Shoshana Baron

The CXCR4 antagonist AMD3100 impairs survival of human AML cells and induces their differentiation

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, participate in the retention of acute myeloblastic leukemia (AML) cells within the bone marrow microenvironment and their release into the circulation. AML cells also constitutively express SDF-1-dependent elastase, which regulates their migration and proliferation. To study the molecular events and genes regulated by the SDF-1/CXCR4 axis and elastase in AML cells, we examined gene expression profiles of the AML cell line, U937, under treatment with a neutralizing anti-CXCR4 antibody or elastase inhibitor, as compared with non-treated cells, using DNA microarray technology. Unsupervised hierarchical clustering analysis demonstrated similar gene expression profiles of anti-CXCR4 antibody or elastase inhibitor-treated cells, as compared with control. Pathway and functional analysis showed a greater tendency toward differentiation in cells under either one of both treatment modalities. Thus given, we further analyzed the effects of CXCR4 inhibition on AML cell growth and differentiation using the antagonist AMD3100. AMD3100 arrested proliferation in AML cell lines and triggered changes that mimicked differentiation, including morphological changes and the expression of myeloid differentiation antigens. Inhibition of elastase also triggered the differentiation of AML cells. Our study defines a new role for the SDF-1/CXCR4 axis in the regulation of leukemic cell survival and differentiation.

Diverse niches within multiple myeloma bone marrow aspirates affect plasma cell enumeration

A basic criterion for the diagnosis of multiple myeloma is plasma cell enumeration within the bone marrow (BM). This report showed that flow cytometry under‐estimated the number of plasma cells in BM aspirates by an average of 60%, compared with morphological evaluation. The discrepancy was partially because BM smears contain cells associated with the lipid‐enriched spicules. In contrast, flow cytometry is performed on the BM fluid, which is depleted of the lipid‐adhesive plasma cells. This discrepancy may point to different plasma cell subpopulations associated with diverse niches within the BM.

Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome.

ZAP‐70 has emerged as a potential pivotal prognostic marker for patients with chronic lymphocytic leukemia (CLL), which could replace immunoglobulin heavy chain mutation status. Although several flow cytometry assays have been described for assessing ZAP‐70 in CLL, certain technical and scientific issues remain unsolved, which have prevented results of this crucial test from being reported, even in the best routine flow cytometry laboratories. In this report, we aimed to solve some of these issues by providing a computerized quantitative flow cytometric assay for ZAP‐70 within the entire CLL population, which would be easy to perform and enable standardization between laboratories.

Photodynamic therapy of colorectal cancer using a new light source : From in vitro studies to a patient treatment

PURPOSE: Photodynamic therapy (PDT) is a relatively new alternative modality for palliation of rectal cancer. Current source of light for PDT are laser systems that are expensive and not necessarily needed for PDT. We evaluated a new nonlaser light source for PDT, Versa-Light®.METHODS AND RESULTS:In vitroPDT—CT26 murine colon carcinoma cells were incubated with aluminum phthalocyanine (AlPcS4) for 48 hours and subjected to photoradiation using Versa-Light®,and viability was assessed. There was a significant decrease in viability of treated cells compared with controls.In vivoPDT—BALB/c mice were injected either subcutaneously or intrarectally with CT26 cancer cells. IP AlPcS4(2.5 mg/kg) was injected when tumors were visible. After 24 hours, mice were subjected to photoradiation. Massive tumor necrosis in response to PDT was observed. PDT also prolonged survival of treated mice. Patient treatment—A 70-year-old woman with recurrent local rectal carcinoma received intravenous Photofrin II®(2 mg/kg). After 48 and 96 hours, she was subjected to direct photoradiation. After the first light session, there was complete macroscopic disappearance of the tumor. Biopsies up to 10 weeks after the treatment showed no cancer cells in the treated area. Sixteen weeks later, a randomized biopsy from previous tumor site showed carcinoma cells. CONCLUSIONS: We believe that Versa-Light®,is a good light source for PDT. It was effective in bothin vitroand animal studies. It can also be safely used for clinical PDT.

The Generation and Regulation of Functional Diversity of Malignant Plasma Cells

Cellular diversity, which is a hallmark of malignancy, can be generated by both genetic and nongenetic mechanisms. We describe here variability in the adhesive and migratory behavior of malignant plasma cell populations, including multiple myeloma-derived lines and primary patient samples. Examination of the plasma cell lines ARH-77, CAG, and AKR revealed two distinct subpopulations of cells, one displaying highly adhesive properties (type A) and the other consisting of poorly adhesive, floating cells (type F). In the ARH-77 cell line, type A cells attach better to fibronectin and to human bone fragments and form paxillin-rich focal adhesions, whereas type F cells are highly motile and exert integrin-dependent bone marrow homing capacity in nonobese diabetic/severe combined immunodeficient mice. Flow cytometry indicated that type A cells express significantly higher levels of CD45 and CD56 and lower levels of CD138 compared with type F cells. Interestingly, culturing of either type A or type F cells under nonselective conditions resulted in the development of mixed cell population similar to the parental ARH-77 cells. Analysis of bone marrow aspirates of multiple myeloma patients revealed that spicules within the aspirates are enriched with type A-like cells. Nonadherent cells within the aspirate fluids express a marker profile similar to type F cells. This study indicates that multiple myeloma patients contain heterogeneous populations of malignant plasma cells that display distinct properties. Diverse subpopulations of malignant plasma cells may play distinct roles in the different biological and clinical manifestations of plasma cell dyscrasias, including bone dissemination and selective adhesion to bone marrow compartments.

Mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6

Multiple myeloma is characterized by the malignant growth of immunoglobulin producing plasma cells, predominantly in the bone marrow. The effects of primary human mesenchymal stromal cells on the differentiation phenotype of multiple myeloma cells were studied by co-culture experiments. The incubation of multiple myeloma cells with bone marrow-derived mesenchymal stromal cells resulted in significant reduction of the expression of the predominant plasma cell differentiation markers CD38 and CD138, and cell surface immunoglobulin light chain. While the down-regulation of CD138 by stromal cells was completely dependent on their adhesive interactions with the multiple myeloma cells, interleukin-6 induced specific down-regulation of CD38. Mesenchymal stromal cells or their conditioned media inhibited the growth of multiple myeloma cell line, thereby reducing the overall amounts of secreted light chains. Analysis of primary multiple myeloma bone marrow samples reveled that the expression of CD38 on multiple myeloma cells was not affected by adhesive interactions. The ex vivo propagation of primary multiple myeloma cells resulted in significant increase in their differentiation markers. Overall, the data indicate that the bone marrow-derived mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6.

MUC1/X Protein Immunization Enhances cDNA Immunization in Generating Anti-MUC1 α/β Junction Antibodies that Target Malignant Cells

MUC1 has generated considerable interest as a tumor marker and potential target for tumor killing. To date, most antibodies against MUC1 recognize epitopes within the highly immunogenic α chain tandem repeat array. A major shortcoming of such antibodies is that the MUC1 α chain is shed into the peripheral circulation, sequesters circulating antitandem repeat array antibodies, and limits their ability to even reach targeted MUC1-expressing cells. Antibodies recognizing MUC1 epitopes tethered to the cell surface would likely be more effective. MUC1 α subunit binding the membrane-tethered β subunit provides such an epitope. By use of a novel protocol entailing immunization with cDNA encoding full-length MUC1 (MUC1/TM) followed by boosting with the alternatively spliced MUC1/X isoform from which the tandem repeat array has been deleted, we generated monoclonal antibodies, designated DMC209, which specifically bind the MUC1 α/β junction. DMC209 is exquisitely unique for this site; amino acid mutations, which abrogate MUC1 cleavage, also abrogate DMC209 binding. Additionally, DMC209 specifically binds the MUC1 α/β junction on full-length MUC1/TM expressed by breast and ovarian cancer cell lines and on freshly obtained, unmanipulated MUC1-positive malignant plasma cells of multiple myeloma. DMC209 is likely to have clinical application by targeting MUC1-expressing cells directly and as an immunotoxin conjugate. Moreover, the novel immunization procedure used in generating DMC209 can be used to generate additional anti-MUC1 α/β junction antibodies, which may, analogously to Herceptin, have cytotoxic activity. Lastly, sequential immunization with MUC1/TM cDNA acting as a nonspecific adjuvant followed by protein of interest may prove to be a generalizable method to yield high-titer specific antibodies. (Cancer Res 2006; 66(23): 11247-53)

Phenotype and function of lymphocytes from the neonatal umbilical cord compared to paired maternal peripheral blood cells isolated during delivery.

In the present study, we analyzed the immunological characteristics of mononuclear cells (MNC) isolated from both neonatal umbilical cord blood (UCB) and maternal peripheral blood (MPB) during the delivery. The in vitro proliferative response of UCB T lymphocytes was significantly reduced compared to the maternal response to phytohemagglutinin A, pokeweed mitogen, and alloantigen stimulation, in correlation with the lower percentage of UCB than MPB lymphocytes, but not with that of B cells. The mean cytotoxic activity level of interleukin-2 (IL-2)-activated natural killer (NK) was higher in UCB than in MBP, whereas the percentage of CD56(+) NK cell count was similar. Our results show differences in the immune reactivity of T and B lymphocytes from neonate and adult isolated under similar physiological conditions.

Inhibition of CT-26 murine adenocarcinoma growth in the rectum of mice treated with recombinant human interleukin-6

In the present study we evaluated the antitumor effects of recombinant human interleukin-6 (rhIL-6), expressed in Chinese hamster ovary cells, in a murine primary tumor model. We showed that treatment with rhIL-6 substantially inhibited the implantation and growth rates of CT-26 adenocarcinoma tumor cells in the rectal submucosa of syngeneic mice. This effect was achieved by injecting rhIL-6 for 7 consecutive days starting 1 day prior to tumor inoculation. No obvious antitumor effect was noted when rhIL-6 injections started 5 days after tumor inoculation. Analysis of the mechanisms by which rhIL-6 exerts its antitumor effects did not reveal a direct antitumor effect on CT-26 tumor cells or the up-regulation of major histocompatibility complex antigens on these cells. However, infiltration of lymphocytes at the tumor site was observed. Increase of carcinoembryonic antigen by IL-6 was clearly seen in human HT-29 colon carcinoma cells. The possible application of these results for adjuvant immunotherapy of selected colorectal patients and prevention of reimplantation of tumor cells disseminated during surgery is discussed.

Divergence in CD19-Mediated Signaling Unfolds Intraclonal Diversity in Chronic Lymphocytic Leukemia, Which Correlates with Disease Progression

Emerging data on intraclonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with chronic lymphocytic leukemia (CLL), where subsets of the CLL clone responding more robustly to external stimuli may gain a growth and survival advantage. In this study, we report intraclonal diversity resolved by responses to CD19 engagement in CLL cells, which can be classified into CD19-responsive (CD19-R) and -nonresponive subpopulations. Engagement of CD19 by anti-CD19 Ab rapidly induced cellular aggregation in the CD19-R CLL cells. The CD19-R CLL cells expressed higher surface levels of CD19 and c-myc mRNA, exhibited distinct morphological features, and were preferentially abolished in rituximab-treated patients. Both subpopulations reacted to sIgM stimulation in a similar manner and exhibited similar levels of Akt and Erk phosphorylation, pointing to functional signaling divergence within the BCR. CD19 unresponsiveness was partially reversible, where nonresponding CD19 cells spontaneously recover their signaling capacity following incubation in vitro, pointing to possible in vivo CD19–signaling attenuating mechanisms. This concept was supported by the lower CD19-R occurrence in bone marrow–derived samples compared with cells derived from the peripheral blood of the same patients. CLL patients with >15.25% of the CD19-R cell fraction had a shorter median time to treatment compared with patients with <15.25% of CD19-R cell fraction. In conclusion, divergence in CD19-mediated signaling unfolds both interpatient and intraclonal diversity in CLL. This signaling diversity is associated with physiological implications, including the location of the cells, their responses to anti-CLL therapeutics, and disease progression.