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Thesis
Peer Reviewed

Studies of the Carcinoma-Associated Fibroblast in Human Oral Squamous Cell Carcinoma

S. Thaddeus Connelly

Abstract

Our understanding of the development and progression of oral squamous cell carcinoma (Oral SCC) remains limited. This is reflected by the fact that oral cancer is associated with one of the poorest 5-year survival rates of any malignancy in the body. It is becoming increasingly evident that the underlying stroma has an important role to play in tumorogenesis. Our studies focus on the central controller of the stroma, the fibroblast and its tumor-associated counterpart, the carcinoma-associated fibroblast (CAF). The molecular and functional characteristics of CAFs associated with Oral SCC remain incomplete. Thus, we studied the influence of the stroma on the tumor microenvironment through a histologic evaluation of cancerous and pre-cancerous lesions. Additionally, we assessed the molecular response that CAFs and fibroblasts display to TGFβ or TGFβ-mediated stimulation by direct measurement of expression levels of downstream genes and in an organotypic co-culture system, respectively. Lastly, we used expression profiling, immunofluorescence and functional assays of CAFs and normal fibroblasts (NFs) in an attempt to classify a CAF specific expression signature, CAF subsets or cellular origin and to identify CAF genes or gene pathways that might contribute to proliferation, migration and invasion. The results demonstrate that the stromal lymphocytic infiltrate increases with lesion severity of pre-cancers with the most in Oral SCC, indicating a causal relationship. Fibroblast response to TGFβ or TGFβ-mediated stimulation was varied based on anatomic site and this heterogeneity was further appreciated in the results of the immunofluorescence and expression profiling, however these studies confirmed that αSMA is a robust CAF marker. Despite the heterogeneity in the CAF/NF expression profiles, a set of genes was found and validated to be differentially expressed between gingiva CAFs and NFs and tongue and gingival fibroblasts in general. Finally, analysis of fibroblast groups defined through functional assays reveal differential gene expression between fibroblasts that are most or least inhibitory to keratinocyte/tumor cell line proliferation and those that increase or decrease invasion/migration. SFRP2 from the proliferation assay and DKK1 from the invasion/migration assay are both differentially expressed negative modulators of WNT signaling, indicating a potentially important pathway in the reciprocal tumor/stroma evolutionary relationship.

Cover page: Studies of the Carcinoma-Associated Fibroblast in Human Oral Squamous Cell Carcinoma

Article
Peer Reviewed

Electroporation technique for joint pain – Pilot feasibility study on TMD patients

UC San Francisco Previously Published Works (2020)

Objective(s)

It is well appreciated that traditional analgesic delivery routes used to treat pain associated with temporomandibular disorder (TMD) often have harmful unintended side effects as a consequence of systemic distribution. Further, localized delivery of analgesic medication via intra-articular injections involves a different set of issues limiting their clinical viability. As an option, transdermal analgesic delivery provides for prolonged pain relief and flexibility in dose administration, while limiting systemic exposure and minimizing adverse events. Incorporation of a novel electroporation technique may further increase transdermal drug penetration into synovial tissue/fluid and enhance pain reduction. The present feasibility study compares the effectiveness of an electroporation-enhanced transdermal application of diclofenac sodium to a conventional intra-articular injection of triamcinolone acetonide suspension (corticosteroids) to treat patients with TMD associated pain.

Methods

Pre- and post-treatment maximal incisal mouth opening (MIO), pain visual analog scale (VAS) and surface electromyography (EMG) of 22 patients treated with electroporation-enhanced diclofenac and 37 patients treated with corticosteroids injections were collected and analyzed.

Results

In general, patients treated with electroporation exhibited better results in terms of pain improvement (corrected p-value = .01) compared to the standard treatment, but both methods were similarly effective for improvement of MIO (corrected p-value = .71) and improvement of all EMG indices (corrected p-values ≥ .05).

Conclusion

The enhancing effect of electroporation in transdermal delivery of diclofenac sodium was demonstrated by decreased pain, increase MIO and EMG improvement to normal values. Its analgesic and inflammatory results are comparable with standard treatment offered by corticosteroids.

Cover page: Electroporation technique for joint pain – Pilot feasibility study on TMD patients

Article
Peer Reviewed

Ethical considerations regarding animal experimentation

UC Davis Previously Published Works (2022)

Animal experimentation is widely used around the world for the identification of the root causes of various diseases in humans and animals and for exploring treatment options. Among the several animal species, rats, mice and purpose-bred birds comprise almost 90% of the animals that are used for research purpose. However, growing awareness of the sentience of animals and their experience of pain and suffering has led to strong opposition to animal research among many scientists and the general public. In addition, the usefulness of extrapolating animal data to humans has been questioned. This has led to Ethical Committees' adoption of the 'four Rs' principles (Reduction, Refinement, Replacement and Responsibility) as a guide when making decisions regarding animal experimentation. Some of the essential considerations for humane animal experimentation are presented in this review along with the requirement for investigator training. Due to the ethical issues surrounding the use of animals in experimentation, their use is declining in those research areas where alternative in vitro or in silico methods are available. However, so far it has not been possible to dispense with experimental animals completely and further research is needed to provide a road map to robust alternatives before their use can be fully discontinued.

Cover page: Ethical considerations regarding animal experimentation

Article
Peer Reviewed

Methodology for clinical research

UC Davis Previously Published Works (2022)

A clinical research requires a systematic approach with diligent planning, execution and sampling in order to obtain reliable and validated results, as well as an understanding of each research methodology is essential for researchers. Indeed, selecting an inappropriate study type, an error that cannot be corrected after the beginning of a study, results in flawed methodology. The results of clinical research studies enhance the repertoire of knowledge regarding a disease pathogenicity, an existing or newly discovered medication, surgical or diagnostic procedure or medical device. Medical research can be divided into primary and secondary research, where primary research involves conducting studies and collecting raw data, which is then analysed and evaluated in secondary research. The successful deployment of clinical research methodology depends upon several factors. These include the type of study, the objectives, the population, study design, methodology/techniques and the sampling and statistical procedures used. Among the different types of clinical studies, we can recognize descriptive or analytical studies, which can be further categorized in observational and experimental. Finally, also pre-clinical studies are of outmost importance, representing the steppingstone of clinical trials. It is therefore important to understand the types of method for clinical research. Thus, this review focused on various aspects of the methodology and describes the crucial steps of the conceptual and executive stages.

Cover page: Methodology for clinical research