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Small extracellular vesicles (sEVs) operate as a signaling platform due to their ability to carry functional molecular cargos. However, the role of sEVs in hypoxic tumor microenvironment-mediated premetastatic niche formation remains poorly understood. Methods : Protein expression profile of sEVs derived from normoxic and hypoxic head and neck squamous cell carcinoma (HNSCC) cells were determined by Isobaric Tagging Technology for Relative Quantitation. In vitro invasion assay and in vivo colonization were performed to evaluate the role of sEV-delivering proteins. Results : We identified lysyl oxidase like 2 (LOXL2) which had the highest fold increase in hypoxic sEVs compared with normoxic sEVs. Hypoxic cell-derived sEVs delivered high amounts of LOXL2 to non-hypoxic HNSCC cells to elicit epithelial-to-mesenchymal transition (EMT) and induce the invasion of the recipient cancer cells. Moreover, LOXL2-enriched sEVs were incorporated by distant fibroblasts and activate FAK/Src signaling in recipient fibroblasts. Increased production of fibronectin mediated by FAK/Src signaling recruited myeloid-derived suppressor cells to form a premetastatic niche. Serum sEV LOXL2 can reflect a hypoxic and aggressive tumor type and can serve as an alternative to tissue LOXL2 as an independent prognostic factor of overall survival for patients with HNSCC. Conclusion : sEVs derived from the hypoxic tumor microenvironment of HNSCC can drive local invasion of non-hypoxic HNSCC cells and stimulate premetastatic niche formation by delivering LOXL2 to non-hypoxic HNSCC cells and fibroblasts to induce EMT and fibronectin production, respectively., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background : Sonodynamic therapy (SDT) is a promising strategy to inhibit tumor growth and activate antitumor immune responses for immunotherapy. However, the hypoxic and immunosuppressive tumor microenvironment limits its therapeutic efficacy and suppresses immune response. Methods: In this study, mitochondria-targeted and ultrasound-responsive nanoparticles were developed to co-deliver oxygen (O 2 ) and nitric oxide (NO) to enhance SDT and immune response. This system (PIH-NO) was constructed with a human serum albumin-based NO donor (HSA-NO) to encapsulate perfluorodecalin (FDC) and the sonosensitizer (IR780). In vitro , the burst release of O 2 and NO with US treatment to generate reactive oxygen species (ROS), the mitochondria targeting properties and mitochondrial dysfunction were evaluated in tumor cells. Moreover, in vivo , tumor accumulation, therapeutic efficacy, the immunosuppressive tumor microenvironment, immunogenic cell death, and immune activation after PIH-NO treatment were also studied in 4T1 tumor bearing mice. Results: PIH-NO could accumulate in the mitochondria and relive hypoxia. After US irradiation, O 2 and NO displayed burst release to enhance SDT, generated strongly oxidizing peroxynitrite anions, and led to mitochondrial dysfunction. The release of NO increased blood perfusion and enhanced the accumulation of the formed nanoparticles. Owing to O 2 and NO release with US, PIH-NO enhanced SDT to inhibit tumor growth and amplify immunogenic cell death in vitro and in vivo . Additionally, PIH-NO promoted the maturation of dendritic cells and increased the number of infiltrating immune cells. More importantly, PIH-NO polarized M2 macrophages into M1 phenotype and depleted myeloid-derived suppressor cells to reverse immunosuppression and enhance immune response. Conclusion: Our findings provide a simple strategy to co-deliver O 2 and NO to enhance SDT and reverse immunosuppression, leading to an increase in the immune response for cancer immunotherapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Aim: Ionizing radiation (IR) can induce local and systemic antitumour immune responses to some degree and augment immunotherapeutic efficacy. IR may also increase residual tumour cell invasion and elicit immunosuppression in the tumour microenvironment (TME). It remains poorly understand, whether IR leads to immune negative response or invasive capacity increases in lung adenocarcinoma (LAC)., Materials and Methods: RNA interference (RNAi) was used to silence pituitary tumour-transforming gene-1 (PTTG1) and SMAD3 expression in LAC cells. A coculture system of tumour cells and PBMCs was constructed. Cells were exposed to different doses (0, 4 and 8 Gy) of X-ray irradiation. Flow cytometric analysis and Transwell assays were applied. An orthotopic Lewis lung cancer (LLC) mouse tumour model was established. Bioluminescence imaging (BLI) was used. LLC tumours were exposed to a single 15 Gy dose of X-ray irradiation., Key Findings: PTTG1 knockdown reinforced the inhibitory effect of IR on the invasive ability of A549 cells and enhanced the antitumour T cell immunity induced by IR via the transforming growth factor-β1 (TGF-β1)/SMAD3 pathway. Positive antitumour immune response and immunosuppression were simultaneously triggered by a single 15 Gy dose of local tumour irradiation. PTTG1 knockdown weakened invasive capacity and promoted the immune response balance induced by IR to tilt towards active immunity, which contributed to reduce metastasis and prolonged overall survival (OS) in orthotopic LLC tumour-bearing mouse., Significance: Targeted blockade of PTTG1 and the TGF-β1/SMAD3 pathway may ameliorate the immunosuppressive TME and enhance the systemic antitumour immune response induced by a single high-dose IR treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Rationale: Iron-saturated Lf (Holo-Lactoferrin, Holo-Lf) exhibits a superior anticancer property than low iron-saturated Lf (Apo-Lf). Ferroptosis is an iron-dependent cell death characterized by the accumulation of lipid peroxidation products and lethal reactive oxygen species (ROS). Radiotherapy also exerts its therapeutic effect through ROS. Methods: The effect of different iron-saturated Lf on ferroptosis and radiotherapy were tested on triple-negative breast cancer (TNBC) cell line MDA-MB-231 and non-TNBC cell line MCF-7. Results: Holo-Lf significantly increased the total iron content, promoted ROS generation, increased lipid peroxidation end product, malondialdehyde (MDA), and enhanced ferroptosis of MDA-MB-231 cells. By contrast, Apo-Lf upregulated SLC7a11 expression, increased GSH generation and inhibited ferroptosis of MDA-MB-231 cells. However, non-TNBC MCF-7 cells were resistant to Holo-Lf-induced ferroptosis because MCF-7 cells have a higher redox balance capacity than MDA-MB-231 cells. More importantly, Holo-Lf downregulated HIF-1α expression, ameliorated the hypoxia microenvironment in subcutaneous MDA-MB-231 tumors, and promoted radiation-induced DNA damage to hypoxic MDA-MB-231 cells. Finally, the efficacy of radiotherapy to MDA-MB-231 tumors was enhanced by Holo-Lf. Conclusion: Holo-Lf could induce ferroptosis in MDA-MB-231 cells and sensitize MDA-MB-231 tumors to radiotherapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)