Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction☆

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Abstract

To confirm whether human cancer-induced stromal cells are derived from bone marrow, bone marrow (BM) cells obtained from β-galactosidase transgenic and recombination activating gene 1 (RAG-1) deficient double-mutant mice (H-2b) were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice (H-2d). The human pancreatic cancer cell line Capan-1 was subcutaneously xenotransplanted into SCID recipients and stromal formation was analyzed on day 14 and on day 28. Immunohistochemical and immunofluorescence studies revealed that BM-derived endothelial cells (X-gal/CD31 or H-2b/CD31 double-positive cells) and myofibroblasts (X-gal/α-smooth muscle actin or H-2b/α-smooth muscle actin double-positive cells) were present within and around the cancer nests. On day 14, the frequencies of BM-derived endothelial cells and BM-derived myofibroblasts were 25.3 ± 4.4% and 12.7 ± 9.6%, respectively. On day 28, the frequency of BM-derived endothelial cells was 26.7 ± 9.7%, which was similar to the value on day 14. However, the frequency of BM-derived myofibroblasts was significantly higher (39.8 ± 17.1%) on day 28 than on day 14 (P<0.05). The topoisomerase IIα-positive ratio was 2.2 ± 1.2% for the H-2b-positive myofibroblasts, as opposed to only 0.3 ± 0.4% for the H-2b-negative myofibroblasts, significant proliferative activity was observed in the BM-derived myofibroblasts (P<0.05). Our results indicate that BM-derived myofibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development.

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Materials and methods

Animals. β-Galactosidase transgenic (β-gal Tg) mice (ROSA 26, C57BL/6 background: H-2b) [18] and recombination activating gene 1 (RAG-1) −/− mice (C57BL/6 background: H-2b) [19] were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The β-gal Tg mice were mated with RAG-1−/− mice to generate F1 offspring that were heterogenous for both genes. The F2 offspring of the F1 interbreeding were screened by Western blot analysis for the absence of serum IgM [19]. Mice identified as

Generation of an animal model of the cancer-induced stromal reaction

Sublethally irradiated SCID (H-2d) mice were injected with 1 × 107 BM cells from β-gal Tg and RAG-1 deficient double-mutant mice (β-gal Tg RAG-1−/−, H-2b). H-2 phenotyping of BM cells from the recipient mice demonstrated that their marrows had been reconstituted by high levels (>90%) of donor cells 4 weeks after BMT (Fig. 1). Histological examination did not reveal any obvious evidences of graft-versus-host disease (GVHD) effects in the liver, lungs, colon, skin, or spleen of a large series of

Discussion

The pathogenesis of the stromal reaction in human cancer has remained unclear because of the lack of a good animal model. The main outcome of our study was the establishment of a SCID mouse model that can be used to analyze the stromal response to invading human cancer cells. We used human pancreatic cancer cell line Capan-1 from the following reasons. First, almost all mouse cancer cell lines show solid growth and only quite a little stroma was seen in these cancers when transplanted into

Acknowledgements

We are grateful to Motoko Suzaki for preparing for the manuscript.

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    ☆

    Supported in part by the Grant-in-Aid for Cancer Research (11–12) from the Ministry of Health, Labour and Welfare, the Grant for Scientific Research Expenses for Health Labour and Welfare Programs, the Foundation for the Promotion of Cancer Research, 2nd-Term Comprehensive 10-year Strategy for Cancer Control, and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. T.S. and N.K. are recipients of Research Resident Fellowships from Foundation for Promotion of Cancer Research. M.E. was a Foreign Research Fellow of the Foundation for Promotion of Cancer Research, Tokyo, from October 15, 1998, to March 14, 1999.

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