Person:

Sun, Yan

Hematopoietic stem cells (HSCs) reside in complex bone marrow microenvironments, where niche-induced signals regulate hematopoiesis. Focal adhesion kinase (Fak) is a nonreceptor protein tyrosine kinase that plays an essential role in many cell types, where its activation controls adhesion, motility, and survival. Fak expression is relatively increased in HSCs compared to progenitors and mature blood cells. Therefore, we explored its role in HSC homeostasis. We have used the Mx1-CreLinducible conditional knockout mouse model to investigate the effects of Fak deletion in bone marrow compartments. The total number as well as the fraction of cycling (Lin^-Sca-1^+c-kit^+) (LSK) cells is increased in (Fak^{-/-}) mice compared to controls, while hematopoietic progenitors and mature blood cells are unaffected. Bone marrow cells from (Fak^{-/-}) mice exhibit enhanced, long-term (i.e., 20-week duration) engraftment in competitive transplantation assays. Intrinsic Fak function was assessed in serial transplantation assays, which showed that HSCs ((Lin^-Sca-1^+c-kit^+CD34^-Flk-2^-) cells) sorted from (Fak^{-/-}) mice have similar self-renewal and engraftment ability on a per-cell basis as wild-type HSCs. When Fak deletion is induced after engraftment of (Fak^{fl/fl}Mx1-Cre^+) bone marrow cells into wild-type recipient mice, the number of LSKs is unchanged. In conclusion, Fak inactivation does not intrinsically regulate HSC behavior and is not essential for steady- state hematopoiesis. However, widespread Fak inactivation in the hematopoietic system induces an increased and activated HSC pool size, potentially as a result of altered reciprocal interactions between HSCs and their microenvironment.

Cancer stem cells (CSCs) are considered to be the root cause for cancer treatment failure. Thus, there remains an urgent need for more potent and safer therapies against CSCs for curing cancer. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against putative CSCs of nasopharyngeal carcinoma (NPC) was fully evaluated in vitro and in vivo. To visualize putative CSCs in vitro by fluorescence imaging, and image and quantify putative CSCs in tumor xenograft-bearing mice by in vivo bioluminescence imaging, NPC cells were engineered with CSC detector vector encoding GFP and luciferase (Luc) under control of Nanog promoter. Our study reported in vitro intense tumor-killing activity of CIK cells against putative CSCs of NPC, as revealed by percentage analysis of side population cells, tumorsphere formation assay and Nanog-promoter-GFP-Luc reporter gene strategy plus time-lapse recording. Additionally, time-lapse imaging firstly illustrated that GFP-labeled or PKH26-labeled putative CSCs or tumorspheres were usually attacked simultaneously by many CIK cells and finally killed by CIK cells, suggesting the necessity of achieving sufficient effector-to-target ratios. We firstly confirmed that NKG2D blockade by anti-NKG2D antibody significantly but partially abrogated CIK cell-mediated cytolysis against putative CSCs. More importantly, intravenous infusion of CIK cells significantly delayed tumor growth in NOD/SCID mice, accompanied by a remarkable reduction in putative CSC number monitored by whole-body bioluminescence imaging. Taken together, our findings suggest that CIK cells demonstrate the intense tumor-killing activity against putative CSCs of NPC, at least in part, by NKG2D-ligands recognition. These results indicate that CIK cell-based therapeutic strategy against CSCs presents a promising and safe approach for cancer treatment.

Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study.

ABSTRACT There is an urgent need for more potent and safer approaches to eradicate cancer stem cells (CSCs) for curing cancer. In this study, we investigate cancer-killing activity (CKA) of cytokine-induced killer (CIK) cells against CSCs of hepatocellular carcinoma (HCC). To visualize CSCs in vitro by fluorescence imaging, and image and quantify CSCs in tumor xenograft-bearing mice by bioluminescence imaging, HCC cells were engineered with CSC detector vector encoding GFP and luciferase controlled by Nanog promoter. We found that CIK cells have a strong CKA in vitro against putative CSCs of HCC, as shown by tumorsphere formation and time-lapse imaging. Additionally, time-lapse recording firstly revealed that putative CSCs were attacked simultaneously by many CIK cells and finally eradicated by CIK cells, indicating the necessity of achieving sufficient effector-to-target ratios. We firstly illustrated that anti-NKG2D antibody blocking partially but significantly inhibited CKA of CIK cells against putative CSCs. More importantly, intravenous infusion of CIK cells remarkably delayed tumor growth in mice with a significant decrease in putative CSC number monitored by bioluminescence imaging. Taken together, these findings demonstrate CKA of CIK cells against putative CSCs of HCC, at least in part, by NKG2D-ligands recognition.

Overexpression of the transcriptional factor Hes1 (hairy and enhancer of split-1) has been observed in numerous cancers, but the precise roles of Hes1 in epithelial-mesenchymal transition (EMT), cancer invasion and metastasis remain unknown. Our current study firstly revealed that Hes1 upregulation in a cohort of human nasopharyngeal carcinoma (NPC) biopsies is significantly associated with the EMT, invasive and metastatic phenotypes of cancer. In the present study, we found that Hes1 overexpression triggered EMT-like cellular marker alterations of NPC cells, whereas knockdown of Hes1 through shRNA reversed the EMT-like phenotypes, as strongly supported by Hes1-mediated EMT in NPC clinical specimens described above. Gain-of-function and loss-of-function experiments demonstrated that Hes1 promoted the migration and invasion of NPC cells in vitro. In addition, exogenous expression of Hes1 significantly enhanced the metastatic ability of NPC cells in vivo. Chromatin immunoprecipitation (ChIP) assays showed that Hes1 inhibited PTEN expression in NPC cells through binding to PTEN promoter region. Increased Hes1 expression and decreased PTEN expression were also observed in a cohort of NPC biopsies. Additional studies demonstrated that Hes1-induced EMT-like molecular changes and increased motility and invasion of NPC cells were mediated by PTEN. Taken together, our results suggest, for what we believe is the first time, that Hes1 plays an important role in the invasion and metastasis of NPC through inhibiting PTEN expression to trigger EMT-like phenotypes.

Traumatic brain injury (TBI), characterized by acute neurological dysfunction, is one of the best known environmental risk factors for chronic traumatic encephalopathy (CTE) and Alzheimer's disease (AD), whose defining pathologic features include tauopathy made of phosphorylated tau (p-tau). However, tauopathy has not been detected in early stages after TBI and how TBI leads to tauopathy is unknown. Here we find robust cis p-tau pathology after sport- and military-related TBI in humans and mice. Acutely after TBI in mice and stress in vitro, neurons prominently produce cis p-tau, which disrupts axonal microtubule network and mitochondrial transport, spreads to other neurons, and leads to apoptosis. This process, termed “cistauosis”, appears long before other tauopathy. Treating TBI mice with cis antibody blocks cistauosis, prevents tauopathy development and spread, and restores many TBI-related structural and functional sequelae. Thus, cis p-tau is a major early driver after TBI and leads to tauopathy in CTE and AD, and cis antibody may be further developed to detect and treat TBI, and prevent progressive neurodegeneration after injury.