Investigating the Role of Tumour-Stroma interaction in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma is the sixth leading cause of cancer-related death worldwide. Although most patients initially respond to treatment, many will eventually recur and die and die of their disease. Understanding the impact of the components of the tumour microenvironment on disease progression is essential to effectively treat HNSCC. The work described in this thesis provides a better understanding of the relationship between tumour cells and fibroblasts and describes novel therapeutic targets for further testing. First, I characterized carcinoma-associated fibroblasts (CAFs) and their effect on tumour cell behaviour in vitro and in vivo. Second, using laser-capture microdissection (LCM), I obtained pure populations of tumour and normal epithelial cells as well as tumour-associated and normal stromal cells. From these cells I obtained transcriptomic profiles and used this data to identify candidate molecular interacting partners utilized by tumour and stromal cells to communicate with each other. Finally, I also use these profiles in combination with publically available data gene expression and clinical data from the The Cancer Genome Atlas (TCGA) to characterize the association between stromal genes and clinical outcomes.Ph.D

Surgical treatment of the fractured and dislocated condylar process of the mandible

Most fractures of the mandible can be managed conservatively. This report is a retrospective evaluation of the clinical and radiological results in 17 patients with 21 dislocated fractures submitted to open reduction and fixation, employing steel wires and maxillomandibular fixation. Follow-up ranged from 7 to 55 months, (mean 29.5). Functional assessment showed good opening movements (mean 41.9 mm). There were no cases of ankylosis, pain, or paralysis of the facial nerve. Radiological assessment was normal when the lateral pterygoid muscle was maintained adherent to the fractured proximal segment. Radiological signs of bone resorption occurred when the fractured segment was detached from the lateral pterygoid muscle. in our opinion, dislocated condylar process fractures can be managed surgically and with steel wire ligatures and maxillomandibular fixation. Whenever possible, the lateral pterygoid muscle should be inserted into the fractured proximal segment, i.e. as an osteomuscular flap.ESCOLA PAULISTA MED,DIV PLAST SURG,São Paulo,BRAZILESCOLA PAULISTA MED,DEPT RADIOL,São Paulo,BRAZILESCOLA PAULISTA MED,DIV PLAST SURG,São Paulo,BRAZILESCOLA PAULISTA MED,DEPT RADIOL,São Paulo,BRAZILWeb of Scienc

Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

<div><p>Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.</p></div

Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies

Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies