BackgroundThe immune checkpoint blockade (ICB) targeting programmed cell death-1 (PD-1) and its ligand (PD-L1) has been proved beneficial for numerous types of cancers, including non-small-cell lung cancer (NSCLC). However, a significant number of patients with NSCLC still fail to respond to ICB due to unfavorable tumor microenvironment. To improve the efficacy, the immune-chemotherapy combination with pemetrexed, cis/carboplatin and pembrolizumab (anti-PD-1) has been recently approved as first-line treatment in advanced NSCLCs. While chemotherapeutic agents exert beneficial effects, the underlying antitumor mechanism(s) remains unclear.MethodsPemetrexed, cisplatin and other chemotherapeutic agents were tested for the potential to induce PD-L1 expression in NSCLC cells by immunoblotting and flow cytometry. The ability to prime the tumor immune microenvironment was then determined by NSCLC/T cell coculture systems and syngeneic mouse models. Subpopulations of NSCLC cells responding differently to pemetrexed were selected and subjected to RNA-sequencing analysis. The key signaling pathways were identified and validated in vitro and in vivo.ResultsPemetrexed induced the transcriptional activation of PD-L1 (encoded by CD274) by inactivating thymidylate synthase (TS) in NSCLC cells and, in turn, activating T-lymphocytes when combined with the anti-PD-1/PD-L1 therapy. Nuclear factor κB (NF-κB) signaling was activated by intracellular reactive oxygen species (ROSs) that were elevated by pemetrexed-mediated TS inactivation. The TS−ROS−NF-κB regulatory axis actively involves in pemetrexed-induced PD-L1 upregulation, whereas when pemetrexed fails to induce PD-L1 expression in NSCLC cells, NF-κB signaling is unregulated. In syngeneic mouse models, the combinatory treatment of pemetrexed with anti-PD-L1 antibody created a more favorable tumor microenvironment for the inhibition of tumor growth.ConclusionsOur findings reveal novel mechanisms showing that pemetrexed upregulates PD-L1 expression and primes a favorable microenvironment for ICB, which provides a mechanistic basis for the combinatory chemoimmunotherapy in NSCLC treatment.

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Diabetic foot ulcers (DFUs) are one of the most costly and troublesome complications of diabetes mellitus. The wound chronicity of DFUs remains the main challenge in the current and future treatment of this condition. Persistent inflammation results in chronic wounds characterized by dysregulation of immune cells, such as M1 macrophages, and impairs the polarization of M2 macrophages and the subsequent healing process of DFUs. The interactive regulation of M1 and M2 macrophages during DFU healing is critical and seems manageable. This review details how cytokines and signalling pathways are co-ordinately regulated to control the functions of M1 and M2 macrophages in normal wound repair. DFUs are defective in the M1-to-M2 transition, which halts the whole wound-healing machinery. Many pre-clinical and clinical innovative approaches, including the application of topical insulin, CCL chemokines, micro RNAs, stem cells, stem-cell-derived exosomes, skin substitutes, antioxidants, and the most recent Phase III-approved ON101 topical cream, have been shown to modulate the activity of M1 and M2 macrophages in DFUs. ON101, the newest clinically approved product in this setting, is designed specifically to down-regulate M1 macrophages and further modulate the wound microenvironment to favour M2 emergence and expansion. Finally, the recent evolution of macrophage modulation therapies and techniques will improve the effectiveness of the treatment of diverse DFUs.

Immune cells are involved in the pathogenesis of osteoarthritis (OA). CD4(+) T cells were activated during the onset of OA and induced macrophage inflammatory protein (MIP)-1γ expression and subsequent osteoclast formation. We evaluated the effects of local knockdown of MIP-1γ in a mouse OA model induced by anterior cruciate ligament transection. The mouse macrophage cell lines and osteoclast-like cells generated from immature hematopoietic monocyte/macrophage progenitors of murine bone marrow were cocultured with either receptor activator of NFκB ligand (RANKL) or CD4(+) T cells. The levels of MIP-1γ and RANKL in cells and mice were examined by enzyme-linked immunosorbent assay (ELISA). The osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase and cathepsin K staining. OA was induced in one hind-leg knee joint of B6 mice. Lentiviral vector encoding MIP-1γ small hairpin RNA (shRNA) and control vector were individually injected intra-articularly into the knee joints, which were histologically assessed for manifestations of OA. The expression of MIP-1γ and matrix metalloproteinase (MMP)-13 and the infiltration of CD4(+) T cells, macrophages, and osteoclastogenesis in tissues were examined using immunohistochemistry. CD4(+) T cells were involved in OA by inducing MIP-1γ expression in osteoclast progenitors and the subsequent osteoclast formation. Neutralizing MIP-1γ with a specific antibody abolishes RANKL-stimulated and CD4(+) T-cell-stimulated osteoclast formation. MIP-1γ levels were significantly higher in synovium and the chondro-osseous junction of joints 90 days postsurgery. The number of infiltrated CD4(+) T cells and macrophages and IL-1β expression were reduced in the synovial tissues of mice treated with MIP-1γ shRNA. Histopathological examinations revealed that mice treated with MIP-1γ shRNA had less severe OA than control mice had, as well as decreased osteoclast formation and MMP-13 expression. Locally inhibiting MIP-1γ expression may ameliorate disease progression and provide a new OA therapy.

Platinum-based drug like cisplatin is the standard first-line drug to treat patients with bladder cancer. However, tumors may recur and develop into multiple-drug resistant characteristics. The acquisition of resistance to conventional chemotherapy is a challenge in the treatment of bladder cancer relapse. Cancer stem cell (CSC) hypothesis has been an attractive theory. CSCs can resist therapeutic assaults, giving rise to multiple drug resistance and promotion of tumor relapse and metastasis. It has been shown that the fraction of cancer cells expressing MDR1 that functions as an energy-dependent drug efflux pump may be associated with Oct-4 expression. We have demonstrated that Oct-4 expression in bladder cancer predicts tumor progression. In the present study, we proposed that Oct-4 expression in bladder cancer increases MDR1 gene expression and thereby results in multiple drug resistance. We found a positive correlation between Oct-4 and MDR1 expression levels in clinical samples of bladder cancer. Furthermore, overexpression of Oct-4 increased MDR1 expression, which resulted in poor response to cisplatin in human bladder transitional cell carcinoma cells. Conversely, knockdown of Oct-4 reduced MDR1 expression and rendered cells hypersensitive to cisplatin. We also verified that Oct-4 transactivated the MDR1 gene promoter by binding to the Oct-4 response element (ORE). More importantly, Oct-4 and MDR1 gene expression could be induced by treatment with cisplatin, suggesting that Oct-4 is a member of the MDR1 enhanceosome. Our data can explain, in part, the high recurrence rate and drug resistance of bladder cancer. For clinical implication, we demonstrated that reduction of Oct-4 expression by all-trans retinoic acid, a derivative of vitamin A, improved sensitivity of bladder cancer cells to gemcitabine and cisplatin. In addition, we found that high expression levels of Oct-4 and MDR1 were associated with high tumor recurrence. Taken together, our results provide evidence that Oct-4 plays an important role in cisplatin-acquired resistance in bladder cancer. They also implicate Oct-4 as a therapeutic target. Note: This abstract was not presented at the meeting. Citation Format: Chia-Sing Lu, Ai-Li Shiau, Gia-Shing Shieh, Bing-Hua Su, Wu-Chou Su, Wen-Horng Yang, Chao-Liang Wu. Acquisition of Oct-4 upregulation transactivates MDR1 and is associated with increased tumor recurrence in bladder cancer. [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 4701. doi:10.1158/1538-7445.AM2014-4701