosal immunity of the GI tract ……………………………………………………………………… 21
Figure 7. Colon length in tumour and non-tumour bearing mice ………………………………………… 29
Figure 8. Small intestinal length in tumour and non-tumour bearing mice …………………………… 30
Figure 9. Caecal weight in tumour and non-tumour bearing mice. ………………………………………. 31
Figure 10. The number of Peyer‟s patches in non-tumour and tumour bearing mice. …………….. 32
Figure 11. The number of caecal patches between tumour and non-tumour bearing groups……. 33
Figure 12. The number of faecal pellets and their dimensions within the dissected colon. ……… 34
Figure 13 The flow of included studies based on the four phases of systematic literature search 36
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List of Tables
Table 1 Summary Characteristics of included articles ………………………………………………………. 37
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Abbreviations
BALB/C mice Bagg Albino mice
n Number
ENS Enteric Nervous System
PNS peripheral nervous system
CNS Central nervous system
CSF-1 Colony Stimulating Factor-1
BMP2 Bone morphogenic protein type 2
GALT Gut associated lymphoid tissues
MALT Mucosa Associated Lymphoid Tissue
ILs interleukins
ECM Extracellular Matrix
VIP vasoactive intestinal peptide
ATCC American Type Culture Collection
NK Natural Killer
NKT Natural killer T cells
PBS Phosphate buffer saline
OCT Optimum cutting temperature
H&E Haematoxylin and Eosin
Iba1 Ionized calcium binding adaptor molecule 1
Hu Human ~ also known as Anti-ANNA-1
(antineuronal nuclear antibodies)
Tuj-1 Tubulin beta III
HH Hypobaric Hypoxia
°C Celsius
Min Minutes
PP Peyer‟s patches
G Gram
Anti Antibody
Cm Centimeter
% Percentage
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Abstract
Breast cancer is the most frequent neoplasm affecting the majority of women around the globe.
As a condition that may prompt immune dysfunction, it is hypothesized in this study that the gut
which is the largest lymphoid tissue might play a potential role in the disease progression. This
thesis aimed at assessing the anatomical, histopathological and neuronal changes in the
gastrointestinal tract of mice during mammary tumour development. To assess this impact, we
studied 14-week-old female adult BALB/C mice consisting of 9 tumour-bearing mice and 3 nontumour bearing mice as the controls. The tumour-bearing group was inoculated with 50,000
metastatic breast cancer cells, derived from a highly metastatic mouse mammary tumour cell
line. There were no observable differences in the length of the intestine in both groups using an
unpaired Student‟s t-test. The average number of Peyer‟s patches from mammary tumourbearing mice (9 ± 0.6, n=9) was significantly greater compared to non-tumour littermates (7 ± 0,
n=3; p=0.01). Also, a trend towards increased numbers of caecal patches in tumour-bearing mice
was observed. There was a significant increase in the average number of faecal pellets within the
dissected colon of the tumour bearing mice (3.77 ± 0.6) compared to non-tumour mice (1.66 ±
0.3; p=0.013). It appears that Peyer‟s patches play an essential role in sensing the distant threat
posed by the developing tumour. Due to the COVID-19 pandemic and resulting lockdown,
sample numbers and experiments were limited, hence reducing the statistical power of the study.
The changes in the gastrointestinal tract could be due to immune disruption triggered in a distant
tissue.
Word count: 262.
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Chapter One: Introduction
1.1 Background
Breast cancer is one of the most common forms of mammary tumours world-wide [1] and
is the most frequent neoplasm affecting the majority of women around the globe. Breast cancer
accounts for at least 32% of all cancer cases among women [1, 2]. It has a lifetime risk of 1 in
10. Moreover, breast cancer is a leading cause of mortality among women at 15% [2]. Metastasis
develops in approximately 75% of breast cancer patients with or without medical malignancy
[1]. Metastatic breast cancer penetrates the gastrointestinal (GI) tract and affects the upper GI
regions, such as the small bowel, the stomach, and the pancreaticobiliary regions [2]. The
occurrence of digestive tract metastases in autopsy varies from 8% to 35% and it appears to be
sporadic, implying that digestive tract metastases occur irregularly [2,3].
Autopsy records show a higher propensity for lobular carcinoma in proximity to GI tract
metastasis [2]. Since such cases are rare in occurrence, there is a lack of research into potential
treatments. Financially, mammary tumours are expensive in terms of operating and maintenance
costs [2, 3,4]. Due to the lack of research on the effects of mammary tumours on the GI tract, this
study focuses on highlighting the impact of immune dysfunction and breast cancer on the
gastrointestinal tract. Therefore, the current study analysed GI parameters in a preclinical mouse
model of mammary tumours to establish the relationship between mammary tumours and
changes in the GI tract.
Generally, breast cancers negatively affect the immune system. The immune system
primarily protects the human body against infection and illnesses caused by viruses, fungi, and
bacteria [3]. Immune responses refer to a collection of responses and reactions produced by the
body to contain diseases. Therefore, breast cancer weakens the immune system by spreading to
regions such as the bone marrow. Bone marrow is primarily responsible for producing blood
cells, which help fight disease-causing pathogens. Since the GI lumen contains commensal and
pathogenic microorganisms, the immune system needs to develop and maintain its presence
along the mucosal barrier, a boundary between the mucosa and the lumen [3] [4]. The tissue of
the GI tract is primarily protected by immune cells such as lymphocytes and macrophages as
well as other cells that participate in the production of immune responses [4].
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1.2 The Enteric Neuroimmune System
The human gastrointestinal tract spans 5 meters long and has a mucosal surface area of
approximately 32 square meters [4]. The gut microbiome consists of approximately 40 trillion
cells from hundreds of microbial species [4]. The GI tract hosts 70-80% of all immune cells in
the body and also contains more than 100 million neurons (known as the enteric nervous system)
with approximately 100,000 nerve endings terminating in the epithelial mucosa [4, 5]. These
statistics show that the human GI tract is a complex system
A wide range of physiological activity occurs in the GI tract including digestive,
metabolic, immune, endocrine, and neurobiological functions. The potential roles of the gut in
health and disease have therefore attracted immense research interest in enteric neurobiology [5].
1.2.1 The Enteric Nervous System
The enteric nervous system (ENS) is the intrinsic nervous system of the GI tract and the
largest segment of the peripheral nervous system (PNS). The ENS is a large, complex neural
network that regulates numerous immune, endocrine, and metabolic functions [5, 6]. Due to its
location and the complexity of the neural networks, the ENS is often referred to as the second
brain or the „brain in the gut‟ [6]. The ENS has extrinsic connectivity to the CNS through
sympathetic and parasympathetic nerves that synapse directly to the neurons [7].
Parasympathetic nerves such as the parasympathetic vagus nerve connect the hindbrain directly
to the GI tract. Other parasympathetic nerves originate from the spinal cord and synapse directly
onto myenteric ganglia [4] to modulate the GI tract activity. The ENS is highly heterogenous,
containing a diverse number of glia and neuronal subpopulations and virtually every
neurotransmitter of the central nervous system including acetylcholine, nitric oxide, serotonin
and glutamate [7, 8].
Neuronal networks in the gut are organized into two layers of ganglia known as the
(Auerbach) myenteric plexus and the (Meissner) submucosal plexus located in the inner
submucosal layer [8, 9]. The myenteric ganglia coordinate functions of the smooth muscle while
the submucosal ganglia control nutrient absorption, blood flow, and gut secretions [8, 9].
Sensory neurons, motor neurons, and interneurons in the ENS produce neuropeptides and
neurotransmitters that contribute to gut homeostasis and gastrointestinal immune systems [9].
Interactions between the ENS and the CNS allow the enteric neural circuits to meet the digestive
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and metabolic needs of the body [10]. The anatomical structure of the ENS is illustrated in
Figure 1 below.
Figure 1. Illustration of the anatomy of the ENS
An overview of ENS anatomy. (A) The myenteric and submucosal plexuses are illustrated and
labelled. The myenteric plexus, located between circular and longitudinal muscles of the gut
wall, coordinates motor movements of the GI. The submucosal plexus, which regulates secretory
functions of the gut, is located closest to the GI mucosal epithelium beneath the muscularis
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mucosa. (B) A cross sectional view showing myenteric and submucosal plexuses, and
innervation of the ENS [6].
The ENS contains reflex circuits that detect the homeostatic and physiological condition
of the GI tract, integrate information, and create outputs to the CNS on the control of gut
movement, local blood flow, and fluid exchange between the lumen and the gut mucosa and
vasculature [6]. Being a part of the autonomic nervous system, the ENS is the only division of
the PNS whose extensive neural circuits are capable of localized, autonomous function [6]. The
ENS maintains intercellular communication with the gut epithelia through mechanisms that
include extracellular vesicle release, soluble molecule secretion, and juxtracrine signalling.
Intercellular epithelial cell communication in the GI tract primarily occurs through synaptic
release of extracellular vesicles [9]. This is achieved by releasing compounds like glutamate
through its neuropods into the synapse with a neuron to aid information transfer. The complex
mechanisms of synaptic connections between gut epithelial cells and neurons remains poorly
understood [10].
1.2.2 Neuroimmune interactions
The human gut contains the largest compartment of immune cells in the body in addition
to a dense neuronal network [6]. The maintenance of intestinal homeostasis involves reciprocal
cross talk between immune cells and neurons of the ENS. Such neuro-immune cellular crosstalk
occurs in distinct anatomical niches and includes exchange between enteric neurons and
macrophages, mast cells, and lymphoid cells [11]. The crosstalk between neuronal and immune
systems of the gut enable the GI tract to control absorption of dietary products, responses to
pathogens, and general regulation of the gut microbiome [4].
Enteric neuroimmune interactions are mediated by cytokines, neurotransmitters and
immune mediators, which create neuroimmune synapses that bridge communications between
the immune cells and neurons [12]. Enteric neurons express immune mediators while immune
cells express neurotransmitter receptors, facilitating the activation of intracellular signalling
between the cells [12]. The bidirectional communication between nerve cells and immune cells
in the enteric environment is amplified during periods of inflammation response [12,13]. The
interactions between enteric neurons and immune cells also ensure immune modulation in the
gut [13].
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A primary effect of neuroimmune cellular interactions in the GI tract is macrophage
activation [14]. Enteric neurons use neurotransmitters to activate muscularis macrophages, a
function that has significant implications in intestinal immune homeostasis and physiological
response to foreign antigens [11, 14]. Macrophages are an integral part of the intestinal innate
immune system, where they are recruited for rapid recognition and phagocytosis of debris,
pathogens, and foreign antigens [12]. The highest density of gut macrophages, which are mainly
heterogenous C-X3-C Motif Chemokine Receptor 1 (CX3CR1+) macrophages, is in the lamina
propria, where they are located close to the intestinal epithelia for immune and secretory
functions [11]. The mechanisms of neuroimmune interactions in the gut are complex, but mainly
consist of reciprocal secretions between neuronal and immune cells [11] (Figure 2).
Figure 2. Neuron-macrophage crosstalk in the GI tract.
Figure 2 shows the interactions between enteric neurons (green) and immune cells (muscularis
macrophages (blue)). Enteric neurons produce colony stimulating factor-1 (CSF-1), which is
required by macrophages for survival. In response, macrophages produce bone morphogenic
protein type 2 (BMP2), which is important for neuronal survival. This interaction is important for
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enteric immune homeostasis. During inflammatory responses, cholinergic enteric neurons
modulate macrophage activation by controlling acetylcholine interaction with nicotinic
acetylcholine receptors. During response to bacterial infection, noradrenaline is produced from
extrinsic sympathetic fibres to promote tissue protection [11]
1.3 Impact of immune dysfunction and cancer on the Gastrointestinal Tract
Immune dysfunction results in the immune system being either underactive or overactive
[11]. A lack of immunity causes the body to lack the ability to defend itself and respond to
various pathogenic attacks. As a result, this dysfunctional resistance causes the body to become
more susceptible to pathogens [12]. Research indicates that the majority of the human immune
system is associated with the GI tract. The GI tract is also a major interface between the immune
system and bacteria [15] and therefore is a significant region through which bacteria can access
the bloodstream and tissues of the host. Therefore, much of the immune system is concentrated
within the GI tract to provide timely responses to the detected pathogens in the body before they
cause detrimental outcomes to the host [14]. First, the digestive tract plays a significant function
in immune homeostasis. The GI tract is the main location whereby the host tissue is in contact
with the external human environment. The GI tract is frequently overloaded with external
stimuli, which may, at times, include toxic pathogens. Hence, the GI tract contains a massive
number of immune cells that reside within the tract walls [18]. The most prominent location of
immune cells is within the gut associated lymphoid tissue (GALT) [18]. The GALT specifically
interacts with GI tract functions in a dynamic way to increase intestinal permeability [19]. The
GALT is also responsible for orienting the immune response. Therefore, it is clear that the GI
tract relies strongly on the reactions of the immune system to interpret and defend the host
against various stimuli. However, in the case of immune dysfunction the immune system’s ability
to mount responses against pathogens are reduced [19].
1.3.1 Cancer and immune dysfunction
Immune deficiency among cancer patients is prevalent and well-documented [20].
Tumour cells within the human body have established several molecular and cellular
mechanisms to avoid antitumour immune responses from the body [20, 21]. Some of these
immune deficiencies include the defective presentation of antigens on the surface of the cell
contributing to the inability of the host to identify components for destruction [20]. Tumourinduced defects have been studied for an extended period and are well known to take place
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across all major sections of the immune system [21]. The continuous supply of vascular
endothelial growth factor, which is created by solid tumours, prevents the normal functional
maturation of dendritic cells [21] which serve to stimulate the differentiation of naïve T cells into
T-cells capable of eliciting immune responses. In the absence of matured dendritic cells, T-cell
secretions to B-cells in the peripheral lymphoid body organs is reduced [20]. Furthermore, it
induces a dramatic and rapid atrophy of the thymus among tumour-bearing animals as shown
below:
Figure 3. The mucosa associated lymphoid tissue (MALT).
A diffuse system that consists of small concentrations of lymphoid tissues located in different
sub mucosal membrane in the human breast and the GI tract. As seen above, MALT is populated
with numerous lymphocytes including the T-cells as well as the B-cells. Each cells is
strategically placed in the MALT to encounter different antigens passing through the mucosal
epithelium as shown in the above figure [21].
Thus, T-cells defects and premature thymic atrophy are frequently observed among breast
cancer patients [21]. Nevertheless, immunotherapy has shown remarkable clinical outcomes for
patients suffering from various types of tumours [22]. However, frequently its potential cannot
be achieved because of the immune dysfunction resulting from different suppressive mechanisms
that play a central role in cancer progression and development. Management and monitoring of
immune dysfunction in cancer patients is a prerequisite for all of the development strategies that
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focus on alleviating cancer-induced immune suppression [23]. Initially, the level to which the
malfunction of the immunity occurs needs to be established. It is currently difficult to accurately
monitor the function and frequency of immune suppressive cells, however, even though it is
quite straight forward to measure the general signs of immune suppression. A lack of specific
indicators and the existing phenotypic complex structures within immune suppressive cells of
similar lineages is a critical challenge [24]. T cells and B cells are important components of the
gut immune defense system. Since breast cancer alters these immune cells it is highly likely that
mammary tumours can affect gut physiology.
1.3.2 Histopathological alterations of the GI tract in cancer and symbiosis
As previously mentioned, breast cancer has metastatic behavior, and can penetrate the
gastrointestinal tract, in particular, the upper GI tract [25]. Once breast cancer cells invade the GI
tract, the immune system is continually suppressed, leading to the development of irritable bowel
syndrome among other gastrointestinal tract disorders among breast cancer patients (25).
Etiopathogenesis seems to be multifactorial, and it incorporates gastrointestinal motor function,
psychosocial factors, and increased sensitivity to visceral stimuli [26]. Histological examination
of the gastrointestinal tract indicates that no mucosal abnormality appears in most cases [26,27].
Instead, quantitative histological, ultrasound and immunohistochemical analyses indicate a subtle
morphological transformation incorporating mast cells, lymphocytes, enteric nerves, and
enterochromaffin cells [28, 29,30]. As a result, these transformations have led to the appreciation
of the new hypothesis that connects the enteric and the central nervous system to the process of
immunity [31]. Studies reveal that mammary tumour development involves multistep processes
that sequentially take place from hyperplasia, carcinoma, and typical hyperplasia until the final
step, which is the invasive stage of the carcinoma [32]. Several factors, both cellular and
molecular, are considered to play a critical role in the carcinogenesis and development of the
mammary gland [33]. Various toxic chemicals such as Dimethylbenz, that induces tumours in
experiments involving animals act as potential carcinogens with the ability to act on numerous
body sites, including the gastrointestinal tract [34].
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Figure 4. Components of the GI system.
A cross-section of the gastrointestinal tract and its components including the stomach, liver,
gallbladder, pancreas, duodenum, rectum, appendix among other organs [31].
1.4 Alterations to the immune system through the introduction of mammary tumour
metastasis
Manipulation of immunity for the treatment of cancer, including breast cancer, is
becoming a common treatment approach [33]. Many attempts are being made to inhibit immune
evasion or strengthen the immune response. Thus, highlighting various biological mechanisms
involved is critical to the potential of improving breast cancer management across different
patients. Studies reveal that the immune response to cancer through the immune system can
either be adaptive or innate [34]. The innate immune response is utilized during the development
of cancer and is typically sustained by the Natural Killer (NK) cells, Natural killer T cells
(NKT), basophils, macrophages, and human intraepithelial lymphocytes [34]. Innate immune
cells differ from typical adaptive lymphocytes because adaptive immune system is more specific
response and longer-lasting than the innate response [35, 36,37]. Unlike innate immunity,
adaptive immunity entails a more flexible and broader repertoire of the immune response.
[38,39].
Tumour diffusion and development is primarily sustained by several altered molecular
pathways [40]. The alterations occur because of the crosstalk between different components of
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cells within the tumour environment, whereby changes in immune cell function are often
mediated by unique cytokines [40]. Some cytokines involved include interleukins (ILs) and
interferons (INFs). ILs and INFs regulate local inflammatory reactions. Since immune
dysfunction is a major component of cancer initiation and progression, immune cell suppression
and inhibition are critical clinical approaches employed in the treatment of breast cancer [42].
Accessible molecular signaling pathways associated with the functioning of the immune system
in breast cancer have been reviewed [43]. Most of these biological pathways (including estrogen
receptors signalling, human epidermal growth factor receptor signalling, and canonical Wnt/βcatenin signaling) are associated with the origin and development of breast cancer, as well as
immune evasion. Thus, drug manufacturing is focused on molecules that act on the specific
molecular pathways that are highly susceptible in breast cancer. These treatments are
administered through adjuvant therapy, an approach involving chemotherapy.
1.4.1 Immune cells at the primary tumour site influence metastatic behaviour of cancer
cells
Immune cell infiltration in the primary tumour can cause a negative or positive effect on
the prognosis of the patient [44]. Tumours can locate away from the immune system. Also,
tumours can co-opt several immune processes. The main mechanisms of co-opting of immune
system functions in tumours is through modification within the stroma region of the cancer
[45,46]. The tumour stroma consists of various cell types that take part in tissue homeostasis
such as endothelial cells, immune cells, and fibroblasts [47]. Typically, the stroma develops
tissue homeostasis by regulating the balance between cell death and cell proliferation through
interactions between fibroblasts and extracellular matrix (ECM) [48]. In cancer, fibroblasts
usually induce a tumour progression through stimulation of the invasive phenotype and spread of
cancerous cells. As a result, it increases the potential for metastasis of the cancer cells [48]. For
example, in pancreatic cancer, desmoplasia or dense fibrosis is postulated to act either in a
protective role by generating survival signals or by impeding the delivery of drugs to the cancer
cells or via an inhibitory role to constrain the growth of the tumour [49]. The tumour stroma can
also encourage the development of new blood vessels through angiogenesis processes. Without
these processes, solid tumours can be limited in size and unable to reach the bloodstream for
dissemination [50]. Hence, this is a critical aspect of metastasis. In general, angiogenesis occurs
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within the tissue of interest due to the imbalance between anti-angiogenic changes and proangiogenic factors, a process widely known as an „angiogenic switch‟ [51] which may be
relevant to the increase in vasculature in the gut mucosa is seen with many GI disorders.
Angiogenesis catalyzes the growth of metastasis and tumours as a result of chemical signals
emanating from the tumour cells in the phase of rapid growth.
1.4.2 The immune interaction with the gut nervous system
Interactions between the immune system and the nervous system assist the GI tract in
responding to various dietary metabolic products within the lumen [51]. The extensive GI
lymphatic system typically mediates the flux of immune cells, which are located adjacent to the
lining of the GI tract. Components of the adaptive and the innate immune system require
stimulating molecules and antigens that are located along the GI tract [52]. Interactions between
the immune system and the gut assists in maintaining general physiology, homeostasis and fight
against pathogens. For example, within the GI tract, a broad range of pathogens are absorbed
within ingested food content and there is a diverse microbiome. Other than the primary
physiological functions like digestion, transport of nutrients, and absorption of nutrients, the gut
provides a critical compartment of neuronal cells and immune cells [53]. The cellular
populations coexist and interact closely in a bidirectional manner. The neural network in the GI,
commonly referred to as “the brain of the gut,” consists of the intrinsic enteric nervous system
(ENS) within the wall of the GI tract and includes afferent fibers that enhance the
communication between the central nervous system and the gut [53]. Thus, the ENS plays a
crucial role in regulating gut motility as well as regulations of sensory neurons and secretions
within the ENS including the mediation of pain signals associated with the central nervous
system [53].
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Figure 5. The mucosal immune system.
The arrows (white) indicate developmental stages of the immune system and its full
operationalization. The plasma cells in the lamina propria synthesize immune agents and
transported into the gut lumen via the epithelial cells. Polymeric IgA binds to the mucus layer
and acts as an antigen specific barrier to pathogens and toxins in the gut lumen [54].
Figure 6. Mucosal immunity of the GI tract
An overview of mucosal immunity anatomy of the GI tract. The microbiota protects the integrity
of the intestinal epithelium (A) and maintains epithelial integrity (B) through the production of
Short Chain Fatty Acids (SCFAs). Microbiota regulates the development of immune system and
function induced by intraepithelial lymphocytes (IELs) and Innate Lymphoid Cells (ILCs) (C).
IELs recognize commensal microbiota (D) while ILCs interact with both the microbiota and
derived metabolites (E) [28].
Alterations to the ENS can lead to functional GI disorders, including severe
inflammatory reactions, ulcerative colitis, and IBS, among other responses [53,54]. Moreover,
the gut contains significant levels of vasoactive intestinal peptide (VIP) [54]. In addition to the
GI tract, VIP is expressed in the peripheral nervous system and the central nervous system [54].
VIP reinforces the survival of T-cells, which is critical in the inhibition of cell death induced by
antigens-. Moreover, VIP attenuates the severity of TNBS colitis by suppressing T Helper Cell
Type 1 and the augmentation of T Helper Cell Type 2 [49, 50, 51]. Therefore, it is widely
acknowledged that neuroendocrine compartments, together with the parasympathetic
components of the autonomic nervous system, act as critical pathways where the GI and the
brain interact [54]. It is now well established that the brain communicates with the gut through
the brain-gut axis including the hypothalamic-pituitary-adrenal axis (HPA).
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The above literature has explored various aspects of immune dysfunction and cancer
impacts on the gastrointestinal tract [54, 53]. In addition, interactions between the GI tract and
the immune system were examined and mechanisms in which the central nervous system and the
ENS of the GI tract interact and work together to maintain physiological homeostasis [54, 55].
Moreover, studies of the effects of breast cancer on the immune system [56] have also been
reviewed. It is acknowledged, however, that there remains a need for a comprehensive study on
the impact of immune dysfunctions and breast tumours on the gastrointestinal tract [57] to
broaden our understanding and identify therapeutic targets to treat GI dysfunction in breast
cancer to improve quality of life for these patients.
1.5 Research Questions

1. What are the anatomical, histopathological, and neuronal differences in the gastrointestinal
tract that occur in non-tumour and tumour-bearing mice?
2. What are the impacts of cancer-related immune dysfunction on the gastrointestinal tract in
mouse models?
1.6 The rationale of the study
The gut houses the largest immune system in the body and therefore employs cellular
mechanisms to constantly survey the environment and defend the host from invasion by
pathogens. The crosstalk between cancer cells and the immune system is crucial for disease
progression and its therapeutic targeting could be vastly improved as we begin to discover new
immunotherapies. It is now well established that inflammation pathways and the nervous system
interact to maintain physiological homeostasis. For example, multiple types of immune cells
express receptors for neurotransmitters and neurons similarly express receptors for cytokines to
facilitate these communications.
Recent studies have shown associations between functional changes in the GI tract and
disease ranging from colitis to cancer, however, research into structural and histopathological
changes of the GI tract in this context are limited. Therefore, the current study will contribute to
the knowledge around changes in GI tissue structure in cancer including potential changes
occurring in the enteric nervous system of the GI tract in the presence of mammary tumours in
mice
23
The current project will investigate the extent to which gut-associated immune responses
are altered in hosts with metastatic breast cancer in mice. This project is focused on
understanding how changes in the host immune system due to cancer affect tissue structure and
the nervous system in the gastrointestinal tract. This objective will be achieved through
investigation of the anatomical, histopathological, and neuronal differences in non-tumour and
tumour-bearing mice.
To study the anatomical, histopathological, and neuronal changes of the GI tract in
tumour and non-tumour models, BALB/C mice will be used. During this project,
histopathological staining and whole mount immunofluorescence techniques will be employed to
study the myenteric neurons and enteric immune cells in mice.
1.7 Hypothesis
This study hypothesizes that:
Alterations to the immune system through the introduction of mammary tumour metastasis
will affect mouse gastrointestinal pathology.
1.8 Research Project Aims
The aims of this study will be:
1. To assess for anatomical differences in the GI tract from non-tumour and mammary
tumour-bearing mice.
2. To assess for histopathological differences in the GI tract from non-tumour and
mammary tumour-bearing mice.
3. To assess for neuronal differences and Iba1 expressing muscular is macrophages in
the GI tract from non-tumour and mammary tumour-bearing mice
4. To undertake a systematic review of the literature relevant to mammary tumours and
GI tract pathology
Note: Due to the covid-19 laboratory shutdown, some of the above components of the project
(such as analysis of potential histopathological and neuronal differences) have been negatively
affected due to a lack of access to the laboratory and are therefore not included in the results
chapter of this thesis.
24
Chapter Two: Materials and Methods
2.1 Animal model
The mice used in this study consisted of 12 female adult BALB/C mice obtained from the Olivia
Newton-John Cancer Research Institute (Heidelberg, Victoria, Australia). All mice were 14
weeks old. Mice were split into two groups consisting of 9 tumour-bearing and 3 non-tumour
bearing mice. To recapitulate metastatic breast cancer in mice, 50,000 cells, derived from a
highly metastatic mouse mammary tumour cell line (4T1, obtained from American Type Culture
Collection (ATCC) were injected into the mammary fat pad of the 4th inguinal mammary gland
(58). Mice in the remaining group were assessed as non -tumour bearing controls. Both groups
were euthanized by cervical dislocation following the Austin Health Animal Ethics Committee
guidelines.
2.1.1 Anatomical evaluation
Mouse gastrointestinal tract tissues were dissected and placed in phosphate buffer saline (PBS,
pH=7.2) for anatomical and histological assessment. Primary tumours were collected and
weighed by the host laboratory (these data are not included in this thesis). Each gastrointestinal
region was separated into small intestine and colon segments. The length of each segment was
measured and the number of faecal pellets in each colon were counted. Images of the colon
tissue containing faecal matter were analysed using ImageJ software. Caecal weights from
individual mice were measured following flushing of caecal content with PBS using a plastic
Pasteur pipette.
2.2 Histopathological Studies
Histopathological investigation was intended to study the cellular structures of tissues and assess
the morphology of the immune cells present. However, due to the COVID-19 pandemic, the
laboratory was shut down and therefore some of these experiments were not completed.
2.2.1 Cryo-preservation
Isolated tissue segments consisting of jejunum, colon and caecum were carefully flushed to rid
them of their lumen contents and cut into approximately 2 cm long pieces. These were fixed
overnight in 4% formaldehyde at 4°C, followed by overnight incubation in 30% sucrose solution.
This is to prevent ice crystal formation during the freezing process. Tissue was well blotted and
25
vertically orientated in a well-labelled crymold then covered in OCT (Optimum Cutting
Temperature; Tissue- Tek Proscitech, Australia) compound. The tissue samples were pre-cooled
in 2-methylbutane before being snap-frozen in liquid nitrogen then stored at -80°C before
sectioning.
2.2.2 Cryosectioning
Frozen tissues were mounted with OCT media onto a cryostat chuck (Leica CM1950 Clinical
Cryostat, Leica Biosystems Nussloch GmbH, Germany) for 10 minutes at -20°C. The block was
trimmed gradually until the embedded tissue was revealed before cutting the sections at 8
micrometres. Sections were collected on a positive charge frosted-glass slide (Superfrost® Plus
Micro Slide, VWR International, Radnor, PA, USA). Slides were air-dried at room temperature
for 24 hours.
2.2.3 Haematoxylin and Eosin Staining
All sections were stained with the conventional H&E method. The staining process can be
summarised into five steps with intermittent rinsing with water. Firstly, tissue sections were
rehydrated and stained in Haematoxylin solution, followed by blueing in Scot‟s water before
counterstaining with Eosin solution. Lastly, sections were dehydrated in ascending grades
alcohol. The alcohol was removed by histolene, then mounted in DPX and coverslipped. The
detailed protocol is listed in the Appendix 1.
Note: Due to the COVID-19 pandemic, the laboratory was shut down and therefore these
experiments were not completed.
2.2.4 Imaging (Brightfield microscopy)
Images from stained tissue slides were captured by an Olympus slide scanner optical microscope
(Olympus Australia Pty. Ltd.; Melbourne, Australia, VS120-S2) using X40 magnification. All
generated data was converted to Tiff format for analysis on the ImageJ software platform
(ImageJ 1.52a, NIH, USA). The software was used to assess and analyse the histopathological
scoring, villus length and height, crypt depth, inter crypt distance, muscle layer thickness and cell
packaging density within the caecal patch.
26
Note: Due to the COVID-19 pandemic, the laboratory was shut down and therefore these
experiments were not completed.
2.3 Immunohistochemistry for muscularis macrophages and neuronal processes.
Immunohistochemistry techniques were planned to be used to determine the distribution of
macrophages and myenteric plexuses of caecal tissue from both tumour and non-tumour bearing
mice (59). However, due to the COVID-19 pandemic, the laboratory was shut down and
therefore some of these experiments were not completed.
2.3.1 Tissue preparation and microdissection
The remaining part of the caecal segment prepared from the dissected mice was prepared for
whole mount immunofluorescence. The caecal content was emptied by carefully flushing with
PBS solution. The tissue was pinned on to the Sylgard dish containing 3 times-filtered
phosphate-buffered saline (3xPBS, filtered 3 times at pH 7.2). This was subsequently cut open
along the mesenteric border and laterally pinned down to reveal the mucosal layer. With the aid
of insect pins, the tissue was stretched flat at each side. The whole tissue was fixed overnight in
4% formaldehyde at 4°C. After fixation, the tissue was washed 3 x10mins with PBS and
refrigerated before peeling (60). The mucosa and submucosa were peeled off to expose the
mesenteric plexus in the circular muscle layer. This process was performed under a stereo
microscope.
The following procedures were planned but were unable to be completed due to lab shutdown in
response to COVID19 pandemic
2.3.2 Primary and secondary antibody
After the dissection, the preparation is treated with 1% triton and 10% CAS Block for 30
minutes, to prevent non-specific binding. This step is followed by incubation of the tissue with
the two antibodies at 4°C, overnight. Anti- Iba1 (Rabbit) and Anti-Tuj-1 (Mouse) were selected
as the two primary antibodies to be utilised for demonstrating all muscularis macrophages and
27
neuronal processes respectively. The Iba1 antibody is an ionized calcium binding adaptor
molecule 1 (Iba1) that binds with macrophage-specific calcium-binding protein that is involved
in modifying their membrane for phagocytic process. Likewise, anti-Tuj1 is an antibody that
binds a structural protein known as beta-tubulin III, present in neurons and aids in the formation
of microtubules.
The secondary antibodies will be added the next day after washing out the primary antibody.
Incubation will be performed in the dark in an enclosed container at room temperature for 150
minutes. Lastly, the slide will be mounted in the fluorescent mounting medium. The detailed
protocol is listed in the Appendix section.
2.3.3 Imaging (florescent microscopy)
Images will be obtained and scanned on a fluorescent microscope for analysis on ImageJ
software. The software will also be used to determine the density, volume and sphericity of IBa1
immunoreactive macrophages within the caecal patch. Also, innervation of nerve processes into
the mucosa and the caecal patch were planned to be assessed.
2.4 Image Analysis
Faecal pellet parameters were determined from images of mouse colon tissue using Image J
software. All values acquired from the images were exported to Microsoft Excel spreadsheet and
saved as a CSV file. All images for histological and immunohistochemistry were planned to be
analysed with ImageJ software [61]. The villus dimensions within a selected the area of interest
were planned to be measured for both tumour and non- tumour bearing groups. Similarly, Iba1
and Tuj–1 immunoreactivity are anticipated to be quantified using ImageJ software. For
immunofluorescence experiments, this can be achieved by adjusting the intensity threshold to
capture the fluorescence labelling on a dark background.
2.5 Statistical analysis
All measurements were collated on excel software and analysed with Graph Prism v8
(California, United States) [62]. Data were expressed as mean and standard deviation. A
Student’s t-test and analysis of variance (ANOVA) were used to determine the statistical
significance between the two groups.
28
2.6 Systematic review methodology
2.6.1 Study design
A systematic review design was used to increase the validity of the results obtained in this
project that evaluated the impact of immune dysfunction and cancer on the gastrointestinal tract
in mice. The systematic review study design was preferred for this study since this approach
ranks high in the hierarchy of evidence and are often used in evidence-based practices [63]. In
this study design, high quality evidence from primary studies will be used. The Preferred
Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used to
guide the review of the included studies.
2.6.2 Search Terms
These are important for the identification and selection of relevant articles for inclusion in the
systematic review. They are used in the search of the articles in databases such as PubMed and
Google Scholar that were searched. In this study, the search terms were: characterising, effect,
impact, immune dysfunction, immune failure, cancer, malignancy, tumour, uncontrolled cell
proliferation, gut, gastrointestinal tract, GIT, mice, rats and murine models. The Boolean
operators AND and OR were used to combine the search terms to enhance the identification of
studies.
2.6.3 Inclusion and Exclusion Criteria
The criteria defined the type of studies that are to be included in the systematic review, and
influence the scope of the study. The inclusion criteria in this review were studies that were
published between 2015-2020 in English, and that focused on the evaluation/ characterisation of
the impact of immune dysfunction and cancer on the GI tract of murine models. The exclusion
criteria were studies that were published before 2015, and that did not evaluate the effects of
immune dysfunction and cancer on the GI tract of murine models.
2.6.4 Data Collection and Analysis
Data collection from the selected studies involved the identification of relevant study
characteristics such as the aims, methodologies, results and conclusion, from all included studies.
29
These characteristics were then combined through narrative synthesis to indicate the results of
the systematic review.
Chapter 3: Results
3.1 Anatomical measures for gut tissues in non-tumour and mammary tumour-bearing
mice
To determine the effect of mammary tumour on the gastrointestinal tract of the mice, this study
compared physical and physiological features of the GI tract in both groups. These features
include the bodyweight, the length of the colon and small intestine, the number and dimension of
faecal pellets, number of Peyer‟s patches, caecal weight and number of caecal patches present.
3.1.1 Body weight
The bodyweight for non-tumour control mice (n=3) was not obtained from the collaborator‟s
laboratory due to Covid19 considerations.
3.1.2 Colon length
The physical effect of neuro-immune activity was investigated by assessing the colon length in
non-tumour (n = 3) and tumour bearing (n= 9) mice. This parameter was measured to determine
if GI gross anatomy is altered due to immune triggers. The result showed no significant
difference in colon length between non-tumour (8.0 ± 0.3cm) and tumour bearing (8.4 ± 0.3 cm;
mean ±SEM) groups. Figure 7
30
Non tumour Tumour 7 8 9 10 Colon length
Colon length(cm)
P=0.363
n=3 Non tumour n=9 Tumour
Mice types

Figure 7. Colon length in tumour and non-tumour bearing mice
The green and red circles represent individual values within the non-tumour and tumour
group of mice, respectively. The colon length (cm) is given along the Y-axis while the
group is presented along the X-axis of the graph. The average colon length for the group
is indicated with the mid-bar of each box plot. Data were compared using unpaired t-test
on GraphPad Prism Software (version 8.0) with the significance of difference taken as p
< 0.05 and at a confidence level (95%).
3.1.3 Small intestinal length
The physical impact of neuroimmune interaction on the upper GI tract was investigated by
determining any changes to the small intestine length between the two groups. The result
showed no statistical significance between the small intestinal length in non-tumour (n = 3) and
tumour bearing (n= 9) group of mice were 34.5 ± 1 cm. and 33.8± 1.0 cm.respectively (p=0.363).
31
Non tumour Tumour 28
30
32
34
36
38 Small intestinal length
Small intestinal length(cm)
P=0.623
n=3 Non tumour n=9 Tumour
Mice types
Figure 8. Small intestinal length in tumour and non-tumour bearing mice.
The green and red circles represent the length of small intestine of each mouse within the
non-tumour and tumour group respectively. The small intestine length (cm) is represented
along the Y-axis while the X-axis of the graph represents the group. The average length
of the small intestine in each group is indicated by the mid bar of each box plot. The
measurement data were analysed and compared using unpaired t-test on GraphPad Prism
Software (version 8.0) with the significance of difference (p < 0.05) and confidence level
(95%).
3.1.4 Caecal weight
The weight of the caecum was measured to detect any physical changes in this gut region. The
caecum is of interest because it provides an environment for fermentation of digesta by the gut
microbiota. This study shows that there was no significant difference between the average caecal
weight of tumour (0.43 ± 0.06 g) and non-tumour bearing mice (0.32 ± 0.01 g; p value = 0.103).
Although caecal weight was not significantly different between the two groups, there was a trend
for heavier caecal in tumour-bearing mice.
32
Non tumour Tumour 0.0
0.2
0.4
0.6
0.8 Caecal weight
Caecal weight(cm)
P=0.103
n=3 Non tumour n=9 Tumour
Mice types
Figure 9. Caecal weight in tumour and non-tumour bearing mice.
The green and red circles represent the weight of the caecum in each mouse within the
non-tumour and tumour group respectively. The caecal weight is represented along the
Y-axis while the X-axis of the graph represents the group. The average weight of the
caecum in each group is indicated with the mid -bar of each box plot. The measurement
data were analysed and compared using unpaired t-test on GraphPad Prism Software
(version 8.0) with the significance of difference (p < 0.05) and confidence level (95%).
3.1.5 Number Peyer’s of patches
Effects of immune stimulation may influence the number of Peyer‟s patches in the small
intestine. Peyer‟s patches are aggregates of lymphoid tissue located predominantly in the ileum
of the small intestine. The average number of Peyer‟s patches from mammary tumor-bearing
mice (9 ± 0.6., n=9) was significantly greater compared to their non-tumor mate mice (7 ± 0,
n=3; p=0.01).
33
Non tumour Tumour 4
6
8
10
12Number of Peyer’s patches
Number of Peyer s’ patches
P=0.0104
** n=3 Non tumour n=9 Tumour
Mice types
Figure 10. The number of Peyer’s patches in non-tumour and tumour bearing mice.
The green and red circles represent the number of Peyer‟s patches in each mouse within
the non-tumour and tumour group respectively. The number of Peyer‟s patches is
represented along the Y-axis while the X-axis of the graph represents the group. The
average number of Peyer‟s patches in each group is indicated with the mid -bar of each
box plot. The measured data were analysed and compared using unpaired t-test on
GraphPad Prism Software (version 8.0) with the significance of difference (p < 0.05) and
confidence level (95%).
3.1.6 Number of caecal patches
The caecal patches within the caecum were counted to determine the physical changes in the
immune tissues of the large intestine. There was no significant difference between non-tumor
control mice and tumor bearing mice in terms of the number of caecal patches. The average
number of caecal patches of non-tumour mice (n=3) and tumour-bearing mice (n=9) was 4 ± 0.6
and 5.56± 0.6 respectively (p=0.102). Although there was no difference between the two groups,
there was a trend towards increased numbers of caecal patches in tumour-bearing mice.
34
Non tumour Tumour 0
2
4
6
8
10 Number of caecal patches
Number of caecal patches
P=0.102
n=3 Non tumour n=9 Tumour
Mice types
Figure 11. The number of caecal patches between tumour and non-tumour bearing groups.
The green and red circles represent the number of caecal patches in each mouse within
the non-tumour and tumour group respectively. The number of patches is represented
along the Y-axis while the X-axis of the graph represents the group. The average caecal
patches in each group is indicated with the mid -bar of each box plot. The measurement
data were analysed and compared using unpaired t-test on GraphPad Prism Software
(version 8.0) with the significance of difference (p < 0.05) and confidence level (95%).
3.1.7 Number of faecal pellets and their dimensions within the dissected colon
To measure the physiological changes in the GI tract in response to mammary pad tumours, the
faecal pellets present in the colon were counted and measured. This information is useful in
evaluating the changes to the absorptive processes of the GI tract. There was a significant
difference in the average number of faecal pellets within the dissected colon of non-tumour mice
(1.66 ± 0.3) and the tumour bearing mice (3.77± 0.6.; p = 0.013; Figure 12A). In contrast, either
faecal pellet length or width showed any significant differences between the two groups. The
average faecal pellet length in non tumor and tumor bearing mice was 8.2 ± 1.1 mm and 6.6 ±
0.6 mm respectively (p=0.27; Figure 12B). The average pellet width for non tumor and tumorbearing mice was 3.2 ± 0.3mm and 2.4± 0.1mm, respectively (p=0.127; Figure 12C).
35
Non tumour Tumour 0
2
4
6
8 Number of pellets
Number of pellets
P= 0.0133
**
Mice types Non tumour Tumour 0
5
10
15 Faecal pellet length
Faecal pellet length (mm)
P= 0.266 Mice types Non tumour Tumour 0
1
2
3
4 Faecal Pellet width
Pellet widthg(mm)
P= 0.127
n=3 Non tumour n=9 Tumour
Mice types
A B C

Figure 12. The number of faecal pellets and their dimensions within the dissected colon.
A: the number of pellets between the two groups; Figure B: faecal pellet length between the two
groups. Figure C: faecal pellet width between the two groups. The green and red colour circles
represent the measurement for each mouse within the non-tumour and tumour group
respectively. Each measurements are represented along the Y-axis while the X-axis of the graph
represents the groups. The measurement data were analysed and compared using unpaired t-test
on GraphPad Prism Software (version 8.0) with the significance of difference (p < 0.05) and
confidence level (95%). The average weight of the caecum in each group is indicated with the
mid -bar of each box plot.
36
Note: Due to the covid19 laboratory shutdown, the following components of the project have
been negatively affected due to a lack of access to the laboratory and are therefore not included
in the results chapter of this thesis.
3.2 Gastrointestinal histopathology in mammary tumour-bearing mice
3.2.1 Villus height in proximal colon
3.2.2 Villus width in proximal colon
3.2.3 Crypt depth in control and mammary tumour-bearing mice proximal colon
Note: Due to the covid19 laboratory shutdown, the following components of the project have
been negatively affected due to a lack of access to the laboratory and are therefore not included
in the results chapter of this thesis.
3.3 Immunofluorescence staining of caecal tissue in mammary tumour-bearing mice
3.3.1 Optimization of immunostaining
3.3.2 Positive and Negative immunofluorescence controls
3.3.3 Detection of Iba1, Hu and Tuj-1 in cross sections of control and mammary tumourbearing mice caecal tissue
3.3.4 Quantification of Iba1 immunoreactivity in caecal tissue of control and mammary
tumour-bearing mice
3.4 Systematic Review Results
During the four phases of the systematic literature search in this study, a total of 727 articles
were retrieved. Out of these, 68 duplicate articles were deleted and the remaining 659 screened
for title and abstract. During this step, a further 596 irrelevant articles were excluded as they did
not report on either cancer, mammary tumour or gut nervous systems. Next, another 63 full-text
articles were assessed culminating to exclusion of 47 articles whose content could not be
accessed due to restricted rights and permission. Of the remaining 16 articles, another 13 were
excluded because they did not satisfy the inclusion/exclusion criteria for this study, resulting in 3
remaining articles. Finally, a total of 5 articles were included for analysis after including 2 other
articles that were obtained from reviewing the reference list of the remaining studies. Figure 13
presents the four phases of the systematic literature search that was used in this study using the
37
PRISMA approach. In addition, a summary of characteristics of the final five studies that were
included for analysis is presented in Table 1
PubMed (n 394) Medline (n 206) Cinahl (n 92) Cochrane (n 35)
Keyword search (n 727) Duplicates excluded (n 68)
Studies screened
for title and
abstract (n 659)
Studies excluded in title and abstract
review; no immune interactions, no
cancer, no mammary tumour, no
gastrointestinal tract, no gut nervous
system (n 596)
Articles selected to read
in full text (n 63)
Articles excluded due to
inaccessible content (n 47)
Full-text studies assessed
for eligibility (n 16)
Articles excluded after full-text review (n)
Reasons for exclusion;
 No immune interactions (n 3)
 No mammary tumour (n 6)
 No gut nervous system (n 4)
Articles included and
analyzed (n 5)
Studies included by reviewing
reference lists of the included
studies (n 2) Screening Eligibility Included Identification
Figure 13 the flow of included studies based on the four phases of systematic literature search
38
The characteristics and description of the five studies that were included for analysis is as
presented in Table 1 below;
Table 1 Summary Characteristics of included articles
Characteristic Description
Year published 2015 – 2020
Study participants Clearly defined human or mice participants
Health conditions of interest Cancer patients with tumours
Differences in non-tumour and tumourbearing mice
Clearly defined for the study
Changes in gastrointestinal pathology Clearly defined for the study
3.4.1 Study Design and Location of the Studies
It was observed that three [64, 65, 66] of the five articles included in the final analysis adopted a
case-control design where rats were categorized into two groups; control and treated. In the study
by Khanna et al. (2019), the treated group was exposed to 7620 m of hypobaric hypoxia (HH) for
different durations before examining them for the extent of intestinal mucosal damage as given
by an increase in mucosal permeability and changes in intestinal villi [66]. On the other hand,
one study [67] adopted a cohort design.
3.4.2 Study Participants
In terms of study participants, two studies [67, 68] were conducted on cancer patients to
determine tumour progression and immune regulation. Similarly, three studies [67, 65, 66]
researched tumorous cells in otherwise healthy individuals and patients with colorectal cancer. A
further two studies [66, 64] conducted research on mammary tumours and a total of three studies
were conducted on mice [64, 65, 66].
39
3.4.3 Measurement of Impact
In one of the reviewed studies the researchers [68] examined change in neuronal splicing factor
neuro-oncological ventral antigen 1 (NOVA1) expression in mice in initial and advanced stages
of cancer relevant to tumour progression and immune regulation [68]. To achieve this, the study
investigated gene expression in a sample of 396 surgically-resected gastric cancer tissues. In
addition, two studies [64, 66] investigated changes in the GI tract in mice due to metastatic
cancer.
3.4.4 Impact of Immune Dysfunction and Cancer on the Gastrointestinal Tract
The study by Kim et al. (2017) anticipated significant association between changes in the
immune system through the introduction of mammary tumour metastasis and gastrointestinal
pathology. These findings showed suppressed NOVA1 expression in tumour cells [68]. In
addition, these findings were independently associated with advanced stages of tumours that
weakened general cellular survival, an observation that the researchers attributed to immune
dysfunction as a result of changes in the composition of immune cells, in particular, T cells and
macrophages [68].
On the other hand, the study by Blomberg et al. (2018) reported changes in the GI tract in mice
due to metastatic cancer, including significant variations in the immune landscape of various
types of tumours [67]. According to these researchers, variations in sensitivity to immunotherapy
were attributed to variations in metabolism and genetic composition of cancer cells [67].
Furthermore, a significant association between hypobaric hypoxia exposure and the
gastrointestinal immune axis was reported by Khanna et al. (2019). Additional findings indicated
that the presence of tumours in mice led to changes in the gastrointestinal tract [64, 65].
In summary, these results indicate that mammary tumours are a leading cause of abnormalities
that affect the gastrointestinal tract.
40
Chapter 4: Discussion
This study assessed the impacts of immune dysfunction and mammary tumours on the
gastrointestinal tract in mice. The objective was to obtain features such as bodyweight, the length
of the colon and small intestine, the number and dimension of faecal pellets, number of Peyer‟s
patches, caecal weight and number of caecal patches present in diseased mice. The
determination of histopathological and neuronal impact in the gastrointestinal tract did not take
place due to the COVID-19 pandemic as it was not possible to access the laboratory.
The findings of this study show no significant changes in all anatomical measurements of
the colon and small intestines of mammary tumour-bearing mice in comparison with the control
group. This is in contrast to some studies that reported elongation of intestinal segments in
certain chronic diseases which is attributed to intrinsic innervation of the intestine [69]. The
small intestine is known to adapt to diseases by changing its structure and how its epithelial cell
divides.
There was a significant difference in the average number of Peyer‟s patches from
mammary tumour-bearing mice and non-tumour mice with diseased mice having three-fold more
Peyer‟s patches than controls. These findings show that mammary tumour metastasis has a direct
link to GI pathology. Peyer’s patches form an important part of the immune system that monitors
bacterial populations and prevent the growth of pathogenic bacteria in the intestines. The
increase in Peyer‟s patches implies an association between mammary tumour metastasis and
changes to the immune system. This view is consistent with other studies that explored the
relationship between breast cancer and gastrointestinal pathology. Generally, breast cancers
negatively affect the immune system. Studies show that breast cancer weakens the immune
system by spreading to regions such as the GI, where disturbances in the microbiota can affect
the immune system [70]. The body needs to obtain immunity against pathogenic microorganisms
present in the lumen. Peyer‟s patches monitor the presence of lymphocytes and macrophages as
well as other cells that participate in the production of immune responses along the mucosal
barrier, a boundary between the mucosa and the lumen. Elevated numbers of these patches may
therefore signify the influence of mammary tumours on cells that participate in the production of
immune responses. Peyer‟s patches, maintain intercellular communication with the gut epithelia
41
through mechanisms that include extracellular vesicle release, soluble molecule secretion, and
juxtracrine signalling [71].
Equivalent to Peyer‟s patches in the large intestine are caecal patches that form part of
the gut-associated lymphoid tissue (GALT). This study shows that there was an increase in the
number of caecal patches in the tumour-bearing group, however, it was not statistically
significant. Nevertheless, it is critical to note that the statistical power might be impacted by the
sample size of each group. The upward trend witnessed might be associated with altered
immune cell activity in this lymphoid tissue. However, unlike the Peyer‟s patches, caecal patches
contained a sparse number of matured M-cells which could be responsible for the statistical
result [72].
Also, significant physiological changes in the GI tract in response to mammary tumours
were noted in the colon with the average number of faecal pellets in tumour-bearing mice more
than two-fold higher in non-tumour mice. An increase in the number of faecal pellets within the
dissected colon has been previously reported in animal models of intestinal dysmotility and
inflammation [73,74]. The results are consistent with previous studies that found metastatic
breast cancer caused physiological changes to the GI tract that result in weight loss, bleeding,
pain, nausea, and early satiety among others. Various studies examining the behaviour of
mammary tumours metastasizing to the GI tract and other organs suggest that the metastasis
occurs due to the small size and shape of invasive lobular carcinoma, with overexpression of the
cell adhesion molecule, E-cadherin [75]. The survival and growth factors in ovaries can explain
the difference in metastatic behaviour of invasive lobular carcinoma [76]. So far, the literature
has not reported the simultaneous spread of metastases from the breast to stomach and intestines.
However, studies have demonstrated the metastasis to the stomach and colon individually [77].
The findings of this study indicate that mammary tumours adversely affect the
gastrointestinal tract. Although the results of the study with respect to potential impacts on GI
histology and the enteric nervous system are inconclusive with some laboratory work not being
able to complete due to the COVID-19 pandemic, available data suggest that mammary tumours
have significant impacts on the immune system, with Peyer‟s patch performing a critical role.
The immune responses elicited by the mammary tumours mediate various physiological changes
to the gastrointestinal tract, including alterations in absorption processes [78]. However, future
42
studies could examine the simultaneous effects of metastasis on the abdomen and the colon to
determine the extent of the damage mammary tumour inflicts on the gastrointestinal tract
pathophysiology, as well as alterations to the enteric nervous system. It is anticipated that the
results of the current study will contribute significantly to the understanding and improvement of
the management of gastrointestinal problems arising in mammary tumour patients.
43
Chapter 5: Conclusion.
The investigation of the impacts of breast cancers cancer shows negative effects on the
immune system and physiological homeostasis. The immune system primarily protects the
human body from disease-causing microorganisms. Breast cancer weakens the immune system
which compromises the ability of the body to fight disease-causing pathogens. This study was
able to demonstrated the that mammary tumours influence the GI tract in mice. The enteric
nervous system contains reflex circuits that detect the homeostatic and physiological condition of
the GI tract, integrate this information, and create outputs to the CNS on the control of gut
movement, local blood flow, and fluid exchange between the lumen and the gut mucosa and
vasculature. Being a part of the autonomic nervous system, the enteric nervous system is the only
division of the peripheral nervous system whose extensive neural circuits are capable of
localized, autonomous function. Although the mechanism of the immune response in the GI tract
as a result of breast cancer is poorly understood, the majority of the immune system is located
within the GI tract where it provides immune defence against pathogens. It is the main location
where the host tissue is in contact with the external human environment and is overloaded with
external stimuli that often include toxic pathogens. Despite the limitations of the current study,
the result of this investigation suggests a direct effect of breast cancer tumours on the intestinal
immune system and pathophysiology. However, further investigations with an increased number
of replicates are needed to fully characterize the GI phenotype including effects on ENS in breast
cancer tumour-bearing mice.
44
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Appendix (optional)
Appendix 1:
Haematoxylin and Eosin staining protocol
Step Solution Duration
1 Wash in running tap water 10 seconds
2 Immerse slides in Mayer‟s Haematoxylin 60 seconds
3 Wash in running tap water 10 seconds
4 Wash in Scott‟s tap water 30 seconds
5 Wash in running tap water 10 seconds
6 Immerse in 0.1% Eosin 30 seconds
7 Wash in running tap water 10 seconds
8 Immerse in 70% Alcohol 2 dips
9 Immerse in absolute Alcohol 2 dips
10 Immerse in absolute Alcohol 4 dips
11 Immerse in absolute Alcohol x2 60 seconds each
12 Place slides in histolene 120 seconds
13 Place slides in histolene 120 seconds
14 Mount slides in DPX and coverslip Air dry under a fume hood overnight