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Tumor-associated macrophages (TAMs) are crucially associated with tumor development and progression; however, it remains unclear how the tumor microenvironment (TME) rewires the metabolic circuits and preferentially induces TAMs to polarize toward a protumoral phenotype. Here, we report that polyunsaturated fatty acids (PUFAs) in malignant ascites promote protumoral M2-like TAMs deposition and facilitate peritoneal metastases of epithelial ovarian cancer (EOC). We demonstrated that PUFAs in the lipid-enriched malignant ascites inactivate RhoA, reducing nuclear YAP1 in macrophages and promoting protumoral M2-like TAMs polarization with OXPHOS metabolism. Conditional Yap1 depletion in murine MΦs leads to skew macrophage polarization toward protumoral M2-like TAMs that, in turn, suppress CD8+ T cell infiltration and aggravate tumor colonization in vivo. Noticeably, the significance of nuclear YAP1 depletion was evinced in the infiltrating TAMs in tumor spheroids of malignant ascites from EOC patients. In contrast, restored nuclear YAP1 expression in TAMs by pharmacological suppression of MST1/2 enhances tumoricidal M1-like TAMs population and CD8+ T cells infiltration, restricting EOC peritoneal metastasis. These results indicate that PUFAs are a key player in promoting tumor-infiltrated TAMs polarization that, in turn, facilitates EOC tumor growth and metastasis.

Refractive disorder is the most prevalent cause of visual impairment worldwide. While treatment of refractive errors can bring improvement to quality of life and socio-economic benefits, there is a need for individualization, precision, convenience, and safety with the chosen method. Herein, we propose using pre-designed refractive lenticules based on poly-NAGA-GelMA (PNG) bio-inks photo-initiated by digital light processing (DLP)-bioprinting for correcting refractive errors. DLP-bioprinting allows PNG lenticules to have individualized physical dimensions with precision achievable to 10 µm (μm). Material characteristics of PNG lenticules in tests included optical and biomechanical stability, biomimetical swelling and hydrophilic capability, nutritional and visual functionality, supporting its suitability as stromal implants. Cytocompatibility distinguished by morphology and function of corneal epithelial, stromal, and endothelial cells on PNG lenticules suggested firm adhesion, over 90% viability, phenotypic maintenance instead of excessive keratocyte-myofibroblast transformation. In-vitro immune response analyzed by illumina RNA sequencing in human peripheral blood mononuclear cells indicated that PNG lenticules activated type-2 immunity, facilitating tissue regeneration and suppressing inflammation. In-vivo performance assessed using intrastromal keratoplasty models in New Zealand white rabbits illustrated that implantation of PNG lenticules maintained stable optical pathway, induced controlled stromal bio-integration and regeneration, avoided complications such as stromal melt, interface scarring, etc, but exerted no adverse effects on the host. Postoperative follow-up examination on intraocular pressure, corneal sensitivity, and tear production remained unaffected by surgery up to 1-month post-implantation of PNG lenticules. DLP-bioprinted PNG lenticule is a bio-safe and functionally effective stromal implants with customizable physical dimensions, providing potential therapeutic strategies in correction of refractive errors.

Programmed cell death 1 ligand (PD-L1) blockade has been used therapeutically in the treatment of ovarian cancer, and potential combination treatment approaches are under investigation to improve the treatment response rate. The increased dependence on glutamine is widely observed in various type of tumors, including ovarian cancer. Kidney-type glutaminase (GLS), as one of the isotypes of glutaminase, is found to promote tumorigenesis. Here, we have demonstrated that the combined treatment with GLS inhibitor 968 and PD-L1 blockade enhances the immune response against ovarian cancer. Survival analysis using the Kaplan–Meier plotter dataset from ovarian cancer patients revealed that the expression level of GLS predicts poor survival and correlates with the immunosuppressive microenvironment of ovarian cancer. 968 inhibits the proliferation of ovarian cancer cells and enhances granzyme B secretion by CD8+ T cells as detected by XTT assay and flow cytometry, respectively. Furthermore, 968 enhances the apoptosis-inducing ability of CD8+ T cells toward cancer cells and improves the treatment effect of anti-PD-L1 in treating ovarian cancer as assessed by Annexin V apoptosis assay. In vivo studies demonstrated the prolonged overall survival upon combined treatment of 968 with anti-PD-L1 accompanied by increased granzyme B secretion by CD4+ and CD8+ T cells isolated from ovarian tumor xenografts. Additionally, 968 increases the infiltration of CD3+ T cells into tumors, possibly through enhancing the secretion of CXCL10 and CXCL11 by tumor cells. In conclusion, our findings provide a novel insight into ovarian cancer cells influence the immune system in the tumor microenvironment and highlight the potential clinical implication of combination of immune checkpoints with GLS inhibitor 968 in treating ovarian cancer.

Malignant ascites in peritoneal metastases is a lipid-enriched microenvironment and is frequently involved in the poor prognosis of epithelial ovarian cancer (EOC). However, the detailed mechanisms underlying ovarian cancer (OvCa) cells dictating their lipid metabolic activities in promoting tumor progression remain elusive. Here, we report that two critical fatty acid desaturases, stearoyl-CoA desaturase-1 (SCD1) and acyl-CoA 6-desaturase (FADS2), are aberrantly upregulated, accelerating lipid metabolic activities and tumor aggressiveness of ascites-derived OvCa cells. Lipidomic analysis revealed that the elevation of unsaturated fatty acids (UFAs) is positively associated with SCD1/FADS2 levels and the oncogenic capacities of OvCa cells. In contrast, pharmaceutical inhibition and genetic ablation of SCD1/FADS2 retarded tumor growth, suppressed cancer stem cell (CSC) formation and reduced platinum resistance in OvCa cells. Mechanistically, inhibition of SCD1/FADS2 directly downregulated GPX4 and the GSH/GSSG ratio, causing disruption of the cellular redox balance and subsequent iron-mediated lipid peroxidation in ascites-derived OvCa cells. Hence, combinational treatment with SCD1/FADS2 inhibitors and cisplatin synergistically repressed tumor cell dissemination, providing a promising chemotherapeutic strategy against EOC platinum resistance and peritoneal metastases.

Purpose The mutation status and prognostic value of PIK3CA in breast cancer were widely investigated, which showed significant difference among the patients from vast areas around the world. In this study, the frequency, distribution, bias, and burden of PIK3CA mutations and their relationships with clinicopathologic variables and prognostic significances were investigated in the breast cancer patients from Central China. Materials and methods Somatic mutations in exon 9 and exon 20 of PIK3CA gene were analyzed using Sanger sequencing combining with targeted next generation sequencing in 494 breast cancer patients from Central China. The correlations between PIK3CA mutations and clinicopathological characteristics and the prognostic values of multiple PIK3CA mutation statuses were evaluated. Results PIK3CA mutations were found in 38% of the patients and associated with estrogen receptor-positive, progesterone receptor-positive, low Ki67 labeling index, and luminal/human epidermal growth factor receptor 2-enriched subtypes. Meanwhile, the prognosis of the total patients and the patients in old diagnostic age, progesterone receptor-negative, low Ki67 labeling index, and luminal/human epidermal growth factor receptor 2-enriched subgroups was significantly related to PIK3CA mutations. Most interestingly, the distribution, bias, and burden of PIK3CA mutations were correlated with different clinical, pathological, and molecular features as well as distinct prognostic implications in multiple breast cancer subgroups. Conclusion The frequency, distribution, bias, and burden of PIK3CA mutations were associated with various clinical, pathological, and molecular characteristics in the breast cancer patients from Central China. These different mutation statuses can be used as potential indicators of prognosis in multiple breast cancer subgroups.

Purpose To study the in vitro effect of vitamin D3 on the healing response of human Tenon's fibroblasts (HTF) and its possible role in preventing excessive postoperative subconjunctival fibrosis. Methods Effect of vitamin D3 on cytotoxicity and cell survival of primary cultured HTF was measured by lactate dehydrogenase and PrestoBlue assays, respectively. Proliferation and migration of vitamin D3-treated HTF (D3-HTF) was determined by CyQUANT proliferation and scratch assay, respectively. The mRNA expression profiles of control-HTF and D3-HTF from six subjects (three with glaucoma and long-term use of topical medications, three with primary pterygium) were assessed by RNA sequencing analyses to identify potential biomarkers for the inhibitory effect on HTF by vitamin D3. Validation of these biomarkers and their potential pathways were performed by quantitative real-time polymerase chain reaction (qRT-PCR) detection. Results Pure monolayers of HTF from controls (retinal detachment or squint surgeries), pterygium, and glaucoma subjects were successfully prepared and passaged. Proliferation and migration of pterygium and glaucoma HTF were inhibited by vitamin D3 in a dose-dependent manner, and without cytotoxicity or decrease in cellular viability with concentrations up to 10 µM. The qRT-PCR results were consistent with the transcriptome analyses, vitamin D3 appears to enhance CYP24A1, SHE, KRT16 but suppresses CILP expression in HTF. Conclusions Vitamin D3 can inhibit the in vitro activity of HTF without compromising cellular survivability at concentration up to 10 µM. This has potential clinical application for improving the outcome of pterygium and filtering surgeries. Translational relevance Vitamin D3 can suppress the in vitro proliferation, migration, and transdifferentiation of human Tenon's fibroblasts, without the cytotoxicity of mitomycin-C, the current standard antifibrotic agent in clinical use.

// Huogang Wang 1, 2, 3, * , Bo Hong 1, 3, * , Xuemin Li 1, 3 , Ke Deng 1, 2, 3 , Hong Li 1, 3 , Vivian Wai Yan Lui 4 and Wenchu Lin 1, 3 1 High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei 230031, Anhui, P.R. China 2 University of Science and Technology of China, Hefei 230036, Anhui, P.R. China 3 Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui, P.R. China 4 School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, P.R. China * Co-first authorship Correspondence to: Wenchu Lin, email: wenchu@hmfl.ac.cn Keywords: small cell lung cancer, N-Myc, Bcl-2, JQ1, ABT-263 Received: April 05, 2017 Accepted: August 26, 2017 Published: September 21, 2017 ABSTRACT Small cell lung cancer (SCLC) is a clinically aggressive cancer with very poor prognosis. Amplification of MYC family genes and overexpression of Bcl-2 protein are common in SCLC, and they are likely therapeutic targets for SCLC. Previous clinical study showed that single agent targeting Bcl-2 with ABT-263 was of limited efficacy in SCLC. In this study, we demonstrated for the first time that co-targeting of N-Myc and Bcl-2 resulted in marked synergistic antitumor effects in MYCN -amplified SCLC. We found that MYCN -amplified SCLC cells were highly sensitive to a Bromodomain and Extra-Terminal domain (BET) inhibitor JQ1, which was able to inhibit N-Myc protein expression. The inhibition of N-Myc by JQ1 induced the expression of Bim, and thereby sensitizing MYCN -amplified SCLC cells to ABT-263. The knockdown on Bim by siRNA reduced this JQ1/ABT-263 induced cell death. ABT-263 and JQ1 co-treatment in MYCN -amplified SCLC cells markedly disrupted Bim/Bcl-2 interaction, and prevented Bim’s interaction with Mcl-1. Importantly, this JQ1/ABT-263 co-targeting substantially inhibited the growth of MYCN -amplified SCLC xenografts in vivo . Our study demonstrates a new JQ-1/ABT-263 co-targeting strategy that can be employed for MYCN -amplified SCLC with high efficacy.

Background Cisplatin-based chemotherapy and radiotherapy are the most commonly used treatments for small cell lung cancer (SCLC). However, despise initially dramatic response, the response duration of SCLC patients is variable and resistance to chemo- and radio-therapy inevitably develops. Objective The aim of the study is to investigate the role of Bcl-2 family proteins in predicting SCLC sensitivity to cisplatin treatment, and to identify the potential sensitizer of cisplatin or ratiation treatment in SCLC. Methods We collected cisplatin sensitivity data from public available database, and evaluated its possible association with mRNA or protein expression of Bcl-2 family members in SCLC cell lines. Results The IC50 value of cisplatin was significantly correlated with the ratio of Bcl-2/Bim mRNA expression in 33 SCLC cell lines (P= 0.041) as well as the ratio of Bcl-2/Bim protein expression in 7 SCLC cell lines (P= 0.0252). Furthermore, a BH3-mimetic ABT-263 was found to be able to sensitize SCLC cells to cisplatin or radiation. The synergistic and additive antitumor activity of ABT-263 combined with cisplatin or radiation was associated with the enhanced apoptosis, which may be caused by the disruption of Bcl-2 binding to Bim by ABT-263. Conclusions Our study indicates that the ratio of Bcl-2/Bim could be a SCLC response predictor to cisplatin, and ABT-263 addition could be an effective strategy to improve the activity of chemo- or radio-therapy in SCLC.

Advanced ovarian cancers with peritoneal metastases are usually accompanied with metastatic recurrence and high mortality. Macrophages, as a primary cell population in the tumor microenvironment, can be active and polarized into heterogeneous tumor-associated macrophages (TAMs). Clinical evidence indicates that a high M2/M1 ratio of TAMs are commonly found in malignant ascites microenvironment (MAM), and is attributed to a poor prognosis in advanced ovarian cancers. However, the molecular mechanisms of macrophage polarization in MAM remain elusive. Here, we report that MAM could modulate Hippo and/or Wnt signaling cascades in facilitating M2 macrophage polarization. In this study, we firstly evidenced the heterogeneity macrophage populations in cancerous omental tissues and ascites cancer cell spheroids of ovarian cancers by multiplex immunohistochemical, flow cytometry and immunofluorescent analyses. Consistently, in vitro polarization model demonstrated that co-cultured monocyte (THP-1 and U937) with omentum conditioned medium (OCM) or ascites significantly led to more CD206+ M2 macrophage than CD86+ M1 macrophage or M2/M1 ratio. Quantitative RT-PCR (qPCR) analysis showed that TAM-related genes: CCL2, IL6, and IL10 genes were significantly upregulated in OCM treated macrophage. Intriguingly, OCM treated macrophage could significantly promotes ovarian cancer cell migratory capacities. Importantly, RNA-seq analysis identified Hippo and Wnt signaling cascades may play important roles in macrophage polarization in malignant OCM and ascites. Further investigations of these pathways in M2/M1 macrophage polarization in MAM are warranted. Citation Format: Huogang Wang, Mingo MH Yung, Arvin FS Chen, Hextan YS Ngan, David W. Chan. Maligant ascites microenvironment skews M2/M1 macrophage polarization in ovarian cancer during peritoneal metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 980.

Small cell lung cancer (SCLC) is a highly malignant cancer with few targeted therapies. In the study, by mining the Cancer Cell Line Encyclopedia (CCLE) database, we found that PI3K/AKT/mTOR pathway was aberrant in 92% of SCLC cell lines. Moreover, we found that the phosphorylation level of 4E-BP1 was significantly correlated with SCLC sensitivity to RAD001 (mTOR inhibitor) and BEZ235 (PI3K/mTOR dual inhibitor). Combination of RAD001 and BEZ235 synergistically inhibited the growth of SCLC cells, which was accompanied by enhanced induction of cell cycle arrest and apoptosis. Such a combination dramatically inhibited the activation of AKT, and strongly reduced the phosphorylation of 4E-BP1 and its downstream target Mcl-1. Knock-down of Mcl-1 enhanced the growth inhibition of SCLC cells induced by RAD001 and BEZ235 co-treatment, whereas over-expression of Mcl-1 reduced the growth inhibitory effect. Furthermore, in vivo study demonstrated that the combination treatment suppressed tumor growth more effectively than RAD001 or BEZ235 treatment alone. In summary, our study suggests that combination of BEZ235 and RAD001 may be an effective regimen for SCLC treatment, and p-4E-BP1 may serve as a predictive biomarker for SCLC response to mTOR inhibitor.