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Figure S1. Cellular senescence, expression profiling and the SASP development in human stromal cells upon genotoxic treatment. Figure S2. SIRT1 decline is not related with single strand breaks, proteasome degradation or autophagy deficiency upon cellular senescence. Figure S3. Senescent stromal sEVs enhance the malignancy of prostate cancer cells. Figure S4. Transcriptomic profiling of prostate cancer cells upon exposure to senescent stromal sEVs and establishment of protein-protein interaction network involving ABCB4. Figure S5. Senescent stromal sEV-conferred malignancy is diminished upon elimination of ABCB4 from cancer cells. Figure S6. Pharmacological targeting SIRT1 minimizes senescent stromal sEV production and prevents ABCB4 upregulation in prostate cancer cells.

Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype, which is implicated in age-related pathologies including cancer. Here, we report that senescent cells actively synthesize and release small extracellular vesicles (sEV) with a distinctive size distribution. Mechanistically, SIRT1 loss supported accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Once released, senescent stromal sEVs significantly altered the expression profile of recipient cancer cells and enhanced their aggressiveness, specifically drug resistance mediated by expression of ATP-binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with agonist SRT2104 prevented development of cancer resistance by restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients were readily detectable by routine biotechniques, presenting an exploitable biomarker to monitor therapeutic efficacy and predict long-term outcome. Together, this study identifies a distinct mechanism supporting pathologic activities of senescent cells and provides a potent avenue to circumvent advanced human malignancies by cotargeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells.Significance:Senescent stromal cells produce a large number of sEVs to promote cancer resistance in therapeutic settings, a process driven by SIRT1 decline in stromal cells and ABCB4 augmentation in cancer cells.See related commentary by Wiley, p. 3193

Mesothelin (MSLN) is an attractive therapeutic target for antibody drug conjugates (ADC) because of significant differences in expression pattern between diseased and normal tissues. RC88-ADC is a novel ADC, targeting MSLN, and inhibits tumor growth significantly in mice xenograft models. We performed an 11-week repeated dose toxicity study of RC88-ADC via intravenous injection in Cynomolgus Monkeys with an 8-Week recovery period according to International Conference on Harmonization (ICH) S9 and S6(R1). RC88-ADC was administered to groups of 5 male and 5 female monkeys at dose levels of 2.5, 5, and 10 mg/kg/2 weeks, meanwhile vehicle, naked antibody, small molecule groups were set up as the control. 4 animals died in 10 mg/kg group of RC88-ADC. The clinical symptoms mainly included ocular toxicity, weight loss and food intake decrease in the middle and high dose groups of RC88-ADC. RC88-ADC caused dose-related reversible myelosuppression, manifested as hematologic toxicity, which was consistent with the small molecule toxicity profile of its coupling. The highest non-severely toxic dose of RC88-ADC was 5 mg/kg in monkeys after repeated dosing. Nonetheless, the integrated analysis showed that RC88-ADC demonstrated an acceptable safety profile and provided an improved treatment window. These results pave the way for further investigation of RC88-ADC in humans.

Antibody-drug conjugated (ADC) plays an important role in oncology indications. The efficacy and toxicity of ADC generally depend on the concentration of the drugs in the body system, and physiologically-based pharmacokinetics (PK) is a quantitative tool to understand the drug concentration in the body. Understanding the PK of the ADC requires a sophisticated bioanalytical approach to carefully characterize the whole drug. ADC bioanalysis generally needs multiple analysis strategies, which can accurately quantify total antibody (TAb), antibody-drud conjugated (ADC), antibody-conjugated payload, free payload. In this work, we mainly described and validated a high throughput capture Liquid Chromatography tandem-Mass Spectrometry (LC-MS/MS) bioanalysis method to detect the concentrations of antibody-conjugated payload. This allowed for the determination of the DAR (Drug to Antibody Ratio) for in vivo samples by of antibody-conjugated payload/ of n TAb. In addition, the method significantly improved throughput by pre-coated antibody on 96-well plate and then the antibody-conjugated payload (ac-MMAE) was released by Cathepsin B. The method had no interference or carryover in endogenous substances and showed linearity (R2≥0.99) in the concentration range within 15.6 - 2000.0 ng/mL. The inter-run accuracy ranged from 75.8 to 120.0%, and precision was within ≤ 20.0%. Meanwhile, selectivity and the benchtop stability of the method were also validated. This optimization method has already been successfully applied to the change of average DAR in PK study.

Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype, which is implicated in age-related pathologies including cancer. Here, we report that senescent cells actively synthesize and release small extracellular vesicles (sEV) with a distinctive size distribution. Mechanistically, SIRT1 loss supported accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Once released, senescent stromal sEVs significantly altered the expression profile of recipient cancer cells and enhanced their aggressiveness, specifically drug resistance mediated by expression of ATP-binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with agonist SRT2104 prevented development of cancer resistance by restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients were readily detectable by routine biotechniques, presenting an exploitable biomarker to monitor therapeutic efficacy and predict long-term outcome. Together, this study identifies a distinct mechanism supporting pathologic activities of senescent cells and provides a potent avenue to circumvent advanced human malignancies by cotargeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells. Significance: Senescent stromal cells produce a large number of sEVs to promote cancer resistance in therapeutic settings, a process driven by SIRT1 decline in stromal cells and ABCB4 augmentation in cancer cells. See related commentary by Wiley, p. 3193

Background Clinical anti-vascular epithelial growth factor (VEGF) therapy trials faced a major challenge due to upregulated expression of other pro-angiogenic factors, such as fibroblast growth factor-2 (FGF-2). RC28, a novel recombinant dual decoy receptor IgG1 Fc-fusion protein, can block VEGFA and FGF-2 simultaneously. It is designed for the treatment of neovascular age-related macular degeneration and other pathological ocular neovascularization. The present study investigated the prevention efficacy of RC28 on choroidal neovascularization (CNV) in a monkey model and compared to the other mono VEGF antagonists; biodistribution and pharmacokinetics performance were also investigated. Methods ELISA and endothelial cell proliferation, migration, and tubule formation assay evaluated the bioactivity of RC28 in vitro, and an initial comparison was made among the mono target antagonists, Bevacizumab (Avastin), Ranibizumab (Lucentis), Aflibercept (EYLEA), Conbercept (KH902), and Ranibizumab (Lucentis). Laser-induced CNV in monkeys, and both VEGF and FGF-2 serum levels were detected in animals before and after the CNV model were induced. RC28 prevention efficacy was compared to other VEGF antagonists on CNV with respect to the incidence of CNV and several ophthalmic examinations. Ocular and systemic levels of RC28 were analyzed by 89Zr-labeled RC28 after single intravitreal administration for the biodistribution and pharmacokinetic profiles. Results RC28 is a unique fusion protein with high affinity to both VEGF and FGF-2, and beneficial to in vitro and in vivo bioactivity. The in vivo pharmacological studies demonstrated that the incidence of CNV formation was largely reduced in RC28 treatment groups with a low dosage as compared to other VEGF antagonist control groups. Furthermore, traces of RC28 were detected as dispersing from eyeballs to the liver after 20 days, and a prolonged half-time pharmacokinetic profile was exhibited.

The HER2 pathway plays a pivotal role in cell proliferation and differentiation, while the receptor overexpression caused by amplification of HER2 gene is associated with the growth of several tumors. Previously published clinical trials have demonstrated that antibody-conjugated drugs (ADCs) remarkably improved clinical effects compared with antibodies alone for the same target. In order to provide more effective drugs, we developed Disitamab vedotin based on ADC. The antibody part was a humanized monoclonal antibody targeting HER2, the small molecule toxin was monomethyl auristatin E (MMAE), a synthetic antineoplastic agent. A protease cleavable linker covalently attached MMAE to the antibody. In this study, we characterized the toxicity profile of Disitamab vedotin through single- and repeat-dose toxicity studies in monkeys. The toxicities of small molecules and naked antibody (Disitamab) were also assessed in these studies. Monkeys were well tolerated with Disitamab vedotin at doses of 6 mg/kg, while equivalent MMAEs resulted in severe myelosuppression. This finding proves that ADCs improve the therapeutic effect. In addition, the safety profiles of Disitamab vedotin and MMAE were similar and consistent with the activation mechanism of MMAE. Toxicology finding included bone marrow/hematology toxicity and lymphoid organ toxicity, while no significant toxicity was observed in animals treated with naked antibody. These side effects were found to be consistent with data acquired from clinical phase I/II patients treated with Disitamab vedotin.

Background: Disease-related injury in any organ triggers a complex cascade of cellular and molecular responses that culminate in tissue fibrosis, inflammation, and angiogenesis simultaneously. Multiple cell angiogenesis is an essential part of the tissue damage response, which is involved in fibrosis development. RC28-E is a novel recombinant dual decoy receptor lgG1 Fc-fusion protein that can block vascular endothelial growth factor (VEGFA), platelet-derived growth factor (PDGF), and fibroblast growth factor-2 (FGF-2) simultaneously. This protein has stepped into clinical trials (NCT03777254) for the treatment of pathological neovascularization-related diseases. Here, we report on the role of RC28-E during anti-fibrosis and its potential multitarget function in regulating fibrosis. Methods: A bleomycin-induced pulmonary fibrosis C57BL/6 mouse model was established. Hematoxylin and eosin staining (HE) and Masson staining (Masson&rsquo, s) were performed to evaluate the pulmonary fibrosis based on the scoring from, Ashcroft score. Fibrosis related factors and inflammatory cytokines including HYP, &alpha, SMA, procollagen, ICAM, IL-6, IL-1, and TNF-&alpha, were also determined at the protein and mRNA levels to characterize the fibrosis. Both mRNA and protein levels of VEGF, FGF, and transforming growth factor (TGF)-&beta, were detected by quantitative real-time PCR (qRT-PCR) and immunohistochemical (IHC) analysis, respectively. Pulmonary fibrosis and related cytokines were re-evaluated in vivo after 3 doses of RC28-E (5 mg/kg, 15 mg/kg, and 50 mg/kg, ip. Tiw &times, 9) in comparison with a mono-target antagonist treatment (VEGF or FGF blocking). RC28-E attenuated the activation of TGF-&beta, induced fibroblasts in vitro. Expression levels of &alpha, SMA and collagen I, as well as proliferation and migration, were determined with the human skin fibroblast cell line Detroit 551 and primary murine pulmonary fibroblast cells. The mechanism of RC28-E via the TGF-&beta, /Smad pathway was also investigated. Results: RC28-E exhibits significant anti-fibrosis effects on Idiopathic pulmonary fibrosis (IPF) in vivo. Moreover, TGF-&beta, induced fibroblast activation in vitro via the inhibition of the TGF-&beta, downstream Smad pathway, thus providing potential therapeutics for clinical disease-related fibrosis-like IPF as well as chemotherapy-induced fibrosis in cancer therapy.

HER2 targeted delivery of ovarian cancer therapy has been beneficial for some patients, although, its efficacy is yet to be confirmed in large populations. We generated a novel anti-HER2 humanized antibody (Hertuzumab) and conjugated it to a microtubule-disrupting drug monomethyl auristatin E conjugate (MMAE) with a lysosomal protease-cleavable valine-citrulline linker. The average drug to antibody ratio (DAR) of Hertuzumab-vc-MMAE was varied by conjugating Hertuzumab antibodies with increasing linker-drugs (LDs) from D0-D8. The resulting conjugates were tested for kinetic affinity for soluble HER2-ECD, cytotoxicity, and in vivo pharmacokinetics. The kinetic binding constant values (KD) were obtained by the bio-layer interference (BLI) method. The half time (t1/2) and clearance (Cl) results of the pharmacokinetic profile in rats were DAR-dependent. Hertuzumab-vc-MMAE with DAR4 was selected for further evaluation. Both Hertuzumab and Hertuzumab conjugates could bind to HER2 antigen, and exhibited significant cytotoxicity on HER2 positive tumor cells after internalization by receptor-mediated endocytosis. Hence, Hertuzumab-vc-MMAE conjugates were significantly selective both in vitro and in vivo as compared to other ovarian cancer clinical therapies that are currently used. Cell signal transduction and cell cycle were also affected, as shown by down regulation of PI3K/AKT pathway and arrested mitosis in the G2/M phase. The pharmacokinetics and pharmacodynamics (PK-PD) of the conjugates in nude mouse xenograft model demonstrated a correlation between efficacy and drug concentration. These results show that Hertuzumab-vc-MMAE is a potential therapeutic agent for HER2 positive ovarian cancer.

To evaluate clinical characteristics and outcome of acute ischemic stroke patients with atrial fibrillation.Consecutive acute ischemic stroke patients who were hospitalized in the neurology department of General Hospital of Jinan Military Region were prospectively recruited from August 2010 to November 2013.The baseline datum including age, sex, National Institute of Health Stroke Scale (NIHSS), type of Oxfordshire Community Stroke Project (OCSP: total anterior circulation infarct, partial anterior circulation infarction, posterior circulation infarction and lacunar infarction), serum creatinine, serum albumin levels etc.were recorded.Atrial fibrillation (AF) was defined as a history of persistent atrial fibrillation or paroxysmal atrial fibrillation, supported by past electrocardiogram or diagnosed by the attending physicians based on physical examination, electrocardiogram and/or 24-hour electrocardiogram monitoring during hospitalization. Outcome was assessed by modified Rankin Scale (mRS) which was obtained 180 days after stroke by telephone interview (mRS ≤ 2 reflected good prognosis, and mRS2 reflected unfavorable prognosis), and death defined as all-cause mortality. Multivariate regression model was used to analyze predictors of mortality and disability.Of the 965 patients included in this study, 113 (11.71%) had AF; valvular AF was observed in 11 patients (9.7%) among them.Only 4 patients with valvular AF and none of the patients with non-valvular AF took warfarin before the stroke event. 14.2% (16/113) acute ischemic stroke patients with AF took aspirin. Compared to patients without AF, patients with AF had a higher NIHSS score on admission (median 11 vs 5, P=0.000); were more often with diabetes (26.55% vs 9.74%, P=0.028), congestive heart failure (12.37% vs 11.03%, P=0.000), prior stroke (31.86% vs 21.83%, P=0.023), total anterior circulation infarct subtype (51.33% vs 19.37%, P=0.000); they were less often smokers (20.35% vs 37.32%, P=0.000), alcohol consumers (13.27% vs 27.58%, P=0.001), partial anterior circulation infarction subtype (24.78% vs 36.74%, P=0.012), lacunar infarct subtype (0 vs 17.61%, P=0.000); they had less often experienced myocardial infarction (11.50% vs 11.74%, P=0.041). AF was a significant independent prognostic factor for long-term poor outcomes (OR=2.227, 95%CI: 1.262-3.933, P=0.006).Oral anticoagulants are underused in AF patients.Brain infarction patients with AF is more severe than patients without AF; have higher frequency of total anterior circulation infarct subtype, prior stroke and lower frequency of lacunar infarct subtype. AF is a significant independent prognostic factor for long-term poor outcome in patients with acute brain infarction.

To evaluate the potential toxicity of refined arachidonic acid-rich oil (RAO) derived from Mortierella alpina (M. alpina) XM027, we performed a 90-day subchronic study in F1 Sprague Dawley (SD) rats. This study was preceded by a 4-week pretreatment period of parental (F0) rats and exposure of the F0 dams throughout mating, gestation, and lactation. The results indicated that RAO, at dose levels of 0.5%, 1.5%, and 5%, did not affect either reproductive performance of the parental rats, or any characteristics of the pups. In the subchronic study with the offspring (F1) rats, no treatment related abnormalities were observed. In summary, no observable adverse effect level (NOAEL) in this study was placed at 5% RAO, the highest level tested. This level corresponds to approximately 3750 mg/kg in F0 females, 2850 mg/kg in F0 males, 4850 mg/kg in F1 females, and 4480 mg/kg in F1 males.

The emphasis in this paper is mainly on the basic principle of Markov chain theory and its application to market occupation rate and promotion strategy. By scientific and reasonable information of the market occupation rate and promotion strategy, we tries to help enterprises achieve the objective of the maximum expected profit according to market analysis and different selling states.

Danshensu (3-(3,4-dihydroxyphenyl) lactic acid), a natural phenolic acid, is isolated from root of Salvia miltiorrhiza, and is widely used as a traditional Chinese medicine for treatment of various cardiovascular diseases. In the present study, toxicity of danshensu was evaluated in male and female dogs after 3-month continuous intravenous infusion. Beagle dogs were treated with danshensu at doses of 17, 50, and 150 mg/kg/day, and observed for 90 days followed by recovery periods. Measurements included clinical observations, body weight, food consumption, temperature, electro-cardiography (EGC), hematology, blood chemistry, urinalysis, gross necropsy, organ weight, and histopathology. No significant adverse effects on these parameters were observed. The only treatment-related finding was a hard knot at injection site observed in the 150 mg/kg group after 2-3 weeks continuous administration, and returned to normal after 3-4 days withdrawal. From these results, it might be concluded that danshensu did not produce any significant cumulative toxicity at the doses administered, as reflected by the various parameters investigated.