Establishment of a 3D co‐culture model to investigate the role of primary fibroblasts in ductal carcinoma in situ of the breast

Abstract Background Ductal carcinoma in situ (DCIS) is a precursor form of breast cancer. 13%–50% of these lesions will progress to invasive breast cancer, but the individual progression risk cannot be estimated. Therefore, all patients receive the same therapy, resulting in potential overtreatment of a large proportion of patients. Aims The role of the tumor microenvironment (TME) and especially of fibroblasts appears to be critical in DCIS development and a better understanding of their role may aid individualized treatment. Methods and results Primary fibroblasts isolated from benign or malignant punch biopsies of the breast and MCF10DCIS.com cells were seeded in a 3D cell culture system. The fibroblasts were cultured in a type I collagen layer beneath a Matrigel layer with MCF10DCIS.com cells. Dye‐quenched (DQ) fluorescent collagen I and IV were used in collagen and Matrigel layer respectively to demonstrate proteolysis. Confocal microscopy was performed on day 2, 7, and 14 to reveal morphological changes, which could indicate the transition to an invasive phenotype. MCF10DCIS.com cells form smooth, round spheroids in co‐culture with non‐cancer associated fibroblasts (NAFs). Spheroids in co‐culture with tumor‐associated fibroblasts (TAFs) appear irregularly shaped and with an uneven surface; similar to spheroids formed from invasive cells. Therefore, these morphological changes represent the progression of an in situ to an invasive phenotype. In addition, TAFs show a higher proteolytic activity compared to NAFs. The distance between DCIS cells and fibroblasts decreases over time. Conclusion The TAFs seem to play an important role in the progression of DCIS to invasive breast cancer. The better characterization of the TME could lead to the identification of DCIS lesions with high or low risk of progression. This could enable personalized oncological therapy, prevention of overtreatment and individualized hormone replacement therapy after DCIS.


| INTRODUCTION
Breast cancer is the most common cancer type in women divided into an invasive and non-invasive histological type(s) according to the WHO classification. 1 The risk of progression to an invasive form varies among pre-invasive lesions. The highest risk for transition to an invasive breast cancer and thus to a potentially life-threatening disease is seen in ductal carcinoma in situ (DCIS) with 13%-50% of the cases. 2 So far, it is not possible to predict which DCIS lesion will become invasive and therefore all patients with a DCIS are treated equally resulting in over-treatment of some patients.
Therapy of DCIS includes surgical treatment either breastconserving or a mastectomy depending on the tumor-size and breast relation. The possibility of radiotherapy after breast-conserving surgery and/or endocrine therapy should be discussed with the patient individually based on a risk-benefit assessment, considering potential adverse effects. [3][4][5] Possible therapy associated morbidity through radiation or surgery can affect the lifestyle of the patient and reduce the quality of life. 6 Therefore, DCIS over-treatment leads to potential adverse effects regarding patients´health and constitutes a burden on the health system. 7 Moreover, there is no data regarding the use of hormone replacement therapy after DCIS, which is usually avoided due to the fear this could trigger an invasive transformation/reformation. 8 Prognostic parameters are needed to assess the patient's individual risk. In case of a low risk, interventional therapy could be replaced by regular follow up. 9 DCIS is considered a non-obligatory precursor to invasive breast cancer 10 and there are two progression theories; the 'genetic' and the 'non-genetic' theory. The genetic theory is supported by studies that have revealed the genetic similarity and the likely common origin of DCIS and invasive carcinoma. [11][12][13] Clinical observations have also shown that the two entities are often located at the same anatomical site or directly next to each other, which suggests an evolutionary continuum. Gene expression analysis has been conducted and provides evidence of a common genetic background. 14 Other studies have shown a significantly different expression pattern for distinct genes, which could indicate driver pathways that play an important role in the progression from DCIS to invasive breast cancer. [15][16][17] However, a single mutation as a cause for the progression seems unlikely. So far, no biomarker could be found to clinically predict the progression from DCIS to invasive breast cancer.
The non-genetic theory suggests that the tumor microenvironment (TME) plays a decisive role in the progression of DCIS to invasive breast cancer. 18 DCIS is surrounded by an outer layer of myoepithelial cells and an intact basement membrane. The layer of normal myoepithelial cells acts as a "gatekeeper" and has tumor suppressive effects on the in situ lesion. 19 Various studies have shown that the loss of this suppressive effect leads to the progression to invasive breast cancer. 19,20 Except from a physical barrier against invasion, myoepithelial cells also secrete various extracellular matrix components (ECM) which inhibit the invasive capacity of DCIS in a paracrine way. 21 Increased stromal cell expression of ECM-modifying enzymes 17,22 as well as glucocorticoids seem to favor the progression of DCIS to invasive breast cancer in vitro and in vivo. 23 Stromal fibroblasts as well as tumor infiltrating lymphocytes (TILs) and their T-to B-cell ratio also seem to play a role in the progression of DCIS. 17,24 Altogether, the current literature leads to the conclusion that the transition from DCIS to invasive breast cancer is largely not dependent on intrinsic mutations. The invasive potential appears to be rather due to extrinsic factors or the TME. 25 These include the ECM, the myoepithelial cell layer, immune cells and fibroblasts. Therefore, there is a need to establish basic novel experimental models of DCIS that take the TME into account. In this study, we describe a novel 3D co-culture model of DCIS and primary fibroblasts as a major component of the TME that can be used to study the progression of DCIS in a convenient laboratory setting. This work primarily focuses on morphological changes potentially representing an invasive phenotype in this model.

| Materials
All supplies and chemicals were from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) unless otherwise stated.

| Cell culture
The human DCIS model cell line MCF10DCIS.com, cloned from a cell culture initiated from a xenograft lesion 26

| 3D co-culture model
The co-culture of DCIS cells and primary fibroblasts was based on the MAME model (mammary architecture and microenvironment engineering) of Sameni et al. and was modified to meet the needs of primary cells. 27 Primary fibroblasts were isolated from the punch biopsies and were then cultured in a Type I collagen layer (Discovery

| Light microscopy
The co-culture was first visualized on day 2, 4, 7, and 14 under the light microscope (Axioscope Carl Zeiss Microscopy GmbH, Jena, Germany) to reveal possible morphological changes between the different wells. (airy unit) so that the signal-to-noise ratio was as low as possible. All images were taken with an identical setting and reconstructed and processed in 3D using the Zen 2 black imaging software (Carl Zeiss Microscopy GmbH, Jena, Germany). In order to be able to present the possible morphological differences between the spheroids in the control and co-culture, consecutive images were taken every 8 μm. Acoordingly, a measuring distance of 3.5 μm between consecutive images was selected for the significantly smaller fibroblasts, so that even small differences could be detected. For the measurement of the distance between fibroblasts and MCF10DCIS.com cells, images were taken every 15 μm.

| Statistical analysis
For statistical analysis a two tailed ttest was performed. The significance level was set equal to 0.05.

| Evaluation of proteolysis and cell morphology of DCIS cells by confocal microscopy
Next, we studied the morphology and proteolytic activity of cell tracker-labeled MCF10DCIS.com cells and fibroblasts with confocal fluorescence microscopy. On day 2 of the 3D-culture the spheroids looked small, smooth-edged and round. Proteolysis was already ongoing during the formation of the spheroids, as indicated by the green signal caused by the degradation of DQ-collagen IV (Figure 2). At this point, there were no morphological differences regardless of the type of TAFs (Figure 2). On day 7, the control spheroids remained smooth and round. Otherwise, the spheroids that were co-cultured with TAFs of type 1 or 2 had an uneven appearance with the cells leaving the formation of the spheroid (Figure 3). The spheroids co-cultured with NAFs had a morphology similar to the spheroids in the control. Spheroids retained their acquired morphology over time, so that on day 14 the spheroids in co-culture with TAFs of type 1 or 2 appeared further irregularly shaped compared to the spheroids co-cultured with NAFs ( Figure 4). Spheroids in co-culture with NAFs showed the same phenotype as the control spheroids. The proteolysis that has taken place is further displayed by the green fluorescence of the DQ collagen IV on day 7 and 12 (Figures 3 and 4). The morphological differences mentioned appeared consistently in all wells with non-TAFs or TAFs respectively, and in the control wells.   (Figures 1 and 7). In the experiment shown, the distance between MCF10DCIS.com cells and NAFs or TAFs is 890 μm and 700 μm on day 2, 760 μm and 560 μm on day 7 and 600 μm and 510 μm on day 14, respectively. This tendency has been observed in several experiments (n = 9). However, there was no significant difference in the mean distance reduction between NAFs and TAFs (day 2 to day 7: 120 ± 10 vs. 115 ± 15, p = .81, day 7 to 14: 130 ± 20 vs. 35 ± 15, p = .14) (Figure 7).  face, irregular shape and cells leaving the structure forming spikes has been described in the literature. [28][29][30][31][32] As so, we interpret the morphological differences of the spheroids in co-culture with TAFs as an indication that a reprogramming of fibroblasts into TAFs plays a role in the conversion of DCIS lesions to an invasive breast cancer. This coincides with the role of fibroblasts in the development of an in-situ to an invasive phenotype in current data. [31][32][33][34][35][36] There is currently an intense debate on TAFs playing a crucial role in the development of DCIS into invasive breast cancer and that these could represent a new therapeutic target. The biology and physiology of the TAFs as well as the full activation mechanism is still subject of research. Various studies have described that TAFs can arise from several cell types; resident tissue fibroblasts, bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, epithelial cells (through epithelial-mesenchymal transition/ EMT) or endothelial cells (through endothelial-mesenchymal transition/ EndMT). 35,36 The aim of systemic therapy, for example, chemotherapy or endocrine therapy, is to destroy tumor cells. In most cases this is very successful in massively reducing the tumor mass. However, once the microenvironment has adopted a tumor-promoting phenotype, restoration of tumor growth of a few cancer cells that may potentially escape first-line therapy is very likely. Over 100 years ago, Paget et al.
already suggested the importance of the TME with the "seed and soil" theory; the TME plays a critical role in the survival of tumor cells. 37 Obviously, the microenvironment can lead to renewed tumor growth.
The targeted treatment of the tumor-promoting activities of TAFs is therefore an important therapeutic strategy. 38 Primary human breast fibroblasts (HMF) stimulated with TGFβ1 have similar characteristics to breast cancer-associated fibroblasts in vivo. 39 It has also been proven that certain miRNAs promote the conversion of resident fibroblasts into TAFs. 40 The elucidation of mechanisms that lead to the reprogramming of TAFs into normal fibroblasts could represent a new, promising therapeutic approach. 38 From this perspective, experimental strategies like our 3D co-culture system that address such issues are of high importance. Evaluating how current treatment strategies may affect cancer-associated fibroblasts, or identifying effective drugs targeting these cancer-associated cells may be the key in prohibiting or forecasting the progression of DCIS to invasive breast cancer.
The 3D mono-culture of DCIS as well as 3D co-culture models in which DCIS cells grow in an architecture resembling the cell: cell interactions or cell:ECM interactions are found in the literature (for comprehensive review see reference 41). These 3D approaches represent preclinical models either to examine different aspects of the tumormicroenvironment in the progression of DCIS to invasive breast cancer 33,34,42 or to predict drug efficacy and toxicity. [43][44][45] Studies on xenograft models have also been conducted to address the same F I G U R E 6 Analysis of the proteolytic activity of fibroblasts in the 3D co-culture system by confocal microscopy. (A) Non-tumor-associated fibroblasts (NAFs) (a) and tumor-associated fibroblasts (TAFs) of type 1 (b) and 2 (c) on day 2. Settings (magnification, laser intensity, gain for channel 1 and 2) are identical for the entire study. The TAFs have a stronger DQ-collagen signal (b2, c2) than the NAFs (a2). The left panels show the cell tracker-labeled cells using the two channels (HeNe543, argon diode 405-30), while the green fluorescent signal isolated in the right panels labels degraded DQ-collagen I. (B) Graphic: The mean fluorescence intensity per cell for NAFs (blue), TAFs of type 1 (orange) and 2 (gray) on day 2, 7, and 14. TAFs of type 2 show the greatest proteolytic activity. The laser intensity and gain for channel 2 was newly set on day 2, 7, and 14 to keep the signal-to-noise ratio as low as possible. In accordance to other research groups 27,33,34 we evaluated proteolytic activity based on degraded DQ collagen signal. However, Yamada et. al showed that aggregated and/or mechanically disrupted collagen can also produce a DQ-collagen type signal. 52 Concequently, the use of controls with directly labeled matrix (e.g., Alexa-labeled collagen) or with protease inhibitors could be used to rule out effects due to interactions with the local physical environment.
A weakness of in vitro cell cultures is that they are grown in the absence of their in vivo environment. In order to further investigate the development of an invasive breast cancer from a DCIS, other cell types, for example, cells of the immune system, could be included.
The 3D-culture is a design that better depicts the in-vivo situation than 2D cultures. Nevertheless, results from the 3D-culture should also be validated in vivo.
In summary, TAFs appear to play a crucial role in the development of DCIS lesions to invasive breast cancer. MCF10DCIS.com cells develop an invasive phenotype in co-culture with TAFs, which have a greater proteolytic activity compared to NAFs. Our study provides the groundwork for follow-up studies to further investigate the role of fibroblasts including the biological behavior of fibroblasts deriving from tumors with different tumor biology (e.g., luminal G3, Her2-positive or triple-negative carcinoma). In addition, we suggest that the morphological differences seen reflect genetic changes in proliferation and / or invasion markers. Our novel 3D-culture model offers a simple in vitro model for the future study of molecular mechanisms underlying the communication of DCIS cells with their TME.

ACKNOWLEDGMENTS
We would like to thank Niki Loges and Burkhard Greve for providing access to the LSM 880 Zeiss microscope.