Takafumi Sangai

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

To confirm whether human cancer-induced stromal cells are derived from bone marrow, bone marrow (BM) cells obtained from beta-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/alpha-smooth muscle actin or H-2b/alpha-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 IIalpha-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.

Stromal MCP-1 in mammary tumors induces tumor-associated macrophage infiltration and contributes to tumor progression

There is growing evidence that tumor‐associated macrophages (TAMs) promote tumor growth and dissemination. Many individual reports have focused on the protumor function of molecules linked to the recruitment of macrophages, but little is known about which factor has the strongest impact on recruitment of macrophages in breast cancer. To elucidate this question, we performed RT‐PCR using species‐specific primers and evaluated tumoral and stromal mRNA expression of macrophage chemoattractants separately in human breast tumor xenografts. The correlation between the tumoral or stromal chemoattractant mRNA expression including monocyte chemoattractant protein‐1 (MCP‐1) (CCL2), MIP‐1α (CCL3), RANTES (CCL5), colony‐stimulating factor 1, tumor necrosis factor α, platelet‐derived growth factor (PDGF)‐BB and macrophage infiltration were compared. There was significant positive correlation between stromal MCP‐1 expression and macrophage number (r = 0.63), and negative correlation between tumoral RANTES expression and macrophage number (r = −0.75). However, no significant correlation was found for the other tumoral and stromal factors. The interaction between the tumor cells and macrophages was also investigated. Tumor cell–macrophage interactions augmented macrophage‐derived MCP‐1 mRNA expression and macrophage chemotactic activity in vitro. Treatment of immunodeficient mice bearing human breast cancer cells with a neutralizing antibody to MCP‐1 resulted in significant decrease of macrophage infiltration, angiogenetic activity and tumor growth. Furthermore, immunohistochemical analysis of human breast cancer tissue showed stromal MCP‐1 had a significant correlation with relapse free survival (p = 0.029), but tumoral MCP‐1 did not (p = 0.105). These findings indicate that stromal MCP‐1 produced as a result of tumor–stromal interactions may be important for the progression of human breast cancer and macrophages may play an important role in this tumor–stroma interaction.

In Vivo Characterization of Bone Marrow–Derived Fibroblasts Recruited into Fibrotic Lesions

Fibroblasts, which are widely distributed and play a key part in tissue fibrosis, are phenotypically and functionally heterogeneous. Recent studies reported that bone marrow can be a source of tissue fibroblast. In the study reported here, we investigated in vivo characterization of bone marrow–derived fibroblasts recruited into various fibrotic lesions. Mice were engrafted with bone marrow isolated from transgenic mice expressing green fluorescent protein (GFP), and fibrotic lesions were induced by cancer implantation (skin), excisional wounding (skin), and bleomycin administration (lung). A small population of GFP+ fibroblast was found even in nonfibrotic skin (8.7% ± 4.6%) and lung (8.9% ± 2.5%). The proportion of GFP+ fibroblasts was significantly increased after cancer implantation(59.7%±16.3%) and excisional wounding (32.2% ± 4.8%), whereas it was not elevated after bleomycin administration (7.1% ± 2.4%). Almost all GFP+ fibroblasts in fibrotic lesions expressed type I collagen, suggesting that bone marrow–derived fibroblasts would contribute to tissue fibrosis. GFP+ fibroblasts expressed CD45, Thy‐1, and α‐smooth muscle actin at various proportions. Our results suggested that bone marrow–derived fibroblasts expressed several fibroblastic markers in vivo and could be efficiently recruited into fibrotic lesions in response to injurious stimuli; however, the degree of recruitment frequency might depend on the tissue microenvironment.

Growth Inhibition of Human Prostate Cancer Cells in Human Adult Bone Implanted into Nonobese Diabetic/Severe Combined Immunodeficient Mice by a Ligand-Specific Antibody to Human Insulin-Like Growth Factors

Advanced prostate cancer frequently involves the bone that has the largest content of insulin-like growth factors (IGFs). However, the role of bone-derived IGFs in bone metastasis of prostate cancer has not been studied extensively because of the lack of a reliable animal model. Therefore, we investigated whether a novel antibody directed against human IGF-I and IGF-II (KM1468) could inhibit the development of new bone tumors and the progression of established bone tumors in nonobese diabetic/severe combined immunodeficient mice implanted with human adult bone. We first confirmed that KM1468 bound specifically to human IGF-I, human IGF-II, and mouse IGF-II but not to insulin. It also blocked autophosphorylation of the type I IGF receptor induced by the binding of IGFs in human-type I IGF receptor-overexpressing BALB/c 3T3 cells, and it inhibited the IGF-stimulated growth of MDA PCa 2b cells in vitro. Then mice were injected intraperitoneally with KM1468 once weekly for 4 weeks either immediately or 4 weeks after inoculation of MDA PCa 2b cells. KM1468 markedly and dose-dependently suppressed the development of new bone tumors and the progression of established tumor foci, as determined by histomorphometry, and it also decreased serum prostate-specific antigen levels, compared with the control. This is the first report of an IGF ligand-specific inhibitory antibody that suppresses the growth of human prostate cancer cells in human adult bone. These results indicate that the IGF signaling axis is a potential target for prevention and treatment of bone metastases arising from prostate cancer.

Blockade of Paracrine Supply of Insulin-Like Growth Factors Using Neutralizing Antibodies Suppresses the Liver Metastasis of Human Colorectal Cancers

Environmental stimuli, such as organ-specific growth factors, can influence the metastatic potential of a tumor. The liver is the main source of insulin-like growth factors (IGFs). The importance of IGF signal in hepatic metastasis has been clarified mainly by IGF-I receptor targeting strategies. This study aims to confirm these precedent reports by novel tool, neutralizing antibodies against IGFs and to show that IGFs are promising therapeutic targets for hepatic metastasis in vivo. Hepatic metastasis was induced by intrasplenic injection of human colorectal cancer cell line, HT29. The antimetastatic effects of three antibodies (anti-mouse IGF-I, anti-mouse IGF-II, and anti-human/mouse IGF-II designated KM1468) were tested singly or in combination in the early phase of metastasis. The dose escalation effect of KM1468 and its survival benefit were examined in the early and late phases of metastasis. The mechanism of IGF neutralization was investigated with immunohistochemistry. Dual neutralization of paracrine IGF-I and IGF-II showed modest additive antimetastatic effects than single neutralization of IGF-I or IGF-II. In any phase of metastasis, neutralization led to significant tumor growth inhibition and longer survival. Dose escalation of KM1468 influenced survival only in the late phase of metastasis. Apoptosis increased significantly in the antibody-treated group compared with the control group (P = 0.0025) In conclusion, IGFs are promising therapeutic targets for hepatic metastases of colorectal cancers. However, the IGF dependency is probably variable in the metastatic process.

Effect of differences in cancer cells and tumor growth sites on recruiting bone marrow-derived endothelial cells and myofibroblasts in cancer-induced stroma

Cancer‐stromal interaction is well known to play important roles during cancer progression. Recently we have demonstrated that bone marrow‐derived vascular endothelial cells (BMD‐VE) and myofibroblasts (BMD‐MF) are recruited into the human pancreatic cancer cell line Capan‐1 induced stroma. To assess the effect of the difference in cancer cell types on the recruitment of BMD‐VE and BMD‐MF, 10 kinds of human cancer cell line were implanted into the subctaneous tissue of the immunodeficient mice transplanted with bone marrow of double‐mutant mice (RAG‐1−/− β‐gal Tg or RAG‐1−/− GFP Tg). The recruitment frequency of BMD‐VE (%BMD‐VE) and BMD‐MF (%BMD‐MF), and tumor‐associated parameters [tumor volume (TV), microvessel density (MVD) and stromal proportion (%St)] were measured. The correlation among them was analyzed. Although %BMD‐VE and %BMD‐MF varied (from 0 to 21.6%, 0 to 29.6%, respectively), depending on the cancer cell line, both parameters were significantly correlated with %St (p < 0.005). Furthermore %BMD‐VE and %BMD‐MF also significantly correlated (p < 0.005). In order to assess the effect of tumor growth sites on the recruitment of the cells of interest, a human pancreatic cancer cell line, Capan‐1, was transplanted into 5 different sites: subcutaneous tissue, peritoneum, liver, spleen and lung. Tumors in the subcutaneous tissue and peritoneum induced desmoplastic stroma (%St = 22.7%, 19.5%, respectively) and contained BMD‐VE (%BMD‐VE = 21.6%, 16.5% respectively) and BMD‐MF (%BMD‐MF = 29.6%, 24.5%, respectively), but weak stromal induction without recruitment of BMD‐VE or ‐MF was observed in the tumors at of the liver, spleen and lung (%St = 9.7%, 9.1%, 5.4%, respectively). cDNA microarray analysis identified the 29 genes that expression was especially up‐ or down‐regulated in the cell line that induced an abundant stromal reaction. However they did not encoded the molecules that were directly involved in stromal cell recruitment (chemokines), differentiation (cytokines) or proliferation (growth factors). These results indicate that the recruitment of BMD‐VE and ‐MF is required for stromal formation during cancer progression and that the cancer microenvironment is important in stromal reaction and the recruitment of BMD‐VE and ‐MF.

Matrix metalloproteinase‐7 triggers the matricrine action of insulin‐like growth factor‐II via proteinase activity on insulin‐like growth factor binding protein 2 in the extracellular matrix

Many growth factors and cytokines are immobilized on the extracellular matrix (ECM) by binding to glycosaminoglycans and are stored in an inactive form in the cellular microenvironment. However, the mechanisms of ECM‐bound growth factor or cytokine activation have not been well documented. We showed that the insulin‐like growth factor type‐1 receptor (IGF‐1R) was rapidly phosphorylated after the addition of matrix metalloproteinase (MMP)‐7 to a serum‐starved human colon cancer cell line (HT29) and that phosphorylation was completely inhibited by an IGF‐II neutralizing antibody. In the ECM of this cell line, IGF‐II and IGF binding protein (BP)‐2 coexisted, but IGFBP‐2 disappeared from the ECM fraction after treatment with MMP‐7 or heparinase III. On the other hand, in a cell line in which IGF‐1R was overexpressed, IGF‐1R was phosphorylated by supernatant from the MMP‐7‐treated ECM fraction of HT29 but not by that from a heparinase‐III‐treated ECM fraction. We also demonstrated that MMP‐7 degrades IGFBP‐2 in vitro at three cleavage sites (peptide bonds E151–L152, G175–L176 and K181–L182), which have not been documented previously. Taken together, these results demonstrate that MMP‐7 generates bioactive IGF‐II by degrading the IGF‐II/IGFBP‐2 complex binding to heparan sulfate proteoglycan in the ECM, resulting in IGF‐II‐induced signal transduction. This evidence indicates that some ECM‐associated growth factors enhance their ability to bind to their receptors by some proteases in the tumor microenvironment. This mechanism of action (‘protease‐triggered matricrine’) represents an attractive model for understanding ECM–tumor interactions. (Cancer Sci 2007; 98: 685–691)

In vivo and in vitro characterization of human fibroblasts recruited selectively into human cancer stroma

Fibroblasts, which are a major component of cancer‐induced stroma, can have a significant impact on the progression of adjacent malignant epithelia. To characterize fibroblasts recruited into cancer‐induced stroma, we examined the recruitment efficiency of 9 human fibroblast cell lines into experimental tumors generated in immunodeficient mice. Green fluorescence protein (GFP)–labeled fibroblast cell lines and human pancreatic cancer cell line Capan‐1 were injected i.p. at different sites; the GFP‐labeled cells within xenografts were then analyzed. KM104GFP (bone marrow) and VA‐13GFP (lung) were selectively recruited into cancer stroma more efficiently than the other cell lines. KM104GFP cells did not affect tumor volume; however, VA‐13GFP cells increased tumor volume by about 2‐fold. After 5 cyclic in vivo passages of KM104GFP in Capan‐1, we selected a subpopulation with an 8.4‐fold higher recruitment efficiency (KM104GFP‐5G) compared to parental KM104GFP. KM104GFP‐5G also exhibited higher chemotaxis and chemoinvasion activity compared to KM104GFP in response to cancer‐released chemoattractant(s). Oligonucleotide microarray analysis identified 8 genes with >3‐fold upregulation and 6 genes with >3‐fold downregulation in KM104GFP‐5G. Immunohistochemistry confirmed that fibroblasts recruited into pancreatic cancer stroma strongly expressed carbonic anhydrase IX and keratin‐8, whose transcripts were upregulated in KM104GFP‐5G by oligonucleotide microarray analysis, whereas their expression in fibroblasts within noncancerous pancreatic stroma were under the detection level. Our results indicate that fibroblast recruitment is not selective with respect to organ origin and that particular fibroblast subpopulations with specific phenotypic characteristics could be recruited efficiently into cancer‐induced stroma.

Accumulation of regulatory T cells in sentinel lymph nodes is a prognostic predictor in patients with node-negative breast cancer.

It has been revealed that sentinel lymph nodes (SLNs) from patients with node-negative breast cancer involve RT-PCR detected micrometastases and isolated tumour cells. However, the prognostic significance of the pathologically undetectable micrometastases is still controversial. In this study, we evaluated Foxp3 positive regulatory T cells (Treg) in SLNs as host-side immune marker that has the potential to detect these micrometastases. In the analyses of training set (n=30), elevated Treg was strongly associated with the pathologically undetectable micrometastases. In the analyses of validation set (n=129) in patients with node-negative, relapse-free survival in patients with elevated Treg was significantly shorter than those with lower Treg (p=0.005). Furthermore, in multivariate analyses, elevated Treg was correlated with relapse-free survival (p=0.012). Our data indicate that Treg may increase in the microenvironment of SLNs along with pathologically undetectable micrometastases and is a prognostic predictor in patients with node-negative breast cancer.

Breast-Conserving Surgery Using Projection and Reproduction Techniques of Surgical-Position Breast MRI in Patients with Ductal Carcinoma In Situ of the Breast

BACKGROUND In this study, we report a breast-conserving surgery (BCS) approach that uses projection and reproduction techniques of breast MRI obtained in the surgical position to the breast surface in patients with ductal carcinoma in situ (DCIS) of the breast. STUDY DESIGN Between February 2005 and January 2007, a total of 104 patients with operable breast cancer at our hospital had surgical-position breast MRI examinations. The 24 patients with relatively localized DCIS received BCS using the projection and reproduction techniques of the surgical-position breast MRI. During the same time period, 28 patients with relatively localized DCIS in whom prone-position breast MRI was performed, had conventional BCS using mammography-guided hookwires. In this study, we compared the surgical outcomes of our surgical approach with those of the conventional approach in a total of 52 patients with relatively localized DCIS. RESULTS Average volume of the pathologic specimens in the new technique group (27.5 cm(3)) was substantially smaller than that in the conventional BCS group (57.6 cm(3), p = 0.0007). In addition, the positive margin rate was substantially lower in the new technique group (12.5%) than in the conventional BCS group (39.3%; p = 0.029). CONCLUSIONS This study demonstrates that BCS can be done guided by the precise projection and reproduction techniques of the lesion obtained by surgical-position breast MRI. To the best of our knowledge, this is the first report of BCS technique for DCIS in this manner. Our surgical approach can be clinically useful in surgical planning and management in patients with DCIS.