Immune-mediated modulation of breast cancer growth and metastasis by the chemokine Mig (CXCL9) in a murine model

Current immunotherapies are limited by several factors, including the failure to recruit sufficient numbers of immune effector cells to tumors. The chemokine monokine induced by gamma-interferon (Mig; CXCL9) attracts activated T cells and natural killer (NK) cells bearing the chemokine receptor CXCR3. We investigated Mig as an immunotherapeutic agent in a syngeneic murine model of metastatic breast cancer. We transfected the highly malignant murine mammary tumor cell line 66.1 to stably express murine Mig cDNA. Immune-competent mice injected with Mig-expressing tumor cells developed smaller local tumors and fewer lung metastases, and they survived longer than mice injected with vector-control tumor cells. Mig-mediated inhibition of local tumor growth was lost in the absence of host T cells. Mig-transduced tumors had increased numbers of CD4 T cells compared with vector-control tumors, consistent with the T-cell chemoattractant property of Mig, and many tumor-infiltrating host cells expressed CXCR3. NK cells had not been examined previously as a possible effector cell in Mig-based therapies. Our studies now show that NK cells are critical to the mechanism by which Mig limits metastasis. Inhibition of angiogenesis was not implicated as a mechanism of Mig-mediated therapy in this model. These studies support the hypothesis that by manipulating the Mig-CXCR3 gradient, it is possible to direct host immune effector cells to tumors, curtailing both local tumor growth and metastasis. These studies also implicate host NK cells as an additional effector cell critical for Mig-mediated control of metastasis.     

Ras- and PI3K-dependent breast tumorigenesis in mice and humans requires focal adhesion kinase signaling

Cancer cells require sustained oncogenic signaling in order to maintain their malignant properties. It is, however, unclear whether they possess other dependencies that can be exploited therapeutically. We report here that in a large fraction of human breast cancers, the gene encoding focal adhesion kinase (FAK), a core component of integrin signaling, was amplified and FAK mRNA was overexpressed. A mammary gland–specific deletion of Fak in mice did not seem to affect normal mammary epithelial cells, and these mice were protected from tumors initiated by the polyoma middle T oncoprotein (PyMT), which activates Ras and PI3K. FAK-deficient PyMT-transformed cells displayed both growth arrest and apoptosis, as well as diminished invasive and metastatic capacity. Upon silencing of Fak, mouse mammary tumor cells transformed by activated Ras became senescent and lost their invasive ability. Further, Neu-transformed cells also underwent growth arrest and apoptosis if integrin β4–dependent signaling was simultaneously inactivated. Human breast cancer cells carrying oncogenic mutations that activate Ras or PI3K signaling displayed similar responses upon silencing of FAK. Mechanistic studies indicated that FAK sustains tumorigenesis by promoting Src-mediated phosphorylation of p130^Cas. These results suggest that FAK supports Ras- and PI3K-dependent mammary tumor initiation, maintenance, and progression to metastasis by orchestrating multiple core cellular functions, including proliferation, survival, and avoidance of senescence.     

Antitumor and antimetastatic activities of chloroquine diphosphate in a murine model of breast cancer

Metastatic breast cancers are hard to treat and almost always fatal. Chloroquine diphosphate, a derivative of quinine, has long been used as a potent and commonly used medicine against different human diseases. We therefore investigated the effects of chloroquine diphosphate on a highly metastatic mouse mammary carcinoma cell line. In vitro treatment of 4T1 mouse breast cancer cells with chloroquine diphosphate resulted in significant inhibition of cellular proliferation and viability, and induction of apoptosis in 4T1 cells in a time- and dose-dependent manner. Further analysis indicated that induction of apoptosis was associated with the loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase. The effect of chloroquine diphosphate was then examined using a mice model in which 4T1 cells were implanted subcutaneously. Chloroquine diphosphate (25 mg/kg and 50 mg/kg, respectively) significantly inhibited the growth of the implanted 4T1 tumor cells and induced apoptosis in the tumor microenvironment. Moreover, the metastasis of tumor cells to the lungs was inhibited significantly and the survival of the mice enhanced. These data suggested that chloroquine diphosphate might have chemotherapeutic efficacy against breast cancer including inhibition of metastasis.     

Inhibition of Tumor Growth and Metastasis by a Combination of Escherichia coli�Cmediated Cytolytic Therapy and Radiotherapy

We have reported that Escherichia coli K-12 colonizes hypoxic and necrotic tumor regions after intravenous injection into tumor-bearing mice. In this study, we established a novel strategy for cancer therapy using engineered bacteria to enhance the therapeutic effects of radiation. E. coli strain K-12 was engineered to produce cytolysin A (ClyA), and its effects on tumor growth in primary and metastatic tumor models were evaluated. A single treatment with E. coli–expressing ClyA significantly decreased tumor growth rates initially (9 days after treatment); however, the tumors tended to grow thereafter. With only radiotherapy (RT; 21 Gy), the tumor growth rates were retarded, but not the tumor sizes. A combination of therapy with E. coli–expressing ClyA and radiation [a total of 5 × 10⁷ colony-forming units (CFU) and 21 Gy] resulted in significant tumor shrinkage and even complete disappearance of tumors in mice with tumors derived from murine CT26 colon cancer. Furthermore, treatment with E. coli–expressing ClyA markedly suppressed metastatic tumor growth and prolonged the survival time in mice. The results described here indicate that therapy with engineered E. coli could significantly improve the results of RT, and could exert a striking inhibitory effect on the development of lung metastasis.     

Enhanced tumorigenesis and lymphatic metastasis of CD133+ hepatocarcinoma ascites syngeneic cell lines mediated by JNK signaling pathway in vitro and in vivo

Cancer stem cells (CSCs), stem-like cells, or tumor-initiating cells (TICs) may initiate tumorigenesis and metastasis, but neither the basic cell biology of CSCs nor the mechanisms of CSC-mediated tumor growth and lymphoid node metastasis are understood. Evidence suggests that CSC phenotype is maintained, at least in part, by altered JNK signaling. In this study, factors influencing the growth and metastatic potential of CSCs were examined by comparing CD133 surface antigen expression, proliferation, clonogenicity, invasive capacity, tumorigenicity, and expression of JNK-associated signaling molecules between the highly metastatic mouse hepatocarcinoma ascites syngeneic cell line Hca-F and the low metastasis potential line Hca-P. The Hca-F line exhibited higher clonogenic, proliferative, and invasive capacities than Hca-P cells, and a greater proportion of Hca-F cells were CD133 positive. In both cell lines, the CD133+ subpopulation showed significantly enhanced tumorigenicity and metastatic potential. An in vivo tumorigenicity assay in nude mice indicated that Hca-F cells possessed significantly higher tumorigenicity than Hca-P cells as indicated by larger tumors after inoculation. Expression levels of E-cadherin (CDH1), annexin VII, and JNK1 proteins were inversely correlated with CD133 expression in both Hca-F and Hca-P cells. These results demonstrate that CD133+ subpopulations of both Hca-F and Hca-P lines show CSC-like properties. However, Hca-F cells showed greater tumorigenicity and invasiveness, consistent with greater lymphatic metastasis capacity. We propose that tumorigenesis and lymphatic metastasis are regulated by JNK/P53/annexin VII and JNK/ATF-2/CDH1/annexin VII signal transduction pathways.     

Preclinical evaluation of the tyrosine kinase inhibitor SU11248 as a single agent and in combination with "standard of care" therapeutic agents for the treatment of breast cancer

SU11248 is an oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities through targeting platelet-derived growth factor receptor, vascular endothelial growth factor receptor, KIT, and FLT3, the first three of which are expressed in human breast cancer and/or its supporting tissues. The purpose of the present studies was to demonstrate the potent anticancer activity of SU11248 alone or in combination with conventional cytotoxic agents against several distinct preclinical models of breast cancer. SU11248 was administered as a monotherapy to (1) mouse mammary tumor virus-v-Ha-ras mice and 7,12-dimethylbenz(a)anthracene-treated rats bearing mammary tumors and (2) mice bearing human breast cancer xenografts of s.c. MX-1 tumors and osseous metastasis of a MDA-MB-435-derived cell line (435/HAL-Luc). SU11248 was also administered in combination with docetaxel both in xenograft models and in combination with 5-fluorouracil and doxorubicin in the MX-1 model. SU11248 treatment potently regressed growth of mammary cancers in mouse mammary tumor virus-v-Ha-ras transgenic mice (82% regression) and 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats (99% regression at the highest dose; P < 0.05 for both). This agent also inhibited MX-1 tumor growth by 52%, with markedly enhanced anticancer effects when administered in combination with docetaxel, 5-fluorouracil, or doxorubicin compared with either agent alone (P < 0.05). SU11248 treatment in combination with docetaxel effectively prolonged survival of mice, with 435/HAL-Luc cancer xenografts established in bone compared with either agent alone (P < 0.05). These results demonstrate that SU11248 is effective in preclinical breast cancer models and suggest that it may be useful in the treatment of breast cancer in the clinic.     

MerTK inhibition in tumor leukocytes decreases tumor growth and metastasis

MerTK, a receptor tyrosine kinase (RTK) of the TYRO3/AXL/MerTK family, is expressed in myeloid lineage cells in which it acts to suppress proinflammatory cytokines following ingestion of apoptotic material. Using syngeneic mouse models of breast cancer, melanoma, and colon cancer, we found that tumors grew slowly and were poorly metastatic in MerTK^–/– mice. Transplantation of MerTK^–/– bone marrow, but not wild-type bone marrow, into lethally irradiated MMTV-PyVmT mice (a model of metastatic breast cancer) decreased tumor growth and altered cytokine production by tumor CD11b⁺ cells. Although MerTK expression was not required for tumor infiltration by leukocytes, MerTK^–/– leukocytes exhibited lower tumor cell–induced expression of wound healing cytokines, e.g., IL-10 and growth arrest-specific 6 (GAS6), and enhanced expression of acute inflammatory cytokines, e.g., IL-12 and IL-6. Intratumoral CD8⁺ T lymphocyte numbers were higher and lymphocyte proliferation was increased in tumor-bearing MerTK^–/– mice compared with tumor-bearing wild-type mice. Antibody-mediated CD8⁺ T lymphocyte depletion restored tumor growth in MerTK^–/– mice. These data demonstrate that MerTK signaling in tumor-associated CD11b⁺ leukocytes promotes tumor growth by dampening acute inflammatory cytokines while inducing wound healing cytokines. These results suggest that inhibition of MerTK in the tumor microenvironment may have clinical benefit, stimulating antitumor immune responses or enhancing immunotherapeutic strategies.     

Angiostatin-mediated Suppression of Cancer Metastases by Primary Neoplasms Engineered to Produce Granulocyte/Macrophage Colony–stimulating Factor

We determined whether tumor cells consistently generating granulocyte/macrophage colony– stimulating factor (GM-CSF) can recruit and activate macrophages to generate angiostatin and, hence, inhibit the growth of distant metastasis. Two murine melanoma lines, B16-F10 (syngeneic to C57BL/6 mice) and K-1735 (syngeneic to C3H/HeN mice), were engineered to produce GM-CSF. High GM-CSF (>1 ng/10⁶ cells)– and low GM-CSF (<10 pg/10⁶ cells)–producing clones were identified. Parental, low, and high GM-CSF–producing cells were injected subcutaneously into syngeneic and into nude mice. Parental and low-producing cells produced rapidly growing tumors, whereas the high-producing cells produced slow-growing tumors. Macrophage density inversely correlated with tumorigenicity and directly correlated with steady state levels of macrophage metalloelastase (MME) mRNA. B16 and K-1735 subcutaneous (s.c.) tumors producing high levels of GM-CSF significantly suppressed lung metastasis of 3LL, UV-2237 fibrosarcoma, K-1735 M2, and B16-F10 cells, but parental or low-producing tumors did not. The level of angiostatin in the serum directly correlated with the production of GM-CSF by the s.c. tumors. Macrophages incubated with medium conditioned by GM-CSF– producing B16 or K-1735 cells had higher MME activity and generated fourfold more angiostatin than control counterparts. These data provide direct evidence that GM-CSF released from a primary tumor can upregulate angiostatin production and suppress growth of metastases.     

Liver metastasis as the initial clinical manifestation of sublingual gland adenoid cystic carcinoma: A case report

BACKGROUNDAdenoid cystic carcinoma (ACC) is a common malignant tumor of salivary gland. The lung and liver are frequent sites of distant metastasis. Liver metastasis as the initial clinical manifestation of sublingual gland ACC is very rare.CASE SUMMARYA 51-year-old Chinese woman presented with a painless mass in the right lobe of liver. The tumor was composed of ductal cells and myoepithelial cells with a morphology including tubiform and cribriform structures. Immunostaining results showed ductal cells positive for CK7, CK14, CK19, CD117, and 34βE12, and negative for MYB, vimentin, ER, PR, and CEA. The myoepithelial cells were positive for p63, calponin and CK5/6. Metastatic salivary ACC was considered, and a sublingual gland mass was revealed by computed tomography. Histological evaluation confirmed primary sublingual gland ACC. Fluorescence in situ hybridization (FISH) did not find an MYB-NFIB fusion gene in specimens from either the primary or metastatic ACC tumors. The sublingual gland ACC relapsed in 20 mo. The recurrent lesion disappeared following local radiation therapy and computed tomography-guided radioactive seed implantation. The patient remains in good condition until now.CONCLUSIONMetastatic sublingual gland ACC with initial clinical manifestation as a liver mass is very rare, and was pathologically confirmed in this patient by its histological appearance. Primary hepatic tumors and metastatic carcinomas should be included in the differential diagnosis. Immunohistochemical detection of MYB protein and MYB-NFIB fusion gene detection by FISH can be helpful, but occasional negative results confuse the diagnosis.     

Interleukin 12–mediated Prevention of Spontaneous Mammary Adenocarcinomas in Two Lines of Her-2/neu Transgenic Mice

The ability of interleukin (IL)-12 to prevent tumors when administered to individuals with a genetic risk of cancer was studied in two lines of transgenic mice expressing rat HER-2/neu oncogene in the mammary gland. Female BALB/c (H-2^d) mice carrying the activated HER-2/ neu oncogene show no morphological abnormalities of the mammary gland until 3 wk of age. They then progress through atypical hyperplasia to in situ lobular carcinoma and at 33 wk of age all 10 mammary glands display invasive carcinomas. Adult FVB mice (H-2^q) carrying the HER-2/neu protooncogene develop mammary carcinomas with a longer latency (38-49 wk) and a lower multiplicity (mean of 2.6 tumors/mice). Treatment with IL-12 (5 daily intraperitoneal injections, 1 wk on, 3 wk off; the first course with 50 ng IL-12/day, the second with 100 ng IL-12/day) begun at 2 wk of age in BALB/c mice and at 21 wk of age in FVB mice markedly delayed tumor onset and reduced tumor multiplicity. Analogous results were obtained in immunocompetent and permanently CD8⁺ T lymphocyte–depleted mice. In both transgenic lines, tumor inhibition was associated with mammary infiltration of reactive cells, production of cytokines and inducible nitric oxide synthase, and reduction in microvessel number, in combination with a high degree of hemorrhagic necrosis.     

Cyclooxygenase-2–dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer

Breast involution following pregnancy has been implicated in the high rates of metastasis observed in postpartum breast cancers; however, it is not clear how this remodeling process promotes metastasis. Here, we demonstrate that human postpartum breast cancers have increased peritumor lymphatic vessel density that correlates with increased frequency of lymph node metastases. Moreover, lymphatic vessel density was increased in normal postpartum breast tissue compared with tissue from nulliparous women. In rodents, mammary lymphangiogenesis was upregulated during weaning-induced mammary gland involution. Furthermore, breast cancer cells exposed to the involuting mammary microenvironment acquired prolymphangiogenic properties that contributed to peritumor lymphatic expansion, tumor size, invasion, and distant metastases. Finally, in rodent models of postpartum breast cancer, cyclooxygenase-2 (COX-2) inhibition during the involution window decreased normal mammary gland lymphangiogenesis, mammary tumor-associated lymphangiogenesis, tumor cell invasion into lymphatics, and metastasis. Our data indicate that physiologic COX-2–dependent lymphangiogenesis occurs in the postpartum mammary gland and suggest that tumors within this mammary microenvironment acquire enhanced prolymphangiogenic activity. Further, our results suggest that the prolymphangiogenic microenvironment of the postpartum mammary gland has potential as a target to inhibit metastasis and suggest that further study of the therapeutic efficacy of COX-2 inhibitors in postpartum breast cancer is warranted.     

Cell therapy with preimmunized effector cells mismatched for minor histocompatible antigens in the treatment of a murine mammary carcinoma

Cell therapy with allogeneic donor cells mismatched for minor histocompatible (MiHC) antigens was applied to a murine mammary carcinoma (4T1) model to test the feasibility of graft versus tumor (GVT) effect against metastatic epithelial tumor cells. BALB/c mice bearing a 4T1 tumor of BALB/c origin were given syngeneic or MiHC-mismatched splenocytes. GVT effects were determined in secondary recipients of adoptively transferred lung cells derived from primary hosts who had previously been inoculated intravenously with 4T1 cells, and injected with one of the following: 1) naive BALB/c splenocytes, 2) naive DBA/2 splenocytes, 3) 4T1-immune DBA/2 splenocytes, or 4) DBA/2 splenocytes immunized with host-derived BABL/c spleen cells. Naive DBA/2 splenocytes inhibited tumor growth only slightly and only slightly prolonged the survival of secondary recipients, in comparison with fully matched tumor/host BALB/c spleen cells. An efficient GVT reaction was demonstrated in vitro and in vivo with MiHC-mismatched DBA/2 splenocytes from mice presensitized by multiple injections of irradiated tumor or BALB/c-derived spleen cells. All 30 mice adoptively inoculated with lung cells from primary hosts that had previously been treated with these presensitized effector cells were tumor free for >250 days. Secondary recipients inoculated with lung cells from mice given naive BALB/c or DBA/2 spleen cells died of metastatic tumors within 33 to 46 days. These results suggest that preimmunized donor cells represent an effective tool against metastatic disease; hence, the next goal should be to control graft-versus-host disease while exploiting the GVT potential.     

Photothermal therapy of tumors in lymph nodes using gold nanorods and near-infrared laser light

Lymph node dissection for regional nodal metastasis is a primary option, but is invasive and associated with adverse effects. The development of non-invasive therapeutic methods in preclinical experiments using mice has been restricted by the small lymph node size and the limited techniques available for non-invasive monitoring of lymph node metastasis. Here, we show that photothermal therapy (PTT) using gold nanorods (GNRs) and near-infrared (NIR) laser light shows potential as a non-invasive treatment for tumors in the proper axillary lymph nodes (proper-ALNs) of MXH10/Mo–lpr/lpr mice, which develop systemic swelling of lymph nodes (up to 13 mm in diameter, similar in size to human lymph nodes). Tumor cells were inoculated into the proper-ALNs to develop a model of metastatic lesions, and any anti-tumor effects of therapy were assessed. We found that GNRs accumulated in the tumor in the proper-ALNs 24 h after tail vein injection, and that irradiation with NIR laser light elevated tumor temperature. Furthermore, combining local or systemic delivery of GNRs with NIR irradiation suppressed tumor growth more than irradiation alone. We propose that PTT with GNRs and NIR laser light can serve as a new therapeutic method for lymph node metastasis, as an alternative to lymph node dissection.     

Blockade of TGF-beta inhibits mammary tumor cell viability,migration, and metastases

TGF-betas are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-beta interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-beta type II receptor fusion protein (Fc:TbetaRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TbetaRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TbetaRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TbetaRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TbetaRII. Therefore, blockade of TGF-beta signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.     

TGF-β upregulates miR-181a expression to promote breast cancer metastasis

Late-stage breast cancer metastasis is driven by dysregulated TGF-β signaling, but the underlying molecular mechanisms have not been fully elucidated. We attempted to recapitulate tumor and metastatic microenvironments via the use of biomechanically compliant or rigid 3D organotypic cultures and combined them with global microRNA (miR) profiling analyses to identify miRs that were upregulated in metastatic breast cancer cells by TGF-β. Here we establish miR-181a as a TGF-β–regulated “metastamir” that enhanced the metastatic potential of breast cancers by promoting epithelial-mesenchymal transition, migratory, and invasive phenotypes. Mechanistically, inactivation of miR-181a elevated the expression of the proapoptotic molecule Bim, which sensitized metastatic cells to anoikis. Along these lines, miR-181a expression was essential in driving pulmonary micrometastatic outgrowth and enhancing the lethality of late-stage mammary tumors in mice. Finally, miR-181a expression was dramatically and selectively upregulated in metastatic breast tumors, particularly triple-negative breast cancers, and was highly predictive for decreased overall survival in human breast cancer patients. Collectively, our findings strongly implicate miR-181a as a predictive biomarker for breast cancer metastasis and patient survival, and consequently, as a potential therapeutic target in metastatic breast cancer.     

Aspirin reduces lung cancer metastasis to regional lymph nodes

Background

Lung cancer is the main cause of cancer-related death worldwide. The high mortality is probably attributable to early metastasis; however, the mechanism underlying metastasis to regional lymph nodes is still unknown. Cyclooxygenase (COX)-derived prostaglandin E₂ (PGE₂) induces tumor growth and metastasis and is associated with a poor prognosis. The present study investigated the effect of an authentic COX inhibitor, aspirin, on regional lymph node metastasis during the development of lung cancer in mice.

Methods

An orthotopic intrapulmonary implantation model based on male C57BL/6 (6–8-weeks-old) mice was used. The lungs were injected with a solution containing Lewis lung carcinoma (LLC) cells overexpressing green fluorescent protein (GFP) and BD Matrigel^®. The effect of aspirin on mediastinal lymph node metastasis of LCC cells from the primary injection sites was then examined.

Results

The implantation process took approximately 30 s per mouse and operative mortality was 10%. Single pulmonary nodules developed at the implanted site in 95% of animals, and regional mediastinal lymph node metastasis was observed at 14 days post-LLC-GFP cell injection in all mice that formed a primary lung tumor. The mean survival time of mice injected with LLC-GFP cells was 15 ± 3 days (range, 12–22 days). Histopathological analysis revealed that no metastatic tumors developed in the regional mediastinal lymph nodes by Day 10–12 post-LLC-GFP cell injection and no metastasis to distant organs or distant lymph nodes was observed by Day 21 post-injection. Oral administration of aspirin (100 mg/kg, twice a day) after LLC-GFP cell injection inhibited metastasis to the regional lymph nodes, with no significant suppression of primary tumor growth in the lungs. Aspirin treatment led to a significant reduction in mortality (P < 0.0001).

Conclusions

The present lymph node metastasis model is useful for evaluating the efficacy of agents that inhibit tumor metastasis to the regional lymph nodes. Aspirin reduced the metastasis of LLC-GFP cells injection to the regional lymph nodes, with a significant reduction in mortality. These findings suggested that COX inhibitors have potential for preventing lymph node metastasis.     

Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy

In human breast carcinomas, overexpression of the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R) correlates with poor prognosis. To establish if there is a causal relationship between CSF-1 and breast cancer progression, we crossed a transgenic mouse susceptible to mammary cancer with mice containing a recessive null mutation in the CSF-1 gene (Csf1(op)) and followed tumor progression in wild-type and null mutant mice. The absence of CSF-1 affects neither the incidence nor the growth of the primary tumors but delayed their development to invasive, metastatic carcinomas. Transgenic expression of CSF-1 in the mammary epithelium of both Csf1(op)/Csf1(op) and wild-type tumor-prone mice led to an acceleration to the late stages of carcinoma and to a significant increase in pulmonary metastasis. This was associated with an enhanced infiltration of macrophages into the primary tumor. These studies demonstrate that the growth of mammary tumors and the development to malignancy are separate processes and that CSF-1 selectively promotes the latter process. CSF-1 may promote metastatic potential by regulating the infiltration and function of tumor-associated macrophages as, at the tumor site, CSF-1R expression was restricted to macrophages. Our data suggest that agents directed at CSF-1/CSF-1R activity could have important therapeutic effects.