PD-L1 blockade TAM-dependently potentiates mild photothermal therapy against triple-negative breast cancer.

The present work was an endeavor to shed light on how mild photothermia possibly synergizes with immune checkpoint inhibition for tumor therapy. We established mild photothermal heating protocols to generate temperatures of 43 °C and 45 °C in both in vitro and in vivo mouse 4T1 triple-negative breast cancer (TNBC) models using polyglycerol-coated carbon nanohorns (CNH-PG) and 808 nm laser irradiation. Next, we found that 1) CNH-PG-mediated mild photothermia (CNH-PG-mPT) significantly increased expression of the immune checkpoint PD-L1 and type-1 macrophage (M1) markers in the TNBC tumors; 2) CNH-PG-mPT had a lower level of anti-tumor efficacy which was markedly potentiated by BMS-1, a PD-L1 blocker. These observations prompted us to explore the synergetic mechanisms of CNH-PG-mPT and BMS-1 in the context of tumor cell-macrophage interactions mediated by PD-L1 since tumor-associated macrophages (TAMs) are a major source of PD-L1 expression in tumors. In vitro, the study then identified two dimensions where BMS-1 potentiated CNH-PG-mPT. First, CNH-PG-mPT induced PD-L1 upregulation in the tumor cells and showed a low level of cytotoxicity which was potentiated by BMS-1. Second, CNH-PG-mPT skewed TAMs towards an M1-like anti-tumor phenotype with upregulated PD-L1, and BMS-1 bolstered the M1-like phenotype. The synergistic effects of BMS-1 and CNH-PG-mPT both on the tumor cells and TAMs were more pronounced when the two cell populations were in co-culture. Further in vivo study confirmed PD-L1 upregulation both in tumor cells and TAMs in the TNBC tumors following treatment of CNH-PG-mPT. Significantly, TAMs depletion largely abolished the anti-TNBC efficacy of CNH-PG-mPT alone and in synergy with BMS-1. Collectively, our findings reveal PD-L1 upregulation to be a key response of TNBC to mild photothermal stress, which plays a pro-survival role in the tumor cells while also acting as a brake on the M1-like activation of the TAMs. Blockade of mPT­induced PD­L1 achieves synergistic anti-TNBC efficacy by taking the intrinsic survival edge off the tumor cells on one hand and taking the brakes off the M1-like TAMs on the other. Our findings reveal a novel way (i.e. mild thermia plus PD-L1 blockade) to modulate the TAMs-tumor cell interaction to instigate a mutiny of the TAMs against their host tumor cells.

Efficient Delivery of Chlorin e6 by Polyglycerol-Coated Iron Oxide Nanoparticles with Conjugated Doxorubicin for Enhanced Photodynamic Therapy of Melanoma.

Chlorin e6 (Ce6) is a promising photosensitizer for tumor photodynamic therapy (PDT). However, the efficacy of Ce6 PDT is limited by Ce6's poor water solubility, rapid blood clearance, and inadequate accumulation in the tumor tissue. This problem is tackled in this work, wherein functionalized superparamagnetic iron oxide nanoparticles (IO-NPs) were used as carriers to deliver Ce6 to melanoma. The IO-NPs were coated with polyglycerol (PG) to afford good aqueous solubility. The chemotherapeutic agent doxorubicin (DOX) was attached to the PG coating via the hydrazone bond to afford affinity to the cell membrane and thereby promote the cell uptake. The hydrophobic nature of DOX also induced the aggregation of IO-NPs to form nanoclusters. Ce6 was then loaded onto the IO nanoclusters through physical adsorption and coordination with surface iron atoms, yielding the final composites IO-PG-DOX-Ce6. In vitro experiments showed that IO-PG-DOX-Ce6 markedly increased Ce6 uptake in mouse melanoma cells, leading to much-enhanced photocytotoxicity characterized by intensified reactive oxygen species production, loss of viability, DNA damage, and stimulation of tumor cell immunogenicity. In vivo experiments corroborated the in vitro findings and demonstrated prolonged blood clearance of IO-PG-DOX-Ce6. Importantly, IO-PG-DOX-Ce6 markedly increased the Ce6 distribution and retention in mouse subcutaneous melanoma grafts and significantly improved the efficacy of Ce6-mediated PDT. No apparent vital organ damage was observed at the same time. In conclusion, the IO-PG-DOX NPs provide a simple and safe delivery platform for efficient tumor enrichment of Ce6, thereby enhancing antimelanoma PDT.

Synergy of nanodiamond-doxorubicin conjugates and PD-L1 blockade effectively turns tumor-associated macrophages against tumor cells.

BACKGROUND: Tumor-associated macrophages (TAMs) are the most abundant stromal cells in the tumor microenvironment. Turning the TAMs against their host tumor cells is an intriguing therapeutic strategy particularly attractive for patients with immunologically "cold" tumors. This concept was mechanistically demonstrated on in vitro human and murine lung cancer cells and their corresponding TAM models through combinatorial use of nanodiamond-doxorubicin conjugates (Nano-DOX) and a PD-L1 blocking agent BMS-1. Nano-DOX are an agent previously proved to be able to stimulate tumor cells' immunogenicity and thereby reactivate the TAMs into the anti-tumor M1 phenotype. RESULTS: Nano-DOX were first shown to stimulate the tumor cells and the TAMs to release the cytokine HMGB1 which, regardless of its source, acted through the RAGE/NF-κB pathway to induce PD-L1 in the tumor cells and PD-L1/PD-1 in the TAMs. Interestingly, Nano-DOX also induced NF-κB-dependent RAGE expression in the tumor cells and thus reinforced HMGB1's action thereon. Then, BMS-1 was shown to enhance Nano-DOX-stimulated M1-type activation of TAMs both by blocking Nano-DOX-induced PD-L1 in the TAMs and by blocking tumor cell PD-L1 ligation with TAM PD-1. The TAMs with enhanced M1-type repolarization both killed the tumor cells and suppressed their growth. BMS-1 could also potentiate Nano-DOX's action to suppress tumor cell growth via blocking of Nano-DOX-induced PD-L1 therein. Finally, Nano-DOX and BMS-1 achieved synergistic therapeutic efficacy against in vivo tumor grafts in a TAM-dependent manner. CONCLUSIONS: PD-L1/PD-1 upregulation mediated by autocrine and paracrine activation of the HMGB1/RAGE/NF-κB signaling is a key response of lung cancer cells and their TAMs to stress, which can be induced by Nano-DOX. Blockade of Nano-DOX-induced PD-L1, both in the cancer cells and the TAMs, achieves enhanced activation of TAM-mediated anti-tumor response.

Doxorubicin-polyglycerol-nanodiamond conjugate is a cytostatic agent that evades chemoresistance and reverses cancer-induced immunosuppression in triple-negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) has the poorest prognosis of all breast cancer subtypes and is one of the most fatal diseases for women. Combining cytotoxic chemotherapy with immunotherapy has shown great promise for TNBC treatment. However, chemotherapy often leads to the development of chemoresistance and severe systemic toxicity compromising the immune functions that are crucial to anti-TNBC immune therapy. Tumor-induced immunosuppression also poses a great hindrance to efficacious anti-TNBC immunotherapy. Nanomedicine holds great promise to overcome these hurdles. RESULTS: Doxorubicin-polyglycerol-nanodiamond conjugate (Nano-DOX) was firstly found to be a cytostatic agent to the 4T1 cells and displayed a lower apparent therapeutic potency than DOX. However, the tumor-bearing animals, particularly some key immune cells thereof, showed good tolerance of Nano-DOX as opposed to the severe toxicity of DOX. Next, Nano-DOX did not induce significant upregulation of P-gp and IL-6, which were demonstrated to be key mediators of chemoresistance to DOX in the 4T1 cells. Then, Nano-DOX was shown to downregulate tumor-derived granulocyte-colony stimulating factor (G-CSF) and suppresses the induction and tissue filtration of myeloid-derived suppressor cells (MDSCs) that are the principal effectors of cancer-associated systemic immunosuppression. Nano-DOX also alleviated the phenotype of MDSCs induced by 4T1 cells. Finally, Nano-DOX induced the 4T1 cells to emit damage associated molecular patterns (DAMPs) that stimulated the tumor immune microenvironment through activating key immune effector cells involved in anti-tumor immunity, such as macrophages, dendritic cells and lymphocytes in the tumor tissue. CONCLUSIONS: Nano-DOX is a cytostatic agent with good host tolerance which is capable of evading chemoresistance and reversing cancer-induced immunosuppression both at the systemic level and in the tumor microenvironment in TNBC. Our work presents Nano-DOX as an interesting example that a chemotherapeutic agent in nano-form may possess distinct biochemical properties from its free form, which can be exploited to join chemotherapy with immunotherapy for better treatment of cancer.

Lipopolysaccharide Enhances Beta2-Glycoprotein I Activation of Nuclear Factor κB in Liver Cancer Cells.

BACKGROUND: Beta2-glycoprotein I (ß2GPI) is a highly abundant glycoprotein in plasma. Our previous study demonstrated strong ß2GPI expression in hepatitis B-related hepatocellular carcinoma (HCC) tissue and the combination of ß2GPI and hepatitis B surface antigen (HBsAg) was shown to significantly activate the nuclear factor kappa B (NF-κB). To investigate whether lipopolysaccharide (LPS) enhances ß2GPI activation of NF-ßB and the expression of downstream factors (e.g., tumor necrosis factor alpha, TNF-α; interleukin-1 beta, IL-1ß; alpha-fetoprotein, AFP) in the human hepatoma cell line, SMMC-7721. METHODS: Experimental samples were divided into 4 groups as follows: Group A--blank cell group (SMMC-7721); group B--low, medium, and high LPS concentration groups (1 ng/mL; 10 ng/mL; and 100 ng/mL, respectively); group C--ß2GPI transfected group; and group D--ß2GPI + low, medium, or high concentrations from the LPS affected group. Activation of NF-κB was evaluated using laser scanning confocal microscopy. Expression of downstream factors was measured by ELISA. RESULTS: Degrees of NF-κB activation in groups B, C, and D were varied. NF-κB activation in group D was the most significant, and the expressions of downstream factors, TNF-α and IL-1ß, were the highest level of activation among the groups (p < 0.05), showing an LPS dose-dependency. CONCLUSIONS: LPS enhanced the signal transduction of ß2GPI in liver cancer cells leading to activation of NF-κB, which triggered downstream signal transduction and increased the expression of downstream factors. This suggests that LPS enhancement of ß2GPI signal transduction may play a role in promoting the development of liver cancer.

Prognostic Value of Postoperative Complication for Gastric Cancer.

Background: The incidence of complications in gastric cancer (GC) patients after surgery was increasing, and it was not clear whether postoperative complications would have an impact on prognosis. The current study attempted to investigate the role of postoperative complication for prognosis on GC patients undergoing radical resection. Materials and Methods: Eligible studies were searched in three databases, including PubMed, Embase, and the Cochrane Library, in accordance with the searching strategy on September 4th, 2022. The survival values were most concerned; then, hazard ratios (HRs) and 95% confidence intervals (CIs) were pooled up. All prognostic values, including overall survival (OS), disease-free survival (DFS), disease-specific survival (DSS), and recurrence-free survival (RFS), were allowed. Subgroup analysis based on complication types was used for further in-depth research. Results: A total of 29 studies involving 33,858 patients were included in this study. Intra-abdominal abscess (19.4%) was the most common complications in the included studies, followed by anastomotic leakage (17.0%) and pneumonia (16.4%). There were 23, 4, 6, and 10 studies that reported OS, DFS, DSS, and RFS, respectively. After analysis, postoperative complication was found to be an independent prognostic factor for OS (HR = 1.52, I2 = 1.14%, 95% CI = 1.42-1.61, P = .00), DFS (HR = 1.71, I2 = 0.00%,95% CI = 1.44-1.98, P < .05), DSS (HR = 1.60, I2 = 54.58%, 95% CI = 1.26-1.93, P < .1), and RFS (HR = 1.26, I2 = 0.00%, 95% CI = 1.11-1.41, P < .05). Subgroup analysis found that noninfectious complication was not significantly associated with OS (HR = 1.39, I2 = 0.00%, 95% CI = 0.96-1.82, P > .05). Conclusion: Surgeons needed to pay more attention to GC patients who developed postoperative complications, especially infectious complications, and take proactive management to improve the prognosis.

Targeted sonodynamic therapy induces tumor cell quasi-immunogenic ferroptosis and macrophage immunostimulatory autophagy in glioblastoma.

In this study, we demonstrated the mechanism of a glioblastoma (GBM)-targeted sonodynamic therapy (SDT) strategy employing platelets loaded with a sonosensitizer based on functionalized boron nitride nanoparticles carrying chlorin e6 (BNPD-Ce6). In the in vitro study, we first found that the BNPD-Ce6-mediated sonodynamic action (SDA) induced remarkable viability loss, DNA damage, and cell death in the GBM cells (GBCs) but not macrophages. Surprisingly, the SDA-exposed GBCs displayed a ferroptotic phenotype while the SDA-exposed macrophages underwent immuno-stimulatory autophagy and potently potentiated the SDA's toxicity to the GBCs. The ferroptotic GBCs induced by the SDA were found to be quasi-immunogenic, characterized by the emission of some alarmins such as ATP, HSP90, and CRT, but absent HMGB1, a potent endogenous adjuvant. As such, the SDA-stressed GBCs were unable to stimulate the BMDMs. This defect, interestingly, could be rescued by platelets as a donor of HMGB1 which markedly enhanced the BNPD-Ce6's sonotoxicity to the GBCs. In the in vivo study, we first employed BNPD-Ce6-loaded platelets to achieve ultrasound-triggered, targeted delivery of BNPD-Ce6 in grafted intra-cranial GBMs and subsequent sonodynamic tumor damage. An SDT regimen designed based on these results slowed the growth of grafted intra-cranial GBMs and significantly increased the survival of the host animals. Pathological examination of the SDT-treated GBMs revealed tissue necrosis and destruction and validated the in vitro observations. Finally, the depletion of macrophages was found to abrogate the efficacy of the SDT in subcutaneous GBC grafts. In conclusion, the BNPD-Ce6@Plt-mediated SDT is a practicable and efficacious anti-GBM therapy. Its therapeutic mechanism critically involves a synergy of tumor cell ferroptosis, macrophage stimulation, and platelet activation induced by the SDA.

Anti-lung cancer synergy of low-dose doxorubicin and PD-L1 blocker co-delivered via mild photothermia-responsive black phosphorus.

We have previously identified a latent interaction mechanism between non-small cell lung cancer cells (NSCLCC) and their associated macrophages (TAM) mediated by mutual paracrine activation of the HMGB1/RAGE/NF-κB signaling. Activation of this mechanism results in TAM stimulation and PD-L1 upregulation in the NSCLCC. In the present work, we found that free DOX at a low concentration that does not cause DNA damage could activate the HMGB1/RAGE/NF-κB/PD-L1 pathway byinducing oxidative stress. It was thus proposed that a combination of low-dose DOX and a PD-L1 blocker delivered in the NSCLC tumor would achieve synergistic TAM stimulation and thereby synergetic anti-tumor potency. To prove this idea, DOX and BMS-202 (a PD-L1 blocker) were loaded to black phosphorus (BP) nanoparticles after dosage titration to yield the BMS-202/DOX@BP composites that rapidly disintegrated and released drug cargo upon mild photothermal heating at 40 °C. In vitro experiments then demonstrated that low-dose DOX and BMS-202 delivered via BMS-202/DOX@BP under mild photothermia displayed enhanced tumor cell toxicity with a potent synergism only in the presence of TAM. This enhanced synergism was due to an anti-tumor M1-like TAM phenotype that was synergistically induced by low dose DOX plus BMS-202 only in the presence of the tumor cells, indicating the damaged tumor cells to be the cardinal contributor to the M1-like TAM stimulation. In vivo, BMS-202/DOX@BP under mild photothermia exhibited targeted delivery to NSCLC graft tumors in mice and synergistic anti-tumor efficacy of delivered DOX and BMS-202. In conclusion, low-dose DOX in combination with a PD-L1 blocker is an effective strategy to turn TAM against their host tumor cells exploiting the HMGB1/RAGE/NF-κB/PD-L1 pathway. The synergetic actions involved highlight the value of TAM and the significance of modulating tumor cell-TAM cross-talk in tumor therapy. Photothermia-responsive BP provides an efficient platform to translate this strategy into targeted, efficacious tumor therapy.

Ruptured small pancreaticoduodenal artery aneurysm-clinical features similar to pancreatitis: A case report.

BACKGROUND: Pancreaticoduodenal artery aneurysm (PDAA) is rare and has high rupture risks. PDAA rupture has a wide range of clinical symptoms, including abdominal pain, nausea, syncope, and hemorrhagic shock, which is difficult to differentiate from other diseases. PATIENT CONCERNS: A 55-year-old female patient was admitted to our hospital due to abdominal pain for 11 days. DIAGNOSIS: Acute pancreatitis was initially diagnosed. The patient's hemoglobin decreased compared to before admission, suggesting that active bleeding may occur. CT volume diagram and maximum intensity projection diagram show that a small aneurysm with a diameter of about 6 mm can be seen at the pancreaticoduodenal artery arch. The patient was diagnosed with a rupture and hemorrhage of the small pancreaticoduodenal aneurysm. INTERVENTIONS: Interventional treatment was performed. After the microcatheter was selected for the branch of the diseased artery for angiography, the pseudoaneurysm was displayed and embolized. OUTCOMES: The angiography showed that the pseudoaneurysm was occluded, and the distal cavity was not redeveloped. CONCLUSION: The clinical manifestations of PDAA rupture were significantly correlated with the aneurysm diameter. Because of small aneurysms, the bleeding is limited around the peripancreatic and duodenal horizontal segments, accompanied by abdominal pain, vomiting, and elevated serum amylase, similar to the clinical manifestations of acute pancreatitis but accompanied by the decrease of hemoglobin. This will help us to improve our understanding of the disease, avoid misdiagnosis, and provide the basis for clinical treatment.

Compound Biejia-Ruangan tablet as an adjunctive therapy to entecavir for chronic hepatitis B complicated with hepatic fibrosis: A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: The compound Biejia-Ruangan tablet (CBRT), as an adjunctive therapy to entecavir, is a potential treatment for hepatic fibrosis (HF) in patients with chronic hepatitis B (HBV). However, the present study yielded inconsistent results. In this systematic review and meta-analysis, we comprehensively investigated the efficacy and safety of CBRT as an adjunctive modality to entecavir for the treatment of HBV infection complicated with HF. METHODS: We searched the Cochrane Library, PubMed, Embase, CNKI, VIP, CBM, and Wangfang databases through April 1, 2022, for randomized controlled trials (RCTs) assessing the effect and safety of CBRT as an adjunctive modality to entecavir for HBV complicated with HF. The primary outcomes were biochemical parameters of serum hyaluronic acid, laminin (LN), pretype-III collagen (PC-III), and type IV collagen (IV-C). The secondary outcomes were liver function indices of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBiL) levels, total effect rate, and occurrence rate of adverse events. Two researchers independently conducted study selection, data extraction, and quality assessment. Statistical analysis was performed using the RevMan 5.3 software. RESULTS: Eight RCTs involving 747 patients were included. Compared with entecavir monotherapy, CBRT as an adjunctive therapy to entecavir exerted more encouraging effect in serum levels of hyaluronic acid (mean difference [MD] = -28.15; 95% confidence interval [CI]: -43.82 to -12.47; P < .001), LN (MD = -29.46; 95% CI: -50.69 to -8.23; P < .001), PC-III (MD = -11.83; 95% CI: -19.43 to -4.23; P < .001), and IV-C (MD = -19.62; 95% CI: -29.76 to -9.49; P < .001); levels of serum ALT (MD = -16.83; 95% CI: -26.30 to -7.36; P < .001), AST (MD = -20.52; 95% CI: -33.11 to -7.93; P < .001), and TBiL (MD = -7.54; 95% CI: -11.58 to -3.49; P < .001); and total effect rate (odds ratio = 3.53; 95% CI: 1.71-7.29; P < .001). Meta-analysis results also showed that CBRT as an adjunctive therapy to entecavir had a lower occurrence rate of adverse events (odds ratio = 0.54; 95% CI: 0.22-1.34; P < .001) than entecavir alone. CONCLUSION: The results of this study showed that CBRT as an adjunctive modality to entecavir may benefit HBV patients complicated with HF. High-quality RCTs are needed to confirm the current findings in the future.

Trastuzumab combined chemotherapy for the treatment of HER2-positive advanced gastric cancer: A systematic review and meta-analysis of randomized controlled trial.

BACKGROUND: This systematic review and meta-analysis aimed to assess the efficacy of trastuzumab combined with chemotherapy for the treatment in HER2-positive advanced gastric cancer (HER2-PAGC). METHODS: This systematic review and meta-analysis was designed using randomized controlled trials that compared trastuzumab in combination with chemotherapy and chemotherapy alone. A comprehensive search was conducted in the following databases from their inception onwards: PubMed, EMBASE, Cochrane Library, WANGFANG, and CNKI. We also searched other literature sources to avoid missing relevant studies. Two reviewers independently performed all record selection, data collection, and methodological assessments. Any confusion was resolved by discussion or referral to a third reviewer. If there were ample data from eligible studies, we performed a fixed-effects meta-analysis. Whenever this was not possible, we conducted a narrative synthesis. RESULTS: Meta-analysis results showed that trastuzumab in combination with chemotherapy achieved better outcomes on response rate (trastuzumab plus CFC vs CFC: odds ratio [OR] = 1.56, 95% confidence interval [CI] [1.17-2.09], I2 = 0%, P < .003; trastuzumab plus OT vs OT: OR = 2.97, 95% CI [1.74-5.09], I2 = 0%, P < .0001; and trastuzumab plus CC vs CC: OR = 2.62, 95% CI [1.84-3.73], I2 = 0%, P < .0001), and disease control rate (trastuzumab plus CFC vs CFC: OR = 1.61, 95% CI [1.17-2.21], I2 = 0%, P = .004; trastuzumab plus OT vs OT: OR = 4.29, 95% CI [2.33-7.90], I2 = 0%, P < .0001; and trastuzumab plus CC vs CC: OR = 2.99, 95% CI [1.99-4.48], I2 = 0%, P < .0001). However, there were no significant differences in the adverse events. CONCLUSIONS: The results of this study revealed that the efficacy of trastuzumab combined with chemotherapy was superior to that of chemotherapy alone for the treatment of HER2-PAGC. The 2 modalities showed similar safety profiles.

Targeted photodynamic therapy of glioblastoma mediated by platelets with photo-controlled release property.

Photodynamic therapy (PDT) has recently emerged as a promising, targeted treatment modality for glioblastoma (GBM) which is the most vicious type of brain tumor. Successful GBM-PDT hinges upon light activation of a photosensitizer accumulated in the tumor. However, inadequate tumor accumulation of photosensitizer severely limits the success of PDT of GBM. To tackle this difficulty, we herein propose a drug delivery strategy of "platelets with photo-controlled release property". This strategy exploits platelets as carriers to deliver a photosensitizer which, in the current study, is a nano-composite (BNPD-Ce6) comprised of chlorine e6 (Ce6) loaded to boron nitride nanoparticles with a surface coating of polyglycerol and doxorubicin. To demonstrate the working mechanism and therapeutic advantage of this strategy, we loaded mouse platelets with BNPD-Ce6 to yield the nano-device BNPD-Ce6@Plt. In vitro experiments showed BNPD-Ce6@Plt to have a high loading capacity and efficiency. Laser irradiation (LI) at a wavelength of 808 nm induced ROS generation in BNPD-Ce6@Plt which displayed rapid activation, aggregation, and speedy discharge of BNPD-Ce6 into co-cultured GL261 mouse GBM cells which in turn, after LI, exhibited marked ROS generation, DNA damage, reduced viability, and cell death. In vivo animal experiments, mice that were intravenously injected with BNPD-Ce6@Plt exhibited rapid and extensive BNPD-Ce6 accumulation in both subcutaneous and intra-brain GL261 tumors shortly after LI of the tumors and the tumors displayed massive tissue necrosis after LI for a second time. Finally, a PDT regimen of two intravenous BNPD-Ce6@Plt injections each followed by multiple times of extracranial LI at the tumor site significantly inhibited the growth of intra-brain GL261 tumors and markedly increased the survival of the host animals. No apparent tissue damage was found in vital organs. Our findings make a compelling case for the notion that platelets are efficient carriers that can photo-controllably deliver nano-photosensitizers to achieve highly targeted and efficacious PDT of GBM. This work presents a novel approach to GBM-PDT with great translational potential.

Esophageal stenosis after chemotherapy for breast cancer: A case report.

RATIONALE: Esophageal stenosis after chemotherapy in breast cancer patients is rare. Distinguishing esophageal stenosis from esophageal metastasis caused by breast cancer is important. PATIENT CONCERNS AND DIAGNOSIS: A 62-year-old woman diagnosed with advanced breast cancer and no distant metastases gradually developed skin changes, oral ulcers and mucosal injures after four cycles of chemotherapy. Dysphagia was the most severe symptom that greatly affected the patient's quality of life. Ultimately, esophageal stenosis and ulceration were confirmed by serial radiological examinations and endoscopic biopsy. INTERVENTIONS: Due to difficulties in eating orally, the patient was initially placed on a nasogastric tube in order to improve her nutritional status. Simultaneously, she was administered powerful proton pump inhibitors. She underwent modified radical mastectomy for breast cancer after her nutritional status improved. However, the patient was still suffering from severe dysphagia after more than 4 months of follow-up. Subsequently, she underwent removable esophageal stent implantation after after unsuccessful attempts to dilate her esophagus. OUTCOMES: The dysphagia symptoms were immediately alleviated to a certain degree, and the dilated cavity of the upper esophagus showed slight retraction. LESSONS: Esophageal stenosis is very infrequent in patients with breast cancer after chemotherapy. It needs to be. distinguished from esophageal metastasis caused by breast cancer. Esophageal stent implantation may provide benefits in terms of both symptom control and survival in patients with severe esophageal structures.

Platelets are highly efficient and efficacious carriers for tumor-targeted nano-drug delivery.

The present work aims to prove the concept of tumor-targeted drug delivery mediated by platelets. Doxorubicin (DOX) attached to nanodiamonds (ND-DOX) was investigated as the model payload drug of platelets. In vitro experiments first showed that ND-DOX could be loaded in mouse platelets in a dose-dependent manner with a markedly higher efficiency and capacity than free DOX. ND-DOX-loaded platelets (Plt@ND-DOX) maintained viability and ND-DOX could be stably held in the platelets for at least 4 hr. Next, mouse Lewis lung cancer cells were found to activate Plt@ND-DOX and thereby stimulate cargo unloading of Plt@ND-DOX. The unloaded ND-DOX was taken up by co-cultured cancer cells which consequently exhibited loss of viability, proliferation suppression and apoptosis. In vivo, Plt@ND-DOX displayed significantly prolonged blood circulation time over ND-DOX and DOX in mice, and Lewis tumor grafts demonstrated infiltration, activation and cargo unloading of Plt@ND-DOX in the tumor tissue. Consequently, Plt@ND-DOX effectively reversed the growth of Lewis tumor grafts which exhibited significant inhibition of cell proliferation and apoptosis. Importantly, Plt@ND-DOX displayed a markedly higher therapeutic potency than free DOX but without the severe systemic toxicity associated with DOX. Our findings are concrete proof of platelets as efficient and efficacious carriers for tumor-targeted nano-drug delivery with the following features: 1) large loading capacity and high loading efficiency, 2) good tolerance of cargo drug, 3) stable cargo retention and no cargo unloading in the absence of stimulation, 4) prolonged blood circulation time, and 5) excellent tumor distribution and tumor-activated drug unloading leading to high therapeutic potency and few adverse effects. Platelets hold great potential as efficient and efficacious carriers for tumor-targeted nano-drug delivery.

Doxorubicin-polyglycerol-nanodiamond conjugates disrupt STAT3/IL-6-mediated reciprocal activation loop between glioblastoma cells and astrocytes.

Astrocytes are key stromal components in glioblastoma (GBM) and have complex interactions with the GBM cells (GBC) promoting the survival, progression and therapy resistance of GBM. In this study, we first demonstrated the existence of a reciprocal activation loop mediated by the STAT3/IL-6 signaling between GBC and astrocytes. This loop of reciprocity was found to be initiated by the constitutive activity of STAT3 and downstream expression of IL-6 in the GBC. GBC-derived IL-6 activated STAT3 and thereby upregulated IL-6 expression in the astrocytes. Astrocyte-derived IL-6 acted back on the GBC causing further activation of STAT3 and leading to enhanced downstream events that promote proliferation, migration, invasion and apoptosis resistance of the GBC. Next, we showed that doxorubicin-polyglycerol-nanodiamond conjugates (Nano-DOX), which could be delivered via GBM-associated macrophages, suppressed STAT3 activity in the GBC reducing their IL-6 output to the astrocytes and thereby abolished the astrocytes' feedback activation of the GBC. Moreover, Nano-DOX also suppressed stimulated activation of STAT3 and IL-6 induced by temozolomide, a first-line anti-GBM chemotherapy, resistance to which critically involves STAT3 activation. In conclusion, Nano-DOX could disrupt the STAT3/IL-6-mediated reciprocal activation loop between the GBC and astrocytes. Nano-DOX also provides a novel approach to therapeutic modulation of the GBM microenvironment.

Doxorubicin conjugated with nanodiamonds and in free form commit glioblastoma cells to heterodromous fates.

AIM: To mechanistically compare the effects of doxorubicin (DOX) and DOX conjugated with nanodiamonds (Nano-DOX) on human glioblastoma cells (GC). MATERIALS & METHODS: GC viablity, proliferation and activation of apoptosis and autophagy was assayed in response to DOX and Nano-DOX. Expression and release of HMGB1 were measured and its role in apoptosis and autophagy probed in response to DOX and Nano-DOX.  Results: DOX induced apoptosis in GC while Nano-DOX induced autophagy. Inhibition of autophagy in Nano-DOX-treated GC promoted apoptosis. DOX suppressed the emission of HMGB1 while Nano-DOX stimulated HMGB1 emission which was attenuated when autophagy was repressed. Blocking of HMGB1 emission mitigated autophagy and enhanced apoptosis in Nano-DOX-treated GC. Exogenously administered HMGB1 promoted autophagy and protected against apoptosis in both Nano-DOX-treated GC and DOX-treated GC. CONCLUSIONS: Nano-DOX is a potent autophagy activator as opposed to DOX as an apoptosis inducer. Nano-DOX initiates a mutual reinforcement loop between autophagy and HMGB1 in GC and thereby protects GC against apoptosis.

Doxorubicin-polyglycerol-nanodiamond composites stimulate glioblastoma cell immunogenicity through activation of autophagy.

Immunosuppression is a salient feature of GBM associated with the disease's grim prognosis and the limited success of anti-GBM immunotherapy. Stimulating immunogenicity of the GBM cells (GC) is a promising approach to subverting the GBM-associated immunosuppression. We had previously devised a drug composite based on polyglycerol-functionalized nanodiamonds bearing doxorubicin (Nano-DOX) and demonstrated that Nano-DOX effectively modulated GBM's immunosuppressive microenvironment through stimulating the immunogenicity of GC and initiated anti-GBM immune responses. The present study now explored the mechanism of Nano-DOX's immunostimulatory action. Nano-DOX was found to induce autophagy rather than apoptosis in GC and stimulated GC to emit antigens and damage-associated molecular patterns (DAMPs) that are potent adjuvants, which resulted in enhanced activation of dendritic cells (DC). Heightened autophagosome release was observed in Nano-DOX-treated GC but was shown not to be a major channel of antigen donation. Blocking autophagy in GC not only reduced Nano-DOX-stimulated GC antigen donation and DAMPs emission, but also efficiently attenuated DC activation stimulated by Nano-DOX-treated GC. Taken together, these findings suggest that activation of autophagy is a central mechanism whereby Nano-DOX stimulates GC's immunogenicity. Our work provides new insight on how nanotechnology can be applied to therapeutically modulate the GBM immune microenvironment by harnessing autophagy in the cancer cells. STATEMENT OF SIGNIFICANCE: Immunosuppression is a salient feature of GBM associated with the grim prognosis of the disease and the limited success of anti-GBM immunotherapy. We demonstrated that Doxorubicin-polyglycerol-nanodiamond composites could activate autophagy in GBM cells and thereby stimulate the immunogenecity of GBM cells. This discovery 1, sheds new light on how nanotechnology could be applied to therapeutically modulate the tumor immune microenvironment, and 2, provides a powerful tool for subverting the GBM's immunosuppressive microenvironment, which has great therapeutic potential for the treatment of GBM.

Dendritic cell-mediated delivery of doxorubicin-polyglycerol-nanodiamond composites elicits enhanced anti-cancer immune response in glioblastoma.

Glioblastoma (GBM) is the deadliest and most common type of primary brain tumor in adults with a grim prognosis despite multimodal treatments. Dendritic cell (DC)-based immunotherapy has emerged as a promising therapeutic modality for GBM, whose efficacy is nonetheless fundamentally undermined by GBM-induced immunosuppression. Inducing emission of damage associated molecular patterns (DAMPs) is a highly effective strategy to subvert tumor-associated immunosuppression. The present work was carried out to explore the idea of subverting the GBM immunosuppressive microenvironment through DC-mediated delivery of doxorubicin-polyglycerol-nanodiamond composites (Nano-DOX), a potent DAMPs inducer demonstrated by our previous study, and thereby eliciting enhanced DC-driven anti-GBM immune response. In the in-vitro work on human cell models, Nano-DOX-loaded DC were shown to be functionally viable and release cargo drug to co-cultured GBM cells (GC). Nano-DOX-treated GC displayed not only profuse DAMPs emission but also antigen release. Enhanced activation and acquisition and presentation of GC-derived antigen were then demonstrated in DC in co-culture with GC and Nano-DOX. Consistently, co-culture with GC and Nano-DOX also activated mouse bone marrow-derived DC (mDC) which in turn stimulated mouse spleen-derived lymphocytes which ultimately suppressed co-cultured GC. Next, athymic mice bearing orthotopic human GBM xenografts were intravenously injected with Nano-DOX-loaded mDC and, 48 h later, spleen-derived lymphocytes. The presence of Nano-DOX, DAMPs emission and enhanced infiltration and activation of mDC and lymphocytes were detected in the GBM xenografts. Taken together, our results demonstrate the efficacy of DC-mediated delivery of Nano-DOX to stimulate GC immunogenicity and elicit anti-cancer immune response in the GBM. By this work, we present a novel approach with great application potential to subverting the GBM immunosuppressive microenvironment and to anti-GBM immunotherapy. Investigation has also been conducted probing the mechanisms by which Nano-DOX stimulates GC immunogenicity, which is described in a follow-up paper.

Harnessing the cross-talk between tumor cells and tumor-associated macrophages with a nano-drug for modulation of glioblastoma immune microenvironment.

Glioblastoma (GBM) is the most frequent and malignant brain tumor with a high mortality rate. The presence of a large population of macrophages (Mφ) in the tumor microenvironment is a prominent feature of GBM and these so-called tumor-associated Mφ (TAM) closely interact with the GBM cells to promote the survival, progression and therapy resistance of the GBM. Various therapeutic strategies have been devised either targeting the GBM cells or the TAM but few have addressed the cross-talks between the two cell populations. The present study was carried out to explore the possibility of exploiting the cross-talks between the GBM cells (GC) and TAM for modulation of the GBM microenvironment through using Nano-DOX, a drug composite based on nanodiamonds bearing doxorubicin. In the in vitro work on human cell models, Nano-DOX-loaded TAM were first shown to be viable and able to infiltrate three-dimensional GC spheroids and release cargo drug therein. GC were then demonstrated to encourage Nano-DOX-loaded TAM to unload Nano-DOX back into GC which consequently emitted damage-associated molecular patterns (DAMPs) that are powerful immunostimulatory agents as well as indicators of cell damage. Nano-DOX was next proven to be a more potent inducer of GC DAMPs emission than doxorubicin. As a result, Nano-DOX-damaged GC exhibited an enhanced ability to attract both TAM and Nano-DOX-loaded TAM. Most remarkably, Nano-DOX-damaged GC reprogrammed the TAM from a pro-GBM phenotype to an anti-GBM phenotype that suppressed GC growth. Finally, the in vivo relevance of the in vitro findings was tested in animal study. Mice bearing orthotopic human GBM xenografts were intravenously injected with Nano-DOX-loaded mouse TAM which were found releasing drug in the GBM xenografts 24h after injection. GC damage was evidenced by the induction of DAMPs emission within the xenografts and a shift of TAM phenotype was detected as well. Taken together, our results demonstrate a novel way with therapeutic potential to harness the cross-talk between GBM cells and TAM for modulation of the tumor immune microenvironment.

Polyglycerol-coated nanodiamond as a macrophage-evading platform for selective drug delivery in cancer cells.

A successful targeted drug delivery device for cancer chemotherapy should ideally be able to avoid non-specific uptake by nonmalignant cells, particularly the scavenging monocyte-macrophage system as well as targeting efficacy to bring the drug preferentially into tumor cells. To this purpose, we developed a platform based on detonation nanodiamond (dND) with hyperbranched polyglycerol (PG) coating (dND-PG). dND-PG was first demonstrated to evade non-specific cell uptake, particularly by macrophages (U937). RGD targeting peptide was then conjugated to dND-PG through multistep organic transformations to yield dND-PG-RGD that still evaded macrophage uptake but was preferentially taken up by targeted A549 cancer cells (expressing RGD peptide receptors). dND-PG and dND-PG-RGD showed good aqueous solubility and cytocompatibitlity. Subsequently, the anticancer agent doxorubicin (DOX) was loaded through acid-labile hydrazone linkage to yield dND-PG-DOX and dND-PG-RGD-DOX. Their cellular uptake and cytotoxicity were compared against DOX in A549 cells and U937 macrophages. It was found that dND-PG-DOX uptake was substantially reduced, displaying little toxicity in either type of cells by virtue of PG coating, whereas dND-PG-RGD-DOX exerted selective toxicity to A549 cells over U937 macrophages that are otherwise highly sensitive to DOX. Finally, dND-PG was demonstrated to have little influence on U937 macrophage cell functions, except for a slight increase of TNF-α production in resting U937 macrophages. dND-PG is a promising drug carrier for realization of highly selective drug delivery in tumor cells through specific uptake mechanisms, with minimum uptake in and influence on macrophages.