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The behavior of cells and tissues in vivo is determined by the integration of multiple biochemical and mechanical signals. Of the mechanical signals, stretch has been studied for decades and shown to contribute to pathophysiological processes. Several different stretch devices have been developed for in vitro investigations of cell stretch. In this work, we describe a new 3D-printed uniaxial stretching device for studying cell response to rapid deformation. The device is a bistable compliant mechanism holding two equilibrium states—an unstretched and stretched configuration—without the need of an external actuator. Furthermore, it allows multiple simultaneous measurements of different levels of stretch on a single substrate and is compatible with standard immunofluorescence imaging of fixed cells as well as live-cell imaging. To demonstrate the effectiveness of the device to stretch cells, a test case using aligned myotubes is presented. Leveraging material area changes associated with deformation of the substrate, changes in nuclei density provided evidence of affine deformation between cells and substrate. Furthermore, intranuclear deformations were also assessed and shown to deform non-affinely. As a proof-of-principle of the use of the device for mechanobiological studies, we uniaxially stretched aligned healthy and dystrophic myotubes that displayed different passive mechanical responses, consistent with previous literature in the field. We also identified a new feature in the mechanoresponse of dystrophic myotubes, which is of potential interest for identifying the diseased cells based on a quick mechanical readout. While some applications of the device for elucidating passive mechanical responses are demonstrated, the simplicity of the device allows it to be potentially used for other modes of deformation with little modifications., Biomaterials Advances, 141, ISSN:2772-9508

Significance Light zone (LZ) B cells are selected in germinal centers (GCs) in a cMyc-dependent manner, before dark zone (DZ) migration. Antigen affinity of B cells is enhanced during GC responses, and some differentiate into plasmablasts or memory B cells (MBCs). Currently, GC selection is presumed as a competitive affinity-dependent process. This cannot explain retention of GC B cells with varied affinities. We identified cMyc+ LZ B cell subpopulations enriched with either higher-affinity plasmablast precursors or lower-affinity MBC precursors and future DZ entrants, which diverged soon after permissive selection. Future DZ entrants’ affinity was enhanced through preferential proliferation of higher-affinity cells, and lower-affinity cells were retained in GCs. These findings elucidate mechanistically how GC selection ensures clonal diversity for broad protection., Affinity maturation depends on how efficiently germinal centers (GCs) positively select B cells in the light zone (LZ). Positively selected GC B cells recirculate between LZs and dark zones (DZs) and ultimately differentiate into plasmablasts (PBs) and memory B cells (MBCs). Current understanding of the GC reaction presumes that cMyc-dependent positive selection of LZ B cells is a competitive affinity-dependent process; however, this cannot explain the production of GC-derived lower-affinity MBCs or retention of GC B cells with varied affinities. Here, by combining single-cell/bulk RNA sequencing and flow cytometry, we identified and characterized temporally and functionally distinct positively selected cMyc+ GC B cell subpopulations. cMyc+ LZ B cell subpopulations enriched with either higher- or lower-affinity cells diverged soon after permissive positive selection. The former subpopulation contained PB precursors, whereas the latter comprised less proliferative MBC precursors and future DZ entrants. The overall affinity of future DZ entrants was enhanced in the LZ through preferential proliferation of higher-affinity cells. Concurrently, lower-affinity cells were retained in GCs and protected from apoptosis. These findings redefine positive selection as a dynamic process generating three distinct B cell fates and elucidate how positive selection ensures clonal diversity for broad protection.

Memory B cells (MBCs) are key for protection from reinfection. However, it is mechanistically unclear how germinal center (GC) B cells differentiate into MBCs. MYC is transiently induced in cells fated for GC expansion and plasma cell (PC) formation, so-called positively selected GC B cells. We found that these cells coexpressed MYC and MIZ1 (MYC-interacting zinc-finger protein 1 [ZBTB17]). MYC and MIZ1 are transcriptional activators; however, they form a transcriptional repressor complex that represses MIZ1 target genes. Mice lacking MYC–MIZ1 complexes displayed impaired cell cycle entry of positively selected GC B cells and reduced GC B cell expansion and PC formation. Notably, absence of MYC–MIZ1 complexes in positively selected GC B cells led to a gene expression profile alike that of MBCs and increased MBC differentiation. Thus, at the GC positive selection stage, MYC–MIZ1 complexes are required for effective GC expansion and PC formation and to restrict MBC differentiation. We propose that MYC and MIZ1 form a module that regulates GC B cell fate.

We previously showed that the formation of protein complexes between MYC and its partner MIZ1 (MYC-interacting zinc finger 1) is critically required for germinal center (GC) B cell expansion (Toboso-Navasa et. al., JEM 2020). MYC and MIZ1 are transcriptional activators; however, they can form a transcriptional repressor complex that represses MIZ1 target genes. High expression of MYC is commonly found in aggressive B cell lymphoma, most notably in Burkitt Lymphoma (BL) and in a fraction of Diffuse B cell lymphomas (DLBCLs). In DLBCL, MYC positivity is associated with poorer prognosis, especially when co-expressed with BCL2, and increased proliferative capacity. However, it remains unclear whether the requirement for MYC-MIZ1 complexes for cell expansion is retained in lymphoma, similar to what we observed in GC B cells. We first investigated MIZ1 expression in primary samples of B cell lymphoma sub-types and found it to be ubiquitous in BL (100% of cases; 14/14), whereas virtually absent in low-grade Follicular Lymphoma (7% of cases, 4/58). Roughly 42% of DLBCL cases (36/85; ) co-expressed MYC and MIZ1 and that was associated with increased cell proliferation assessed by Ki67. To investigate the role of MYC-MIZ1 complexes in lymphomagenesis, we generated compound mutant mice overexpressing in GC B cells wild-type MYC or a MYC mutant that cannot interact with MIZ1 (MYC VD) in combination with PI3K. MYC VD cannot repress MIZ1 target genes but displays normal interaction with MAX and transcriptional activation. As shown previously (Sander et. al., Cancer Cell 2012) MYC synergised with PI3K for BL development; however, overexpression of MYC VD plus PI3K had significantly delayed disease development and developed instead plasma cell hyperplasia. Analysis of pre-tumoral cells by single cell RNAseq revealed massive expansion of a unique GC B cell cluster only in mice carrying MYC and PI3K and this cluster had the highest suppression of MYC-MIZ1 target genes among all GC B cell clusters. Taken together, this data indicates that the fraction of cells in which MYC and MIZ1 are co-expressed represents a "sweet spot" for B cell lymphomagenesis; and that the transcriptional repressive complex formed by MYC and MIZ1 is crucial for GC B lymphomagenesis. Disclosures Toboso-Navasa: Benevolent AI: Current Employment. Calado: Myricx Pharma: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties: Cancer Treatments. WO patent WO 2020/128475 A1 (2020).

In hematopoietic malignancies, oncogenic alterations interfere with cellular differentiation and lead to tumoral development. Identification of the proteins regulating differentiation is essential to understand how they are altered in malignancies. Chronic myelogenous leukemia (CML) is a biphasic disease initiated by an alteration taking place in hematopoietic stem cells. CML progresses to a blast crisis (BC) due to a secondary differentiation block in any of the hematopoietic lineages. However, the molecular mechanisms of CML evolution to T-cell BC remain unclear. Here, we have profiled the changes in DNA methylation patterns in human samples from BC-CML, in order to identify genes whose expression is epigenetically silenced during progression to T-cell lineage-specific BC. We have found that the CpG-island of the ENGRAILED-2 (EN2) gene becomes methylated in this progression. Afterwards, we demonstrate that En2 is expressed during T-cell development in mice and humans. Finally, we further show that genetic deletion of En2 in a CML transgenic mouse model induces a T-cell lineage BC that recapitulates human disease. These results identify En2 as a new regulator of T-cell differentiation whose disruption induces a malignant T-cell fate in CML progression, and validate the strategy used to identify new developmental regulators of hematopoiesis., Research at C.C.’s lab was partially supported by FEDER, Fondo de Investigaciones Sanitarias, CSIC P.I.E., Junta de Castilla y León, and from an institutional grant from the Fundación Ramón Areces. Research in ISG group is partially supported by FEDER and by MICINN (SAF2012-32810), by MEC OncoBIO Consolider-Ingenio 2010 (Ref. CSD2007-0017), by NIH grant (R01 CA109335-04A1), the ARIMMORA project (FP7-ENV-2011, European Union Seventh Framework Program), by Junta de Castilla y León (BIO/SA06/13) and by “Proyecto en red de investigación en células madre tumorales” supported by Obra Social Kutxa y Consejería de Sanidad de la Junta de Castilla y Leon. C.V.D.’s research is supported by Junta de Castilla y León (proyecto de investigación en biomedicina SAN/39/2010). J.A.M.C.’s research is supported by the Instituto de Salud Carlos III (ISCIII), grants FIS-PI12/00202 and RTICC-RD12/0036/0063. All Spanish funding is co-sponsored by the European Union FEDER program. I.S.G. is an API lab of the EuroSyStem project and a partner of DECIDE European network. F.A.-J. and E.C.S. were supported by Spanish Ministry of Science and Innovation fellowships. E.C.-S. was a “Residencia de Estudiantes” Fellow. A.T.N. was the recipient of a “Beca de Postgrado de la Fundación Ramón Areces/UAM.”

This is an open access article., The latest scientific findings in the field of cancer research are redefining our understanding of the molecular and cellular basis of the disease, moving the emphasis toward the study of the mechanisms underlying the alteration of the normal processes of cellular differentiation. The concepts best exemplifying this new vision are those of cancer stem cells and tumoral reprogramming. The study of the biology of acute lymphoblastic leukemias (ALLs) has provided seminal experimental evidences supporting these new points of view. Furthermore, in the case of B-cells, it has been shown that all the stages of their normal development show a tremendous degree of plasticity, allowing them to be reprogrammed to other cellular types, either normal or leukemic. Here we revise the most recent discoveries in the fields of B-cell developmental plasticity and B-ALL research, and discuss their interrelationships and their implications for our understanding of the biology of the disease. © 2011 Landes Bioscience., Research in C.C. lab was partially supported by FEDER, Fondo de Investigaciones Sanitarias (PI080164), CSIC P.I.E., 200920I055 and 201120E060, from the ARIMMORA project (FP7-ENV-2011, European Union Seventh Framework Program) and from an institutional grant from the “Fundación Ramón Areces.” Research in I.S.G. group was partially supported by FEDER and by MICINN (SAF2009-08803 to I.S.G.), by Junta de Castilla y León (REF. CSI007A11-2 and Proyecto Biomedicina 2009–2010), by MEC OncoBIO Consolider-Ingenio 2010 (Ref. CSD2007-0017), by NIH grant (R01 CA109335-04A1), by Sandra Ibarra Foundation, by Group of Excellence Grant (GR15) from Junta de Castilla y Leon, and the ARIMMORA project (FP7-ENV-2011, European Union Seventh Framework Program) and by Proyecto en Red de Investigación en Celulas Madre Tumorales en Cancer de Mama, supported by Obra Social Kutxa y Conserjería de Sanidad de la Junta de Castilla y Leon. All Spanish funding is co-sponsored by the European Union FEDER program. I.S.G. is an API lab of the EuroSyStem project. E.C.S. and M.B.D. are recipients of JAE-predoc fellowships from CSIC. I.R.C. is recipient of a FPU fellowship from MICINN. E.C.S. is recipient of a “Residencia de Estudiantes” fellowship.