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A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma

Julia Kirshner, Kyle J. Thulien, Lorri D. Martin, Carina Debes Marun, Tony Reiman, Andrew R. Belch and Linda M. Pilarski

Article Figures & Data

Figures

  • Figure 1

    3-D culture model is designed to mimic the in vivo microenvironment of the BM. (A) Diagram of the 3-D BM culture setup with fibronectin/collagen I surface coating to reconstruct rEnd, overlaid with BMCs mixed with Matrigel/fibronectin ECM, covered with growth medium (left panel). Cellular composition of the 3-D culture assessed after 3 weeks (right panel). Cytospins were prepared from the preculture BM and from the same samples after culture (n = 6 individually analyzed patients) and stained with May-Grunwald-Giemsa. Nuclear and cytoplasmic staining and morphology were analyzed, and cells of various lineages were counted and reported as percentage of total cellularity. Blast indicates all blasts; PMN, polymorphonuclear (bands, neutrophils, eosinophils, basophils); pre-PMN, promyelocyte, myelocyte, metamyelocyte; lymphocyte, B and T cells; NRC, nucleated red cells (proerythroblast, basophilic erythroblast, polychromatophilic erythroblast, orthochromatic erythroblast); other, macrophages, stromal cells, reticular cells, osteoclastic cells, osteoblastic cells. (B) BMCs grown in rBM recapitulate multicellularity of the BM. BMCs were grown in 3-D for the indicated number of days, and the overall view of the culture was analyzed using brightfield microscopy (original magnification ×200; see “Microscopy”). Open arrows and inset represent migrating cells; large black arrows, B-cell colonies; large white arrows, PC colonies; striped arrows, stromal cells. Representative images from 48 individually analyzed patients. (C) Proliferation of the various cellular compartment of the rBM was measured by labeling BMCs with CFSE (n = 4 individually analyzed patients) and culturing in 3-D for the indicated number of days. Cells were isolated from 3-D, stained with fluorescent tagged antibodies as indicated, and proliferation capacity of the aggregate rBM (*P = .001), CD20+ B cells (*P = .024, **P = .006), and CD138+ PCs (*P = .004) was measured by multicolor flow cytometry (inset, proliferation profile of CD138+CD56+ cells). Percentage CFSE labeled cells represents cells with average amount of retained CFSE above the unlabeled control. Error bars in panels B and D represent SEM.

  • Figure 2

    rBM maintains the architecture of in vivo BM. (A) BMCs were grown in rBM for 21 days and stained with DAPI (pseudocolored red) to mark nuclei. The stratification of the 3-D culture was assessed by confocal microscopy as a 3-D reconstruction from the confocal stack (original magnification 100×). Representative images from BM of 48 individually analyzed patient samples. (B) BM aspirated particle grown in rBM for 21 days and stained with DAPI (pseudocolored red) to mark nuclei. The stratification of the 3-D culture was assessed by confocal microscopy as a 3-D reconstruction from the confocal stack (original magnification 100×). Representative image from analysis of aspirated particles from 28 individually analyzed patients. (C) BM stromal cells redistribute to rEnd. The rBM layer was removed from 3-D culture, and the cells at rEnd were stained with markers to identify stromal components observed at rEnd (adipocytes, Oil Red; fibroblasts, fibronectin and phallodin; osteoblasts, alkaline phosphatase [ALP] and Alizarin Red [inset]; osteoclasts, tartarate-resistant acidic phosphatase [TRAP]). Brightfield microscopy (original magnification 200×). Representative images from 7 individually analyzed patients; see “Immunohistochemistry.” (D) BMCs were grown for 14 days and the percentage of CD34+ HPCs, B cells, or PCs in each layer (black represents fraction in bottom layer; gray, fraction in middle; white, fraction in top layer) of the 3-D culture was calculated based on the immunohistochemical staining for CD34, CD20, or CD138 (n = 4 individually analyzed patients). Error bars represent SEM. For complete image acquisition information, see “Microscopy.”

  • Figure 3

    MM rBM has an abnormal tissue architecture and exhibits clonal expansion of MM cells with chromosomal abnormalities. (A) BMCs from normal donors (n = 5) and MM patients (n = 10) were grown in rBM for 21 days followed by assessment of the overall culture architecture by brightfield microscopy (top, original magnification 50×; bottom, original magnification 200×). Cells localized to “tracks” are TRAP+ osteoclasts (not shown). Representative images are shown with the plane of focus set to the bottom of the plate representing reconstructed endosteum. (B) BM cells (n = 16 patients) were grown in rBM for the indicated number of days, and the extent of the malignant outgrowth of the MM clone was measured by RQ-PCR using patient specific primers. Each malignant cell has only one copy of the IgH VDJ rearranged template; RQ-PCR determines the number of rearranged IgH VDJ templates compared with a β2-microglobulin standard curve and an IgH VDJ positive control curve. Percent clonal cells corresponds to the percentage clonal VDJ templates present in the sample normalized to the β2-microglobulin gene (*P = .006, **P = .001, ***P = .002, ****P = .003). Error bars represent SEM. (C) Absolute numbers of BMCs and PCs, which were originally placed into and recovered from rBM (mean values for 3 different patients). (D) FISH analysis of cells harvested from 3-D culture. Cells from 3-D cultures were screened for the chromosomal abnormalities (marked with arrows, and percentage plasma cells with each abnormality stated at the top left) detected in preculture BM PCs, using cytospins from the matching D21 rBM (n = 5 patients) stained by interphase FISH. Abnormal cells are indicated by arrows. (Left panel) PCs with a deletion of 13q34 locus (1 green signal) and a normal cell (2 green signals). (Middle panel) PCs with an amplification of 1q21 (3 red signals) and a normal cell (2 red signals). (Right panel) PCs with t(11;14) translocation with 2 derivative chromosomes (2 yellow signals), 1 normal chromosome 11 (1 red signal), and 1 normal chromosome 14 (1 green signal; original magnification 630×). For additional image acquisition information, see “Microscopy” and “Fluorescent in situ hybridization.”

  • Figure 4

    Melphalan and bortezomib affect the hematopoietic, but not the stromal, compartment of rBM. (A-D) MM BMCs were grown in 3-D for 14 days and treated with melphalan or bortezomib (“Preclinical drug treatment studies”). (A) The reduction of the clonal cells was measured by RQ-PCR with patient specific primers (melphalan, P = .016; bortezomib, P = .033; n = 5 individually analyzed patients). (B) Apoptosis within rBM was assessed by flow cytometry measuring annexin V reactivity after treatment with melphalan (left panel) or bor-tezomib (middle panel). MM specific cell killing by bor-tezomib was monitored in the CD138+/CD56+ population by annexin V reactivity (right panel; n = 5 individually analyzed patients). Error bars represent SEM. (C) rBM was grown and treated with melphalan as in panel A. Cell kill was monitored by brightfield microscopy (original magnification 200×; see “Microscopy”). Representative images from 5 individually analyzed patients. (D) The rBM layer was removed, and the cells at rEnd were stained with TRAP (osteoclasts), Oil Red (adipocytes), and ALP (osteoblasts). Brightfield microscopy (original magnification 200×; see “Immunohistochemistry”). Representative images from 5 individually analyzed patients.

  • Figure 5

    LRCs redistribute to rEnd. (A) Mononuclear cells from MBA (n = 3 patients) were treated with melphalan (“Preclinical drug treatment studies”) and grown in rBM for 21 days, and the extent of the malignant outgrowth was measured by RQ-PCR. MBA generated MM progeny and LRCs comparable with those from BM; the majority of MBAs also generated stromal layers. Preculture ex vivo BMCs and MBA cells were used as controls. Horizontal lines represent medians. (B,C) BMCs were labeled with CFSE (green) and cultured for 21 days. (B) rBM was stained with DAPI (blue inset) to detect total cells in the cultures, and the position of the brightly staining CFSE+ LRCs was analyzed by confocal microscopy (original magnification 100×) as indicated in “Microscopy.” Representative images from 8 individually analyzed patient samples. (C) The rBM layer was removed, and the cells at rEnd were fixed and stained with N-cadherin. CFSE+ LRCs (green) shown in contact with an N-cadherin+ stromal cell (red), DAPI stained nuclei (blue). Representative 10 images from 3 individually analyzed patients.

  • Figure 6

    LRCs include drug-resistant progenitors able to generate clonotypic progeny. (A-D) Mononuclear cells were isolated from the mobilized blood preparations, labeled with CFSE, grown in 3-D for 14 days, and treated with melphalan (“Preclinical drug treatment studies”). (A) Flow cytometric profile of the LRCs. The gates for sorting were set to isolate cells with lymphocyte scatter properties (scatter plot shown is gated on the lymphocytes) and CFSEhigh fluorescence, comparable with that in the stained population preculture, as expected for nonproliferating or slowly proliferating cells. Percentage of LRCs was calculated as the percentage of CFSEhigh cells in the lymphocyte fraction of the 3-D culture. Representative FACS plots from 5 individually analyzed patients. (B) May-Grunwald-Giemsa (MGG) staining of the sorted LRCs (top) and immunohistochemical staining of sorted LRCs for CD20 (bottom; original magnification ×400); see “Immunohistochemistry.” Representative images from 3 individually analyzed patients. (C) Representative colonies from the CFU assay (original magnification 40×; n = 10 individually analyzed patients) as indicated in “Microscopy.” (D) Progeny generated from sorted LRCs from both untreated and melphalan-treated cultures were analyzed by RQ-PCR to quantify the number of clonotypic cells. These c(T) values, compared with control values, indicate that approximately 100% of the progeny were clonotypic. No RQ-PCR amplification was detectable for nonclonotypic colonies. Similar c(T) values were observed for clonotypic colonies from both treatment groups (n = 8 colonies from untreated group and n = 11 colonies from melphalan group). Horizontal lines represent medians. (E) Quantification of the LRCs as a percentage of total cells in the culture, CFU as a percentage of LRCs, and clonal CFU as a percentage of total colonies obtained in the CFU assay (mean ± SD of 8 individually analyzed patients).

  • Figure 7

    LRCs can be serially passaged and can generate MM plasma cells. (A) Mononuclear cells were isolated from the mobilized blood preparations, labeled with CFSE, grown in 3-D for 14 days, after which CFSEhigh lymphocytic cells were sorted from the harvested cells, and cultured in a CFU assay (no morphologic differences between clonotypic and nonclonotypic colonies were observed). Clonotypic or nonclonotypic progeny were generated from LRCs in CFU assay and tested for clonotypic IgH VDJ signatures by PCR and by RQ-PCR. Representative single-stage PCR analysis of the clonotypic IgH VDJ and β2-microglobulin from representative CFU progeny is shown; these results were confirmed using RQ-PCR (Figure 6D and data not shown). (B) LRCs were sorted and placed into CFU culture, followed by serial replating for 6 generations. Each panel is a representative image from the consecutive passage of a colony (p1) from a single patient (n = 6 patients). (C) FACS analysis of the LRC progeny after the sixth serial passage of a clonotypic colony (culture p6 from panel B). The entirety of p4 and p5 was used for clonal expansion to obtain sufficient cells for the p6 FACS analysis. Histograms are representative of those obtained for 6 different patients. All of the secondary colonies from clonotypic CFU were clonotypic. In contrast, secondary colonies from the nonclonotypic CFU were all nonclonotypic. Self-renewal capability within colonies was further supported by the morphology of the CFU arising from LRCs, which at all passages included small secondary colonies scattered throughout the primary colony, as shown in the figure.