Background and Objectives: Conventional ALA-PDT (C-PDT) has limited efficacy in cutaneous squamous cell carcinoma (cSCC), and there is obvious pain during treatment, which limits its clinical application. We sought to modify photodynamic therapy into a more painless and effective treatment. Methods: We modified C-PDT by reducing the incubation time of the pro-sensitizer and increasing the light dose; we named this method modified ALA-PDT (M-PDT). We compared the pain response and curative effect between C-PDT and M-PDT in cSCC mouse models. Pain-related proteins were examined by western blot analysis and immunohistochemistry. Tumor progression-associated signaling pathways were analyzed by RNA-seq and western blot analysis. Reactive oxygen species (ROS) generation was measured with a ROS test kit and Microplate reader. Results: M-PDT greatly reduced pain during treatment. Interestingly, when the cSCC tumor volume increased to 150-200 mm 3 , M-PDT almost completely eliminated the tumors, while C-PDT did not. The better curative effect of M-PDT might be due to the stronger suppression of the Stat3, Erk1/2, and mTOR signaling pathways. Moreover, flow cytometry demonstrated that M-PDT could recruit CD8 + T cells to inhibit cSCC progression. Further investigation determined that the different mechanisms of C-PDT and M-PDT were related to more ROS generation induced by M-PDT. Conclusions: Our results suggest that M-PDT, which is more painless and effective than C-PDT, is expected to provide a solution for the treatment of cSCC.

Non‐invasively diagnosis of actinic keratoses (AK) is important for preventing cutaneous squamous cell carcinoma (cSCC). Optical coherence tomography (OCT) can be used to detect the cross‐sectional skin micromorphology with sufficient resolution and imaging depth. It has the capability to reveal the changes in skin microstructure during the development of AK. Therefore, OCT can serve as a tool for diagnosing AK. This study explores the feasibility of OCT in evaluating the structural changes in mouse skin at the different stages following exposure to ultraviolet radiation. The performance of OCT is compared with histology, the gold standard in this context. The imaging results demonstrate that a wave‐shaped irregular dermo‐epidermal junction (DEJ), as well as the continuous thickening of the epidermis, are useful diagnostic parameters for diagnosing AK. Histological examinations confirm these observations. These findings emphasize the need for effective skin protection or medical treatment once changes in the DEJ and epidermis are detected. OCT shows strong potential for non‐invasive evaluation of such changes and AK development, and can be used for the prevention of cSCC without the necessity of taking biopsies.

Background UV-induced cutaneous squamous cell carcinoma (cSCC) is the most common skin cancer. The constant alterations of the lymphatic-centered immune microenvironment are essential in transforming from photoaging to cSCC. Studying the mechanism will be beneficial for finding new targets for the early prediction of cSCC.Aims To investigate the dynamic changes and mechanism of the lymphatic-centered immune microenvironment in transforming from photoaging to cSCC induced by ultraviolet irradiation (UVR).Methods TIMER2.0 was used to analyze whether YAP1/VEGFC signaling pathway is involved in lymphangiogenesis in head and neck squamous cell carcinoma (HNSCC). Meanwhile, lymphatic-centered immune microenvironments alterations and the related cumulative survival time were also analyzed. With accumulated UVR at 8, 16–18, and 20–24 weeks, skin photoaging developed and gradually progressed into actinic keratosis and cSCC on SKH-1 hairless mice. The skin lymphatic-centered immune microenvironment was evaluated at the 0th, 8th, 12th, 16-18th, and 20-24th week of UVR. Skin phenotype was assessed using optical coherence tomography (OCT) and skin image. The structure of lymphatic vessels (LVs), blood vessels, and different types of T cells was evaluated by immunohistochemistry staining. The expression of Piezo1, whose deletion in adult lymphatics led to substantial valve degeneration, VE-cadherin, which maintained the drainage function of LVs, and YAP1 were evaluated by immunohistochemistry staining as well. Besides, the drainage function of LVs was assessed by Evans Blue assay in vivo. H&E and Masson’s trichrome staining evaluated the skin dermis and collagen structure.Results The lymphatic function and immune cell infiltration have adapted under continuous UVR. TIMER2.0 analysis indicated that YAP1 and VEGFC genes high expressed in HNSCC. LV density increased in human cSCC. More LVs in HNSCC were beneficial to prolong the survival time. VEGFC and YAP1 gene overexpression was correlated to CD8+T cell infiltration. More CD8A+ T cells and CD8B+ T cell infiltration in HNSCC also extended survival time. However, YAP1 gene overexpression and more CD4+T cell infiltration at the same time might be against survival time. In animal studies, UVR-induced eight weeks (photoaging skin) and 16–18 weeks (actinic keratosis) were two turning points in the lymphatic-centered immune microenvironment. LVs in UV-8w and UV-12w groups were the lowest in density and lower than that in normal mice. When normal skin developed into AK lesions (UV-16-18w), LV slightly exceeded healthy skin and proliferated sharply in cSCC (UV-20-24w). YAP1 expression was consistent with LV but rose after the photoaging stage. The drainage of cSCC induced by UVR was better than that of photoaging skin and worse than that of normal skin. The dynamic alterations of LVs number, Piezo1 expression, and collagen content were reasons for it. Piezo1 expression represented the highest point in the UV-8w group, then gradually descended to the platform. The dermis slowly diminished after chronic UVR, especially the collagen. The total T cells maintained steady, but the number of CD4+T cells increased, and CD8+T cells decreased after eight weeks of UVR. The whole T cells and CD4+T cells increased sharply in UV-16-18w and UV-20-24w groups.Conclusion The lymphatic-centered immune microenvironment has adapted under continuous UVR via regulating YAP1/VEGFC and Piezo1. UVR-induced eight weeks (photoaging) and 16–18 weeks (precancerous), two turning points. YAP1, Piezo1, LVs, and immune cells constantly changed with the skin state induced by UVR. According to changes in photoaging, UV-12w, and precancerous, identify the process of cSCC in advance and intervene timely.