Abstract
The uncontrolled proliferation of genetically mutated cells is the commonly understood mechanism for cancer growth and invasion, with accumulation of new mutations in daughter cells leading to clonal diversity of cancer derived from a single founding event. The genetic alterations are passed to new generations by cell division and vertical gene transfer. Viral transmission of oncogenes represents a known mechanism of lateral gene transfer in cancer initiation. Some experimental systems have also suggested that circulating DNA or micro-vesicles may contribute to lateral oncogene transfer in tumorigenesis. We hypothesized that interactions between leukemic cells and adjacent normal hematopoietic stem or progenitor cells may provide an alternative mechanism for the accumulation of mutated genes and the multiplicity of distinct clones in leukemia.
To test this hypothesis, we performed experiments to determine whether tumorigenic properties could be transferred from a tumor cell line to normal mouse bone marrow cells using both in vivo and in vitro and systems. B6-GFP+ mice were injected i.v. with 200,000 C1498-Luc cells (a B6-derived NKT-cell-like mouse tumor cell line expressing luciferase and DSRed). Bioluminescent imaging was used to monitor the progression of tumor cell growth in recipients. At 1 month after tumor-cell inoculation, marrow from these mice was harvested and FACS-sorted for GFP+ cells (to eliminate C1498 cells), and then cultured on irradiated stromal cell layers in 96-well plates in a limiting dilution analysis for Poisson analysis of GFP+ clonogenic precursor frequency on day 9. On day 10, cells were harvested from culture and GFP+ cells resorted onto fresh stromal layers for second and third determinations of GFP+ clonogenic precursor frequency on days 15 and 18. As shown in Figure 1, the frequency of clonogenic precursors increased with each successive determination for marrow from C1498-injected mice, while control cultures from non-injected mice showed no increase in precursor frequency, suggesting that exposure to C1498 cells conferred a growth advantage to the marrow cells in the tumor-cell injected mice. Similar results were obtained using an in vitro system of co-culture using C1498 cells and GFP+ bone marrow cells, followed by serial rounds of GFP+ sorting and Poisson analysis, showing increases in clonogenic frequency over 5 successive sorts and re-cultures over a 2-month period, while control cultures showed decreased clonogenic frequencies over the course of the experiment.
To confirm these observations in vivo, B6-GFP mice were injected with C1498-Luc and marrow was harvested after a month and sorted for GFP+ cells. The sorted marrow was transplanted into 11Gy-irradiated (FVB x B6albino)F1 recipients (5 x 106 cells per recipient, n=5). Control recipients were irradiated and transplanted with GFP+ marrow from non-injected donors. All recipients developed full hematopoietic engraftment with GFP+ cells. At 6 months post-transplant, a tumor was observed near the left shoulder of one of the recipients of C1498-exposed GFP+ marrow. Figure 2 shows IVIS GFP imaging of this mouse with the GFP+ tumor along with control animals. The tumor was not positive for luciferase expression. The mouse was sacrificed and the tumor excised and a portion was dissociated for flow cytometric analysis and culturing (with other segments reserved for subsequent histological and genetic analysis). Both GFP+ and non-GFP cells were found in the dissociated tumor cell suspension. The GFP+ cells were hematopoietic in origin (CD45+) and exhibited a mixed phenotype containing markers expressed on C1498 (DX5+) and myeloid lineage cells (CD11b+) as well as Sca-1, a stem cell marker. Cultures of the GFP+ tumor yielded a population of GFP+ mononuclear cells.
These data are consistent with a model in which growth-promoting or transforming genes from cancer cells become incorporated within a healthy hematopoietic stem or progenitor cell, which contributes to the genetic diversity of the cancer through the initiation a new transformed clone. Genetic analysis with deep sequencing will compare the DNA sequences between the parental C1498 cell line, sorted populations of clonogenic GFP+ cells obtained from the in vitro and in vivo experiments, and the GFP+ tumor cells to confirm the transformation of healthy bone marrow hematopoietic stem cells with genetic sequences derived from the C1498 cells.
No relevant conflicts of interest to declare.
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