Anti-PD1 antibody and not anti-LAG-3 antibody improves the antitumor effect of photodynamic therapy for treating metastatic breast cancer

Photodynamic therapy(PDT)has limited effects in treating metastatic breast cancer.Immune checkpoints can deplete the function of immune cells;however,the expression of immune checkpoints after PDT is unclear.This study investigates whether the limited e±cacy of PDT is due to upregulated immune checkpoints and tries to combine the PDT and immune checkpoint inhibitor to observe the e±cacy.A metastatic breast cancer model was treated by PDT mediated by hematoporphyrin derivatives(HpD-PDT).The anti-tumor effect of HpD-PDT was observed,as well as CD4þT,CD8þT and calreticulin(CRT)by immunohistochemistry and immunofluorescence.Immune checkpoints on T cells were analyzed byflow cytometry after HpD-PDT.When combining PDT with immune checkpoint inhibitors,the antitumor effect and immune effect were assessed.For HpD-PDT at 100 mW/cm2 and 40,60 and 80 J/cm2,primary tumors were suppressed and CD4þT,CD8þT and CRT were elevated;however,distant tumors couldn't be inhibited and survival could not be prolonged.Immune checkpoints on T cells,especially PD1 and LAG-3 after HpD-PDT,were upregulated,which may explain the reason for the limited HpD-PDT effect.After PDT combined with anti-PD1 antibody,but not with anti-LAG-3 antibody,both the primary and distant tumors were signi-cantly inhibited and the survival time was prolonged,additionally,CD4þT,CD8þT,IFN-þCD4þT and TNF-þCD4þT cells were signi-cantly increased compared with HpD-PDT.HpD-PDT could not combat metastatic breast cancer.PD1 and LAG-3 were upregulated after HpD-PDT.Anti-PD1 antibody,but not anti-LAG-3 antibody,could augment the antitumor effect of HpD-PDT for treating metastatic breast cancer.

Tissue optical clearing enhances efficacy of vascular targeted photodynamic therapy of mouse dorsal skin

Vascular-targeted photodynamic therapy(V-PDT)is an effective treatment for port wine stains(PWS).However,repeated treatment is usually needed to achieve optimal treatment outcomes,possibly due to the limited treatment light penetration depth in the PWS lesion.The optical clearing technique can increase light penetration in depth by reducing light scattering.This study aimed to investigate the V-PDT in combination with an optical clearing agent(OCA)for the therapeutic enhancement of V-PDT in the rodent skinfold window chamber model.Vascular responses were closely monitored with laser speckle contrast imaging(LSCI),optical coherence tomography angiography,and stereo microscope before,during,and after the treatment.We further quantitatively demonstrated the effects of V-PDT in combination with OCA on the blood flow and blood vessel size of skin microvasculature.The combination of OCA and V-PDT resulted in significant vascular damage,including vasoconstriction and the reduction of blood flow.Our results indicate the promising potential of OCA for enhancing V-PDT for treating vascular-related diseases,including PWS.

Effects of pulse width and repetition rate of pulsed laser on kinetics and production of singlet oxygen luminescence

Pulsed and continuous-wave(CW)lasers have been widely used as the light sources for photodynamic therapy(PDT)treatment.Singlet oxygen(^(1)O_(2))is known to be a major cytotoxic agent in type-II PDT and can be directly detected by its near-infrared luminescence at 1270 nm.As compared to CW laser excitation,the effiects of pulse width and repetition rate of pulsed laser on the kinetics and production of^(1)O_(2)luminescence were quantitatively studied during photosensitization of Rose Bengal.Significant di®erence in kinetics of^(1)O_(2)luminescence was found under the excitation with various pulse widths of nanosecond,microsecond and CW irradiation with power of 20mW.The peak intensity and duration of^(1)O_(2)production varied with the pulse widths for pulsed laser excitation,while the^(1)O_(2)was generated continuously and its production reached a steady state with CW excitation.However,no significant di®erence(P>0:05)in integral^(1)O_(2)production was observed.The results suggest that the PDT efficacy using pulsed laser may be identical to the CW laser with the same wavelength and the same average°uence rate below a threshold in solution.

Photodynamic therapy for high-grade dysplasia of bile duct via a choledochoscope

When a distal common bile duct neoplasm is at the stage of carcinoma in situ or high-grade dysplasia,it is difficult for the surgeon to decide whether to perform pancreaticoduodenectomy.Here we describe a patient with a progressive dysplastic lesion in the common bile duct,which developed from moderate-high to highgrade dysplasia in approximately 2 mo.The patient refused major surgery.Therefore,endoscopic-assisted photodynamic therapy was performed.The result at follow-up using a trans-T-tube choledochoscope showed that the lesion was completely necrotic.This report is the first to describe the successful treatment of highgrade dysplasia of the distal bile duct using photodynamic therapy via a choledochoscope.

AUTOMATED ASSESSMENT OF EPIDERMAL THICKNESS AND VASCULAR DENSITY OF PORT WINE STAINS OCT IMAGE

Optical coherence tomography(OCT)enables in vivo imaging of port wine stains(PWS)lesions.The knowledge of vascular struct ure and epidermal thickness(ET)of PWS may aid the objectivediagnosis and optimal treatment.To obtain the structural parameters more rapidly and avoiduser intervention,an automated algorithm of energy map is introduced based on intensity andedge information to extract the skin surface using dynamic programming method.Subsequently,an averaged A-scan analysis is performed to obtain the mean ET and the relative intensity ofdermis indicating the corresponding vascular density.This approach is currently successfullyapplied in clinical diagnosis and shows promising guidance and assessment of PDT treatment.

Improvement in Resolution of Multiphoton Scanning Structured Illumination Microscopy via Harmonics

We describe a multiphoton(mP)-structured illumination microscopy(SIM)technique,which demonstrates substantial improvement in image resolution compared with linear SIM due to the nonlinear response of fluorescence.This nonlinear response is caused by the effect of nonsinusoidal structured illumination created by scanning a sinusoidally modulated illumination to excite an mP fluorescence signal.The harmonics of the structured fluorescence illumination are utilised to improve resolution.We present an mP-SIM theory for reconstructing the super-resolution image of the system.Theoretically,the resolution of our m P-SIM is unlimited if all the high-order harmonics of the nonlinear response of fluorescence are considered.Experimentally,we demonstrate an 86 nm lateral resolution for two-photon(2P)-SIM and a 72 nm lateral resolution for second-harmonic-generation(SHG)-SIM.We further demonstrate their application by imaging cells stained with F-actin and collagen fibres in mouse-tail tendon.Our method can be directly used in commercial mP microscopes and requires no specific fluorophores or high-intensity laser.

COMBINED EFFECTS OF SELECTIVE PHOTOTHERMAL THERAPY AND IMMUNOADJUVANT AGAINST STAGE IV BREAST CANCER

Metastasis to distant sites is a severe treatment challenge and a major cause of death for breast cancer patients.Laser immunotherapy(LIT)is a novel technique,combining a selective photothermal therapy with local application of glycated chitosan,a potent immunoadjuvant.The pre-clinical studies of LIT have shown its unique characteristics in generating specific antitumor immunity.The clinical application of LIT in the treatment of melanoma patients has achieved preliminary success.Recently,LIT has been used to treat late-stage breast cancer patients.Here we report for the first time the clinical results of this combination therapy in breast cancer patients.The LIT treatment procedures are presented and the medical history of two stage Ⅳ breast cancer patients is reviewed.Most of the breast cancer lesions and the metastasis of lung and brain disappeared after repeated treatments of LIT.One patient achieved complete response;the other achieved partial response at the time of this report.Although there is still a long way for LIT to become a standard modality for breast cancer treatment,the results of this study indicated its promising future.

A COMPARATIVE STUDY OF DNA FLOW CYTOMETRY AND MICROSPECTROPHOTOMETRY IN GASTRIC CARCINOMAS

Flow cytometric (FCM) and microspectrophotometric (MSP) measurements of cell nuclear DNA content were made in 53 fresh gastric carcinoma specimens and in 30 gastric mucosal specimens with chronic gastritis. DNA aneuploidy was found in 32/53 (60%) of gastric carcinomas, and appeared more frequently in wellor moderately differentiated tubular adenocarcinomas (90%) than in undifferentiated and mucousa cell carcinomas (23.6%) (P【0.001). No aneuploidy was found in chronic gastritis samples, but their proliferative cell fractions were higher than in normal control gastric mucosa samples (P【0.01). A comparison was made between FCM and MSP analyses of DNA content in 29 cases of gastric carcinoma, a high correlation rate (r=0.90) was found. The advantages and limitations of both methods are discussed, they may be used in combination for more precise cytochemical analysis.

从细胞水平研究激光焊接大鼠神经的机制

目的 探讨激光焊接神经的基本机制。方法 SD大白鼠 2 4只 ,随机分为激光焊接组和传统外膜缝合组 ,每组 12只。游离大鼠坐骨神经 ,从中段剪断行神经断端对合后 ,激光组用氩激光 (波长 5 14 5nm ,石英光纤芯径 6 0 0 μm ,功率 70 0mW ,光斑直径 1mm) ,照射对合处一圈 ;缝合组用 11 0号尼龙缝线缝合对合处的神经外膜。于术后 10天、1个月和 3个月对再生神经用光镜、电镜进行组织形态学观察 ,术后 3个月用电生理仪进行功能测试 ,比较两组间的差异。结果 组织形态学观察 :术后 10天 ,激光组的神经膜细胞明显多于缝合组 ;术后 1个月再生神经纤维生长 ,数量比缝合组多 ;术后 3个月 ,激光组的再生神经纤维密度大 ,且分布均匀 ,吻合部位的神经纤维连续性良好且平直 ,明显好于缝合组 ,神经电生理测试 :术后 3个月测定再生神经的传导速度 (CV)和复合动作电位峰值 (AP) ,激光组优于缝合组。结论 在激光焊接神经外膜的同时 ,透过神经外膜进入神经组织内的漫射激光刺激了神经膜细胞 ,加速其不断分裂增殖 。

Aligned fibers enhance nerve guide conduits when bridging peripheral nerve defects focused on early repair stage

Nerve conduits enhance nerve regeneration in the repair of long-distance peripheral nerve defects. To help optimize the effectiveness of nerve conduits for nerve repair, we developed a multi-step electrospinning process for constructing nerve guide conduits with aligned nanofibers. The alignment of the nerve guide conduits was characterized by scanning electron microscopy and fast Fourier transform. The mechanical performance of the nerve guide conduits was assessed by testing for tensile strength and compression resistance. The biological performance of the aligned fibers was examined using Schwann cells, PC12 cells and dorsal root ganglia in vitro. Immunohistochemistry was performed for the Schwann cell marker S100 and for the neurofilament protein NF200 in PC12 cells and dorsal root ganglia. In the in vivo experiment, a 1.5-cm defect model of the right sciatic nerve in adult female Sprague-Dawley rats was produced and bridged with an aligned nerve guide conduit. Hematoxylin-eosin staining and immunohistochemistry were used to observe the expression of ATF3 and cleaved caspase-3 in the regenerating matrix. The recovery of motor function was evaluated using the static sciatic nerve index. The number of myelinated fibers, axon diameter, fiber diameter, and myelin thickness in the distal nerve were observed by electron microscopy. Gastrocnemius muscle mass ratio was also determined. The analyses revealed that aligned nanofiber nerve guide conduits have good mechanical properties and can induce Schwann cells, PC12 cells and dorsal root ganglia to aggregate along the length of the nanofibers, and promote the growth of longer axons in the latter two(neuronal) cell types. The aligned fiber nerve conduits increased the expression of ATF3 and cleaved caspase-3 at the middle of the regenerative matrix and at the distal nerve segment, improved sciatic nerve function, increased muscle mass of the gastrocnemius muscle, and enhanced recovery of distal nerve ultrastructure. Collectively, the results show that highly aligned nanofibers improve the performance of the nerve conduit bridge, and enhance its effectiveness in repairing peripheral nerve defects.

Immune response induced by hematoporphyrin derivatives mediated photodynamic therapy:Immunogenic cell death and elevated costimulatory molecules

Photodynamic therapy(PDT)not only destroys tumor cells directly but also induced anti-tumor immune response through damage-associated molecular patterns(DAMPs).It is reported that anti-tumor response was associated with light dose and photosensitizer used in PDT.In this study,4T1 tumor cells were implanted on both the right and left flanks of mice.Only the right tumor was treated by HpD-PDT,while the left tumor was not irradiated.The anti-tumor immune response induced by HpD-PDT was investigated.The expression of DAMPs and costimulatory molecules induced by HpD-PDT were tested by immuno fluorescence and flow cytometry in vivo.Different light doses of PDT were designed to treat 4T1 cells.The killing effect was assessed by CCK-8 kit and apoptosis kit.The expression of DAMPs on 4T1 cells after HpDPDT were evaluated by flow cytometry,western blot and ATP kit.This study showed that CD4^(+)T,CD8^(+)T and the production of IFN-γwere increased significantly on day 10 in righttumor after PDT treatment compared with control group.HpD-PDT enhanced the expression of calreticulin(CRT)on tumor tissue.Importantly,co-stimulatory molecular OX-40 and 4-1BB were elevated on CD8^(+)T cells.In vitro,immunogenic death of 4T1 cells was induced after PDT.Besides,the expression of DAMPs increased with the increasing of energy density.This study indicates that anti-tumor immune effect was induced by HpD-PDT.The knowledge of the involvement of CRT,ATP and co-stimulatory molecules uncovers important mechanistic insight into the anti-tumor immunogenicity.It was the first time that co-stimulatory molecules were investigated and found to elevate after PDT.

Comparative study on Photobiomodulation between 630 nm and 810 nm LED in diabetic wound healing both in vitro and in vivo

Photobiomodulation(PBM)promoting wound healing has been demonstrated by many studies.Currently,630 nm and 810 nm light-emitting diodes(LEDs),as light sources,are frequently used in the treatment of diabetic foot ulcers(DFUs)in clinics.However,the dose-effect relationship of LED-mediated PBM is not fully understood.Furthermore,among the 630 nm and 810 nm LEDs,which one gets a better effect on accelerating the wound healing of diabetic ulcers is not clear.The aim of this study is to evaluate and compare the effects of 630 nm and 810 nm LED-mediated PBM in wound healing both in vitro and in vivo.Our results showed that both 630 nm and 810 nm LED irradiation significantly promoted the proliferation of mouse fibroblast cells(L929)at different light irradiances(1,5,and 10 mW/cm^(2)).The cell proliferation rate increased with the extension of irradiation time(100,200,and 500 s),but it decreased when the irradiation time was over 500 s.Both 630 nm and 810nm LED irradiation(5 mW/cm^(2))significantly improved the migration capability of L929 cells.No difference between 630 nm and 810 nm LED-mediated PBM in promoting cell proliferation and migration was detected.In vivo results presented that both 630 nm and 810 nm LED irradiation promoted the wound healing and the expression of the vascular endothelial growth factor(VEGF)and transforming growth factor(TGF)in the wounded skin of type 2 diabetic mice.Overall,these results suggested that LED-mediated PBM promotes wound healing of diabetic mice through promoting fibroblast cell proliferation,migration,and the expression of growth factors in the wounded skin.LEDs(630 nm and 810 nm)have a similar outcome in promoting wound healing of type 2 diabetic mice.

Influence of laser power density on damage of comb by photodynamic therapy—simulation and validation of mathematical models

Photodynamic therapy （PDT） has poor therapeutic outcomes for the treatment of port-wine stain （PWS） lesions with long drug-light intervals （DLIs）. This letter investigates the possibility of improving the treatment efficacy through increasing the laser power density using a computer simulation and a cock comb model. The computational model includes a Monte Carlo simulation for the laser distribution and a calculation of the singlet oxygen concentrations （102）. Both simulation and experimental results show that increasing the power density from i00 to 140 mW/cm^2 not only improves the PDT efficacy, but also results in the unwanted skin damage.

SINGLET OXYGEN DETECTION DURING PHOTOSENSITIZATION

Singlet oxygen(^(1)O_(2))is a highly reactive oxygen species involved in numerous chemical and photochemical reactions in diferent biological systems and in particular,in photodynamic therapy(PDT).However,the quantifcation of^(1)O_(2) generation during in vitro and in vivo pho-tosensitization is still technically challenging.To address this problem,indirect and direct methods for^(1)O_(2)detection have been intensively studied.This review presents the available methods currently in use or under development for detecting and quantifying^(1)O_(2) generation during photosensitization.The advantages and limitations of each method will be presented.Moreover,the future trends in developing PDT-^(1)O_(2) dosimetry will be briefly discussed.

Elsinochrome A photosensitizers:Alternative drugs for photodynamic therapy

Photodynamic therapy(PDT)has already been a multifunctional modality for various tumors and nontumorous diseases.However,the development of photosensitizers is relatively delayed,compared with the tremendous progress in laser technology.Elsinochrome A(EA),a per-ylenequinonoid pigment from China,has all the typical advantages of perylenequinones.More-over,singlet oxygen quantum yield of EA is superior to other kinds of photosensitizers and EA could be artifially biosynthesized at present,which make it an alternative candidate for PDT.In.this review,the photophysics,photochemistry,photobiology and chemical or biological syntheses of EA photosensitizers are brielty presented.Besides,the future prospects of EA photosensitizers are also proposed.

Optimization of hypocrellin B derivative amphiphilicity and biological activity

To satisfy the dual requirements of the fluent transportation in blood and the affinity to the target tissues of vascular diseases, hypocrellin derivatives with optimized amphiphilicity are expected. In this work, 3-amino-1-propanesulfonic acid and 4-amino-1-butanesulfonic acid substituted hypocrellin B, named compounds 1 and 2, were designed, synthesized in high yields and characterized. Besides greatly strengthened red absorption, the maximum solubility of compound 2 in phosphate buffered saline (PBS) is 4.2 mg/mL which is just enough to prepare an aqueous solution for intravenous injection in clinically acceptable concentration, while the partition coefficient between n-octanol and PBS, 5.6, benefits the cell-uptake and biological activity as well. Furthermore, EPR measurements reveal that the photosensitization activities of the two compounds to generate semiquinone anion radicals, superoxide anion radicals and singlet oxygen are a little bit higher than those of taurine substituted hypocrellin B (THB), but the photodynamic activities to human lung cancer A549 cells are several times that of THB, mainly due to increases in lipophilicity and cell-uptake.

Handheld optical coherence tomography device for photodynamic therapy

Photodynamic therapy (PDT) is a very effective treatment for port wine stains (PWS). To guide and assess PDT treatment, a handheld optical coherence tomography (OCT) probe was designed for real-time imaging of the PWS patient. The system uses a light source with a center wavelength of 1310 nm, -3 dB bandwidth of 90 nm and an optical power of 9 mW. The system also has a spatial resolution of 8 μm (lateral) × 7 μm (axial), an imaging rate of 4 frames per second, and a 102 dB sensitivity. We then demonstrate that the OCT imaging system can clearly distinguish between normal and PWS tissues. Therefore, the system can provide valuable guidance for PDT treatment.