The tumor stroma includes many nonneoplastic cells and the extracellular matrix, making up the microenvironment in which neoplastic cells grow. The non-neoplastic cells are predominantly stromal fibroblasts, sometimes called “tumor associated fibroblasts”. These cells are important for the maintenance and remodeling of the microenvironment, providing the appropriate conditions for neoplastic cell growth and invasion. The role of stromal fibroblasts in promoting tumor invasion has recently been highlighted in a number of cancers such as breast cancer, gastric carcinoma, non-small cell lung carcinoma, and colorectal cancer. This interaction between the stromal fibroblasts and the neoplastic cells can occur indirectly through secreted paracrine factors, or directly by physical cell-cell contact. Desmoid tumors (DTs) are characterized by proliferating and invading fibroblastic cells embedded in depositions of extracellular matrix driven by mutations that activate β-catenin signaling. We hypothesized that desmoid tumor cells interact with the stromal fibroblasts and that this interaction is responsible for maintaining the neoplastic phenotype of tumor cells. To test our hypothesis, we proposed to answer the following questions:
Aim 1: How do the neoplastic and stromal populations of desmoid tumors differ?
Our main goal is to study the composition and potential cross-talk of sub-populations within DTs. However, it is difficult to distinguish the neoplastic, mutant DT cells from the non-neoplastic stromal, “wild-type” fibroblasts due to the invasive nature of the tumor and due to both cellular populations exhibiting a mesenchymal fibroblastic phenotype. In the past year, we utilized a single cell-based method we developed to isolate individual cells from DT patient samples to establish several individual clones in fibroblastic colony forming culture media. We characterized individual clones to study whether it is a mutant neoplastic cell, or a non-mutant stromal cell. We also characterized differences in the expression of cell surface markers. This helped us identify a unique cell surface marker that can accelerate our isolation efforts and can more sensitively quantify tumor composition of heterogeneous samples. In the coming year, we aim to take advantage of our identified markers to establish more mutant and non-mutant pairs from more samples. We also aim to perform gene expression analyses which will be used to further characterize similarities and differences between the different populations.
Aim 2: Do desmoid tumor cells interact with the surrounding stromal cells by paracrine signals?
In the past year, we analyzed the differential expression of secreted factors between mutant and nonmutant populations and found several factors that are uniquely expressed by either subpopulation. Modulation of the activity of selected factors altered the growth of DT cultures. We also co-cultured DT cells with normal cells separately using a permeable co-culture system that allows the sharing of released factors but without cell contact. We measured the effect of co-culturing on cells by viability, proliferation and migration assays to study how the two subpopulations can influence cell behavior. This work allowed us to optimize our assays and decide on readouts for subsequent studies. In the coming year, we aim to study gene expression differences to study the underlying changes in molecular biology due to secreted factors. We also aim to further study the role of candidate secreted factors on DT culture growth. The selected pathways will be disrupted by selective drugs or neutralizing antibodies and the effect on the co-culturing experiment will be measured by the same outlined assays that we have established in the last year. Our work so far has identified potentially novel pathways for further studies, and we expect that we will identify more pathways that modulate tumor-stromal interaction which can be targeted for therapy.
Aim 3: Do desmoid tumor cells interact with the surrounding stromal cells by direct cell-cell contact?
In addition to paracrine signals, invading tumor cells can also interact with the microenvironment via direct cell-cell adhesion. Molecules involved in this process include integrins, cadherins, and Notch signaling pathway components. Since β-catenin is known to interact with these molecules, directly or indirectly, we anticipate that direct cell contact will have an effect on the mixed populations. In the past year, we conducted a high throughput cell surface antigen screen to study the expression of proteins that can play a role in cell-cell interaction and how they differ between mutant and non-mutant populations. Our preliminary analysis has identified a number of integrin molecules that exhibit aberrant expression in the mutant populations. We anticipate that these molecules play a role in direct cell-cell contact. In the coming year, we will label DT cells by generating cell lines that stably express a marker, such as GFP (green fluorescence protein). We will then mix DT cells with normal cells and allow the two cell populations to grow together in direct cell contact. We will measure the effect of direct co-culturing on each cell population, taking advantage of our labeling method, by conducting viability and proliferation assays in addition to gene expression analyses. We will take a candidate approach at inhibiting these molecules that are likely to play a role in this process based on our surfaceomics data to date, in addition to any selected pathways from Aim 1, and we expect that we will identify a number of potential therapeutic targets.
By elucidating the composition of desmoid tumors, and how the tumor cells interact with the normal stromal cells, we can greatly enhance our knowledge of how they grow. DTs are invasive tumors characterized by abundant extracellular matrix deposition. Therefore, it is very likely that tumorstromal interactions are important in the way they grow and infiltrate the surrounding tissue. By isolating and characterizing individual cell populations from the tumor mass, we can gain a better understanding of what distinguishes the mutant cells from the normal cells. Our method of separating individual cells will also allow us to study variability between patient samples at the cellular level, without the confounding factor of having a different proportion of normal cells in each tumor sample. Since DTs are known to be heterogeneous, this method is key at understanding the source of this heterogeneity, which can influence treatment decisions. Elucidating what signaling pathways are involved in the process of tumor-stroma interaction will also allow us to potentially identify novel therapeutic targets that we can use to inhibit tumor growth and infiltration. We anticipate that the established single cell-derived clones, and the knowledge gained from this project of desmoid tumor biology, will be the basis for future experiments that build on our knowledge of desmoid tumor heterogeneity and tumor-stromal interactions.
LAY VERSION OF ABSTRACT- “Single cell-derived clonal analysis of desmoid tumors to investigate tumor-stroma interactions.”