IntroductionHematopoiesis in bone marrow (BM) occurs in distinct microenvironmental niches. Discrete extracellular matrix (ECM) microenvironments within the BM help to separate endosteum, an interface between bone and BM, from the central marrow. Methods for studying hematopoiesis include clonal culture systems in semisolid media, 1 short-term 2 and long-term 3,4 liquid cultures, and tissue culture systems where hematopoietic cells grow on feeder layers of BM stromal cells. 5,6 However, cell culture systems involving growth on the surface of tissue culture plastic do not accurately represent tissue architecture 7 or the complex interactions between cells and their micro-environment. Recently, a stromal spheroid coculture model 8 and various scaffolds 9,10 have been developed to recreate the three-dimensional (3-D) environment of the BM, but these models fail to recapitulate the physiologic conditions of the BM. To adequately study B-cell development, 11 pathogenesis, 12,13 and neoplasia, 14 a culture system that places BM cells within their physiologic environment is required. For BM-localized malignancies, more effective culture systems must incorporate all compartments of the malignant clone, including cancer stem and progenitor cells, to identify their therapeutic vulnerabilities.Multiple myeloma (MM), an incurable cancer with 3-to 5-year survival despite the development of potent new therapies, 15 is characterized by monoclonal immunoglobulin (Ig), lytic bone lesions, 16 Here we present a robust 3-D tissue culture model in which the human BM microenvironment is reconstructed in vitro. In 3-D, the MM clone expands within its native microenvironment providing a valuable preclinical model within which conventional (melphalan) and novel (bortezomib) therapeutics selectively kill their target cells. In 3-D cultures, nonproliferating cells from MM BM concentrate at a reconstructed endosteummarrow junction (rEnd). Purified nonproliferating MM BM cells include MM-CSCs, as defined by their ability in a secondary culture to generate B/PC progeny harboring the unique MM clonotypic signature. Three-dimensional cultures of BM or mobilized blood autografts (MBAs) offer a preclinical model within which new therapies can be tested for their impact on all compartments of the MM clone, as well as providing access to the MM-CSC that underlie disease progression. An Inside Blood analysis of this article appears at the front of this issue.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 8, 2018. by guest www.bloodjournal.org From Methods MaterialsAfter approval from the Health Research Board (University of Alberta) and the Alberta Cancer Board, and after informed consent was obtained in accordance with the Declaration of Helsinki, BM samples (n ϭ 48) were provided from patients undergoing BM biopsies at the Cross Cancer Institute. Al...
Gene organization in nonmalignant B cells from t(4;14) and t(11;14) multiple myeloma (MM) patients differs from that of healthy donors. Among recurrent IGH translocations in MM, the frequency of t(4;14) (IGH and FGFR3) or t(11;14) (IGH and CCND1) is greater than the frequency of t(14;16) (IGH and MAF). Gene organization in t(14;16) patients may influence translocation potential of MAF with IGH. In patients, three-dimensional FISH revealed the positions of IGH, CCND1, FGFR3, and MAF in nonmalignant B cells that are likely similar to those when MM first arose, compared with B cells from healthy donors. Overall, IGH occupies a more central nuclear position while MAF is more peripherally located. However, for B cells from t(4;14) and t(11;14) patients, IGH and FGFR3, or IGH and CCND1 are found in spatial proximity: IGH and MAF are not. This differs in B cells from t(14;16) patients and healthy donors where IGH is approximately equidistant to FGFR3, CCND1, and MAF, suggesting that gene organization in t(14;16) patients is different from that in t(4;14) or t(11;14) patients. Translocations between IGH and MAF may arise only in the absence of close proximity to the more frequent partners, as appears to be the case for individuals who develop t(14;16) MM.
Accumulating evidence suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. It is not known, however, whether genome organization differs in nonmalignant cells from patients as compared to their cellular counterparts from healthy donors. This could contribute to translocation potential causing cancer. Multiple myeloma is a hematopoietic cancer of the B‐lineage, characterized by karyotypic instability, including chromosomal translocations involving the IGH locus and several translocation partners. Utilizing 3‐D FISH and confocal imaging, we investigate whether nuclear spatial positioning of the translocation‐prone gene loci, IGH, FGFR3, and CCND1 differs in nonmalignant cell subsets from multiple myeloma patients as compared to positioning in their corresponding healthy donor cell subsets. 3‐D analysis software was used to determine the spatial proximity of potential translocation pairs and the radial distribution of each gene. We observed that in all cell subsets, the translocation‐prone gene loci are intermediately located in the nucleus, while a control locus occupies a more peripheral position. In nonmalignant B‐cells from multiple myeloma patients, however, the translocation‐prone gene loci display a more central nuclear position and close spatial proximity. Our results demonstrate that gene positioning in nonmalignant B‐cells from multiple myeloma patients differs from that in healthy donors, potentially contributing to translocation probability in patient cells. We speculate that genome reorganization in patient B‐cells may closely reflect gene positioning at the time the multiple myeloma‐specific translocation initially formed, thus influencing translocation probability between proximal loci in the B‐cell population from which the malignancy emerged. © 2012 Wiley Periodicals, Inc.
Many B-cell malignancies are characterized by chromosomal translocations involving IGH and a proto-oncogene. For translocations to occur, spatial proximity of translocation-prone genes is necessary. Currently, it is not known how such genes are brought into proximity with one another. Although decondensed chromosomes occupy definitive, non-random spaces in the interphase nucleus known as chromosome territories (CTs), chromatin at the edges of CTs can intermingle, and specific genomic regions from some chromosomes have been shown to "loop out" of their respective CTs. This extra-territorial positioning of specific genomic regions may provide a mechanism whereby translocation-prone genes are brought together in the interphase nucleus. FGFR3 and MAF recurrently participate in translocations with IGH at different frequencies. Using 3D, 4-color FISH, and 3D analysis software, we show frequent extra-territorial positioning of FGFR3 and significantly less frequent extra-territorial positioning of MAF. Frequent extra-territorial positioning may be characteristic of FGFR3 in B-cells from healthy adult donors and non-malignant B-cells from patients, but not in hematopoietic stem cells from patients with translocations. The frequency of extra-territorial positioning of FGFR3 and MAF in B-cells correlates with the frequency of translocations in the patient population. Most importantly, in patient B-cells, we demonstrate a significant proportion of extra-territorial FGFR3 participating in close loci pairs and/or colocalizing with IGH. This preliminary work suggests that in patient B-cells, extra-territorial positioning of FGFR3 may provide a mechanism for forming close loci pairs and/or colocalization with IGH; indirectly facilitating translocation events involving these two genes. © 2016 Wiley Periodicals, Inc.
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