# Obesity and breast cancer: impact of vitamin D on the inflammatory adipose tumor microenvironment in obesity in a 3D co-culture model

AOUN Jessy, HABANJAR Ola, NEHME Rawan, DECOMBAT Caroline, DIAB-ASSAF Mona, CALDEFIE-CHEZET Florence, DELORT Laetitia

Clermont-Auvergne University, INRAE, UNH, Unité de Nutrition Humaine, Clermont-Ferrand

## Introduction

### Breast cancer progression

<figure><img src="image_1.png" /></figure>

### Breast tumor microenvironment in obese women

<figure><img src="image_2.png" /></figure>

The tumor microenvironment is composed of adipose tissue containing macrophages and adipose cells. In obesity, the percentage of resident macrophages increases and becomes M1-like pro-inflammatory macrophages.

Adipose cells are preadipocytes that can differentiate into mature adipocytes. In obesity, a hyperplasia and hypertrophy of adipose cells are observed

<figure><img src="image_3.png" /></figure>

Vit D can act on different cell types in the microenvironment including cancer cells. Obese people are more likely deficient which puts them at higher risk of developing breast cancer

## Results

<figure><img src="image_4.png" /></figure>

Vit D reduced the differentiation of preadipocytes, the inflammation of M1 type macrophages and the proliferation of cancer cells

## Objective

<figure><img src="image_5.png" /></figure>

To evaluate the effects of vitamin D on the progression of breast cancer in the context of obesity

## Materials and methods

### 1. Formation of spheroids

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

INCUCYTE®

<figure><img src="image_9.png" /></figure>

Spheroids of adipose cells

Spheroids of
cancer cells

### 2. Polarization of macrophages

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

THP1 monocytic
cell line

72h

24h

M0 macrophages

M1 macrophages

### 3. Co-culture model

<figure><img src="image_12.png" /></figure>

A co-culture model was used where spheroids of preadipocytes or mature adipocytes and spheroids of breast cancer cells were formed in agarose molds and put on top of a 6 well plate where M1 type macrophages were adherent. After 72h, RT-qPCR and ELISA tests were performed on the different cell types

## Results

<figure><img src="image_13.png" /></figure>

Vit D increased the expression of leptin in adipospheroids and had no effect on the proliferation of cancer cells

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

## Conclusion

Vit D can reduce inflammation by reducing differentiation of preadipocytes and by reducing inflammation in macrophages of adipose tissue