The escape of bladder cancer from immunosurveillance causes monotherapy to exhibit poor efficacy; therefore, designing a multifunctional nanoparticle that boosts programmed cell death and immunoactivation has potential as a treatment strategy. Herein, we developed a facile one-pot coprecipitation reaction to fabricate cluster-structured nanoparticles (CNPs) assembled from Fe3O4 and iron chlorophyll (Chl/Fe) photosensitizers. This nanoassembled CNP, as a multifunctional theranostic agent, could perform red-NIR fluorescence and change the redox balance by the photoinduction of reactive oxygen species (ROS) and attenuate iron-mediated lipid peroxidation by the induction of a Fenton-like reaction. The intravesical instillation of Fe3O4@Chl/Fe CNPs modified with 4-carboxyphenylboronic acid (CPBA) may target the BC wall through glycoproteins in the BC cavity, allowing local killing of cancer cells by photodynamic therapy (PDT)-induced singlet oxygen and causing chemodynamic therapy (CDT)-mediated ferroptosis. An interesting possibility is reprogramming of the tumor microenvironment from immunosuppressive to immunostimulatory after PDT-CDT treatment, which was demonstrated by the reduction of PD-L1 (lower “off” signal to the effector immune cells), IDO-1, TGF-β, and M2-like macrophages and the induction of CD8+ T cells on BC sections. Moreover, the intravesical instillation of Fe3O4@Chl/Fe CNPs may enhance the large-area distribution on the BC wall, improving antitumor efficacy and increasing survival rates from 0 to 91.7%. Our theranostic CNPs not only demonstrated combined PDT-CDT-induced cytotoxicity, ROS production, and ferroptosis to facilitate treatment efficacy but also opened up new horizons for eliminating the immunosuppressive effect by simultaneous PDT-CDT.

The prognosis of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer has considerably improved. However, no reliable treatment besides anti-HER2 strategies has been available. FTY720, a small-molecule compound used for treating refractory multiple sclerosis, has been reported to have beneficial effects against cancers. We therefore evaluated the efficacy of FTY720 in trastuzumab-resistant breast cancer cells and investigated the possible mechanism involved. This study evaluated morphological changes after FTY720 treatment. Antiproliferative WST-1 assays and LDH Cytotoxicity Assay Kits were used to determine the treatment effects of drugs, whereas Western blot analysis was used to evaluate protein expression. Apoptotic events were investigated through annexin V staining and TUNEL assays using flow cytometry. FTY720 was effective in trastuzumab-resistant breast cancer cell lines despite the presence of PIK3CA mutation. Studied on a xenograft mouse model, FTY720-treated groups had statistically significantly poorer HCC1954 xenograft growth in vivo compared with the control group. Our findings suggest that FTY720 can overcome resistance to trastuzumab therapy in patients with HER2-positive breast cancer, with FTY720 plus trastuzumab might offer even better efficacy in vitro and in vivo.

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Interleukin-6 (IL-6) is overexpressed in various cancer cells, intriguingly contributing to both tumor growth and modulating anti-tumor immunity. Cell-type-specific glycosylations on IL-6 have been deduced in early studies; however, the glycosylation pattern and biological function of cancer cell-secreted IL-6 remain unclear. Here, we describe the glycosylation pattern of lung cancer cell-secreted IL-6 and its impact on the activation of JAK/STAT pathway. IL-6 molecules with different molecular weights were detected in the conditional media of IL-6-overexpressed lung cancer cell lines by immunoblot. Because in NetNGlyc 1.0 Server, one possible N-glycosylation site has been predicted at N73 on IL-6, we used treatment of N-glycosylation-specific inhibitors and site-directed mutagenesis on N73 to demonstrate that lung cancer cell-secreted IL-6 is modified by N-glycosylation. To examine which glycotransferases may participate in the modification, we had screened the expression of glycosyltransferases using qPCR and found that the expression of fucosyltransferase 8 (FUT8), responsible for core fucosylation on N-glycosylated proteins, was higher in lung cancer cells compared to normal bronchial cell. We then reduced core fucosylation on lung cancer cell-secreted IL-6 by silencing FUT8 with shRNA transduction. Subsequently, cells were treated with conditional media containing fully-glycosylated or core fucose-depleted IL-6 to uncover the potential influences on cellular signaling from glycosylation on IL-6. The fully-gycosylated IL-6 induced prolonged STAT3Y705 phosphorylation and distinct gene population compared to core fucose-depleted IL-6. The nuclear retention of STAT3 was concordant with the prolonged STAT3 activation in cells treated with fully-gycosylated IL-6. In paired normal (N) and tumor (T) tissues from lung cancer patients, higher FUT8 mRNA was detected in tumor part than normal part. Besides, we found similar glycosylation pattern in the secreted IL-6 of short-term cultured lung cancer cells derived from malignant pleural effusions. Together, we report the presence of specific IL-6 glycoforms secreted from lung cancer cell lines and lung cancer cells from clinical samples. Moreover, the glycosylation on IL-6 changes its activity on the regulation of JAK/STAT pathway. Citation Format: Chun-Hua Hung, Hsuan-Heng Yeh, Hao-Chen Wang, Chien-Chung Lin, Tsung-Lin Tsai, Wei-Lun Huang, Chuan-Fa Chang, Wu-Chou Su. N-glycosylation on lung cancer cell-secreted IL-6 prolongs its activation on JAK/STAT pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3452. doi:10.1158/1538-7445.AM2014-3452

Current research suggests that on tumor-related extracellular vesicles (EV) play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms including angiogenesis. Our previous study demonstrated that IL6 induced secretion of VEGF and contributing angiogenesis via activation of Stat3. To further clarify the biological activities of the lung cancer derived EVs, we analyzed the content of IL6 and VEGF in exosome using ELISA kit. We found that IL-6 and VEGF were more enriched in EVs than non-EVs in AS2 and CL1-5 cells. And the isolated EV from AS2 and CL1-5 cells increased vascular permeability in miles assay and promotes angiogenesis in tubular formation assay. We further investigated if the inhibition of exosome secretion affects the autocrine IL6/Stat3/VEGF pathway. GW4869 inhibits secretion of exosome by playing an inhibitor of neutral sphingomyelinase (nSMase). Treatment of either cell with GW4869 significantly decreased the levels of released exosomes in AS2 but not in CL1-5 cell. Consistent with the result, the treatment GW4869 dampened autocrine IL6/Stat3/VEGF pathway in AS2 but not in CL1-5 cell. Bevacizumab, the recombinant anti- VEGF monoclonal antibody, were approved for treating patients with advanced NSCLC, has been shown to be efficient in suppressing the accumulation of pleural fluid (malignant pleural effusion). To investigate if the exosomal VEGF has a different affinity for bevacizumab, we treated AS2, CL1-5 and MPE-derived cancer cells with bevacizumab and EV or non-EV part from cell-free medium. We found VEGF dimer form were more abundant in EV part and cannot be neutralized by bevacizumab. Conversely, VEGF mono-form was more abundant non-EV part and can be neutralized by bevacizumab. In animal model, AS2 was injected intraperitoneal and then malignant ascites developed as follow. We found the administration of bevacizumab after AS2 injection i.p. inhibited peritoneal seeding and ascites formation compared to control group. But the inhibition was reversed when we injected EV as well as bevacizumab in combination group. In breast cancer cell, microvesicles (MVs) activates VEGF receptors through a unique 90 kDa form of VEGF (VEGF90K) which has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor. And an Hsp90 inhibitor (17-AAG) was demonstrated to release VEGF90K from MVs without interaction with VEGF restoring the sensitivity of VEGF90K to Bevacizumab. However, our study found 17-AAG alone inhibited VEGF secretion in EV as well as the activity of upstream Stat3, and Akt pathway in lung cancer. Our preliminary data implied EV-VEGF and the existence of different isoform of VEGF in EV contribute to tumor angiogenesis and limit the effectiveness of bevacizumab therapy which is not dependent on Hsp90. Citation Format: Chien-Chung Lin, Yu-Ting Huang, Chun-Hua Hung, Mei-Ling Tsai, Wu-Chou Su. The extracellular vesicles from lung cancer cells activates Stat3 and tumor angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 192.