Many cancers feature cellular hierarchies that are driven by tumor-initiating, cancer stem cells (CSCs) and rely on complex interactions with the tumor microenvironment. Standard cell culture conditions fail to recapitulate the original tumor architecture or microenvironmental gradients, and are not designed to retain the cellular heterogeneity of parental tumors. Here, we describe a three-dimensional culture system that supports the long-term growth and expansion of tumor organoids derived directly from glioblastoma specimens, including patient-derived primary cultures, xenografts, genetically engineered glioma models, or patient samples. Organoids derived from multiple regions of patient tumors retain selective tumorigenic potential. Furthermore, organoids could be established directly from brain metastases not typically amenable to in vitro culture. Once formed, tumor organoids grew for months and displayed regional heterogeneity with a rapidly dividing outer region of SOX2+, OLIG2+, and TLX+ cells surrounding a hypoxic core of primarily non-stem senescent cells and diffuse, quiescent CSCs. Notably, non-stem cells within organoids were sensitive to radiation therapy, whereas adjacent CSCs were radioresistant. Orthotopic transplantation of patient-derived organoids resulted in tumors displayed histological features, including single cell invasiveness, that were more representative of the parental tumor compared with those formed from patient-derived sphere cultures. In conclusion, we present a new ex vivo model in which phenotypically diverse stem and non-stem glioblastoma cell populations can be simultaneously cultured to explore new facets of microenvironmental influences and CSC biology.

The nucleosome is generally found to be a strong barrier to transcript elongation by RNA polymerase II (pol II) in vitro. The elongation factors TFIIF and TFIIS have been shown to cooperate in maintaining pol II in the catalytically competent state on pure DNA templates. We now show that although TFIIF or TFIIS alone is modestly stimulatory for nucleosome traversal, both factors together increase transcription through nucleosomes in a synergistic manner. We also studied the effect of TFIIF and TFIIS on transcription of nucleosomes containing a Sin mutant histone. The Sin point mutations reduce critical histone-DNA contacts near the center of the nucleosome. Significantly, we found that nucleosomes with a Sin mutant histone are traversed to the same extent and at nearly the same rate as equivalent pure DNA templates if both TFIIS and TFIIF are present. Thus, the nucleosome is not necessarily an insurmountable barrier to transcript elongation by pol II. If unfolding of template DNA from the nucleosome surface is facilitated and the tendency of pol II to retreat from barriers is countered, transcription of nucleosomal templates can be rapid and efficient.It is now appreciated that control of transcript elongation is an important aspect of gene regulation (recently reviewed in Ref. 1). A major checkpoint occurs as RNA polymerase II (pol II) 2 passes from initiation into productive transcript elongation, ϳ50 bp downstream of the transcription start site in many genes (2-6). This checkpoint is roughly coincident with the initial contact between pol II and the first nucleosome of the transcription unit (7-9). Once pol II crosses this initial checkpoint, it can elongate transcripts efficiently over hundreds of kilobases of predominantly nucleosomal template (10). The rate of transcript elongation in vivo (10) slightly exceeds the maximum rates reported for transcript elongation by pol II on pure DNA templates in vitro (11,12). To understand both the initial elongation checkpoint and the exceptional efficiency of transcript elongation downstream of that point, it is essential to develop in vitro systems that duplicate these processes. Progress toward this goal has been somewhat limited because even single nucleosomes have generally proven to be strong barriers to transcribing pol II in vitro (11,(13)(14)(15)(16)(17).Given the tight association of all 146 bp of nucleosomal DNA with the histone octamer surface, one might initially imagine that the nucleosome presents an essentially continuous barrier to transcript elongation by pol II. However, studies with templates bearing single, precisely positioned nucleosomes indicated that the transcriptional barrier for pol II is more discrete. The strongest pauses for human pol II typically occur 45-55 bp within the nucleosome. After pol II crosses this barrier and the nucleosome dyad, transcription continues largely uninhibited through the remainder of the nucleosome (13). This suggests that traversal is controlled primarily by the unfolding of the template DNA from the octamer ...

Type 2 CXC chemokine receptor CXCR2 plays roles in development, tumorigenesis and inflammation. CXCR2 also promotes demyelination and decreases remyelination by actions toward hematopoietic cells and non-hematopoietic cells. Germline CXCR2 deficient (Cxcr2−/−) mice reported in 1994 revealed the complexity of CXCR2 function and its differential expression in varied cell-types. Here, we describe Cxcr2fl/fl mice for which the targeting construct was generated by recombineering based on homologous recombination in E. coli. Without recombination Cxcr2fl/fl mice have CXCR2 expression on neutrophils in peripheral blood, bone marrow and spleen. Cxcr2fl/fl mice were crossed to Mx-Cre mice in which Cre recombinase is induced by type I interferons, elicited by injection with polyinosinic-polycytidylic acid (poly(I:C)). CXCR2-deficient neutrophils were observed in poly(I:C) treated Cxcr2fl/fl::Mx-Cre+ (Cxcr2-CKO) mice, but not in poly(I:C) treated Cxcr2f//+::Mx-Cre+ mice. CXCR2 deletion was mainly observed peripherally but not in the CNS. Cxcr2-CKO mice showed impaired neutrophil migration in sterile peritonitis. Cxcr2-CKO mice reported here will provide a genetic reagent to dissect roles of CXCR2 in the neutrophil granulocyte lineage. Furthermore Cxcr2fl/fl mice will provide useful genetic models to evaluate CXCR2 function in varied cell populations.

Background:Residual CXCR2 expression on CNS cells in Cxcr2+/−→Cxcr2−/− chimeric animals slowed remyelination after both experimental autoimmune encephalomyelitis and cuprizone-induced demyelination.Methods:We generated Cxcr2fl/−:PLPCre-ER(T) mice enabling an inducible, conditional deletion of Cxcr2 on oligodendrocyte lineage cells of the CNS. Cxcr2fl/−:PLPCre-ER(T) mice were evaluated in 2 demyelination/remyelination models: cuprizone-feeding and in vitro lysophosphatidylcholine (LPC) treatment of cerebellar slice cultures.Results:Cxcr2fl/−:PLPCre-ER(T)+ (termed Cxcr2-cKO) mice showed better myelin repair 4 days after LPC-induced demyelination of cerebellar slice cultures. Cxcr2-cKOs also displayed enhanced hippocampal remyelination after a 2-week recovery from 6-week cuprizone feeding.Conclusion:Using 2 independent demyelination/remyelination models, our data document enhanced myelin repair in Cxcr2-cKO mice, consistent with the data obtained from radiation chimerism studies of germline CXCR2. Further experiments are appropriate to explore how CXCR2 function in the oligodendrocyte lineage accelerates myelin repair.

<p>Wide-field mosaic image of SOX2 and OLIG2 immunofluorescence in an IN528 organoid.</p>

<p>Extended Organoid Culture Methods</p>

<div>Abstract<p>Many cancers feature cellular hierarchies that are driven by tumor-initiating cancer stem cells (CSC) and rely on complex interactions with the tumor microenvironment. Standard cell culture conditions fail to recapitulate the original tumor architecture or microenvironmental gradients and are not designed to retain the cellular heterogeneity of parental tumors. Here, we describe a three-dimensional culture system that supports the long-term growth and expansion of tumor organoids derived directly from glioblastoma specimens, including patient-derived primary cultures, xenografts, genetically engineered glioma models, or patient samples. Organoids derived from multiple regions of patient tumors retain selective tumorigenic potential. Furthermore, organoids could be established directly from brain metastases not typically amenable to <i>in vitro</i> culture. Once formed, tumor organoids grew for months and displayed regional heterogeneity with a rapidly dividing outer region of SOX2⁺, OLIG2⁺, and TLX⁺ cells surrounding a hypoxic core of primarily non-stem senescent cells and diffuse, quiescent CSCs. Notably, non-stem cells within organoids were sensitive to radiotherapy, whereas adjacent CSCs were radioresistant. Orthotopic transplantation of patient-derived organoids resulted in tumors displaying histologic features, including single-cell invasiveness, that were more representative of the parental tumor compared with those formed from patient-derived sphere cultures. In conclusion, we present a new <i>ex vivo</i> model in which phenotypically diverse stem and non-stem glioblastoma cell populations can be simultaneously cultured to explore new facets of microenvironmental influences and CSC biology. <i>Cancer Res; 76(8); 2465–77. ©2016 AACR</i>.</p></div>

<p>Growth of a brain metastasis sample in organoid format.</p>