Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways.

Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth.

Malignant transformation of Slp65-deficient pre-B cells involves disruption of the Arf-Mdm2-p53 tumor suppressor pathway.

The adapter protein Slp65 is a key component of the precursor-B (pre-B) cell receptor. Slp65-deficient mice spontaneously develop pre-B cell leukemia, but the mechanism by which Slp65(-/-) pre-B cells become malignant is unknown. Loss of Btk, a Tec-family kinase that cooperates with Slp65 as a tumor suppressor, synergizes with deregulation of the c-Myc oncogene during lymphoma formation. Here, we report that the presence of the immunoglobulin heavy chain transgene V(H)81X prevented tumor development in Btk(-/-)Slp65(-/-) mice. This finding paralleled the reported effect of a human immunoglobulin heavy chain transgene on lymphoma development in Emu-myc mice, expressing transgenic c-Myc. Because activation of c-Myc strongly selects for spontaneous inactivation of the p19(Arf)-Mdm2-p53 tumor suppressor pathway, we investigated whether disruption of this pathway is a common alteration in Slp65(-/-) pre-B cell tumors. We found that combined loss of Slp65 and p53 in mice transformed pre-B cells very efficiently. Aberrations in p19(Arf), Mdm2, or p53 expression were found in all Slp65(-/-) (n = 17) and Btk(-/-)Slp65(-/-) (n = 32) pre-B cell leukemias analyzed. In addition, 9 of 10 p53(-/-)Slp65(-/-) pre-B cell leukemias manifested significant Mdm2 protein expression. These data indicate that malignant transformation of Slp65(-/-) pre-B cells involves disruption of the p19(Arf)-Mdm2-p53 tumor suppressor pathway.

A mouse model for chronic lymphocytic leukemia based on expression of the SV40 large T antigen.

The simian virus 40 (SV40) T antigen is a potent oncogene able to transform many cell types and has been implicated in leukemia and lymphoma. In this report, we have achieved sporadic SV40 T-antigen expression in mature B cells in mice, by insertion of a SV40 T antigen gene in opposite transcriptional orientation in the immunoglobulin (Ig) heavy (H) chain locus between the D and J(H) segments. SV40 T-antigen expression appeared to result from retention of the targeted germline allele and concomitant antisense transcription of SV40 large T in mature B cells, leading to chronic lymphocytic leukemia (CLL). Although B-cell development was unperturbed in young mice, aging mice showed accumulation of a monoclonal B-cell population in which the targeted IgH allele was in germline configuration and the wild-type IgH allele had a productive V(D)J recombination. These leukemic B cells were IgD(low)CD5(+) and manifested nonrandom usage of V, D, and J segments. V(H) regions were either unmutated, with preferential usage of the VH11 family, or manifested extensive somatic hypermutation. Our findings provide an animal model for B-CLL and show that pathways activated by SV40 T antigen play important roles in the pathogenesis of B-CLL.

Ceramic hydroxyapatite coating on titanium implants drives selective bone marrow stromal cell adhesion.

The aim of this study was to determine the cell characteristics that regulate implant osseointegration. The heterogeneity of bone marrow stromal cells obtained from 11 donors was assessed by measuring the expression of a large panel of adhesion molecules. Large differences in expression of adhesion molecules were detected depending on the culture conditions used. Cells cultured in fetal bovine serum induced the expression of different adhesion molecules from cells cultured in human serum. Donor-to-donor variation was determined by measuring the expression of adhesion molecules for stromal cells obtained from different donors that were processed identically. Fat adherent cells but also loose bone marrow cells showed large differences in expression of some but not all adhesion molecules. The flow cytometric data demonstrated large heterogeneity in expression of adhesion molecules, and this heterogeneity was influenced by culture conditions and varied from donor to donor. This demonstrates that the implant encounters different cell types, which could lead to different levels of integration. Surprisingly, in vitro only a subfraction of bone marrow stromal cells attached to titanium coated with ceramic hydroxyapatite. Adaptation of all cell types present in heterogeneous bone marrow to a coated surface is apparently not possible. Differential binding was not caused by aberrant staining of the stromal cells as the results were confirmed with bone marrow cells obtained from transgenic GFP mice. These results demonstrate that hydroxyapatite ceramics are selective in cell recruitment from the bone marrow, explaining the differences found in vivo for these coatings compared with titanium.

Effect of calcium phosphate coating composition and crystallinity on the response of osteogenic cells in vitro.

The aim of this study was to investigate the effect of calcium phosphate coating crystallinity and composition on the proliferation and differentiation of rat bone marrow (RBM) cells. Grit-blasted titanium substrates were provided with thin sputter-coated calcium phosphate (Ca-P) films of different composition. The Ca-P-coated substrates were used as-sputtered or were heat-treated. XRD measurements showed that the as-sputtered coatings had an amorphous structure, whereas the heat-treated substrates showed an amorphous-crystalline structure. RBM cells were cultured on these substrates and on noncoated titanium substrates. After specific culture times, the expression of osteogenic markers by the cells was studied. On the amorphous-crystalline coatings as well as on titanium substrates, RBM cells proliferated, expressed alkaline phosphatase and showed mineralization. More mineralization was found on the amorphous-crystalline coatings than on the titanium substrates. Some precipitation was also found on substrates that were incubated in complete culture medium without cells. This precipitate disappeared after prolonged incubation. Alkaline phosphatase expression differed on the various amorphous-crystalline Ca-P-coated substrates, but no difference was found in the mineralization on these substrates. The amorphous Ca-P coatings showed extensive dissolution and some signs of precipitation after longer culture periods. Proliferation and differentiation of RBM cells was not seen on the amorphous coatings, regardless of Ca-P composition. We conclude that amorphous-crystalline Ca-P coatings stimulate differentiation of RBM cells, with only limited differences between coatings of various composition. In contrast, Ca-P coatings with an amorphous structure inhibit the growth and differentiation of RBM cells. This effect was found on all amorphous substrates, regardless of Ca-P composition.