The tumor stroma includes many non-neoplastic 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 hypothesize 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 propose to answer the following questions:
Aim 1: How do the neoplastic and stromal populations of desmoid tumors differ?
Aim 2: Do desmoid tumor cells interact with the surrounding stromal cells by paracrine signals?
Aim 3: Do desmoid tumor cells interact with the surrounding stromal cells by direct cell-cell contact?
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 tumor- stromal 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.
In neoplasia, tumor cells interact with the normal stromal cells, such as the surrounding fibroblasts. This makes studying desmoid tumors difficult as both tumor and stromal cells display a mesenchymal phenotype and there are no well-established markers to distinguish the two populations. To elucidate the composition of desmoid tumors, we isolated and expanded single cells derived from patient desmoid tumor samples. Sequencing of individual clones derived from the same patient sample revealed that desmoid tumors do consist of mutant sub-populations carrying the beta-catenin activating mutation, and normal sub-populations lacking the mutation. To improve our isolation methodology, we performed a high throughput flow cytometry surface antigen screen to study the expression pattern of over 300 surface markers on mutant and non-mutant cells. From this analysis, we identified CD142 as a unique positive marker for the mutant population. Having isolated mutant and non-mutant cell populations, we next looked into the expression of secreted factors that could potentially play a role in cell-cell communication. From this study, we found that CTHRC1 is a factor secreted by the mutant cells. Gain- and loss-of-function experiments showed that CTHRC1 can influence the proliferation rate of desmoid tumor primary cultures. Studying desmoid tumors at the clonal level will enhance our understanding of the intratumoral heterogeneity of these tumors. Identifying unique surface markers allows for the rapid isolation of mutant and non-mutant subpopulations while minimizing cell divisions. In addition to studying tumor-stroma communication, measuring tumor composition may also be useful for ongoing drug screening efforts and as a potential post-treatment readout.
LAY VERSION OF ABSTRACT- Single cell-derived clonal analysis of desmoid tumors to investigate tumor-stroma interactions