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Hemogenic endothelial (HE) cells are specialized endothelial cells to give rise to hematopoietic stem/progenitor cells during hematopoietic development. The underlying mechanisms that regulate endothelial-to-hematopoietic transition (EHT) of human HE cells are not fully understand. Here, we identified platelet endothelial aggregation receptor-1 (PEAR1) as a novel regulator of early hematopoietic development in human pluripotent stem cells (hPSCs). We found that the expression of PEAP1 was elevated during hematopoietic development. A subpopulation of PEAR1

Ruibin Wang,1 Yuchen Li,2 Yanjie Zhao,3 Feng Shi,4 Quan Zhou,5 Jiangping Wu,6 Shuzhen Lyu,7 Qingkun Song8,9 1Department of Emergency, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 2Cell and Molecular Biology, Sid Faithfull Brain Cancer Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia; 3Department of Medical Oncology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 4Department of Pathology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 5Department of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, People’s Republic of China; 6Department of Cancer Research, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 7Department of Breast Surgery, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 8Department of Clinical Epidemiology, Beijing Youan Hospital, Capital Medical University, Beijing, People’s Republic of China; 9Department of Clinical Epidemiology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of ChinaCorrespondence: Qingkun Song, Department of Clinical Epidemiology, Beijing Youan Hospital, Capital Medical University, Xitoutiao 8, Fengtai Dist, Beijing, People’s Republic of China, Email songqingkun@aliyun.comBackground: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Metformin has been shown to have the potential to inhibit the proliferation of malignant cells. This study aimed to investigate the regulatory effect of metformin on phenotypic tumor-infiltrated lymphocytes (TILs) and mechanisms in TNBC.Methods: Microarray analysis was performed on 4T1 cells post metformin treatment. BALB/c mice were inoculated with 4T1 cells with knockdown/overexpression of C-Jun N-terminal kinase (JNK), and administered with metformin. Phenotypic TILs in the tumor microenvironment (TME) were visualized by immunofluorescence staining.Results: Metformin inhibited 4T1 cell proliferation and increased expression of JNK by 21% in vitro. In vivo, Metformin increased cell counts of CD4+ and CD8+TILs by 100% and 85%, respectively, and the increase of TILs was associated with JNK pathway. Cell counts of CD4+/PD-1+ and CD8+/PD-1+TILs were reduced by 64% and 58%, respectively, post metformin treatment, but the reduction of exhausted TILs was not associated with JNK pathway. Metformin induced a 11% and 20% reduction of IL-6 and TNF-α level in the TNBC model.Conclusion: Our study demonstrated that metformin increased the functional phenotype of TILs and associated with JNK pathway, and suppressed the exhausted phenotype of TILs independently to JNK pathway in TNBC microenvironment. Further studies are needed to explore the basic mechanism of action of the drug. Metformin has potentially enhanced efficacy when used in combination with immunotherapy against TNBC.Keywords: triple-negative breast cancer, metformin, phenotypic tumor-infiltrated lymphocytes, C-Jun N-terminal kinase

In this contribution, a series of ionic liquids (ILs) with tri-n-butylethyl-phosphonium cation ([P4442]) and functional anions are designed and prepared, including pyridinolate anion ([PyO]), pyrid...

Purpose. The immune checkpoint inhibitor is approved for breast cancer treatment, but the low expression of PD-L1 limits the immunotherapy. CD155 is another immune checkpoint protein in cancers and interacts with ligands to regulate immune microenvironment. This study is aimed at investigating the expression of CD155 and the association with prognosis and pathological features of breast cancer. Methods. 126 patients were recruited this cohort study consecutively, and CD155 expression on tumor cells was detected by immunohistochemistry. The Kaplan-Meier survival curve and Cox hazard regression model were used to estimate the association. Results. 38.1% patients had an overexpression of CD155, and the proportion of tumor cells with CD155 overexpression was 17%, 39%, 37%, and 62% among Luminal A, Luminal B, HER2-positive, and triple negative breast cancer cases, respectively (p<0.05). Patients with CD155 overexpression had the Ki-67 index significantly higher than that of patients with low expression (42% vs. 26%). Though the number of tumor-infiltrating lymphocytes was higher among patients with CD155 overexpression (144/HPF vs. 95/HPF), the number of PD-1+ lymphocytes was significantly higher (52/HPF vs. 25/HPF, p<0.05). Patients of CD155 overexpression had the disease-free and overall survival decreased by 13 months and 9 months, respectively (p<0.05). CD155 overexpression was associated with an increased relapse (HR=13.93, 95% CI 2.82, 68.91) and death risk for breast cancer patients (HR=5.47,1.42,20.99). Conclusions. Overexpression of CD155 was correlated with more proliferative cancer cells and a dysfunctional immune microenvironment. CD155 overexpression introduced a worse relapse-free and overall survival and might be a potential immunotherapy target for breast cancer.

Hematopoietic differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic cell and gene regulatory networks but often generates blood cells that lack natural function. Here, we performed extensive single-cell transcriptomic analyses to map fate choices and gene expression patterns during hematopoietic differentiation of hPSCs and showed that oxidative metabolism was dysregulated during in vitro directed differentiation. Applying hypoxic conditions at the stage of endothelial-to-hematopoietic transition in vitro effectively promoted the development of arterial specification programs that governed the generation of hematopoietic progenitor cells (HPCs) with functional T cell potential. Following engineered expression of the anti-CD19 chimeric antigen receptor, the T cells generated from arterial endothelium-primed HPCs inhibited tumor growth both in vitro and in vivo. Collectively, our study provides benchmark datasets as a resource to further understand the origins of human hematopoiesis and represents an advance in guiding in vitro generation of functional T cells for clinical applications.

The cover image is based on the Original Manuscript Vitronectin‐activated αvβ3 and αvβ5 integrin signalling specifies haematopoietic fate in human pluripotent stem cells by Jun Shen et al., https://doi.org/10.1111/cpr.13012. [Image: see text]

Objectives Vitronectin (VTN) has been widely used for the maintenance and expansion of human pluripotent stem cells (hPSCs) as feeder‐free conditions. However, the effect of VTN on hPSC differentiation remains unclear. Here, we investigated the role of VTN in early haematopoietic development of hPSCs. Materials and Methods A chemically defined monolayer system was applied to study the role of different matrix or basement membrane proteins in haematopoietic development of hPSCs. The role of integrin signalling in VTN‐mediated haematopoietic differentiation was investigated by integrin antagonists. Finally, small interfering RNA was used to knock down integrin gene expression in differentiated cells. Results We found that the haematopoietic differentiation of hPSCs on VTN was far more efficient than that on Matrigel that is also often used for hPSC culture. VTN promoted the fate determination of endothelial‐haematopoietic lineage during mesoderm development to generate haemogenic endothelium (HE). Moreover, we demonstrated that the signals through αvβ3 and αvβ5 integrins were required for VTN‐promoted haematopoietic differentiation. Blocking αvβ3 and αvβ5 integrins by the integrin antagonists impaired the development of HE, but not endothelial‐to‐haematopoietic transition (EHT). Finally, both αvβ3 and αvβ5 were confirmed acting synergistically for early haematopoietic differentiation by knockdown the expression of αv, β3 or β5. Conclusion The established VTN‐based monolayer system of haematopoietic differentiation of hPSCs presents a valuable platform for further investigating niche signals involved in human haematopoietic development., Compared with Matrigel (MTG), vitronectin (VTN) was required for the mesoderm to acquire higher endothelial‐hematopoietic potential. The promoting effect of VTN on early hematopoiesis was dependent on αvβ3 and αvβ5 integrins. Inhibition of αvβ3 and αvβ5 impaired HE development without affecting EHT.

A series of pyridinolate anion-functional ionic liquids (ILs) containing different kinds of cations were designed and prepared, including tri-n-hexyltetradecylphosphonium [P66614], tri-n-butylethylphosphonium [P4442], and 1,1,3,3-tetramethylguandinium [TMG] cations and pyridinolate [PyO] anions with different N positions. Densities and viscosities at different temperatures were measured because of their importance for the absorption of SO2. Effect of different structures on the absorption of SO2 was studied, including the type and the alkyl chain length of cations, and the N positions of [PyO] anions (2-, 3-, and 4-). Additionally, effects of SO2 concentration and temperature for absorption were investigated. Especially, up to 6.00 and 2.53 mol SO2 per mole IL could be obtained by [P66614][4-PyO] at 1 bar and 0.1 bar of SO2, respectively, when absorptions performed at 20 °C. The results indicate that (1) the aprotic cations weakened the cation⋯anion interaction and resulted in the released active sites on the anions, and (2) N position has an influence on charge distribution of [PyO] anions and resulted in the tunable absorption of SO2. Reversible SO2 absorption and [P66614][4-PyO] regeneration could be performed several times without losing efficiency. Furthermore, the mechanism of absorption was studied through FT-IR and NMR methods and the results showed that the absorption of SO2 by [P66614][4-PyO] includes physisorption and chemisorption.

Deep eutectic solvents (DESs) have been identified as optimal gas absorbents. A tertiary amine-containing task-specific DES AChBr+Gly and a conventional DES ChBr+Gly have been designed, prepared and used to capture SO2. It is found that absorption capacity of 3.74 and 2.97 mol of SO2 per mole of DES can be obtained by AChBr+Gly and ChBr+Gly, respectively, at 293.2 K and 1 bar. When SO2 partial pressure was decreased to 0.01 bar, the capacity decreased to 0.93 and 0.13 mol SO2 of per mole of AChBr+Gly and ChBr+Gly, respectively. Mechanism and thermodynamic studies show that absorption by former absorbent includes physisorption, quasi-chemisorption (a strong physisorption) and chemisorption, while absorption by later absorbent only includes physisorption and quasi-chemisorption. Two equations were first proposed for the description of SO2 absorption. In these equations, the physisorption capacity is correlated with Henry's law, and the quasi-chemisorption and chemisorption are correlated with the quasi-chemical equilibrium constant of halogen···SO2 interaction and the chemical equilibrium constant of tertiary amine···SO2 interaction, respectively. In addition, the corresponding enthalpy change, entropy change, and Gibbs energy change have been calculated and discussed.

Human pluripotent stem cells (hPSCs) provide a powerful platform for generating functional hematopoietic cells for blood disease modeling and therapeutic testing. However, the quantity and quality of hPSC-derived blood cells remain to be improved. Here, by performing extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during hematopoietic differentiation of hPSCs, we construct the first hematopoietic landscape of hPSCs at the single-cell level and identify strategies to promote hematopoietic progenitor (HP) generation from hPSCs with functional T cell potential. By focusing specifically on cell populations and molecular events involved in endothelial-to-hematopoietic transition (EHT), we compared the difference of early hematopoiesis between hPSCs and human embryos (Yang Zeng et al. Cell Research. 2019) and found aerobic metabolism was dysregulated during in-vitro-directed differentiation. The decreased oxygen metabolism program was further deciphered as a key molecular event occurred during the EHT. Providing hypoxia at the stage of EHT enhanced hematopoietic differentiation of hPSCs via specifying arterial programs, including arterial hemogenic endothelium (AHE) and arterial endothelium cells (AE). To further determine the effect of AE on hematopoietic development, we isolated AE, venous endothelium and mesenchymal cells identified in our single-cell transcriptomic analyses and cocultured them with AHE respectively for HP generation. AE were finally validated as a critical regulator of definitive HP specification with more T cell potential. T cells generated from AE-primed HPs (AE-T) were highly functional and exhibited polyfunctional production of interferon (IFN)-γ, tumor necrosis factor alpha (TNF-α), and IL-2 in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin. To further evaluate the function of AE-T, we engineered T cells with CD19-CAR. The in vitro cytotoxicity of CAR-engineered AE-T was performed both in CD19+ cell lines (Nalm-6 and Raji) and human primary B-ALL samples. The efficacy of CAR-engineered AE-T in vivo was evaluated in a mouse xenograft model inoculated intravenously with luciferase-expressing Nalm-6 cells. Similar to CD19 CAR-transduced peripheral blood T cells, the AE-T potently inhibited tumor growth both in vitro and in vivo. Collectively, our study provides benchmark datasets to understand the origins of human hematopoiesis and presents an advance for guiding the generation of functional T cells in vitro for clinical applications. Disclosures No relevant conflicts of interest to declare.

Human pluripotent stem cells (hPSCs) provide a powerful platform for studying the dynamic molecular network towards hematopoiesis. To date, a comprehensive roadmap at single-cell level for hPSC-derived hematopoietic differentiation has not been established. Here, we performed single-cell transcriptomic analyses to map gene expression programs during hematopoietic differentiation of hPSCs, from which new strategies to improve in vitro hematopoietic differentiation may be thus developed. We firstly established a serum-free, stroma-free and chemically defined monolayer system for hematopoietic differentiation of hPSCs, which enabled single-cell transcriptomic analysis. Through identifying sequential molecular events, we generated a time-course gene expression profile revealing the temporally restricted expression dynamics of stage-specific genes associated with hematopoietic cell fate choices. To better understand the lineage trajectories and gene regulatory networks governing diversification of cell fates, we established transcription factor regulatory networks predicting developmental fate choices during hematopoietic differentiation. By pseudotime analysis, we constructed the endothelial-hematopoietic trajectory revealing a novel hematopoietic development model that hemogenic endothelium initiated first and then differentiated into arteries and veins, whereas arteries had the ability to promote endothelial-to-hematopoietic transition. Moreover, we found that the genes related to aerobic metabolism were significantly decreased in hematopoietic cells and hypoxia-primed hematopoietic differentiation showed higher potency for generating more hematopoietic lineages. Further studies showed that hypoxia enhanced hematopoietic differentiation by promoting the development of arterial endothelium, rather than directly acting on hemogenic endothelium.