IR-780 Dye-based Targeting of Cancer-associated Fibroblasts Improves Cancer Immunotherapy by Increasing Intra-tumoral T Lymphocytes Infiltration.

BACKGROUND: Immune-checkpoint inhibitors (ICIs) against programmed death (PD)-1/PD-L1 pathway immunotherapy have been demonstrated to be effective in only a subset of patients with cancer, while the rest may exhibit low response or may develop drug resistance after initially responding. Previous studies have indicated that extensive collagen-rich stroma secreted by cancer-associated fibroblasts (CAFs) within the tumor microenvironment is one of the key obstructions of the immunotherapy for some tumors by decreasing the infiltrating cytotoxic T cells. However, there is still a lack of effective therapeutic strategies to control the extracellular matrix by targeting CAFs. METHODS: The enhanced uptake of IR-780 by CAFs was assessed by using in vivo or ex vivo nearinfrared fluorescence imaging, confocal NIR fluorescent imaging, and CAFs isolation testing. The fibrotic phenotype down-regulation effects and in vitro CAFs killing effect of IR-780 were tested by qPCR, western blot, and flow cytometry. The in vivo therapeutic enhancement of anti-PD-L1 by IR-780 was evaluated on EMT6 and MC38 subcutaneous xenograft mice models. RESULTS: IR-780 has been demonstrated to be preferentially taken up by CAFs and accumulate in the mitochondria. Further results identified low-dose IR-780 to downregulate the fibrotic phenotype, while high-dose IR-780 could directly kill both CAFs and EMT6 cells in vitro. Moreover, IR-780 significantly inhibited extracellular matrix (ECM) protein deposition in the peri-tumoral stroma on subcutaneous EMT6 and MC38 xenografts, which increased the proportion of tumor-infiltrating lymphocytes (TILs) in the deep tumor and further promoted anti-PD-L1 therapeutic efficacy. CONCLUSION: This work provides a unique strategy for the inhibition of ECM protein deposition in the tumor microenvironment by targeted regulating of CAFs, which destroys the T cell barrier and further promotes tumor response to PD-L1 monoclonal antibody. IR-780 has been proposed as a potential therapeutic small-molecule adjuvant to promote the effect of immunotherapy.

Characterization of cellular senescence in radiation ulcers and therapeutic effects of mesenchymal stem cell-derived conditioned medium.

Background: Radiation ulcers are a common and severe injury after uncontrolled exposure to ionizing radiation. The most important feature of radiation ulcers is progressive ulceration, which results in the expansion of radiation injury to the nonirradiated area and refractory wounds. Current theories cannot explain the progression of radiation ulcers. Cellular senescence refers to as irreversible growth arrest that occurs after exposure to stress, which contributes to tissue dysfunction by inducing paracrine senescence, stem cell dysfunction and chronic inflammation. However, it is not yet clear how cellular senescence facilitates the continuous progression of radiation ulcers. Here, we aim to investigate the role of cellular senescence in promoting progressive radiation ulcers and indicate a potential therapeutic strategy for radiation ulcers. Methods: Radiation ulcer animal models were established by local exposure to 40 Gy X-ray radiation and continuously evaluated for >260 days. The roles of cellular senescence in the progression of radiation ulcers were assessed using pathological analysis, molecular detection and RNA sequencing. Then, the therapeutic effects of conditioned medium from human umbilical cord mesenchymal stem cells (uMSC-CM) were investigated in radiation ulcer models. Results: Radiation ulcer animal models with features of clinical patients were established to investigate the primary mechanisms responsible for the progression of radiation ulcers. We have characterized cellular senescence as being closely associated with the progression of radiation ulcers and found that exogenous transplantation of senescent cells significantly aggravated them. Mechanistic studies and RNA sequencing suggested that radiation-induced senescent cell secretions were responsible for facilitating paracrine senescence and promoting the progression of radiation ulcers. Finally, we found that uMSC-CM was effective in mitigating the progression of radiation ulcers by inhibiting cellular senescence. Conclusions: Our findings not only characterize the roles of cellular senescence in the progression of radiation ulcers but also indicate the therapeutic potential of senescent cells in their treatment.

A small-molecule inhibitor of Keap1-Nrf2 interaction attenuates sepsis by selectively augmenting the antibacterial defence of macrophages at infection sites.

BACKGROUND: Macrophages at infection sites are considered as the promising therapeutic targets to prevent sepsis development. The Nrf2/Keap1 system acts as a critical modulator of the antibacterial activity of macrophages. Recently, Keap1-Nrf2 protein-protein interaction (PPI) inhibitors have emerged as safer and stronger Nrf2 activators; however, their therapeutic potential in sepsis remains unclear. Herein, we report a unique heptamethine dye, IR-61, as a Keap1-Nrf2 PPI inhibitor that preferentially accumulates in macrophages at infection sites. METHODS: A mouse model of acute lung bacterial infection was used to investigate the biodistribution of IR-61. SPR study and CESTA were used to detect the Keap1 binding behaviour of IR-61 in vitro and in cells. Established models of sepsis in mice were used to determine the therapeutic effect of IR-61. The relationship between Nrf2 levels and sepsis outcomes was preliminarily investigated using monocytes from human patients. FINDINGS: Our data showed that IR-61 preferentially accumulated in macrophages at infection sites, enhanced bacterial clearance, and improved outcomes in mice with sepsis. Mechanistic studies indicated that IR-61 potentiated the antibacterial function of macrophages by activating Nrf2 via direct inhibition of the Keap1-Nrf2 interaction. Moreover, we observed that IR-61 enhanced the phagocytic ability of human macrophages, and the expression levels of Nrf2 in monocytes might be associated with the outcomes of sepsis patients. INTERPRETATIONS: Our study demonstrates that the specific activation of Nrf2 in macrophages at infection sites is valuable for sepsis management. IR-61 may prove to be a Keap1-Nrf2 PPI inhibitor for the precise treatment of sepsis. FUNDING: This work was supported by the National Natural Science Foundation of China (Major program 82192884), the Intramural Research Project (Grants: 2018-JCJQ-ZQ-001 and 20QNPY018), and the Chongqing National Science Foundation (CSTB2022NSCQ-MSX1222).

Mitochondrial-targeting fluorescent small molecule IR-780 alleviates radiation-induced brain injury.

Radiation-induced brain injury (RIBI) is a common complication of radiation therapy for brain tumors. Vascular damage is one of the key factors closely related to the severity of the RIBI. However, effective vascular target treatment strategies are lacking. Previously, we have identified a fluorescent small molecule dye, IR-780, which shows the properties of injury tissue targeting and provided protection against various injuries by modulating oxidative stress. This study aims to validate the therapeutic effect of IR-780 on RIBI. The effectiveness of IR-780 against RIBI has been comprehensively evaluated through techniques such as behavior, immunofluorescence staining, quantitative real-time polymerase chain reaction, Evans Blue leakage experiments, electron microscopy, and flow cytometry. Results show that IR-780 improves cognitive dysfunction, reduces neuroinflammation, restores the expression of tight junction proteins in the blood-brain barrier (BBB), and promotes the recovery of BBB function after whole brain irradiation. IR-780 also accumulates in injured cerebral microvascular endothelial cells, and its subcellular location is in the mitochondria. More importantly, IR-780 can reduce the levels of cellular reactive oxygen species and apoptosis. Moreover, IR-780 has no significant toxic side effects. IR-780 alleviates RIBI by protecting vascular endothelial cells from oxidative stress, reducing neuroinflammation, and restoring BBB function, suggesting IR-780 as a promising treatment candidate for RIBI therapy.

Near-infrared Nrf2 activator IR-61 dye alleviates radiation-induced lung injury.

Oxidative stress injury and subsequent inflammatory response are considered to play critical roles in radiation-induced lung injury (RILI). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates oxidative stress response and represses inflammation, but its therapeutic value in RILI remains elusive. Our previous studies have shown that the near-infrared (NIR) IR-61 dye evokes intracellular antioxidant defense by enhancing Nrf2 signaling and promoting anti-inflammatory effects. We established a model of RILI in mice exposed to whole-thoracic irradiation. The results showed that IR-61 treatment notably improved pulmonary functions by decreasing lung density and diminishing airway resistance. In addition, IR-61 significantly ameliorated radiation-induced inflammatory cell infiltration and proinflammatory cytokine (IL-1ß, IL-6, and TNF-α) release, thereby mitigating inflammatory response. Furthermore, IR-61 mitigated radiation-induced lung fibrosis by decreasing the collagen deposition and the levels of fibrogenesis-related factors (collagen I, collagen III, α-SMA, and fibronectin). More importantly, IR-61 was found to accumulate in the mitochondria of macrophages in irradiated lung tissues. Therefore, the functions of IR-61 in macrophages were further studied in irradiated macrophage cell lines, MH-s and RAW 264.7 in vitro. The results indicated that IR-61 upregulated the expression of Nrf2 and heme oxygenase-1 (HO-1) and decreased the levels of reactive oxygen species (ROS) and pro-inflammatory cytokines (IL-1ß and IL-6) in macrophages after radiation. In summary, our study suggests that IR-61 effectively mitigates RILI by activating Nrf2 signaling in irradiated lung tissues. In particular, Nrf2-mediated anti-inflammatory and antioxidant effects in irradiated lung tissue macrophages play critical roles in protecting against RILI.

Increased Expression of Integrin Alpha 6 in Nucleus Pulposus Cells in Response to High Oxygen Tension Protects against Intervertebral Disc Degeneration.

The destruction of the low oxygen microenvironment in nucleus pulposus (NP) cells played a critical role in the pathogenesis of intervertebral disc degeneration (IVDD). The purpose of this study was to determine the potential role of integrin alpha 6 (ITG α6) in NP cells in response to high oxygen tension (HOT) in IVDD. Immunofluorescence staining and western blot analysis showed that the levels of ITG α6 expression were increased in the NP tissue from IVDD patients and the IVDD rat model with mild degeneration, which were reduced as the degree of degeneration increases in severity. In NP cells, the treatment of HOT resulted in upregulation of ITG α6 expression, which could be alleviated by blocking the PI3K/AKT signaling pathway. Further studies found that ITG α6 could protect NP cells against HOT-induced apoptosis and oxidative stress and protect NP cells from HOT-inhibited ECM protein synthesis. Upregulation of ITG α6 expression by HOT contributed to maintaining NP tissue homeostasis through the interaction with hypoxia-inducible factor-1α (HIF-1α). Furthermore, silencing of ITG α6 in vivo could obviously accelerate puncture-induced IVDD. Taken together, these results revealed that the increase of ITG α6 expression by HOT in NP cells might be a protective factor in IVD degeneration as well as restore NP cell function.

Pharmaceutical targeting of succinate dehydrogenase in fibroblasts controls bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix in the lung with fibroblast-to-myofibroblast transition, leading to chronically compromising lung function and death. However, very little is known about the metabolic alterations of fibroblasts in IPF, and there is still a lack of pharmaceutical agents to target the metabolic dysregulation. Here we show a glycolysis upregulation and fatty acid oxidation (FAO) downregulation in fibroblasts from fibrotic lung, and perturbation of glycolysis and FAO affects fibroblasts transdifferentiation. In addition, there is a significant accumulation of succinate both in fibrotic lung tissues and myofibroblasts, where succinate dehydrogenase (SDH) operates in reverse by reducing fumarate to succinate. Then succinate contributes to glycolysis upregulation and FAO downregulation by stabilizing HIF-1α, which promotes the development of lung fibrosis. In addition, we identify a near-infrared small molecule dye, IR-780, as a targeting agent which stimulates mild inhibition of succinate dehydrogenase subunit A (SDHA) in fibroblasts, and which inhibits TGF-ß1 induced SDH and succinate elevation, then to prevent fibrosis formation and respiratory dysfunction. Further, enhanced cell retention of IR-780 is shown to promote severe inhibition of SDHA in myofibroblasts, which may contribute to excessive ROS generation and selectively induces myofibroblasts to apoptosis, and then therapeutically improves established lung fibrosis in vivo. These findings indicate that targeting metabolic dysregulation has significant implications for therapies aimed at lung fibrosis and succinate dehydrogenase is an exciting new therapeutic target to treat IPF.

Reactive Oxygen Species Mediate Low Back Pain by Upregulating Substance P in Intervertebral Disc Degeneration.

Reactive oxygen species (ROS) are thought to have a strong correlation with a number of intervertebral disc (IVD) diseases. Here, we aimed to determine whether ROS represent an etiology of low back pain (LBP) during IVD degeneration. Thirty degenerated intervertebral disc samples were obtained from patients, and ROS levels were quantified using dihydroethidium (DHE) staining. The results suggested a significant correlation between the ROS level and the severity of LBP. Subsequently, a puncture-induced LBP model was established in rats, and ROS levels significantly increased compared with those in the sham surgery group, accompanied with severe puncture-induced IVD degeneration. In addition, when ROS levels were increased by H2O2 administration or decreased by NAC treatment, the rats showed increased or decreased LBP, respectively. Based on this evidence, we further determined that stimulation with H2O2 in nucleus pulposus cells (NPCs) in vivo or in vitro resulted in upregulation of substance P (SP), a peptide thought to be involved in the synaptic transmission of pain, and that the severity of LBP decreased when SP levels were increased by exogenous SP administration or neutralized via aprepitant treatment in the IVDs of rats. In conclusion, ROS are primary inducers of LBP based on clinical and animal data, and the mechanism involves ROS stimulation of NPCs to secrete SP, which is a critical neurotransmitter peptide, to promote LBP in IVDs. Therefore, reducing the level of ROS with specific drugs and inhibiting SP may be alternative methods to treat LBP in the clinic.

Knockdown of PC4 increases chemosensitivity of Oxaliplatin in triple negative breast cancer by suppressing mTOR pathway.

As a multifunctional nuclear protein, the human positive cofactor 4 (PC4) is highly expressed in various tumors including breast cancer and has potential roles in cancer development and progression. However, the functional signatures and molecular mechanisms of PC4 in triple negative breast cancer (TNBC) progression and chemotherapeutic response are still unknown. In this study, we found that PC4 is significantly upregulated in TNBC cells compared with non-TNBC cells, implying its potential role in TNBC. Then, in vivo and in vitro studies revealed that knockdown of PC4 increased chemosensitivity of Oxaliplation (Oxa) in TNBC by suppressing mTOR pathway. Therefore, our findings demonstrated the signatures and molecular mechanisms of PC4 in TNBC chemotherapeutic response, and indicated that PC4 might be a promising therapeutic target for TNBC.

Mitigation of radiation-induced pulmonary fibrosis by small-molecule dye IR-780.

Radiation-induced pulmonary fibrosis (RIPF) is a common complication during thoracic radiotherapy, but there are few effective treatments. Here, we identify IR-780, a mitochondria-targeted near-infrared (NIR) dye, can selectively accumulate in the irradiated lung tissues. Besides, IR-780 significantly alleviates radiation-induced acute lung injury and fibrosis. Furthermore, our results show that IR-780 prevents the differentiation of fibroblasts and the release of pro-fibrotic factors from alveolar macrophages induced by radiation. Besides, IR-780 downregulates the expression of glycolysis-associated genes, and 2-Deoxy-d-glucose (2-DG) also prevents the development of fibrosis in vitro, suggesting radioprotective effects of IR-780 on RIPF might be related to glycolysis regulation. Finally, IR-780 induces tumour cell apoptosis and enhances radiosensitivity in representative H460 and A549 cell lines. These findings indicate that IR-780 is a potential therapeutic small-molecule dye during thoracic radiotherapy.

Bi-needle technique versus transforaminal endoscopic spine system technique for percutaneous endoscopic lumbar discectomy in treating intervertebral disc calcification: a propensity score matched cohort analysis.

OBJECTIVE: The aim of this study was to evaluate the clinical results of a Bi-needle technique and conventional transforaminal endoscopic spine system (TESSYS) technique for percutaneous endoscopic lumbar discectomy (PELD) in treating patients with intervertebral disc calcification (IDC). BACKGROUND: PELD has gained acceptance for treating patients with IDC. The Bi-needle technique was designed to improve the efficiency and safety of PELD. METHOD: Bi-needle and TESSYS group within each cohort were balanced using 1:1 propensity score matching. Finally, 32 patients with IDC treated by Bi-needle technique from December 2015 to September 2017 were enrolled and 25 patients treated by TESSYS technique from the same spine surgery center between January 2013 and October 2017 were enrolled as controls. RESULTS: Propensity score matching generated 22 Bi-needle and 22 TESSYS patients. There were no significant differences in visual analog scale and lumbar Japanese Orthopaedic Association scores between Bi-needle and TESSYS group. Operative time and rate of complications in the Bi-needle was significantly better than the TESSYS group (p < 0.01). CONCLUSIONS: Both surgical methods achieved good clinical outcomes. However, compared with the TESSSY technique, operative time of the Bi-needle technique is shorter, and rate of complications is lower.

[Effect of penetration of mini-plate lateral mass screws into facet joint on axial symptoms in cervical laminoplasty].

OBJECTIVE: To investigate the effect of the penetration of mini-plate mass screws into facet joint on axial symptoms in cervical laminoplasty. METHODS: A retrospective analysis was made on the clinical data of 52 patients who underwent unilateral open-door cervical expansive laminoplasty fixed with Centerpiece mini-plate between September 2009 and December 2011. There were 42 males and 10 females, with a mean age of 61.2 years (range, 34-83 years). Seventeen patients exhibited simple degeneration cervical canal stenosis, 25 patients had multilevel cervical disc protrusion, and 10 patients had ossification of posterior longitudinal ligaments. Disease duration ranged 1-120 months (median, 11 months). The Japanese Orthopedic Association (JOA) score was used to assess neurological function, and JOA recovery rates were calculated. The visual analogue score (VAS) and the neck disability index (NDI) were used to evaluate the axial pain and neck daily activities. The axial symptoms and other complications were recorded. The cervical canal diameter, cervical curvature, cervical canal cross area, and open angle were measured according to the X-ray films, CT scans, and MRI scans. The postoperative CT three dimensional (3-D) reconstruction images were used to identify whether the screws penetrated into the facet joints. All the patients were divided into 3 groups according to involved facet joints: no joint penetrating group (no penetrated facet joint), oligo-joint penetrating group (one or two penetrated facet joints), and multi-joint penetrating group (three or more penetrated facet joints). RESULTS: Five patients suffered from C5 nerve palsy, and 2 patients had cerebrospinal fluid leakage. The follow-up time ranged 3-35 months (mean, 15.7 months). At the final follow-up, the JOA scores, NDI, cervical canal diameter, and cervical canal cross area were significantly improved when compared with preoperative ones (P < 0.05). At 1 week after operation, CT 3-D reconstruction showed that 16 patients had no penetrated facet joint, 23 patients had one or two penetrated facet joints, and 13 patients had three or more penetrated facet joints. There was no significant difference in age, gender, disease duration, operation time, intraoperative blood loss, and follow-up time among 3 groups (P > 0.05). And at the final follow-up, there was no significant difference in JOA score, VAS score, cervical curvature, cervical canal diameter, cervical canal cross area, the JOA recovery rates, and lamiae open angle among 3 groups (P > 0.05). The NDI of the multi-joint penetrated group was significantly higher than that of other 2 groups (P < 0.05). Axial pain occurred in 1 case of no penetrating group, in 4 cases of oligo-joint penetrating group, and in 5 cases of multi-joint penetrating group, showing no significant difference among 3 groups (Chi(2)=4.881, P=0.087). CONCLUSION: The penetrations of lateral mass screws into articular surface of facet joint may contribute to the axial symptoms after cervical laminoplasty. The risk of axial symptom raises accompany with increased penetrated facet joints.