Effect of a 12-Week Mediterranean Diet on Rectal Submucosa White Adipose Tissue (WAT) Inflammation and Fat Droplet Size in Patients with an Advanced Adenoma or Early colorectal Adenocarcinoma: A Pilot Study
The minivan- study

Globally, colorectal cancer (CRC) is the third most commonly diagnosedmost
diagnosed cancer and the second most common cause of cancer death.1 The number
of new cases is projected to increase from more than 1.9 million of new cases
in 2020 to more than 3.5 million by 2040. The Netherlands has the fourth
highest age-standardized incidence rate in Europe, at 41.0 cases per 100 000
persons behind Hungary (45.3 cases), Slovakia (43.9 cases), and Norway (41.9
cases).2 The development of CRC is multifactorial and involves interactions
between genetics, epigenetics, and environmental and lifestyle-related factors
such as poor diet.2,17

Overweight and obesity are important risk factors for CRC.3,4 However, the
mechanisms underlying the association between overweight/obesity and the
development of CRC are still not fully understood. However, current research
evidence suggests that chronic-low grade inflammation in white adipose tissue
(WAT) and in peripheral target organs plays a key role. In particular,
excessiveExcessive accumulation of visceral fat due to overnutrition is
associated with changes that lead to an inflammatory state: pathogenic
hypertrophy and dysfunction of adipocytes; increased levels of free fatty
acids; and deregulation of adipokines (eg, adiponectin and leptin),
pro-inflammatory mediators, growth factors, and reactive oxygen species.5

Consequences of an inflammatory state in the adipose tissue microenvironment
WAT consists of many different cells such as adipocytes, immune cells,
fibroblasts, and endothelial cells, which all play a role in maintaining tissue
homeostasis. Together, these cells comprise the adipose tissue microenvironment
(ATME).

Under normal weight conditions, the ATME exists as an anti-inflammatory immune
state. However, when (excessive) weight gain occurs and accumulates
particularly around the viscera such as the large intestine,18 adipocyte
hypertrophy and hyperplasia occur. Adipocyte hypertrophy can lead to adipocyte
death, which triggers an (innate) immune response and shifts the ATME towards a
pro-inflammatory state. The pro-inflammatory state is characterized by an
increased release of pro-inflammatory cytokines [eg, tumor necrosis factor
(TNF)-alpha, interleukin (IL)-1-beta] and adipokines (eg, adiponectin and
leptin). CD4+ T cells and macrophages also accumulate.19,20 Through cellular
crosstalk, this obesity-driven inflammatory state may increase tumor growth and
progression as visceral adipose tissue can exist as peritumoral fat.21,22

Adipocytes and inflammatory cells are sources of potential carcinogens. In
breast cancer, for example, the pro-inflammatory adipokine, leptin, was
identified as an important biomarker of breast cancer prognosis. Leptin and its
receptor are overexpressed in breast cancer cells compared to normal mammillary
epithelial cells23 and by binding to its receptor, leptin activates STAT3, ERK,
and P13K/AKT pathways, which triggers breast cancer cell proliferation.24,25
Compared with adjacent normal colon tissue, both leptin and its receptor have
also been found to be overexpressed in CRC tissue.26

Crown-like structures
When hypertrophic adipocytes in the WAT begin to die, their cellular content is
released into the ATME. This cellular content acts as a signal for macrophages
to surround and scavenge the dying adipocytes, thereby creating so-called
crown-like structures (CLS).6-10 Multiple receptors on the membranes of
macrophages (eg, toll-like receptors) are stimulated and lead to the activation
of inflammatory pathways. The presence of CLS in obese WAT is viewed as a
biomarker of WAT inflammation.10 In cancer patients, CLS have been linked to
worse prognosis.

The association between WAT inflammation and cancer has previously been shown
for breast,27-29 prostate,30 and tongue31 cancers. For example, prevalence of
CLS in breast adipose tissue was 40-50% in breast cancer patients,27,28 with a
higher prevalence in overweight and obese patients compared to lean patients.
Moreover, there existed a linear correlation between adipocyte size and CLS
density in breast adipose tissue.27 Mechanistically, this may be explained by
hypertrophy leading to adipocyte death, which triggers accumulation of
macrophages into CLS. Thus, next to systemic elevations in pro-inflammatory
factors, obesity may also promote tumorigenesis through local adipose tissue
inflammation. In case of prostate cancer, prevalence of periprostatic WAT
inflammation was around 50%, and periprostatic WAT inflammation was associated
with a higher BMI, a larger adipocyte size and higher grades of tumors.30

M1 and M2 macrophages
Macrophages may be divided into pro-inflammatory M1 or anti-inflammatory M2
classifications. In the context of WAT inflammation, both M1 and M2 macrophages
appear to have a role in the pathogenesis of CRC. First, findings from a
combined retrospective cohort study with a mouse model of CRC, indicated that
CRC patients who consumed >100g red meat daily over the past year (which was
considered an HFD) had an increased expression of MCP-1 and its receptor CCR2
and an increased incidence of more advanced stages of CRC. The HFD in mice
induced microbial dysbiosis, promoted intestinal carcinogenesis, upregulated
the MCP-1/CCR2 axis, and recruited M2 tumor-associated macrophages.32

Second, Pinto et al33 found the predominance of anti- or pro-inflammatory
macrophages can differ depending on tumor-associated location and tumor stage.
For instance, anti-inflammatory (CD163+) macrophages were predominant at the
tumor invasive front and pro-inflammatory ones (CD80+) in the adjacent normal
mucosa. Also, anti-inflammatory macrophages were more abundant in T2 tumors and
pro-inflammatory ones in T1 tumors. In T3 tumors, a higher overall macrophage
infiltration and a lower pro-inflammatory/anti-inflammatory ratio was
associated with worse survival. It is hypothesized that when a tumor initially
develops, newly recruited macrophages are pro-inflammatory and in later stages,
may switch to an anti-inflammatory phenotype.

Third, a study of 20 patients with CRC and mean BMI of 27.2 kg/m2 demonstrated
that M2-like phenotype macrophages were predominant in peritumoral visceral
adipose tissue and pro-inflammatory M1 macrophages in CLS of visceral and
subcutaneous adipose tissue. M2 macrophages may induce angiogenesis in
peritumoral tissue, which favors tumor growth. An in vitro co-culture
experiment in the same study that used adipocytes and a colon cancer cell line
suggested evidence for adipocyte-cancer cell crosstalk in CRC.34

Colorectal submucosal WAT deposits
More recently, next to the visceral and subcutaneous adipose tissue, also
colorectal submucosal WAT deposits have been found. This submucosal WAT, inside
a tissue normally not accommodating fat, maycan have pathogenic effects.
Submucosal fat may act locally as an endocrine and paracrine organ acting on
adjacent anatomic organs. Submucosal fat accumulation in the colon and/or
rectum may play a role in the metabolic syndrome or even in the development of
CRC and is associated with an increased BMI.35 In this study we will take
rectum biopsies both from patients in whom the lesions is located in the rectum
and in whom it is located outside of the rectum.


The potential of Mediterranean diet for CRC prevention
Since weight gain and the ensuing low-grade inflammation appear to have key
roles in obesity-related cancers such as CRC, an anti-inflammatory
Mediterranean diet may represent a relevant strategy to reduce the incidence of
CRC.

Indeed, the World Cancer Research Fund (WCRF) reported that diets high in red
meat and low in fruit and non-starchy vegetables might increase the risk for
CRC. Consumption of vitamin C rich foods, fish, and vitamin D and multivitamin
supplements might decrease the risk for CRC.36 A recent meta-analysis concluded
that a pro-inflammatory western diet (high intake of meat and processed foods)
was associated with an increased CRC risk. Conversely, more *healthy* diets
(rich in fruits and vegetables) were associated with a decreased CRC risk.37

Furthermore, a meta-analysis concluded that consuming a more pro-inflammatory
diet was associated with a 12-65% increased risk for CRC compared to consuming
a more anti-inflammatory diet. This anti-inflammatory diet was characterized by
a high intake of plant-based foods and a low intake of animal-based foods.12 A
large cohort study also found that more pro-inflammatory dietary patterns were
associated with an increased risk for the development of CRC.13

In this pilot study, we aim to investigate whether a 12-week Mediterranean diet
(MeD) intervention alters fat droplet size or the degree of inflammation in the
rectal submucosa of overweight patients who have undergone an endoscopic
resection of an advanced adenoma or early (T1) colorectal adenocarcinoma.

Primary Objective:
- To examine whether a 12-week Mediterranean Diet (MeD) intervention reduces
white adipose tissue (WAT) inflammation or fat droplet size in the rectal
submucosa in patients with BMI >= 25 kg/m2 who have undergone an endoscopic
resection of an advanced adenoma or early (T1) colorectal adenocarcinoma.

Secondary Objective(s):
- To explore the relationship between rectal submucosal WAT characteristics
(inflammation and fat droplet size) and body composition measures (height,
weight, waist-hip ratio, bio-impedance) in patients with BMI >= 25 kg/m2 who
have undergone an endoscopic resection of an advanced adenoma or early (T1)
colorectal adenocarcinoma.
- To explore the relationship between rectal submucosal WAT characteristics
(inflammation and fat droplet size) and insulin resistance in patients with BMI
>= 25 kg/m2 who have undergone an endoscopic resection of an advanced adenoma or
early (T1) colorectal adenocarcinoma.
- To examine the relationship between rectal submucosal WAT characteristics
(inflammation and fat droplet size) and liver fat (as a measure of ectopic fat
stores) in patients with BMI >= 25 kg/m2 who have undergone an endoscopic
resection of an advanced adenoma or early (T1) colorectal adenocarcinoma.
- To assess whether a 12-week MeD intervention alters the ratio between M1 and
M2 macrophages in the rectal submucosa of patients with BMI >= 25 kg/m2 who have
undergone an endoscopic resection of an advanced adenoma or early (T1)
colorectal adenocarcinoma.
- To explore the relationship between changes in rectal submucosal WAT
characteristics (inflammation and fat droplet size) to that of changes in the
fecal microbiome of patients with BMI >= 25 kg/m2 who have followed a 12-week
MeD intervention after undergoing an endoscopic resection of an advanced
adenoma or early (T1) colorectal adenocarcinoma.
- To compare rectal submucosal WAT characteristics (inflammation and fat
droplet size) between subjects in whom the colorectal lesion is located within
and outside of the rectum.

We will perform a single-center open-label intervention study. The study
procedures and the 12-week Mediterranean diet (MeD) intervention are integrated
into the standard care timelines for endoscopic resection or endoscopic
submucosal dissection (ESD) of an advanced adenoma or early-stage (T1)
colorectal adenocarcinoma.

In general, the study comprised 3 phases: baseline & recovery, intervention,
and follow-up. Figure 1 provides an overview of the study design and main
procedures. First, the baseline & recovery phase consists of the endoscopic
resection of the cancerous tissue, baseline biopsy of submucosal tissue, and
nonsurgical assessment procedures (ie, baseline blood and fecal samples,
anthropometric and body composition measurements, a liver ultrasound
elastography, and completion of questionnaires). The standard postoperative
recovery period after an endoscopic resection is 3 months. Second, the
intervention phase consists of the 12-week MeD intervention. Last, the
follow-up phase consists of a postoperative colonoscopy to check healing after
the resection and to take a follow-up biopsy of the submucosal tissue. In
addition, the nonsurgical assessments will be repeated.

The intervention is a 12-week, anti-inflammatory Mediterranean diet (MeD). MeD
is characterized by the following:14,15
- a high intake of vegetables, fruit, pulses, grains, and nuts
- moderate consumption of yogurt, cheese, egg, poultry, fish, and seafood
- moderate intake of wine with meals
- little or no red or processed meats
- olive oil being the predominant source of fat

The burden for patients includes the time investment to undergo measurements at
baseline, before the start of the intervention, and at follow-up; and to
receive dietary counselling before the start of the intervention and at 4-weeks
and 8-weeks.

To minimize the burden for patients, the study procedures are embedded as much
as possible within the standard care protocol and timelines for endoscopic
resection of of an advanced adenoma or early-stage (T1) colorectal
adenocarcinoma. The first 2 biopsies will be performed during the endoscopic
resection of the cancerous tissue; the second set of 2 biopsies will be taken
during the follow-up colonoscopy that is necessary in order to assess the
presence of any residual or recurring dysplastic tissue
The risks associated with taking the biopsy include minor bleeding, and
functional or mechanical changes after the procedure

There are also risks associated having blood withdrawn: pain at the injection
site and bruising. The pain and bruising are self-limiting. The discomfort
during the anthropometric and bio-impedance assessments and the liver
ultrasound elastography are minimal.

Benefits for participating patients are related to receiving an intervention
that may reduce risk factors for (the progression of) CRC and thereby improve
their prognosis. The knowledge and healthier eating habits gained from the
dietary counselling remain in the hands of the patients so they may experience
continued benefits of following a MeD beyond the study period.