Efficacy Of Phototherapy Combined With Immunotherapy For Primary And Secondary Lung Malignancies

Background:Lung cancer has become one of the highest incidence of cancer in the world and is the leading cause of cancer death.Due to their physiological characteristics,many cancers are prone to metastasize and colonize the lungs.Sentinel lymph nodes(SLNs)are key nodes for cancer cell metastasis,and their removal can effectively reduce the risk of metastasis.However,in patients who have developed distant metastases,surgical treatment is no longer appropriate and other treatment options need to be explored.In recent years,immunotherapy has made remarkable progress in the treatment of lung malignant tumors,especially the application of immune checkpoint inhibitors,which has greatly improved the prognosis and survival of patients with primary and secondary lung malignant tumors.However,the inhibitory tumor microenvironment(TME)limits the effectiveness of single immunotherapy,so multi-modal combination therapy is the key to improving efficacy.Phototherapy,including PDT and PTT,has been gradually recognized in clinical practice as a new tumor treatment method.PDT was approved by the National Comprehensive Cancer Network(NCCN)and the Chinese Expert Consensus on the Clinical Application of Photodynamic Therapy for Respiratory Tumors in 2019 for the treatment of tracheal obstruction and severe hemoptysis caused by local symptoms or local recurrence in patients with advanced lung cancer.Photosensitizer is one of the three elements of phototherapy.The traditional photosensitizers limit the use of phototherapy in clinic due to their poor water solubility and limited targeting of cancer cells.In order to overcome these shortcomings,researchers are gradually applying nano-sized photosensitizers to anti-tumor therapy.Due to the enhanced permeability and retention(EPR)effect of nanoparticles,nano photosensitizers can better achieve targeted cancer therapy.In addition,nano photosensitizer-mediated phototherapy targeting lymph nodes has inhibitory effects on both primary tumor and cancer cells in lymph nodes.Phototherapy can directly cause cancer cell death,and further induce immunogenic cell death(ICD),stimulate the maturation of dendritic cells(DCs),induce T cell differentiation,and improve TME.At the same time,it also increased the expression of PD-L1 in cancer cells,activated the specific anti-tumor immune response,and further induced the"abscopal effect".Therefore,combining phototherapy with ICIs is a potential strategy for the treatment of primary lung cancer as well as secondary lung tumors.Objective:1.Porphyrin cholesterol nanoparticles(TPPC NPs)were prepared to explore the anti-tumor effects of TPPC NPs combined with ICIs at cellular and animal levels.It provides a new idea for the clinical application of PDT combined with ICIs in the treatment of primary lung cancer.2.To explore the photothermal therapeutic effect of clinically approved lymph node tracer carbon nanoparticle suspension injection(CNSI)at the cellular and animal levels,and to study the mechanism of CNSI combined with ICIs in inhibiting secondary lung tumors.It provides a new strategy for the treatment of secondary lung tumors.Methods:1.For primary lung cancer,we designed and synthesized a porphyrin cholesterol nanoparticle(TPPC NPs)for photodynamic combined immunotherapy.Specific research methods are as follows:(1)The structure of TPPC was confirmed by H nuclear magnetic resonance spectroscopy and mass spectrometry.The basic characterization of TPPC NPs was identified by particle size meter,transmission electron microscope and ultraviolet absorption spectrum,and the photodynamic properties of TPPC NPs were tested by singlet oxygen experiment.(2)The endocytosis of TPPC NPs was observed by confocal microscopy(CLSM).The effect of PDT on lung cancer cell viability was evaluated by cytotoxicity assay,and the effect of PDT on metastasis and invasion of lung cancer cells was detected by scratch assay and Transwell assay.The expression level of PD-L1 was detected by Western Blot.(3)CLSM observed the changes of ICD-related molecules HMGB1 and CRT after TPPC NPs treatment,and the content of ATP in the cell supernatant was detected.DCs maturation was evaluated by flow cytometry,Actin-Tracker labeling of DCs morphology and ELISA detection of related molecules.(4)A mouse transplanted tumor model was constructed,and the accumulation of TPPC NPs in tumor tissues was observed by animal imaging system,and the inhibitory effect of TPPC NPs-mediated PDT combined with aPD-1 on tumor was evaluated according to the changes in tumor volume during treatment in mice.(5)The immune activation of tumor tissues and important immune organs was observed by flow cytometry.A mouse model of distant tumor was used to observe the inhibitory effect of PDT combined with aPD-1.2.For secondary lung tumors,in view of the inability of lymph node tracing with TPPC NPs,we used the clinically approved lymph node tracer CNSI for photothermal combined immunotherapy.Specific research methods are as follows:(1)Mice were injected subcutaneously with CNSI to evaluate its lymph node targeting ability in vivo,and the composition of CNSI was confirmed by X-ray photoelectron spectroscopy and Fourier transform infrared spectrometer.The basic characterization of CNSI was identified by particle size analyzer,transmission electron microscopy and ultraviolet absorption spectrum,and its photothermal conversion ability was detected by photothermal performance test.(2)Cytotoxicity assay was used to detect the effect of CNSI-mediated PTT on cancer cell viability,and scratch assay was used to detect the migration of cancer cells after CNSI treatment;For example,the changes of ICD-related molecules HMGB1,CRT and ATP after CNSI treatment were detected by the same method of TPPC NPs,and the maturation of DCs was further detected by flow cytometry.(3)The mouse model of distant tumor was constructed to observe the inhibitory effect of CNSI-mediated PTT combined with aPD-1 on distant tumor,and the immune activation in vivo was observed by flow cytometry.(4)A mouse model of trans-lymph node lung metastasis was established.In the treatment experiment,primary tumor and SLNs dual irradiation combined with ICIs were used to evaluate lung metastasis through lung H&E staining and the number of lung metastasis nodules,and the immune activation in SLNs was further detected by flow cytometry.Results:1.Application of TPPC NPs in photodynamic combined immunotherapy in primary lung cancer:(1)Small molecules of porphyrin cholesterol(TPPC)were designed and synthesized with porphyrin and cholesterol as raw materials.TPPC was self-assembled in water by nano-precipitation method to form nanoparticles TPPC NPs,whose particle size was about 197.8±2.6 nm.Physical and chemical experiments confirmed that TPPC NPs had a good 1O2 generation efficiency.(2)In vitro experiments proved that cholesterol in TPPC NPs could mediate its endocytosis by cancer cells.Under laser irradiation,when the TPPC NPs was 25μg/m L,the cell viability was only 31.76%.PDT inhibited the metastasis and invasion of lung cancer cells,and upregulated the expression level of PD-L1.(3)PDT mediated by TPPC NPs induced the release of HMGB1 and the exposure of CRT,and the content of ATP in the cell supernatant was significantly increased;In the PDT group,the expression of mature DCs was significantly increased,and the contents of TNF-αand IFN-γwere significantly increased,and the DCs with Actin-Tracker staining showed obvious dendritic structure.(4)In the transplanted tumor model of mice,24 hours after intravenous injection of nanoparticles,their accumulation in tumor tissues reached a peak.The results showed that the relative inhibitory rate of TPPC NPs mediated PDT and aPD-1 was91.4%.(5)In the combined treatment group,the infiltrated and mature DC in lymph nodes increased significantly,and the proportion of CD4~+T cells in tumor tissues and spleen tissues also increased;In the experiment of distant tumor in mice,there was no difference between the PDT group and the PBS group,but the growth of distant tumor was significantly inhibited in the PDT group combined with aPD-1 group.2.Study on CNSI for photothermal combined immunotherapy in secondary lung tumors:(1)CNSI,as a clinically approved lymph node tracer,also has the effect of lymph node aggregation in mice;At the same time,CNSI has a wide absorption in the near infrared window,so its maximum temperature change under 808 nm irradiation reaches43.9(CNSI is 50μg/m L),and its photothermal conversion efficiency is 42.06%.(2)Under the condition of laser irradiation,the cellular survival rate was significantly reduced,and PTT could effectively inhibit the metastasis of cancer cells.ICD-related molecules HMGB1,CRT and ATP were changed in PTT group,and the expression of mature DCs was increased,and the contents of TNF-αand IFN-γwere significantly increased.(3)In the mouse distant tumor experiment,CNSI combined with aPD-1 can effectively inhibit the growth of primary tumor and distant tumor.In the combined treatment group,DCs maturation rate in the draining lymph nodes was the highest(about 52.5%),and the number of CD8~+T cells in the spleen and the number of CD4~+T cells infiltrating the tumor were also significantly increased.(4)In a mouse model of trans-lymph node lung metastasis,the volume of metastatic SLNs and the number of pulmonary nodules were significantly inhibited in the CNSI-mediated photothermal combined immunotherapy group,and the proportion of mature DCs and CD4~+T cells in the SLNs in the combined therapy group were significantly increased.Conclusion:1.Design and synthesize TPPC NPs,which mediate PDT to reduce the viability of lung cancer cells and induce ICD to stimulate DCs maturation.2.TPPC NPs mediated PDT combined with aPD-1 can effectively inhibit tumor growth in mice,reshape TME in vivo,and inhibit the growth of distant tumors.3.CNSI also has the effect of lymph node aggregation in mice,and its mediated PTT inhibits the activity of cancer cells and induces ICD to stimulate DCs maturation.4.PTT combined with aPD-1 inhibited the growth of primary tumor and distant tumor,and activated TME.5.CNSI-mediated PTT combined with aPD-1 inhibited the growth of cancer cells in SLNs,activated the intrinsic immune response,and reduced the occurrence of secondary lung tumors.