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Background: Cancer stem-like cells in triple-negative breast cancer (TNBC-SLCs) are the tumorigenic core for malignancy. Aberrant expression of the RON receptor tyrosine kinase has implications in TNBC tumorigenesis and malignancy. Objective: In this study, we identified the RON receptor as a pathogenic factor contributing to TNBC cell stemness and validated anti-RON antibody-drug conjugate Zt/g4-MMAE for eradication of RONexpressing TNBC-SLCs. Methods: Immunofluorescence and Western blotting were used for analyzing cellular marker expression. TNBC-SLCs were isolated by magnetic-immunofluorescence cell-sorting techniques. Spheroids were generated using the ultralow adhesion culture methods. Levels of TNBC-SLC chemosensitivity were determined by MTS assays. TNBC-SLC mediated tumor growth was determined in athymic nude mice. The effectiveness of Zt/g4-induced RON internalization was measured by immunofluorescence analysis. Efficacies of Zt/g4-MMAE in killing TNBC-SLCs in vitro and in eradicating TNBC-SLCmediated tumors were determined in mouse models. All data were statistically analyzed using the GraphPad Prism 7 software. Results: Increased RON expression existed in TNBC-SLCs with CD44+/CD24- phenotypes and ALDH activities and facilitated epithelial to mesenchymal transition. RON-positive TNBC-SLCs enhanced spheroid-formatting capability compared to RON-negative TNBC-SLCs, which were sensitive to small molecule kinase inhibitor BMS-777607. Increased RON expression also promoted TNBC-SLC chemoresistance and facilitated tumor growth at an accelerated rate. In vitro, Zt/g4-MMAE caused massive TNBC-SLC death with an average IC50 value of ~1.56 μg per/ml and impaired TNBC cell spheroid formation. In mice, Zt/g4-MMAE effectively inhibited and/or eradicated TNBC-SLC mediated tumors in a single agent regimen. Conclusions: Sustained RON expression contributes to TNBC-SLC tumorigenesis. Zt/g4-MMAE is found to be effective in vivo in killing TNBC-SLC-mediated xenograft tumors. Our findings highlight the feasibility of Zt/g4-MMAE for the eradication of TNBC-SLCs in the future.

This work was to identify the function and mechanism of miR-219a-5p in regulating knee osteoarthritis (KOA).Rat fibroblast-like synoviocytes (FLSs) were isolated to construct KOA cell model by lipopolysaccharide and adenosine triphosphate treatment. miR-219a-5p and FBXO3 expression in FLSs was modulated by transfection. Flow cytometry was executed to research FLSs apoptosis. Caspase-1 and IL-1β expression in FLSs was researched by immunofluorescence. The binding between miR-219a-5p and FBXO3 was identified by dual luciferase reporter gene assay. KOA rat model and miR-219a-5p up-modulation KOA rat model were constructed. Step size of rats was analyzed. Knee joints of rats were experienced Safranin O-fast green staining to evaluate the knee joint injury. FBXO3, pyroptosis-associated proteins, and IL-1β and IL-18 expression in FLSs and articular cartilage tissues of rats were assessed by Western blot, qRT-PCR and Enzyme-linked immunosorbent assay.KOA cell model had higher apoptosis percentage, expression of pyroptosis-associated proteins, and IL-1β and IL-18 level. miR-219a-5p up-modulation decreased the above indicators, whereas miR-219a-5p down-modulation increased the above indicators. FBXO3 expression was directly repressed by miR-219a-5p. Loss of FBXO3 suppressed the above indicators. FBXO3 counteracted the suppression of miR-219a-5p on the above indicators. miR-219a-5p agomir attenuated knee joint injury, increased step size of KOA rats, and reduced FBXO3, pyroptosis-associated proteins and level of IL-1β and IL-18 in the articular cartilage tissues of KOA rats.miR-219a-5p suppressed the pyroptosis in KOA by inactivating the NLRP3 signaling via targeting FBXO3, which might be a promising target for ameliorating KOA in the clinic.

Fig. S4 is about the verification of cell surface markers in isolated TNBC stem-like cells.

PDF - 424K, Supplementary Figure 1. Effect of BMS-777607 alone or in combination with AZD8055 on polyploidy and viability of Panc1 and BxPC3 cells; Supplementary Figure 2. Effect of siRNA-mediated knockdown of RON expression on BMS-777607-induced polyploidy; Supplementary Figure 3. Effect of BMS-777607 on DNA/chromosome content in L3.6pl cells and CSCs+24/44/ESA; Supplementary Figure 4. Effect of BMS-777607 alone or in combination with AZD8055 on apoptosis of L3.6pl cells; Supplementary Figure 5. Effect of BMS-777607 alone or plus AZD8055 on MCL1 and cyclin B expression in L3.6pl cells.

Table S3 is about the sensitivity of TNBC stem-like cells to a different chemotherapeutics.

Fig. S1 is about the HIC analysis of Zt/g4-MMAE and its stability in PBS

PDF file - 59K, Figure S3: Effect of PHA665752, Compound-I, and BMS-777607 on induction of polyploidy in breast cancer cells.

Table S1 is about the levels of RON expression in 168 primary TNBC samples analyzed by anti-RON mAb IHC staining

Fig. S3 is about the detection of spontaneous activation of RON in TNBC cell lines

PDF - 79K, Supplementary Table 2. Sensitivities of L3.6pl cells, CSCs+24/44/ESA and their derived polyploid cells in response to different chemotherapeutics.

Table S2 is about the pathological features of a panel of human TNBC cell lines

PDF file - 63K, Figure S1: Expression of RON and MET by breast cancer MCF-7, T-47D and ZR-75-1 cells.

Fig. S2 is about the cytotoxic effect of Zt/g4-MMAE on a panel of TNBC cell lines

PDF - 73K, Supplementary Table 1. Effect of BMS-777607 and AZD1152 on changes in DNA content in L3.6pl cells and pancreatic CSCs+24/44/ESA.

Triple-negative breast cancer (TNBC) is a highly diverse group of malignant neoplasia with poor outcome. Currently, the lack of effective therapy has fostered a major effort to discover new targets to treat this malignant cancer. Here we identified the RON receptor tyrosine kinase as a therapeutic target for potential TNBC treatment. We analyzed RON expression in 168 primary TNBC samples via tissue microarray using anti-RON IHC staining and demonstrated that RON was widely expressed in 76.8% TNBC samples with overexpression in 76 cases (45.2%). These results provide the molecular basis to target RON for TNBC therapy. To this end, anti-RON monoclonal antibody Zt/g4-drug monomethyl auristatin E conjugate (Zt/g4-MMAE) was developed with a drug to antibody ratio of 3.29 and tested in a panel of TNBC cell lines with different phenotypes. In vitro, Zt/g4-MMAE rapidly induced RON internalization, resulted in cell-cycle arrest followed by massive cell death. The calculated IC50 values ranged from 0.06 to 3.46 μg/mL dependent on individual TNBC cell lines tested. Zt/g4-MMAE also effectively killed TNBC stem-like cells with RON+/CD44+/CD24− phenotypes and RON-negative TNBC cells through the bystander effect. In vivo, Zt/g4-MMAE at 10 mg/kg in a Q12 × 2 regimen completely eradicated TNBC xenografts without the regrowth of xenograft tumors. In conclusion, increased RON expression is a pathogenic feature in primary TNBC samples. Zt/g4-MMAE is highly effective in eradicating TNBC xenografts in preclinical models. These findings lay the foundation for using anti-RON Zt/g4-MMAE in clinical trials as a novel strategy for TNBC treatment.

PDF file - 68K, Figure S2: Effect of BMS-777607 on breast cancer cell growth and apoptotic death.

PDF - 64K, Supplementary Table 3. Minimum Inhibitory Concentrations* of BMS-777607 and Chemoagents in L3.6pl Cells and L3.6pl Cell-Derived CSCs+24/44/ESA.

Fig. S5 is about the effect of Zt/g4-MMAE on tumor weight and number.

PDF file - 57K, Supplementary Table 2 Increased resistance of BMS-777607-induced breast cancer polyploid cells in response to cytotoxic chemotherapy agents.

PDF file - 66K, Supplementary Table 1 Inductive Effect of BMS-777607 on Breast Cancer Polyploid Cells with Different Numbers of Chromosomes.

Background: Aberrant expression of the MET receptor tyrosine kinase is an oncogenic determinant and a drug target for cancer therapy. Currently, antibody-based biotherapeutics targeting MET are under clinical trials. Objective: Here, we report the preclinical and therapeutic evaluation of a novel anti-MET antibody- drug conjugate PCMC1D3-duocarmycin SA (PCMC1D3-DCM) for targeted cancer therapy. Methods: The monoclonal antibody PCMC1D3 (IgG1a/κ), generated by a hybridoma technique and specific to one of the MET extracellular domains, was selected based on its high specificity to human MET with a binding affinity of 1.60 nM. PCMC1D3 was conjugated to DCM via a cleavable valine-citrulline dipeptide linker to form an antibody-drug conjugate with a drug-to-antibody ratio of 3.6:1. PCMC1D3-DCM in vitro rapidly induced MET internalization with an internalization efficacy ranging from 6.5 to 17.2h dependent on individual cell lines. Results: Studies using different types of cancer cell lines showed that PCMC1D3-DCM disrupted the cell cycle, reduced cell viability, and caused massive cell death within 96h after treatment initiation. The calculated IC50 values for cell viability reduction were 1.5 to 15.3 nM. Results from mouse xenograft tumor models demonstrated that PCMC1D3-DCM in a single dose injection at 10 mg/kg body weight effectively delayed xenograft tumor growth up to two weeks without signs of tumor regrowth. The calculated tumoristatic concentration, a minimal dose required to balance tumor growth and inhibition, was around 2 mg/kg body weight. Taken together, PCMC1D3-DCM was effective in targeting the inhibition of tumor growth in xenograft models. Conclusion: This work provides the basis for the development of humanized PCMC1D3-DCM for MET-targeted cancer therapy in the future.

Legends for supplementary figures

Supplementary figure 2. Effect of anti-RON ADCs in vitro on reduction of viability of CRC cells expressing no or low levels of RON

Supplementary figure 4. Effect of anti-RON ADCs Zt/g4-DM1 and Zt/c1-DM1 in vitro on reduction of the number of viable CRC cells

Purpose: The receptor tyrosine kinase RON is critical in epithelial tumorigenesis and a drug target for cancer therapy. Here, we report the development and therapeutic efficacy of a novel anti-RON antibody Zt/g4–maytansinoid (DM1) conjugates for targeted colorectal cancer (CRC) therapy.Experimental Design: Zt/g4 (IgG1a/κ) was conjugated to DM1 via thioether linkage to form Zt/g4–DM1 with a drug-antibody ratio of 4:1. CRC cell lines expressing different levels of RON were tested in vitro to determine Zt/g4–DM1-induced RON endocytosis, cell-cycle arrest, and cytotoxicity. Efficacy of Zt/g4–DM1 in vivo was evaluated in mouse xenograft CRC tumor model.Results: Zt/g4–DM1 rapidly induced RON endocytosis, arrested cell cycle at G2–M phase, reduced cell viability, and caused massive cell death within 72 hours. In mouse xenograft CRC models, Zt/g4–DM1 at a single dose of 20 mg/kg body weight effectively delayed CRC cell-mediated tumor growth up to 20 days. In a multiple dose-ranging study with a five injection regimen, Zt/g4–DM1 inhibited more than 90% tumor growth at doses of 7, 10, and 15 mg/kg body weight. The minimal dose achieving 50% of tumor inhibition was approximately 5.0 mg/kg. The prepared Zt/g4–DM1 is stable at 37°C for up to 30 days. At 60 mg/kg, Zt/g4–DM1 had a moderate toxicity in vivo with an average of 12% reduction in mouse body weight.Conclusion: Zt/g4–DM1 is highly effective in targeted inhibition of CRC cell-derived tumor growth in mouse xenograft models. This work provides the basis for development of humanized Zt/g4–DM1 for RON-targeted CRC therapy in the future. Clin Cancer Res; 20(23); 6045–58. ©2014 AACR.

Supplementary figure 1. Effect of free DM1 in vitro on reduction of viability of CRC cells

Supplementary Table 3. Effect of Anti-RON ADCs on cell cycles of Three Colorectal Cancer Cell Lines

Supplementary figure 3. Effect of Zt/g4-DM1 on reduction of cell viability in CRC cell lines expressing different numbers of RON receptor

Supplementary Table 1. Biochemical and biological features of anti-RON mAbs Zt/g4

The Artificial Intelligence of Things (AIoT) turns passive fiber sensors into learning machines. It can be used to integrate intelligent nodes into a multi-dimensional sensing system. In this study, the temperature measurement system based on Brillouin Gain Spectrum (BGS) test setup is creatively implemented with the AIoT architecture; the training and testing stages of the neural network are divided into different layers of equipment to improve performance and reduce the network traffic. To enable the lightweight and low-power consumption edge computing device to extract accurate temperature from the BGS during testing, we have integrated the post-processing method inspired by curve fitting into the machine learning and proposed the efficient digital resampling filter. The resampling filter approach is achieved by the peak range algorithm with Gauss differential operator and digital band-pass filter. The evaluation results for different methods on the BGS datasets show the superior performance of our approach. Notably, the approach can increase temperature extraction accuracy and compress the sampling data. The RMSEA of the extraction temperature is 0.5635, which can bring an almost 21% accuracy increase over the classic method. Compared with the classic method of processing the original data on the same hardware platform, the amount of data post-processed by the filter is reduced by 75%; the time consumption is reduced by 22%.