Abstract
Objectives During malignant transformation, remodeling of the extracellular matrix creates a stromal tumor microenvironment (TME) essential for cancer progression. Collagen is a main component of this TME and its relevance as an indicator of tumor transformation is highlighted in research on breast and prostate cancer. The most sensitive reported technology to study these collagenic architectural transformations is multiphoton microscopy (MPM), monitoring 2-photon excited fluorescence and second harmonic generation. The main objective of this work is to establish a proof of concept of MPM to better understand the TME processes underlying oral cancer (OC) and to support novel effective therapeutic strategies. The relevance of the topic gains its evidence from the current lack of effective prevention and curative treatment for this devastating cancer. Methods Cryostat-sectioned and paraffin-embedded samples were analyzed and the generated SHG images were recorded using a custom-made MPM based on a Tsunami laser and an upright SliceScope microscope furbished with a multiphoton galvanometer scan head. 38 samples, pertaining to 15 types of lesions representative of all tumor stages, were analyzed. 44% of samples (n=168) were usable. Results Results showed that collagen, normally well organized, deteriorates with tumour progression and gradually transforms into an increasingly disordered network, with fibers firstly dissociating and thinning. Analysis by vectorization of perilesional collagen fibers suggests, in invasive tumors, an organization of these fibers perpendicularly to the tumor, creating "gateways" for migration into surrounding tissues of tumor cells. The identification of the evolution of collagen density and organization seems to allow, as for other tumor localizations, to define a tumor-associated collagen signatures (TACS) gradation scale for OC, statistically correlated with overall and recurrence-free survival. Conclusions MPM could be a reliable tool in the identification of tumor progression markers of OC, which would be complementary to pathological examination and useful for identifying target areas for biopsy or surgical margins.