Trainee Project
Title:
Development of 3D model in co-culture for Metvixia-mediated photodiagnosis of VIN
Dates:
2022/01/10 - 2022/06/24
Supervisor(s): 
Other supervisor(s):
Description:
According to the National Cancer Institute, approximately 0.3% of women in the United States will develop vulvar cancer during their lifetime. It is one of twelve cancers whose incidence continues to rise. This trend is correlated with the increase in HPV (Human Papillomavirus) infections. Nearly 40% of women will be infected with an oncogenic HPV and 10% of them will develop a persistent HPV infection, responsible for precancerous or cancerous lesions of the female genital tract, despite an effective HPV vaccination, due to insufficient vaccination coverage.
High-grade vulvar intraepithelial neoplasia (VIN), a vulvar precancerous condition, includes high-grade vulvar squamous intraepithelial lesions (HSILv, vulvar High Squamous Intraepithelial Lesion (HSILv), which is most common in young HPV-infected women, and Differentiated Vulvar Intraepithelial Neoplasia (dVIN), which is more common in older women and is associated with lichen scleroventrum.
The diagnosis of high-grade vulvar intraepithelial neoplasia is a real challenge. Indeed, the natural history of vulvar dysplasia, which is mostly asymptomatic, the diversity of clinical presentations, and their often multifocal and multicentric nature are all obstacles to an early and exhaustive diagnosis of the disease. The improvement of lesion detection is therefore a decisive step in their management insofar as it allows the definition of adequate treatment, the detection of subclinical infiltrating forms and the limitation of the risk of local recurrence.
Indeed, conservative treatments are preferred because they cause few local sequelae and are considered less mutilating than surgical management. However, it is necessary to treat all sites or risk exposing the patient to a high risk of recurrence, estimated at 35% at 1 year (Leufflen et al, 2013). Overall, the 10-year recurrence rate for vulvar cancers is estimated to be 43% (Te Grootenhuis et al, 2019) with a high secondary risk of non-conservative surgical treatment, which is a source of major post-operative sequelae.
These treatments are not indicated in invasive vulvar cancer. It is critical to ensure the absence of invasive cancer before initiating conservative treatment.
If surgical excision is performed, the presence of HSIL or VIN puts patients at high risk for local recurrence.

Diagnostic tools for vulvar intraepithelial neoplasia are limited because of the variety of clinical presentations and their often asymptomatic nature. Vulvoscopy, a binocular loupe examination coupled with multiple biopsies, is useful to localize vulvar lesions and to ensure the absence of infiltrating cancer but it remains burdensome for the patient, non-specific and operator dependent. Andersson and Block estimate the correlation between vulvoscopy and histological examination at 66% in trained teams (Andreasson and Bock, 1985). In the presence of high-grade vulvar neoplasia, the risk of clinically unrecognized vulvar invasive cancer is estimated in the literature to be between 3 and 9% (Jones et al, 2005).
Photodynamic diagnosis (PDD) based on 5-aminolevulinic acid (5-ALA) used as a photosensitizer) has been tested by several teams to detect vulvar intraepithelial neoplasia (Nowakowski et al, 2005, Maździarz et al, 2013, Akoel et al, 2003). Although the diagnostic performance of 5-ALA appears to be excellent, the data in the literature on 5-ALA are from small series published by the same teams. Moreover, 5-ALA is only marketed in an oral form, which is not very lipohilic and whose transcutaneous passage must be potentiated by the addition of other drugs, which is why one of its derivatives, methyl aminolevulinate hydrochloride, marketed in France under the name of Metvixia, has been preferred for use in skin tumours.
The objective of this work will be to develop a preclinical model to evaluate the diagnostic possibilities of VIN lesions thanks to the study of fluorescence after application of m-ALA (methylaminolevulinate). Indeed, cancer cells metabolize m-ALA into protoporphyrin IX (PPIX), which fluoresces after excitation in blue or red light. Photodiagnosis has been evaluated in animal models (Leufflen et al 2018).

In a first step, we will design a spheroid model from A431 cells (derived from vulvar squamous cell carcinomas): elaboration of the model, evaluation of the kinetics and the growth curve then study of the morphology of the spheroid (HES labeling, immunohistochemistry, size, morphological characterization) as a function of the number of days in culture to design the model.
In a second step, we wish to realize a co-culture of tumor cells / healthy cells in order to get closer to the clinical model (keratinocytes). In the same way this model will be studied morphologically, its growth and evolution controlled with immunohisto characterization and optimization of the model according to these characteristics.
We will then test the selectivity of m-ALA and PPIX production in this model.
Department(s): 
Biology, Signals and Systems in Cancer and Neuroscience