Efficient tumour formation by single human melanoma cells

A fundamental question in cancer biology is whether cells with tumorigenic potential are common or rare within human cancers. Studies on diverse cancers, including melanoma, have indicated that only rare human cancer cells ( 0.1 - 0.0001%) form tumours when transplanted into non- obese diabetic/ severe combined immunodeficiency ( NOD/ SCID) mice. However, the extent to which NOD/ SCID mice underestimate the frequency of tumorigenic human cancer cells has been uncertain. Here we show that modified xenotransplantation assay conditions, including the use of more highly immunocompromised NOD/ SCID interleukin- 2 receptor gamma chain null (Il2rg(-/-)) mice, can increase the detection of tumorigenic melanoma cells by several orders of magnitude. In limiting dilution assays, approximately 25% of unselected melanoma cells from 12 different patients, including cells from primary and metastatic melanomas obtained directly from patients, formed tumours under these more permissive conditions. In single- cell transplants, an average of 27% of unselected melanoma cells from four different patients formed tumours. Modifications to xenotransplantation assays can therefore dramatically increase the detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.Howard Hughes Medical Institute ; Allen H. Blondy Research Fellowship ; Lewis and Lillian Becker ; University of Michigan Comprehensive Cancer Center ; National Institutes of Health [CA46592]; University of Michigan Flow Cytometry Core Facility ; N. McAnsh and the University of Michigan Cancer Centre Histology Core ; National Institute of Diabetes, Digestive, and Kidney Diseases [NIH5P60- DK20572]; Michigan Diabetes Research and Training Center ; Spanish Ministry of Education ; Marie Curie Outgoing International Fellowship from the European Commission ; Australian National Health and Medical Research Council ; Human Frontiers Science Program and Australia PostThis work was supported by the Howard Hughes Medical Institute and by the Allen H. Blondy Research Fellowship. The University of Michigan Melanoma Bank was supported by a gift from Lewis and Lillian Becker. Flow cytometry was partly supported by the University of Michigan Comprehensive Cancer Center grant from the National Institutes of Health CA46592. We thank: D. Adams, M. White and the University of Michigan Flow Cytometry Core Facility for support; N. McAnsh and the University of Michigan Cancer Centre Histology Core for histological studies; G. K. Smyth for assistance with statistics; and Z. Azizan for support with tissue collection. Antibody production was supported in part by the National Institute of Diabetes, Digestive, and Kidney Diseases, grant NIH5P60- DK20572 to the Michigan Diabetes Research and Training Center. Some antibodies were provided by Caltag or by eBioscience to screen for cancer stem- cell markers. Human primary melanocyte cultures were provided by M. Soengas. Human mesenchymal stem cells were provided by Z. Wang and P. Krebsbach. E. Q. was supported by the Spanish Ministry of Education and the Marie Curie Outgoing International Fellowship from the European Commission. M. S. was supported by the Australian National Health and Medical Research Council, the Human Frontiers Science Program and Australia Post.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62970/1/nature07567.pd

Differences in nuclear positioning of 1q12 pericentric heterochromatin in normal and tumor B lymphocytes with 1q rearrangements.

The frequent rearrangement of chromosome band 1q12 constitutive heterochromatin in hematologic malignancies suggests that this rearrangement plays an important pathogenetic role in these diseases. The oncogenic mechanisms linked to 1q12 heterochromatin are unknown. Constitutive heterochromatin can epigenetically regulate gene function through the formation of transcriptional-silencing compartments. Thus, as a first step toward understanding whether 1q12 rearrangements might compromise such activity in tumor cells, we investigated the 3-D organization of the 1q12 heterochromatin domain (1q12HcD) in normal and tumor B lymphocytes. Strikingly, in normal B cells, we showed that the 1q12HcD dynamically organizes to the nuclear periphery in response to B-cell receptor engagement. Specifically, we observed an almost twofold increase in 1q12Hc domains at the extreme nuclear periphery in activated versus resting B lymphocytes. Remarkably, 1q12Hc organization was noticeably altered in tumor cells that showed structural alterations of 1q12; the 1q12Hc domains were significantly displaced from the extreme nuclear periphery compared to normal activated B lymphocytes (P > 0.0001), although overall peripheral localization was maintained. In a case in which there was a translocation of IGL enhancer to 1q, the altered nuclear positioning of the 1q12HcD was even more pronounced (5% of the 1q12Hc domains at the nuclear periphery compared to 20% in other lymphoma lines), and we were able to mimic this effect in two additional B-cell tumor lines by treatment with trichostatin A, a histone deacetylase (HDAC) inhibitor. Taken together, these results point to the 1q12HcD having a specific, nonrandom, and regulated peripheral organization in B lymphocytes. This organization is significantly disrupted in lymphoma cells harboring 1q rearrangements

Development of autologous cytotoxic CD4+ T clones in a human model of B-cell non-Hodgkin follicular lymphoma.

Immunotherapy for cancer aims to generate cytotoxic cells that are capable of eradicating tumour cells. It has been well demonstrated that helper, non-cytotoxic CD4(+) T cells are important for the induction and maintenance of anti-tumour immunity exerted by cytotoxic CD8(+) T cells. In contrast, the existence of direct anti-tumour, effector cytotoxic CD4(+) T cells remains elusive, mainly due to the paucity of reliable experimental data, especially in human B-cell non-Hodgkin lymphomas. This study developed an appropriate, autologous follicular B-cell non-Hodgkin follicular lymphoma model, including the in vitro establishment of a malignant, human leucocyte antigen class I (HLA-I) deficient B-cell line, and the generation of three autologous anti-tumour cytotoxic CD4(+) T-cell clones originating from the peripheral blood of the same patient. These three clones were considered as tumour specific, because they were capable of killing the malignant, HLA-I-deficient B-cell line through a classical HLA-II restricted perforin-mediated pathway, but did not lyse the Epstein-Barr virus-infected autologous normal B lymphocytes. All three CD4(+)clones were T-cell receptor Vbeta17-Dbeta1-Jbeta1.2 and exhibited an identical complementarity-determining region 3, suggesting the immunodominance of a single peptide antigen presented by tumour cells. Such lymphoma models would provide a useful tool for in vivo expansion and the adoptive transfer of selected CD4(+) cytotoxic cells in immunotherapeutic strategies