Characterising the impact of immune dysfunction and cancer on the gastrointestinal tract in mice

 

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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                                                                                        14

1.3.         Impact of immune dysfunction and cancer on the Gastrointestinal Tract                       17

1.3.1.             Cancer and immune dysfunction                                                                              17

1.3.2.             Histopathological alterations of the GI tract in cancer and symbiosis                      19

1.4.         Alterations to the immune system through the introduction of mammary tumour metastasis        20

1.4.1. Immune cells at the primary tumour site influence metastatic behavior of cancer cells        21

1.4.2. The immune interaction with the gut nervous system                                                            22

1.5.         Research Questions                                                                                                          25

1.6.         The rationale of the study                                                                                                25

1.7.         Hypothesis                                                                                                                       26

1.8.         Research Project Aims                                                                                                     27

Chapter2: Materials and Method                                                                                                    28

2.1. Animal model                                                                                                                          28

2.1.1              Anatomical evaluation                                                                                              28

2.2. Histopathological Studies                                                                                                        28

2.2.1              Cryo-preservation                                                                                                     29

2.2.2              Cryosectioning                                                                                                          29

2.2.3              Haematoxylin and Eosin Staining                                                                             29

2.2.4              Imaging (Brightfield microscopy)                                                                             30

2.3          Immunohistochemistry for muscularis macrophages and neuronal processes.                30

2.3.1              Tissue preparation and microdissection                                                                   30

2.3.2              Primary and secondary antibody                                                                              31

2.3.3              Imaging (florescent microscopy)                                                                               32

2.4. Image Analysis                                                                                                                        32

2.5. Statistical analysis                                                                                                                    32

2.6. Systematic review methodology                                                                                             32

Chapter 3: Results                                                                                                                          35

3.1.         Anatomical measures for gut tissues in non-tumour and mammary tumour-bearing mice 35

3.1.1.             Body weight                                                                                                              35

3.1.2.             Colon length                                                                                                             35

3.1.3.             Small intestinal length                                                                                               36

3.1.4.              Caecal weight                                                                                                          37

3.1.5.             Number Peye’s of patches                                                                                        37

3.1.6.             Number of caecal patches                                                                                         38

3.1.7.             Number of faecal pellets and their dimensions within the dissected colon                39

3.2      Gastrointestinal histopathology in mammary tumour-bearing mice                                    40

3.2.1.             Villus height in proximal colon                                                                                 40

3.2.2.             Villus width in proximal colon                                                                                  41

3.2.3.             Crypt depth in control and mammary tumour-bearing mice proximal colon           41

3.3.         Immunofluorescence staining of caecal tissue in mammary tumour-bearing mice          41

3.3.1.             Optimization of immunostaining                                                                               41

3.3.2.             Positive and Negative immunofluorescence controls                                                41

3.3.3.             Detection of Iba1, Hu and Tuj-1 in cross sections of control and mammary tumour-bearing mice caecal tissue                                                                                                                             41

3.3.4.             Quantification of Iba1 immunoreactivity in caecal tissue of control and mammary tumour-bearing mice       41

3 Systematic Review Results 41

3.3.1 Study Design and Location of the Studies                                                                           43

3.3.2 Study Participants                                                                                                                 43

3.3.3 Measurement of Impact                                                                                                         44

3.3.4 Impact of Immune Dysfunction and Cancer on the Gastrointestinal Tract                          44

Chapter 4: Discussion                                                                                                                   46

Chapter 5: Conclusion                                                                                                                    49

References                                                                                                                                      50

 

 

 

 

Figure 1 Illustration of the anatomy of the ENS [6].                                                                     13

Figure 2 An illustration of neuron-macrophage crosstalk in the GI tract (11).                              16

 Figure 3 The mucosa associated lymphoid tissue (MALT) (21)                                                    18

Figure 4 An image showing parts of the GI system (31).                                                              20

Figure 5 The mucosal immune system [54].                                                                                   23

Figure 6  Mucosal immunity of the GI tract (28).                                                                          24

Figure 7 Colon length in non-tumour and tumour bearing mice                                                    35

Figure 8 A graphical presentation of the length of small intestine between non-tumour and tumour bearing groups.                                                                                                                                            36

Figure 9 Caecal weight in non-tumour and tumour bearing mice.                                                 37

Figure 10 The number of Peyer’s patches in non-tumour and tumour bearing mice.                     38

Figure 11 The number of caecal patches between non-tumour and tumour bearing groups.         39

Figure 12 The number of faecal pellets and their dimensions within the dissected colon.            40

Figure 13 The flow of included studies based on the four phases of systematic literature search 42

 

 

 

Table 1  Summary Characteristics of included articles                                                                   43
 

GI
 gastrointestinal

ENS
Enteric Nervous System

PNS
peripheral nervous system

 CSF-1
Colony Stimulating Factor-1

GALT
Gut associated lymphoid tissues

MALT
Mucosa Associated Lymphoid Tissue

 ECM
Extracellular Matrix

 ATCC
American Type Culture Collection

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

 BMP2
 Bone Morphogenic Protein type 2

 HH
Hypobaric Hypoxia

°C
Celsius

min
Minutes

µm
Micrometre

PP
:  Peyer’s patches

DC
Dendritic cells

g
gram

SI
Small intestine

Anti
Antibody

 
 

 
 

 
 

 
 

 
 

 
 

 

 

 

Breast cancer is the most frequent neoplasm affecting the majority of women around the globe. This thesis aimed at characterizing the impacts of immune dysfunction and cancer on the gastrointestinal tract in mice. The study used randomized controlled trials on twelve 14-weeks-old female adult BALB/C mice obtained from the Olivia Newton-John Cancer Research Institute. Mice were split into two groups consisting of 9 tumour-bearing and 3 non-tumour bearing mice. The intervention group was inoculated with 50,000 metastatic breast cancer cells, derived from a highly metastatic mouse mammary tumour cell line. Mice in the remaining group were assessed as non -tumour bearing controls. The results show 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). 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). Generally, breast cancers negatively affect the immune system. Breast cancer weakens the immune system by spreading to the GI, where disturbances in the microbiota affect the immune system. The body needs immunity against pathogenic microorganisms present in the lumen, and Peye’s patches plays the essential role of monitoring the presence of lymphocytes and macrophages. The study concludes that mammary tumours have adverse effects on the GI pathophysiology. The limitation of the study includes inconclusive results due to the COVID-19 as some laboratory work was not concluded. The study recommends simultaneous assess of gastric and colon.

 

 

1.1.            Background

Breast cancer is one of the most common forms of mammary tumours world-wide [1]. Breast cancer 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 occurs 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 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].

1.2.            The Enteric Neuroimmune System

The human gastrointestinal tract spans 5 meters long and has a mucosal surface area 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 has 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 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 muscles 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 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 [6].

This figure presents an overview of ENS anatomy. (A) The myenteric and submucosal plexuses are illustrated and labelled. The myenteric plexus, which coordinates motor movements of the GI, is located between the circular and longitudinal muscle layers of the gut wall. The submucosal plexus, which regulates secretory functions of the gut, is located closest to the GI mucosal epithelium beneath the muscularis mucosa. (B) A cross sectional view showing myenteric and submucosal plexuses, and innervation of the ENS.

The ENS contains reflex circuits that detect the homeostatic and physiological condition of the GI tract, integrate the 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]. 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, response to the 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].

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 An illustration of neuron-macrophage crosstalk in the GI tract (11).

Figure 2 shows the interactions between enteric neurons and immune cells (muscularis macrophages). 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 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.

1.3.            Impact of immune dysfunction and cancer on the Gastrointestinal Tract

Immune dysfunction results in the immune system to be 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, 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 overloaded with external stimuli, which may, at times, include toxic pathogens. Hence, the GI tract contains a massive amount 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]. Tumour-induced defects have been studied for an extended period and are well known to take place 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]. This reduces T-cell secretions to B-cells in the peripheral lymphoid body organs [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) (21)

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 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, an ecological factor 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].

Figure 4 An image showing parts of the GI system (31).

A cross-section of the gastrointestinal tract and its components including the stomach, liver, gallbladder, pancreas, duodenum, rectum, appendix among others 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 (NK) cells (NKT), basophils, macrophages, and human intraepithelial lymphocytes [34]. Innate immune cells differ from typical adaptive lymphocytes because they usually express somatically [35] [36] [37]. Unlike innate immunity, adaptive immunity entails a more flexible and a 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 cells within the tumour environment, whereby changes in immune cell function are often mediated by unique cytokines [40]. Some of the cytokines involved include interleukins (ILs) and interferon (INFs). Since immune manipulation is a major component for cancer treatment, 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 are associated with the origin and development of breast cancer, as well as immune evasion. Thus, drugs manufacturing is focused on molecules that act on the specific molecular pathways in breast cancer that are highly susceptible. These treatments are administered through adjuvant therapy.

1.4.1. Immune cells at the primary tumour site influence metastatic behavior 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 chief mediator of the co-opting of the immune system 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 is developed within the tissue of interest when there is a balance between anti-angiogenic changes and the pro-angiogenic factors, a process widely known as an angiogenic switch [51] and may be relevant to the increase in vasculature in the gut mucosa is seen with many GI disorders. Since cancer promotes angiogenesis, detrimental effects on gut could be greater because angiogenesis catalyzes the growth of metastasis and tumours as a result of the 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 help the GI tract respond to various dietary metabolic products [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 helps in maintaining general physiology, homeostasis and fight against pathogens. For example, within the GI tract there is a broad range of different pathogens absorbed food and 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 which include the 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 controls sensory neurons and the secretions within the ENS where necessary mediation for painful signals associated with the central nervous system [53].

Figure 5 The mucosal immune system [54].

The figure depicts the mucosal immune system and the arrow shown above indicates development stages of the immune system and its full operationalization. This is synthesized by plasma cells in the lamina propria and transported into the lumen of the gut through epithelial cells at the base of the crypts. Polymeric IgA binds to the mucus layer overlying the gut epithelium and acts as an antigen specific barrier to pathogens and toxins in the gut lumen.

 

Figure 6  Mucosal immunity of the GI tract (28).

An overview of mucosal immunity anatomy of the GI tract. It illustrates interactions between the microbiota and innate lymphocytes. The microbiota prevent pathogens from occupying specific niches, hence protecting the integrity of the intestinal epithelium (A). Moreover, through the production of SCFAs, the epithelial integrity is also maintained (B). Additionally, the microbiota modulates the immune system development and immune function elicited not only by IELs, but also by cells located in the lamina propria, such as ILCs (C). IELs recognize harmless antigens including dietary antigens, such as SCFAs, and commensal microbiota (D). Similarly, ILCs interact extensively with both the microbiota and derived metabolites (E).

 

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 various body tissues, including the peripheral nervous system and the central nervous system [54]. VIP reinforces the survival of T-cells, which is critical in the inhibition of antigen induced apoptosis. 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).

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. However, it is acknowledged 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 the quality of life for these patients,.

1.5.            Research Questions

        This research study will answer the following 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 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

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 wholemount 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:

To assess for anatomical differences in the GI tract from non-tumour and mammary tumour-bearing mice.
To assess for histopathological differences in the GI tract from non-tumour and mammary tumour-bearing mice.
To assess for neuronal differences and Iba1 expressing muscular is macrophages in the GI tract from non-tumour and mammary tumour-bearing mice

Note: “Due to the covid-19 laboratory shutdown, some of the above 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”. Such as histopathological and neuronal differences

 

 

 

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. Each gastrointestinal region was separated into small intestine and colon segments. The length 1g) 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. Individual mouse caecal weight was measured following flushing of caecal content with PBS using a plastic 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 vertically orientated in a well-labelled cryomold 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.

 

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 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 with the software. This can be achieved by adjusting the threshold to capture the fluorescence on a dark background.

2.5. Statistical analysis

All measurements were collated on excel software and were 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.

 

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. These characteristics were then combined through narrative synthesis to indicate the results of the systematic review.

 

 

 

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 3.1.

 

Figure 7 Colon length in non-tumour and 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).

Figure 8 A graphical presentation of the length of small intestine between non-tumour and tumour bearing groups.

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 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%).

 

3.1.4.   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 caecae in tumor-bearing mice.

Figure 9 Caecal weight in non-tumour and 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 Peye’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).

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 at the ileocaecal valve of 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 tumor mice (n=3) and tumor-bearing mice (n=9) were   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 tumor-bearing mice.

 

Figure 11 The number of caecal patches between non-tumour and 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 3.6A). 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=0.266; Figure 3.6 B). The average pellet width for non tumor and tumor-bearing mice was 3.2 ± 0.3mm and 2.4± 0.1mm, respectively (p=0.127; Figure 3.6 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.

 

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

 

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 tumour-bearing 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

Based on the four phases of the systematic literature search that was used 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 1 presents the four phases of the systematic literature search that was used in this study using the PRISMA approach. In addition, a summary of characteristics of the final five studies that were included for analysis is presented in Table 1.

Figure 13 The flow of included studies based on the four phases of systematic literature search

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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 tumour-bearing 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 on tumour on healthy individuals and patients with colorectal cancer. A further two studies [66, 64] were conducted on mammary tumours and a total of three studies were conducted on mice [64, 65, 66].

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. Its findings showed that there was suppressed NOVA1 expression in tumour cells [68]. 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 therefore, the results indicate that mammary tumours are a leading cause of abnormalities that affect the gastrointestinal tract.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Chapter 4: Discussion

This study assessed the impacts of immune dysfunction and mammary tumour on the gastrointestinal tract in mice. The objective was to obtain features 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 some of these parameters 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 significant differences in the average number of Peyer’s patches from mammary tumor-bearing mice and non-tumor mice with diseased mice having mover Peye’s patches thrice as many. These findings show that mammary tumour metastasis has a direct link to GI pathology. Peye’s patches 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 Peye’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 [69]. The body needs immunity against pathogenic microorganisms present in the lumen. Peye’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 signify the influence of mammary tumor on cells that participate in the production of immune responses. Peye’s patches, as part of the enteric nervous system, maintain 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.

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 twice in non-tumour mice. The results are consistent with previous studies that found metastatic breast cancer to cause physiological changes to the GI that result in weight loss, bleeding, pain, nausea, and early satiety among others. Various studies explain the behaviour of mammary tumor metastasizing to the GI tract and other organs. These studies suggest that the metastasis occur because of the small size and shape of invasive lobular carcinoma, with overexpression E-cadherin [70]. The survival and growth factors in ovaries can explain the difference in metastatic behavior of invasive lobular carcinoma [71]. So far, the literature has not reported the simultaneous spread of metastatic spread from the breast cancer to stomach and the intestines. However, studies have demonstrated the metastasis to the stomach and colon individually [72].

The findings of this study indicate that mammary tumours adversely affect the gastrointestinal tract. Although the results of the study are inconclusive with some laboratory work skipped due to the COVID-19 pandemic, available data suggest that mammary tumours have significant impacts on the immune system, with Peye’s patch performing a critical role. The immune responses elicited by the mammary tumors mediate various physiological changes to the gastrointestinal tract, including absorption. However, the future studies need to examine the simultaneous gastric and colon metastasis to determine the extent of the damage mammary tumour inflict on the gastrointestinal tract. The results of this study will play a significant role in management of gastrointestinal problems arising from mammary tumor patients.

 

 

 

 

 

 

 

The investigation of the impacts of breast cancers cancer shows negative effects on the immune system and physiological balance. The immune system primarily protects the human body 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 demonstrate the influence of mammary tumours on the GI via the enteric nervous system. The enteric nervous system contains reflex circuits that detect the homeostatic and physiological condition of the GI tract, integrate the 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 immune response in the GI as a result of breast cancer is poorly understood, the most of the immune system is located within the GI tract where it provides immune responses to 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 includes toxic pathogens.

 

 

 

 

 

 

 

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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

 

 

 

 

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Clarify if all of this was actually done