Cord blood stem cell­derived Angptl7 ameliorates the severity of bronchopulmonary dysplasia via anti­inflammatory and proangiogenic effects.

Perinatal exposure of the neonatal lung to inflammation leads to decreased lung angiogenesis and the development of bronchopulmonary dysplasia (BPD). Notably, autologous cord blood mononuclear cells (ACBMNCs) can substantially prevent severe BPD and decrease the inflammatory response in surviving very preterm neonates. Angiopoietin­like protein 7 (Angptl7) is one of the main paracrine cytokines in cord blood stem cells, and is capable of stimulating human hematopoietic stem and progenitor cell expansion. The present study compared Angptl7 levels between the ACBMNCs infusion and control groups (cohort 1). Subsequently, the association between cord blood Angptl7 levels and BPD incidence in a cohort of very preterm neonates was assessed (cohort 2). The hypothesis was further verified in a lipopolysaccharide (LPS)­induced lung injury mouse model. The mRNA expression levels and protein concentrations of inflammatory cytokines in the lung tissue and mouse serum were measured using reverse transcription­quantitative PCR and ELISA, respectively. The number and diameter of lung vessels and macrophage infiltration were assessed using immunofluorescence staining. Compared with in the control group, Angptl7 levels were significantly higher in the ACBMNCs infusion group in cohort 1. In cohort 2, the cord blood Angptl7 levels were significantly lower in infants who later developed BPD. Multiple linear regression analysis showed that higher Angptl7 level was an independent protective factor for BPD. The concentrations of interleukin­6 and monocyte chemoattractant protein­1 were negatively correlated with cord blood Angptl7 level; whereas, vascular endothelial growth factor­A levels were positively correlated with Angptl7 levels. In the LPS­induced lung injury mouse model, the LPS group presented with a significant loss of pulmonary vessels and smaller vessel diameters, which were ameliorated in the Angptl7 treatment group. Furthermore, LPS­induced lung inflammation and macrophage infiltration were alleviated by Angptl7 treatment (P<0.05). In conclusion, the anti­inflammatory and proangiogenic effects of Angptl7 derived from cord blood stem cells may ameliorate BPD severity. The trial for cohort 1 was registered at ClinicalTrials.gov (trial registration no. NCT02999373; date registered, December 21, 2016).

[SWI/SNF Complex Gene Mutations Promote the Liver Metastasis 
of Non-small Cell Lung Cancer Cells in NSI Mice].

BACKGROUND: The switch/sucrose nonfermentable chromatin-remodeling (SWI/SNF) complex is a pivotal chromatin remodeling complex, and the genomic alterations (GAs) of the SWI/SNF complex are observed in several cancer types, correlating with multiple biological features of tumor cells. However, their role in liver metastasis of non-small cell lung cancer (NSCLC) remains unclear. Our study aims to investigate the role and potential mechanisms underlying NSCLC liver metastasis induced by the GAs of SWI/SNF complex. METHODS: The GAs of SWI/SNF complex in NSCLC cell lines (H1299, H23 and H460) were identified by whole-exome sequencing (WES). ARID1A knockout H1299 cell was constructed with the CRISPR/Cas9 technology. The mouse model of liver metastasis from NSCLC was established to simulate lung cancer liver metastasis and observe the metastasis rate under different gene mutation conditions. RNA sequencing and Western blot were conducted for differential gene expression analysis. Immunohistochemistry (IHC) analysis was used to assess protein expression levels of SWI/SNF-regulated target molecules in mouse liver metastases. RESULTS: WES analysis revealed intracellular gene mutations. The animal experiments demonstrated a correlation between the GAs of SWI/SNF complex and a higher liver metastasis rate in immunodeficient mice. Transcriptome sequencing and Western blot analysis showed upregulated expression of ALDH1A1 and APOBEC3B in SWI/SNF-mut cells, particularly in ARID1A-deficient H460 and H1299 sgARID1A cells. IHC staining of mouse liver metastases further demonstrated elevated expression of ALDH1A1 in the H460 and H1299 sgARID1A group. CONCLUSIONS: This study underscores the critical role of the GAs of SWI/SNF complex, such as ARID1A and SMARCA4, in promoting liver metastasis of lung cancer cells. The GAs of SWI/SNF complex may promote liver-specific metastasis by upregulating ALDH1A1 and APOBEC3B expression, providing novel insights into the molecular mechanisms underlying lung cancer liver metastasis.

T lymphocytes expressing the switchable chimeric Fc receptor CD64 exhibit augmented persistence and antitumor activity.

Chimeric antigen receptor (CAR) T cell therapy lacks persistent efficacy with "on-target, off-tumor" toxicities for treating solid tumors. Thus, an antibody-guided switchable CAR vector, the chimeric Fc receptor CD64 (CFR64), composed of a CD64 extracellular domain, is designed. T cells expressing CFR64 exert more robust cytotoxicity against cancer cells than CFR T cells with high-affinity CD16 variant (CD16v) or CD32A as their extracellular domains. CFR64 T cells also exhibit better long-term cytotoxicity and resistance to T cell exhaustion compared with conventional CAR T cells. With trastuzumab, the immunological synapse (IS) established by CFR64 is more stable with lower intensity induction of downstream signaling than anti-HER2 CAR T cells. Moreover, CFR64 T cells exhibit fused mitochondria in response to stimulation, while CARH2 T cells contain predominantly punctate mitochondria. These results show that CFR64 T cells may serve as a controllable engineered T cell therapy with prolonged persistence and long-term antitumor activity.

Chimeric CTLA4-CD28-CD3z T Cells Potentiate Antitumor Activity Against CD80/CD86-Positive B Cell Malignancies.

The adoptive transfer of chimeric antigen receptor T (CAR T) cells have been recognized as a promising therapeutic strategy for the treatment of hematological malignancies; however, clinical success using CAR T cells for the treatment of solid tumors are still limited since the T-cell function is inhibited by negative signals in the microenvironment of solid tumors. CTLA4 is a well-known immune checkpoint molecule, thus we developed a novel CAR by converting this negative signal to positive signal. The CAR developed consists of the extracellular and transmembrane domains of CTLA4 and the cytoplasmic domains of CD28 and CD3z (CTLA4-CAR T). CTLA4-CAR T cells exhibited superior cytokine secreting activities and cytotoxic to tumor cells in vitro and in xenograft models. CTLA4-CAR T cells were found to accumulate in tumors and are toxic to myeloid-derived suppressor cells (MDSCs) without signs of severe GVHD and CRS in preclinical models. Thus, this chimeric CTLA4-CAR can enhance the antitumor activity of CAR T cells and shed light on the strategy of using armed CAR T cells to target the immunomodulatory tumor microenvironment.

Umbilical cord blood CD34+ cells administration improved neurobehavioral status and alleviated brain injury in a mouse model of cerebral palsy.

PURPOSE: Cerebral palsy (CP) is the most common neuromuscular disease in children, and currently, there is no cure. Several studies have reported the benefits of umbilical cord blood (UCB) cell treatment for CP. However, these studies either examined the effects of UCB cell fraction with a short experimental period or used neonatal rat models for a long-term study which displayed an insufficient immunological reaction and clearance of human stem cells. Here, we developed a CP model by hypoxia-ischemic injury (HI) using immunodeficient mice and examined the effects of human UCB CD34+ hematopoietic stem cells (HSCs) on CP therapy over a period of 8 weeks. METHODS: Sixty postnatal day-9 (P9) mouse pups were randomly divided into 4 groups (n = 15/group) as follows: (1) sham operation (control group), (2) HI-induced CP model, (3) CP model with CD34+ HSC transplantation, and (4) CP model with CD34- cell transplantation. Eight weeks after insult, the sensorimotor performance was analyzed by rotarod treadmill, gait dynamic, and open field assays. The pathological changes in brain tissue of mice were determined by HE staining, Nissl staining, and MBP immunohistochemistry of the hippocampus in the mice. RESULTS: HI brain injury in mice pups resulted in significant behavioral deficits and loss of neurons. Both CD34+ HSCs and CD34- cells improved the neurobehavioral statuses and alleviated the pathological brain injury. In comparison with CD34- cells, the CD34+ HSC compartments were more effective. CONCLUSION: These findings indicate that CD34+ HSC transplantation was neuroprotective in neonatal mice and could be an effective therapy for CP.

Myeloid-derived suppressor cells promote lung cancer metastasis by CCL11 to activate ERK and AKT signaling and induce epithelial-mesenchymal transition in tumor cells.

Myeloid-derived suppressor cells (MDSCs) suppress antitumor immune activities and facilitate cancer progression. Although the concept of immunosuppressive MDSCs is well established, the mechanism that MDSCs regulate non-small cell lung cancer (NSCLC) progression through the paracrine signals is still lacking. Here, we reported that the infiltration of MDSCs within NSCLC tissues was associated with the progression of cancer status, and was positively correlated with the Patient-derived xenograft model establishment, and poor patient prognosis. Intratumoral MDSCs directly promoted NSCLC metastasis and highly expressed chemokines that promote NSCLC cells invasion, including CCL11. CCL11 was capable of activating the AKT and ERK signaling pathways to promote NSCLC metastasis through the epithelial-mesenchymal transition (EMT) process. Moreover, high expression of CCL11 was associated with a poor prognosis in lung cancer as well as other types of cancer. Our findings underscore that MDSCs produce CCL11 to promote NSCLC metastasis via activation of ERK and AKT signaling and induction of EMT, suggesting that the MDSCs-CCL11-ERK/AKT-EMT axis contains potential targets for NSCLC metastasis treatment.

Efficient gene correction of an aberrant splice site in ß-thalassaemia iPSCs by CRISPR/Cas9 and single-strand oligodeoxynucleotides.

ß-thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin ß (HBB) gene. Among them, the HBB IVS2-654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing ß-thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double-strand break (DSB), which can be combined with the single-stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2-654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of ß-Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.

Establishment of peripheral blood mononuclear cell-derived humanized lung cancer mouse models for studying efficacy of PD-L1/PD-1 targeted immunotherapy.

Animal models used to evaluate efficacies of immune checkpoint inhibitors are insufficient or inaccurate. We thus examined two xenograft models used for this purpose, with the aim of optimizing them. One method involves the use of peripheral blood mononuclear cells and cell line-derived xenografts (PBMCs-CDX model). For this model, we implanted human lung cancer cells into NOD-scid-IL2Rg-/- (NSI) mice, followed by injection of human PBMCs. The second method involves the use of hematopoietic stem and progenitor cells and CDX (HSPCs-CDX model). For this model, we first reconstituted the human immune system by transferring human CD34+ hematopoietic stem and progenitor cells (HSPCs-derived humanized model) and then transplanted human lung cancer cells. We found that the PBMCs-CDX model was more accurate in evaluating PD-L1/PD-1 targeted immunotherapies. In addition, it took only four weeks with the PBMCs-CDX model for efficacy evaluation, compared to 10-14 weeks with the HSPCs-CDX model. We then further established PBMCs-derived patient-derived xenografts (PDX) models, including an auto-PBMCs-PDX model using cancer and T cells from the same tumor, and applied them to assess the antitumor efficacies of anti-PD-L1 antibodies. We demonstrated that this PBMCs-derived PDX model was an invaluable tool to study the efficacies of PD-L1/PD-1 targeted cancer immunotherapies. Overall, we found our PBMCs-derived models to be excellent preclinical models for studying immune checkpoint inhibitors.

CD215+ Myeloid Cells Respond to Interleukin 15 Stimulation and Promote Tumor Progression.

Interleukin 15 (IL-15) regulates the development, survival, and functions of multiple innate and adaptive immune cells and plays a dual role in promoting both tumor cell growth and antitumor immunity. Here, we demonstrated that the in vivo injection of recombinant human IL-15 (200 µg/kg) or murine IL-15 (3 µg/kg) to tumor-bearing NOD-SCID-IL2Rg-/- (NSI) mice resulted in increased tumor progression and CD45+ CD11b+ Gr-1+ CD215+ cell expansion in the tumors and spleen. In B16F10-bearing C57BL/6 mice model, we found that murine IL-15 has antitumoral effect since the activation and expansion of CD8+ T cells with murine IL-15 treatment. But no enhanced or reduced tumor growth was observed in mice when human IL-15 was used. However, both murine and human IL-15 promote CD45+ CD11b+ Gr-1+ CD215+ cells expansion. In xenograft tumor models, CD215+ myeloid cells, but not CD215- cells, responded to human IL-15 stimulation and promoted tumor growth. Furthermore, we found that human IL-15 mediated insulin-like growth factor-1 production in CD215+ myeloid cells and blocking IGF-1 reduced the tumor-promoting effect of IL-15. Finally, we observed that higher IGF-1 expression is an indicator of poor prognosis among lung adenocarcinoma patients. These findings provide evidence that IL-15 may promote tumor cell progression via CD215+ myeloid cells, and IGF-1 may be an important candidate that IL-15 facilitates tumor growth.

GZD824 suppresses the growth of human B cell precursor acute lymphoblastic leukemia cells by inhibiting the SRC kinase and PI3K/AKT pathways.

Available therapeutic options for advanced B cell precursor acute lymphoblastic leukemia (pre-B ALL) are limited. Many lead to neutropenia, leaving patients at risk of life-threatening infections and result in bad outcomes. New treatment options are needed to improve overall survival. We previously showed that GZD824, a novel BCR-ABL tyrosine kinase inhibitor, has anti-tumor activity in Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia cells and tumor models. Here, we show that GZD824 decreases cell viability, induces cell-cycle arrest, and causes apoptosis in pre-B ALL cells. Furthermore, Ph- pre-B ALL cells were more sensitive to GZD824 than Ph+ pre-B ALL cells. GZD824 consistently reduced tumor loads in Ph- pre-B ALL xenografts but failed to suppress Ph+ pre-B ALL xenografts. GZD824 decreased phosphorylation of SRC kinase, STAT3, RB and C-myc. It also downregulated the expression of BCL-XL, CCND1 and CDK4 and upregulated expression of CCKN1A. Expression of IRS1 was decreased in GZD824-treated pre-B ALL cells, blocking the PI3K/AKT pathway. These data demonstrate that GZD824 suppresses pre-B ALL cells through inhibition of the SRC kinase and PI3K/AKT pathways and may be a potential therapeutic agent for the management of pre-B ALL.

Current status and perspectives of patient-derived xenograft models in cancer research.

Cancers remain a major public health problem worldwide, which still require profound research in both the basic and preclinical fields. Patient-derived xenograft (PDX) models are created when cancerous cells or tissues from patients' primary tumors are implanted into immunodeficient mice to simulate human tumor biology in vivo, which have been extensively used in cancer research. The routes of implantation appeared to affect the outcome of PDX research, and there has been increasing applications of patient-derived orthotopic xenograft (PDOX) models. In this review, we firstly summarize the methodology to establish PDX models and then go over recent application and function of PDX models in basic cancer research on the areas of cancer characterization, initiation, proliferation, metastasis, and tumor microenvironment and in preclinical explorations of anti-cancer targets, drugs, and therapeutic strategies and finally give our perspectives on the future prospects of PDX models.

CD34 and CD38 are prognostic biomarkers for acute B lymphoblastic leukemia.

CD34 and CD38 proteins are usually used as surface markers to identify HSCs and Leukemic stem cells. However, there have been cases that lacked CD34 or CD38 protein but still had leukemia initiating capacity in B-ALL suggesting the restrictive of these two markers. CD34 and CD38 expression were detected in most B-ALL and can serve as a specific biomarker for the prognosis of this subset of leukemia. Lack of CD34 or high CD38 expression is associated with favorable prognosis.

Mechanisms determining the fate of hematopoietic stem cells.

Successful in vitro expansion of hematopoietic stem cells (HSCs) will facilitate the application of HSC transplantation for the treatment of various diseases, including hematological malignancies. To achieve this expansion, the molecular mechanisms that control the fate of HSCs must be deciphered. Leukemia-initiating cells (LICs) or leukemia stem cells (LSCs) may originate from normal HSCs, which suggest that the dysregulation of the mechanisms that regulate the cell fate of HSCs may underlie leukemogenesis. Here we review the recent progress in the application of HSCs, the regulatory mechanisms of the fate of HSCs, and the origins of leukemia.