15-Methylene-Eburnamonine Kills Leukemic Stem Cells and Reduces Engraftment in a Humanized Bone Marrow Xenograft Mouse Model of Leukemia.

Recent studies suggest that leukemia stem cells (LSCs) play a critical role in the initiation, propagation, and relapse of leukemia. Herein we show that (-)-15-methylene-eburnamonine, a derivative of the alkaloid (-)-eburnamonine, is cytotoxic against acute and chronic lymphocytic leukemias (ALL and CLL) and acute myelogenous leukemia (AML). The agent also decreases primary LSC frequency in vitro. The cytotoxic effects appear to be mediated via the oxidative stress pathways. Furthermore, we show that the compound kills AML, ALL, and CLL stem cells. By the use of a novel humanized bone marrow murine model of leukemia (huBM/NSG), it was found to decrease progenitor cell engraftment.

1α,25-Dihydroxyvitamin D Inhibits the Metastatic Capability of MCF10CA1a and MDA-MB-231 Cells in an In Vitro Model of Breast to Bone Metastasis.

Breast cancer metastasis to the bone continues to be a major health problem, with approximately 80% of advanced breast cancer patients expected to develop bone metastasis. Although the problem of bone metastasis persists, current treatment options for metastatic cancer patients are limited. In this study, we investigated the preventive role of the active vitamin D metabolite, 1α,25-dihydroxyvitamin D (1,25(OH)2D), against the metastatic potential of breast cancer cells using a novel three-dimensional model (rMET) recapitulating multiple steps of the bone metastatic process. Treatment of MCF10CA1a and MDA-MB-231 cells inhibited metastasis in the rMET model by 70% (±5.7%) and 21% (±6%), respectively. In addition, 1,25(OH)2D treatment decreased invasiveness (20 ± 11% of vehicle) and decreased the capability of MCF10CA1a cells to survive in the reconstructed bone environment after successful invasion through the basement membrane (69 ± 5% of vehicle). An essential step in metastasis is epithelial-mesenchymal transition (EMT). Treatment of MCF10CA1a cells with 1,25(OH)2D increased gene (2.04 ± 0.28-fold increase) and protein (1.87 ± 0.20-fold increase) expression of E-cadherin. Additionally, 1,25(OH)2D treatment decreased N-cadherin gene expression (42 ± 8% decrease), a marker for EMT. Collectively, the present study suggests that 1,25(OH)2D inhibits breast cancer cell metastatic capability as well as inhibits EMT, an essential step in the metastatic process.

Maytansinoid immunoconjugate IMGN901 is cytotoxic in a three-dimensional culture model of multiple myeloma.

Environmental-mediated drug-resistance (EM-DR) presents a major challenge for therapeutic development. Tissue microenvironment in the form of extracellular matrix, soluble factors, and stroma contribute to EM-DR. In multiple myeloma (MM), drug-resistance has hindered treatment success with 5-year survival rates remaining <50%. Here we evaluated IMGN901, a maytansinoid immunoconjugate, for its ability to overcome EM-DR alone or in combination with lenalidomide or dexamethasone. We show that while adhesion of MM cells to the extracellular matrix reduces potency of IMGN901, it remains cytotoxic with an average LC50=43 nM. However, only a combination of IMGN901, lenalidomide, and dexamethasone was able to overcome drug-resistance arising from the direct contact between MM and stromal cells. We demonstrate that multi-drug resistance protein-1 (MDR-1) was upregulated in MM cells grown in contact with stroma, likely responsible for the observed resistance. This study emphasizes the importance of incorporating the elements of tumor microenvironment during preclinical testing of novel therapeutics.

A reconstructed metastasis model to recapitulate the metastatic spread in vitro.

Metastasis remains a leading cause of morbidity and mortality from solid tumors. Lack of comprehensive systems to study the progression of metastasis contributes to the low success of treatment. We developed a novel three-dimensional in vitro reconstructed metastasis (rMet) model that incorporates extracellular matrix (ECM) elements characteristic of the primary (breast, prostate, or lung) and metastatic (bone marrow, BM) sites. A cytokine-rich liquid interphase separates the primary and distant sites, further recapitulating circulation. Similar to main events underlying the metastatic cascade, the rMet model fractionated human tumor cell lines into sub-populations with distinct invasive and migratory abilities: (i) a primary tumor-like fraction mainly consisting of non-migratory spheroids; (ii) an invasive fraction that invaded through the primary tumor ECM, but failed to acquire anchorage-independence and reach the BM; and (iii) a highly migratory BM-colonizing population that invaded the primary ECM, survived in the "circulation-like" media, and successfully invaded and proliferated within BM ECM. BM-colonizing fractions successfully established metastatic bone lesions in vivo, whereas the tumor-like spheroids failed to engraft the bones, showing the ability of the rMet model to faithfully select for highly aggressive sub-populations with a propensity to colonize a metastatic site. By applying the rMet model to study real-time ECM remodeling, we show that tumor cells secrete collagenolytic enzymes for invading the primary site ECM but not for entering the BM ECM, indicating possible differences in ECM remodeling mechanisms at primary tumor versus metastatic sites.

A three-dimensional tissue culture model to study primary human bone marrow and its malignancies.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

Dkk-1 and IL-7 in plasma of patients with multiple myeloma prevent differentiation of mesenchymal stem cells into osteoblasts.

Bone disease is the leading cause of morbidity associated with multiple myeloma (MM). Lytic bone lesions have been detected in 90% of patients diagnosed with MM and present a great therapeutic challenge. After the removal of the tumor burden, the bone lesions persist and the bone remodeling homeostasis is not restored even in patients in clinical remission. To determine whether systemic factors generated by malignant MM cells can skew the osteoblast (OB) differentiation program of normal mesenchymal stem cells (MSCs), we generated an immortalized bone marrow MSC line (hTERT-MSC). The hTERT-MSCs were exposed to plasma from healthy donors and patients with MM. Cells grown in media supplemented with plasma from MM patients failed to differentiate into OBs, while the hTERT-MSCs grown in the presence of normal human plasma generated OB clusters that mineralized calcium, expressed Runx2, and were positive for alkaline phosphatase, fibronectin, collagen I, osteocalcin, and osteopontin. Blocking Dickkopf-1 (Dkk-1) and interleukin-7 (IL-7) in MM plasma restored proper OB differentiation of hTERT-MSCs. Finally, we show that hTERT-MSCs cultured in the presence of MM plasma adopt a cancer-associated stroma phenotype. Thus, we show, that systemic factors present in the plasma of patients with MM affect the behavior of non-malignant MSCs and contribute to the sustained bone disease reported in MM.

Differentiation of cancer cells in two-dimensional and three-dimensional breast cancer models by Raman spectroscopy.

We demonstrate the first application of Raman spectroscopy in diagnosing nonmalignant, premalignant, malignant, and metastatic stages of breast cancer in a three-dimensional (3-D) cell culture model that closely mimics an in vivo environment. Comprehensive study comparing classification in two-dimensional (2-D) and 3-D cell models was performed using statistical methods composed of principal component analysis for exploratory analysis and outlier removal, partial least squares discriminant analysis, and elastic net regularized regression for classification. Our results show that Raman spectroscopy with an appropriate classification tool has excellent resolution to discriminate the four stages of breast cancer progression, with a near 100% accuracy for both 2-D and 3-D cell models. The diversity in chemical groups related to nucleic acids, proteins, and lipids, among other chemicals, were identified by appropriate peaks in the Raman spectra that correspond to the correct classification of the different stages of tumorigenesis model comprising of MCF10A, MCF10AneoT, MCF10CA1h, and MCF10CA1a cell lines. An explicit relationship between wavenumber and the stages of cancer progression was identified by the elastic net variable selection.

Synthesis of 15-methylene-eburnamonine from (+)-vincamine, evaluation of anticancer activity, and investigation of mechanism of action by quantitative NMR.

The biological role of installing a critical exocyclic enone into the structure of the alkaloid, (-)-eburnamonine, and characterization of the new chemical reactivity by quantitative NMR without using deuterated solvents are described. This selective modification to a natural product imparts potent anticancer activity as well as bestows chemical reactivity toward nucleophilic thiols, which was measured by quantitative NMR. The synthetic strategy provides an overall conversion of 40%. In the key synthetic step, a modified Peterson olefination was accomplished through the facile release of trifluoroacetate to create the requisite enone in the presence of substantial steric hindrance.

The systemic cytokine environment is permanently altered in multiple myeloma.

Multiple myeloma (MM) is an incurable bone marrow malignancy of the B cell lineage. Utilizing multiplex Luminex technology we measured levels of 25 cytokines in the plasma of normal donors (n = 177), those with monoclonal gammopathy of undetermined significance (n = 8), and MM patients (n = 55) with either active disease, on treatment, or in remission. The cytokine levels were compared between normal donors and MM patients as well as between various phases of MM, and discriminant analysis was used to create a predictive classification model based on the differentially expressed cytokines. Evaluating age- and gender-dependence of cytokine expression, we determined that with age there is a shift toward a pro-inflammatory environment. Moreover, we observed a strong gender bias in cytokine expression. However, the profile of differentially expressed cytokines was heavily skewed toward an anti-inflammatory, pro-tumorigenic response in patients with MM. Significantly, our predictive model placed all patients in remission in the same category as those with active disease. Thus, our study demonstrates that the homeostasis of systemic cytokines is not restored when MM patients enter remission, suggesting that once an individual has cancer, the microenvironment is permanently altered and the system is primed for a relapse.

N-cadherin impedes proliferation of the multiple myeloma cancer stem cells.

Multiple myeloma (MM) is an incurable malignancy of the plasma cells localized to the bone marrow. A rare population of MM cancer stem cells (MM-CSCs) has been shown to be responsible for maintaining the pull of residual disease and to contribute to myeloma relapse. The stem cells are found in a bone marrow niche in contact with the stromal cells that are responsible for maintaining the proliferative quiescence of the MM-CSC and regulate its self-renewal and differentiation decisions. Here we show that both MM and bone marrow stromal cells express N-cadherin, a cell-cell adhesion molecule shown to maintain a pool of leukemic stem cells. Inhibition of N-cadherin using a neutralizing antibody led to an increase in the MM cell proliferation. A decrease in MM cell adhesion to the bone marrow stroma was observed in the first 24 hours of co-culture followed by a 2.3-30-fold expansion of the adherent cells. Moreover, inhibition of N-cadherin led to a 4.8-9.6-fold expansion of the MM-CSC population. Surprisingly, addition of the N-cadherin antagonist peptide resulted in massive death of the non-adherent MM cells, while the viability of the adherent cells and MM-CSCs remained unaffected. Interestingly, the proliferative effects of N-cadherin inhibition were not mediated by the nuclear translocation of ß-catenin. Taken together, our findings demonstrate the crucial role of N-cadherin in regulating MM cell proliferation and viability and open an interesting avenue of investigation to understand how structural modifications of N-cadherin can affect MM cell behavior. Our findings suggest that targeting N-cadherin may be a useful therapeutic strategy to treat MM in conjunction with an agent that has anti-MM-CSC activity.

Exploiting the facile release of trifluoroacetate for the α-methylenation of the sterically hindered carbonyl groups on (+)-sclareolide and (-)-eburnamonine.

An efficient method for the α-methylenation of carbonyl groups is reported, and this transformation is accomplished by a facile elimination of trifluoroacetate during the formation of the olefin. This method represents an improvement beyond existing protocol in cases of steric hindrance, and we have demonstrated the utility of the process across a series of ketones, lactams, and lactones. Additionally, we have applied this method to produce semisynthetic derivatives of the natural products (+)-sclareolide and (-)-eburnamonine, in which the carbonyl group is proximal to bulky functional groups. Mechanistic insight is also provided from a time course of (19)F NMR. Biological evaluation of the natural-product-derived enones led to the identification of a derivative of (-)-eburnamonine with significant cytotoxicity (LC(50) = 14.12 µM) in drug-resistant MDA-MB-231 breast cancer cells.

The natural products parthenolide and andrographolide exhibit anti-cancer stem cell activity in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy where nearly all patients succumb to a relapse. The current preclinical models of MM target the plasma cells, constituting the bulk of the tumor, leaving the cancer stem cells to trigger a relapse. Utilizing a three-dimensional tissue culture system where cells were grown in extracellular matrix designed to reconstruct human bone marrow, we tested the anti-multiple myeloma cancer stem cell (MM-CSC) potential of two natural product inhibitors of nuclear factor κB (NFκB). Here we show that parthenolide and andrographolide are potent anti-MM-CSC agents. Both natural products demonstrated preferential toxicity toward MM-CSCs over non-tumorigenic MM cells. Addition of the bone marrow stromal compartment abrogated andrographolide activity while having no effect on parthenolide cytoxicity. This is the first report of a natural product with anti-CSC activity in myeloma, suggesting that it has the potential to improve the survival of patients with MM by eliminating the relapse-causing MM-CSCs.

In a patient with biclonal Waldenstrom macroglobulinemia only one clone expands in three-dimensional culture and includes putative cancer stem cells.

A small percentage of cases of Waldenstrom macroglobulinemia (WM) present with biclonality, defined here as the rearrangement of two distinct VDJ gene segments. Here we investigated the expansion of two clones from a patient with WM expressing molecularly detectable clonotypic gene rearrangements, one V(H)3 and one V(H)4. Biclonality was determined in blood and bone marrow mononuclear cells using real-time quantitative PCR (RQ-PCR). V(H)4 expressing cells but not V(H)3 expressing cells underwent clonal expansion in 3-D culture of reconstructed WM bone marrow. After 3-D culture, secondary culture in a colony forming unit assay, and RQ-PCR, only the V(H)4 clone was shown to harbor a subpopulation with characteristics of cancer stem cells, including proliferative quiescence, self-regeneration, and the ability to generate clonotypic progeny, suggesting that the V(H)4, but not the V(H)3, clone is clinically significant. Enrichment of potential WM stem cells in 3-D cultures holds promise for monitoring their response to treatment and for testing new therapies.

Altered expression of fibronectin and collagens I and IV in multiple myeloma and monoclonal gammopathy of undetermined significance.

Multiple myeloma (MM) is an incurable B-cell malignancy that arises in the bone marrow (BM). The malignant cells within the BM have extensive interaction with the structural components of their microenvironment. It has been previously shown that the interactions between MM cells and the BM extracellular matrix (ECM) proteins contribute to drug resistance. To understand the underlying causes of adhesion-mediated drug resistance in MM, the components of human BM ECM available for interactions with MM cells must be characterized. We analyzed the expression and localization of fibronectin, laminin, and collagens I and IV in the core biopsies of normal donors and patients with monoclonal gammopathy of undetermined significance (MGUS) or MM. In addition, we compared the patterns of ECM expression in MM patients with low-, mid-, and high-level plasmacytosis of the BM. Although expression of laminin was the same for all groups tested, levels of fibronectin and collagen I were reduced in MM patients with high-level plasmacytosis. Expression of collagen IV in the BM of MGUS and MM patients was higher than in the BM from normal donors. Compared with the plasma cells isolated from the patients with low- and mid-level plasmacytosis, sorted CD138(+) plasma cells from MM patients with high-level plasmacytosis overexpressed collagen IV. Our findings show that, compared with normal controls, the ECM composition of the bone, endosteum, and BM is aberrant in patients with MM, further establishing ECM as a key player in the MM disease process.

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

Although the in vitro expansion of the multiple myeloma (MM) clone has been unsuccessful, in a novel three-dimensional (3-D) culture model of reconstructed bone marrow (BM, n = 48) and mobilized blood autografts (n = 14) presented here, the entire MM clone proliferates and undergoes up to 17-fold expansion of malignant cells harboring the clonotypic IgH VDJ and characteristic chromosomal rearrangements. In this system, MM clone expands in a reconstructed microenvironment that is ideally suited for testing specificity of anti-MM therapeutics. In the 3-D model, melphalan and bortezomib had distinct targets, with melphalan targeting the hematopoietic, but not stromal com-partment. Bortezomib targeted only CD138(+)CD56(+) MM plasma cells. The localization of nonproliferating cells to the reconstructed endosteum, in contact with N-cadherin-positive stroma, suggested the presence of MM-cancer stem cells. These drug-resistant CD20(+) cells were enriched more than 10-fold by melphalan treatment, exhibited self-renewal, and generated clonotypic B and plasma cell progeny in colony forming unit assays. This is the first molecularly verified demonstration of proliferation in vitro by ex vivo MM cells. The 3-D culture provides a novel biologically relevant preclinical model for evaluating therapeutic vulnerabilities of all compartments of the MM clone, including presumptive drug-resistant MM stem cells.

Mutation analysis of the short cytoplasmic domain of the cell-cell adhesion molecule CEACAM1 identifies residues that orchestrate actin binding and lumen formation.

CEACAM1-4S (carcinoembryonic antigen cell adhesion molecule 1, with 4 ectodomains and a short, 12-14 amino acid cytoplasmic domain) mediates lumen formation via an apoptotic and cytoskeletal reorganization mechanism when mammary epithelial cells are grown in a three-dimensional model of mammary morphogenesis. We show by quantitative yeast two-hybrid, BIAcore, NMR HSQC and STD, and confocal analyses that amino acids phenylalanine (Phe(454)) and lysine (Lys(456)) are key residues that interact with actin orchestrating the cytoskeletal reorganization. A CEACAM1 membrane model based on vitamin D-binding protein that predicts an interaction of Phe(454) at subdomain 3 of actin was supported by inhibition of binding of actin to vitamin D-binding protein by the cytoplasmic domain peptide. We also show that residues Thr(457) and/or Ser(459) are phosphorylated in CEACAM1-transfected cells grown in three-dimensional culture and that mutation analysis of these residues (T457A/S459A) or F454A blocks lumen formation. These studies demonstrate that a short cytoplasmic domain membrane receptor can directly mediate substantial intracellular signaling.

Inhibition of transforming growth factor-beta1 signaling attenuates ataxia telangiectasia mutated activity in response to genotoxic stress.

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGFbeta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGFbeta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgfbeta1 null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGFbeta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gammaH2AX radiation-induced foci; and increased radiosensitivity compared with TGFbeta competent cells. We determined that loss of TGFbeta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGFbeta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgfbeta1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGFbeta may be used to advantage in cancer therapy.

Cell-cell adhesion molecule CEACAM1 is expressed in normal breast and milk and associates with beta1 integrin in a 3D model of morphogenesis.

CEA cell adhesion molecule-1 (CEACAM1) is a cell-cell adhesion molecule that, paradoxically, is expressed in an apical location in normal breast epithelium. Strong lumenal membrane staining is observed in 100% of normal glands (11/11), low in atypical hyperplasia (2/6), high in cribiform ductal carcinoma in situ (DCIS) (8/8), but low in other types of DCIS (2/15). Although most invasive ductal carcinomas express CEACAM1 (21/26), the staining pattern tends to be weak and cytoplasmic in tumours with minimal lumena formation (grades 2-3), while there is membrane staining in well-differentiated tumours (grade 1). The 'normal' breast epithelial line MCF10F forms acini with lumena in Matrigel with apical membrane expression of CEACAM1. MCF7 cells that do not express CEACAM1 and fail to form lumena in Matrigel, revert to a lumen forming phenotype when transfected with the CEACAM1-4S but not the -4L isoform. CEACAM1 directly associates with and down-regulates the expression of beta1-integrin. Immuno-electron microscopy reveals numerous vesicles coated with CEACAM1 within the lumena, and as predicted by this finding, CEACAM1 is found in the lipid fraction of breast milk. Thus, CEACAM1 is a critical molecule in mammary morphogenesis and may play a role in the absorption of the lipid vesicles of milk in the infant intestinal tract.

Characterization of recombinant soluble carcinoembryonic antigen cell adhesion molecule 1.

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a type 1 transmembrane, homotypic cell adhesion protein expressed on epithelial and hematopoietic cells. CEACAM1 has four major isoforms with three or four immunoglobulin (Ig)-like ectodomains and either long or short cytoplasmic domains. In a 3D model of breast epithelial cell morphogenesis, CEACAM1 plays an essential role in lumen formation [J. Cell Sci. 112 (1999) 4193]. Two soluble ectodomain isoforms of CEACAM1 expressed in myeloma cells were immunologically active and highly glycosylated. The molecular weights of the 3-ecto- and 4-ectodomain isoforms were 90 and 110kDa, respectively, and monomers by sedimentation equilibrium centrifugation. Both isoforms were prolate ellipsoids with axial ratios of 6 for the 3-ecto- and 8 for 4-ectodomain isoforms, respectively, by size exclusion chromatography and analytical ultracentrifugation. Both isoforms caused a significant reduction in lumen formation when tested in the 3D model culture system.

CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis.

The epithelial cell adhesion molecule CEACAM1 (carcinoembryonic antigen cell adhesion molecule-1) is down-regulated in colon, prostate, breast, and liver cancer. Here we show that CEACAM1-4S, a splice form with four Ig-like ectodomains and a short cytoplasmic domain (14 amino acids), directly associates with annexin II, a lipid raft-associated molecule, which is also down-regulated in many cancers. Annexin II was identified using a glutathione S-transferase pull-down assay in which the cytoplasmic domain of CEACAM-4S was fused to glutathione S-transferase, the fusion protein was incubated with cell lysates, and isolated proteins were sequenced by mass spectrometry. The interaction was confirmed first by reciprocal immunoprecipitations using anti-CEACAM1 and anti-annexin II antibodies and second by confocal laser microscopy showing co-localization of CEACAM1 with annexin II in mammary epithelial cells grown in Matrigel. In addition, CEACAM1 co-localized with p11, a component of the tetrameric AIIt complex at the plasma membrane, and with annexin II in secretory vesicles. Immobilized, oriented peptides from the cytoplasmic domain of CEACAM1-4S were shown to directly associate with bovine AIIt, which is 98% homologous to human AIIt, with average KD values of about 30 nM using surface plasmon resonance, demonstrating direct binding of functionally relevant AIIt to the cytoplasmic domain of CEACAM1-4S.