| Background:cutaneous Squamous cell cancer (SCC) is a common human skin cancer, accounting for about 20% of all non-melanocytic skin cancer. SCC most occurs in elderly patients and more often occur in head and face, vulva and external genitalia. Traditional treatments included surgery, laser, freezing and radiotherapy can remove or destroy tumor tissue directly, but they have disadvantages for example organizational structure destructive, recurrence, difficultity in treating again. Due to the weak immunogenicity of SCC, host functional defects in immunity or poor cannot induce effective anti-tumor immune response. This is main reason for recurrence and metastasis for SCC. Therefore, It is necessary to look for the ideal tumor therapy that cloud not only kill tumor cells but also stimulate the body’s anti-tumor immune response by enhance the immunogenicity of the tumor cells.Photodynamic therapy involves the accumulation of photosensitizer in diseased tissues followed by illumination of disease site with light of an appropriate wavelength to induce photochemical reactions that result in tissue destruction. Topical PDT is becoming an established treatment for actinic keratosis, Bowen’s Disease and superficial basal cell carcinoma. PDT-based anti-tumour effects are multifactorial and include:(1) direct tumour cell kill, (2) damage to the vasculature, (3) cytotoxic effects towards tumour-infiltrating immune cells and (4) rapid recruitment and activation of immune cells that can facilitate development of anti-tumour adaptive immunity. Our team take the lead in ALA-PDT treatment of superficial skin tumors and precancerous lesions and achieved satisfactory clinical efficacy. Further studies have also found that ALA-PDT significantly inhibited tumor growth and could prevent cancer.In recently, "immunogenic cell death" the concept make the medical profession have new understanding of antigen expression on the surface of tumor cells or release from them. Immunogenic cell death (ICD) relies on molecular signals called damage-associated molecular patterns (DAMPs), that activate innate immunity and induce specific anti-tumor immune response. "Immunogenic death" is different with normal death ways, while at killing tumor cells and stimulate the body to produce a powerful anti-tumor immune response. The study found that when PDT treating local tumor lesions, induce local or systemic anti-tumor immune response, and even lead to suppression of distant metastases and reduce the recurrence rate.Our previous studies have confirmed that ALA-PDT in the treatment of tumor lesions induced tumor cell ICD, but the mechanism is not clear. This study investigate whether ALA-PDT can induce cutaneous SCC tumor cells express DAMPs enhance immunity and its molecular mechanism.Objective:This study investigate ALA-PDT induce cutaneous SCC induce tumor cells express DAMPs enhance immunity and its molecular mechanism.1. To explore the optimal time and illuminated dose of ALA-PDT-induced mouse skin squamous cell expression DAMPs.2. To confirmed DAMPs play a important in enhancing immunity of SCC by ALA-PDT.3. To explore molecular mechanism of DAMPs enhancing immunity of SCC by ALA-PDT.Method:1. The expression of DAMPs of tumor cell at different light dose and time points after ALA-PDT:The mouse skin SCC celline PECA were incubated with 0.5mM of ALA for 5h, we collected cells and cell supernatant at doses (0.25J/cm2,0.5J/cm2, 1 J/cm2 and 2J/cm2) and different time points(1h,3h,6h,9h,12h after PDT). HSP70, HMGB1 and CRT expression of intracellular and cell membrane were determined by western blot. HMGB1 and CRT released from PECA were detected by Elisa assay.2. DAMPs play an important role in inducing phenotype and functional maturation of DC that pulse ALA-PDT treatmented PECA were confirmed:Isolating and culturing mouse bone marrow dendritic cells (DCs), DCs were incubated with PECA treated by ALA-PDT(0.5mM,5h,0.5J/cm2). The inhibitors were added in DAMPs suppression group.24h after incubation, DCs and cell supernatants were collected. Flow cytometry was used to detect MHCⅡ, CD80 and CD86 of DCs surface. Elisa assay was used to detect IL-12 and INF-y secreted by DCs.3. Analysis weather ALA-PDT mediated by DAMPs whether enhanced immunogenicity by TLRs-NF-κB pathway:Mouse myeloid DCs were isolated and cultured. DCs were incubated with PECA treated by ALA-PDT(0.5mM,5h,0.5J/cm2). The inhibitors were added in DAMPs suppression group.24h after incubation, DCs were collected. Expression of TLR2 and TLR4 and IκB, plκB and nucleus NF-κB of DCs were determined by western blot.Result:1. Expression of DAMPs of tumor cell at different light dose and time points after ALA-PDT:Expression level of PECA and cell membranes that treated with ALA-PDT were enhanced and reach a peak at 0.5J/cm2 after PDT 6h. HSP70 and HMGB1 secreted by PECA treated with ALA-PDT increased and reach a peak at 2J/cm2 PDT after 6h.2. DAMPs play an important role in inducing phenotype and functional maturation of DC that pulse ALA-PDT treatmented PECA:The level of MHC II, CD80 and CD86 of ALA-PDT treated PECA pulse DCs (PDT-DCs) significantly increased compared with non-sensitized group, and still lower than LPS stimulation group. When HSP70, HMGB1 and CRT were inhibited, the level of MHC, CD80 and CD86 of PDT-DCs significantly decreased. The secretion of IL-12 and INF-y of PDT-DCs significantly increased, compared with non-sensitized group. When HSP70, HMGB1 or CRT inhibited separately, secretion of INF-y of PDT-DCs decreased. When HSP70, HMGB1 and CRT were inhibited, secretion of ESF-γ of PDT-DCs significantly decreased. When HSP70, HMGB1 or CRT was inhibited separately, secretion of IL-12 of PDT-DCs slightly decreased. When HSP70, HMGB1 and CRT were inhibited, secretion of IL-12 of PDT-DCs decreased.3. DAMPs mediated ALA-PDT enhanced immunogenicity thought TLRs-NF-κB pathway:The expression of TLR2 and TLR4 on PDT-DCs surface significantly increased, when HSP70, HMGB1 or CRT was inhibited separately, the expression of TLR2 on PDT-DCs surface decreased. When HSP70 or HMGB1 was inhibited separately, the expression of TLR4 on PDT-DCs surface decreased, when HSP70, HMGB1 and CRT were inhibited, TLR2 and TLR4 of expression on PDT-DCs surface significantly decrease, when the CRT was inhibited, the level of TLR4 expression did not change significantly. The expression level of I-κB, pI-κB on PDT-DCs surface and NF-κB in the nucleus upregulated. When HSP70 or HMGB1 was inhibited separately, expression level I-κB, pI-κB on PDT-DCs surface and NF-κB in the nucleus decrease, when the CRT was inhibited, the expression level I-κB, pI-κB on PDT-DCs surface and NF-κB in the nucleus did not change significantly. When HSP70, HMGB1 and CRT were inhibited, the expression levels of I-κB, pI-κB NF-κB in the nucleus of PDT-DCs reduced significantly.Conclusion:1. ALA-PDT can induce the expression of DAMPs of SCC cell PECA. Expression level of PECA and cell membranes that treated with ALA-PDT and reach a peak at 0.5J/cm2 PDT after 6h. HSP70 and HMGB1 secreted by PECA treated with ALA-PDT increased and reach a peak at 2J/cm2 PDT after 6h.2. DAMPs play a vital role in enhancing the immunogenicity of skin SCC and stimulate DCs maturation process.3. Activating DCs of TLRs-NF-κB signaling pathway maybe main molecular mechanism of DAMPs enhancing the immunogenicity cutaneous SCC. |
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