技术资料

Inflammation is a common medical complication in colorectal cancer (CRC) patients,which plays significant roles in tumor progression and immunosuppression. However,the influence of inflammatory conditions on the tumor response to immune checkpoint inhibitors (ICI) is incompletely understood. Here we show that in a patient with high microsatellite instability (MSI-H) CRC and a local inflammatory condition,the primary tumor progresses but its liver metastasis regresses upon Pembrolizumab treatment. In silico investigation prompted by this observation confirms correlation between inflammatory conditions and poor tumor response to PD-1 blockade in MSI-H CRCs,which is further validated in a cohort of 62 patients retrospectively enrolled to our study. Inhibition of local but not systemic immune response is verified in cultures of paired T cells and organoid cells from patients. Single-cell RNA sequencing suggests involvement of neutrophil leukocytes via CD80/CD86-CTLA4 signaling in the suppressive immune microenvironment. In concordance with this finding,elevated neutrophil-to-lymphocyte ratio indicates inhibited immune status and poor tumor response to ICIs. Receiver operating characteristic curve further demonstrates that both inflammatory conditions and a high NLR could predict a poor response to ICIs in MSI- CRCs,and the predictive value could be further increased when these two predictors are combined. Our study thus suggests that inflammatory conditions in MSI-H CRCs correlate with resistance to ICIs through neutrophil leukocyte associated immunosuppression and proposes both inflammatory conditions and high neutrophil-to-lymphocyte ratio as clinical features for poor ICI response. View Publication

Three-dimensional (3D) culture systems have been developed that can re-capitulate organ level responses,simulate compound diffusion through complex structures,and assess cellular heterogeneity of tissues,making them attractive models for advanced in vitro research and discovery. Organoids are a unique subtype of 3D cell culture that are grown from stem cells,are self-organizing,and closely replicate in vivo pathophysiology. Organoids have been used to understand tissue development,model diseases,test drug sensitivity and toxicity,and advance regenerative medicine. However,traditional organoid culture methods are inadequate because they are low throughput and ill-suited for single organoid imaging,phenotypic assessment,and isolation from heterogenous organoid populations. To address these bottlenecks,we have adapted our tissue culture consumable and instrumentation to enable automated imaging,identification,and isolation of individual organoids. Organoids grown on the 3D CytoSort? Array can be reliably tracked,imaged,and phenotypically analyzed using brightfield and fluorescent microscopy as they grow over time,then released and transferred fully intact for use in downstream applications. Using mouse hepatic and pancreatic organoids,we have demonstrated the use of this technology for single-organoid imaging,clonal organoid generation,parent organoid subcloning,and single-organoid RNA extraction for downstream gene expression or transcriptomic analysis. The results validate the ability of the CellRaft AIR? System to facilitate efficient,user-friendly,and automated workflows broadly applicable to organoid research by overcoming several pain points: 1) single organoid time-course imaging and phenotypic assessment,2) establishment of single cell-derived organoids,and 3) isolation and retrieval of single organoids for downstream applications. View Publication

The multikinase inhibitors sunitinib,sorafenib,and axitinib have an impact not only on tumor growth and angiogenesis,but also on the activity and function of immune effector cells. In this study,a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. Tyrosine kinase inhibitor treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib,axitinib did not affect the mitochondrial membrane potential but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl-1),leading to an irreversible arrest in the G2/M cell cycle phase and delayed apoptosis. Furthermore,the sorafenib-mediated suppression of immune effector cells,in particular the reduction of the CD8(+) T cell subset along with the down-regulation of key immune cell markers such as chemokine CC motif receptor 7 (CCR7),CD26,CD69,CD25,and CXCR3,was not observed in axitinib-treated immune effector cells. Therefore,axitinib rather than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy. View Publication

Redirecting Ag specificity by transfer of TCR genes into PBLs is an attractive method to generate large numbers of cytotoxic T cells for immunotherapy of cancer and viral diseases. However,transferred TCR chains can pair with endogenous TCR chains,resulting in the formation of mispaired TCR dimers and decreased or unspecific reactivity. TCR gene transfer into hematopoietic stem cells (HSCs) is an alternative to create T cells with desired Ag specificity,because in this case expression of endogenous TCR chains is then less likely owing to allelic exclusion. We generated TCR-transduced T cells from peripheral T cells using the lymphocytic choriomeningitis virus-specific P14 TCR. After transfer of the P14 TCR genes into HSCs and subsequent reconstitution of irradiated mice,TCR-engineered HSC-derived T cells were produced. We then compared the Ag-specific T cell populations with P14 TCR-transgenic T cells for their therapeutic efficiency in three in vivo models. In this study,we demonstrate that TCR-transduced T cells and TCR-engineered HSC-derived T cells are comparable in controlling lymphocytic choriomeningitis virus infection in mice and suppress growth of B16 tumor cells expressing the cognate Ag in a comparable manner. View Publication

Respiratory syncytial virus (RSV) is a common childhood infection that in young infants can progress into severe bronchiolitis and pneumonia. Disease pathogenesis results from both viral mediated and host immune processes of which alveolar macrophages play an important part. Here,we investigated the role of different types of alveolar macrophages on RSV infection using an in vitro co-culture model involving primary tissue-derived human bronchial epithelial cells (HBECs) and human blood monocyte-derived M0-like,M1-like,or M2-like macrophages. It was hypothesized that the in vitro model would recapitulate previous in vivo findings of a protective effect of macrophages against RSV infection. It was found that macrophages maintained their phenotype for the 72-hour co-culture time period and the bronchial epithelial cells were unaffected by the macrophage media. HBEC infection with RSV was decreased by M1-like macrophages but enhanced by M0- or M2-like macrophages. The medium used during the co-culture also impacted the outcome of the infection. This work demonstrates that alveolar macrophage phenotypes may have differential roles during epithelial RSV infection,and demonstrates that an in vitro co-culture model could be used to further investigate the roles of macrophages during bronchial viral infection. View Publication

Ab-targeted vaccination involves targeting a receptor of choice expressed by dendritic cells (DCs) with Ag-coupled Abs. Currently,there is little consensus as to which criteria determine receptor selection to ensure superior Ag presentation and immunity. In this study,we investigated parameters of DC receptor internalization and determined how they impact Ag presentation outcomes. First,using mixed bone marrow chimeras,we established that Ag-targeted,but not nontargeted,DCs are responsible for Ag presentation in settings of Ab-targeted vaccination in vivo. Next,we analyzed parameters of DEC205 (CD205),Clec9A,CD11c,CD11b,and CD40 endocytosis and obtained quantitative measurements of internalization speed,surface turnover,and delivered Ag load. Exploiting these parameters in MHC class I (MHC I) and MHC class II (MHC II) Ag presentation assays,we showed that receptor expression level,proportion of surface turnover,or speed of receptor internalization did not impact MHC I or MHC II Ag presentation efficiency. Furthermore,the Ag load delivered to DCs did not correlate with the efficiency of MHC I or MHC II Ag presentation. In contrast,targeting Ag to CD8(+) or CD8(-) DCs enhanced MHC I or MHC II Ag presentation,respectively. Therefore,receptor expression levels,speed of internalization,and/or the amount of Ag delivered can be excluded as major determinants that dictate Ag presentation efficiency in setting of Ab-targeted vaccination. View Publication

Sulfated glycosaminoglycans (GAGs),or synthetic mimetics thereof,are not favorably viewed as orally bioavailable drugs owing to their high number of anionic sulfate groups. Devising an approach for oral delivery of such highly sulfated molecules would be very useful. This work presents the concept that conjugating cholesterol to synthetic sulfated GAG mimetics enables oral delivery. A focused library of sulfated GAG mimetics was synthesized and found to inhibit the growth of a colorectal cancer cell line under spheroid conditions with a wide range of potencies ( 0.8 to 46). Specific analogues containing cholesterol,either alone or in combination with clinical utilized drugs,exhibited pronounced in vivo anticancer potential with intraperitoneal as well as oral administration,as assessed by ex vivo tertiary and quaternary spheroid growth,cancer stem cell (CSC) markers,and/or self-renewal factors. Overall,cholesterol derivatization of highly sulfated GAG mimetics affords an excellent approach for engineering oral activity. View Publication

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress,which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1? (IRE1?) is activated to splice X-box binding protein 1 (XBP1) mRNA,thereby increasing XBP1s protein,which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study,we examined whether IRE1?-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1? endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines,without toxicity in normal mononuclear cells. Importantly,it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG,even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress,evidenced by induction of XBP1s,which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946,associated with increased CHOP. Finally,MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo,associated with significant growth inhibition of MM cells. Taken together,our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1? endoribonuclease domain is a potential therapeutic opt View Publication

BACKGROUND AND AIMS The intestinal mucosa undergoes a continual process of proliferation,differentiation,and apoptosis. An imbalance in this highly regimented process within the intestinal crypts is associated with several intestinal pathologies. Although metabolic changes are known to play a pivotal role in cell proliferation and differentiation,how glycolysis contributes to intestinal epithelial homeostasis remains to be defined. METHODS Small intestines were harvested from mice with specific hexokinase 2 (HK2) deletion in the intestinal epithelium or LGR5+ stem cells. Glycolysis was measured using the Seahorse XFe96 analyzer. Expression of phospho-p38 mitogen-activated protein kinase,the transcription factor atonal homolog 1,and intestinal cell differentiation markers lysozyme,mucin 2,and chromogranin A were determined by Western blot,quantitative real-time reverse transcription polymerase chain reaction,or immunofluorescence,and immunohistochemistry staining. RESULTS HK2 is a target gene of Wnt signaling in intestinal epithelium. HK2 knockout or inhibition of glycolysis resulted in increased numbers of Paneth,goblet,and enteroendocrine cells and decreased intestinal stem cell self-renewal. Mechanistically,HK2 knockout resulted in activation of p38 mitogen-activated protein kinase and increased expression of ATOH1; inhibition of p38 mitogen-activated protein kinase signaling attenuated the phenotypes induced by HK2 knockout in intestinal organoids. HK2 knockout significantly decreased glycolysis and lactate production in intestinal organoids; supplementation of lactate or pyruvate reversed the phenotypes induced by HK2 knockout. CONCLUSIONS Our results show that HK2 regulates intestinal stem cell self-renewal and differentiation through p38 mitogen-activated protein kinase/atonal homolog 1 signaling pathway. Our findings demonstrate an essential role for glycolysis in maintenance of intestinal stem cell function. View Publication

Chimerism testing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represents a promising tool for predicting disease relapse,although its precise role in this setting remains unclear. We investigated the predictive value of T lymphocyte chimerism analysis at 90 to 120 days after allo-HSCT in 378 patients with AML/MDS who underwent busulfan/fludarabine-based myeloablative preparative regimens. Of 265 (70%) patients with available T lymphocyte chimerism data,43% of patients in first or second complete remission (CR1/CR2) at the time of transplantation had complete (100%) donor T lymphocytes at day +90 to +120 compared with 60% of patients in the non-CR1/CR2 cohort (P = .005). In CR1/CR2 patients,donor T lymphocyte chimerism ?85% at day +90 to +120 was associated with a higher frequency of 3-year disease progression (29%; 95% confidence interval [CI],18% to 46% versus 15%; 95% CI,9% to 23%; hazard ratio [HR],2.1; P = .04). However,in the more advanced,non-CR1/CR2 cohort,mixed T lymphocyte chimerism was not associated with relapse (37%; 95% CI,20% to 66% versus 34%; 95% CI,25% to 47%; HR,1.3; P = .60). These findings demonstrate that early T lymphocyte chimerism testing at day +90 to +120 is a useful approach for predicting AML/MDS disease recurrence in patients in CR1/CR2 at the time of transplantation. View Publication

The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented stirred-tank bioreactors,we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 X 10(6) cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry,quantitative reverse-transcriptase polymerase chain reaction,and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures,underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly,physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism,suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture. SIGNIFICANCE Human pluripotent stem cells (hPSCs) are a unique source for the,in principle,unlimited production of functional human cell types in vitro,which are of high value for therapeutic and industrial applications. This study applied single-use,clinically compliant bioreactor technology to develop advanced,matrix-free,and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy,unexpected physiological features of hPSCs were discovered. These data allow a more rational process development,providing significant progress in the field of translational stem cell research and medicine. View Publication

Microtubule-associated protein (MAP) 2 has been perceived as a static cytoskeletal protein enriched in neuronal dendritic shafts. Emerging evidence indicates dynamic functions for various MAPs in activity-dependent synaptic plasticity. However,it is unclear how MAP2 is associated with synaptic plasticity mechanisms. Here,we demonstrate that specific silencing of high-molecular-weight MAP2 in vivo abolished induction of long-term potentiation (LTP) in the Schaffer collateral pathway of CA1 pyramidal neurons and in vitro blocked LTP-induced surface delivery of AMPA receptors and spine enlargement. In mature hippocampal neurons,we observed rapid translocation of a subpopulation of MAP2,present in dendritic shafts,to spines following LTP stimulation. Time-lapse confocal imaging showed that spine translocation of MAP2 was coupled with LTP-induced spine enlargement. Consistently,immunogold electron microscopy revealed that LTP stimulation of the Schaffer collateral pathway promoted MAP2 labeling in spine heads of CA1 neurons. This translocation depended on NMDA receptor activation and Ras-MAPK signaling. Furthermore,LTP stimulation led to an increase in surface-expressed AMPA receptors specifically in the neurons with MAP2 spine translocation. Altogether,this study indicates a novel role for MAP2 in LTP mechanisms and suggests that MAP2 participates in activity-dependent synaptic plasticity in mature hippocampal networks. View Publication