Intracellular delivery of oncolytic viruses with engineered Salmonella causes viral replication and cell death.

As therapies, oncolytic viruses regress tumors and have the potential to induce antitumor immune responses that clear hard-to-treat and late-stage cancers. Despite this promise, clearance from the blood prevents treatment of internal solid tumors. To address this issue, we developed virus-delivering Salmonella (VDS) to carry oncolytic viruses into cancer cells. The VDS strain contains the PsseJ-lysE delivery circuit and has deletions in four homologous recombination genes (ΔrecB, ΔsbcB, ΔsbcCD, and ΔrecF) to preserve essential hairpins in the viral genome required for replication and infectivity. VDS delivered the genome for minute virus of mice (MVMp) to multiple cancers, including breast, pancreatic, and osteosarcoma. Viral delivery produced functional viral particles that are cytotoxic and infective to neighboring cells. The release of mature virions initiated new rounds of infection and amplified the infection. Using Salmonella for delivery will circumvent the limitations of oncolytic viruses and will provide a new therapy for many cancers.

Intracellular Salmonella delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity.

Introduction: Immunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. Methods: To test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. Results: We showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. Discussion: This response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients.

A poly(ethylene glycol) three-dimensional bone marrow hydrogel.

Three-dimensional (3D) hydrogels made from synthetic polymers have emerged as in vitro cell culture platforms capable of representing the extracellular geometry, modulus, and water content of tissues in a tunable fashion. Hydrogels made from these otherwise non-bioactive polymers can be decorated with short peptides derived from proteins naturally found in tissues to support cell viability and direct phenotype. We identified two key limitations that limit the ability of this class of materials to recapitulate real tissue. First, these environments typically display between 1 and 3 bioactive peptides, which vastly underrepresents the diversity of proteins found in the extracellular matrix (ECM) of real tissues. Second, peptides chosen are ubiquitous in ECM and not derived from proteins found in specific tissues, per se. To overcome this critical limitation in hydrogel design and functionality, we developed an approach to incorporate the complex and specific protein signature of bone marrow into a poly (ethylene glycol) (PEG) hydrogel. This bone marrow hydrogel mimics the elasticity of marrow and has 20 bone marrow-specific and cell-instructive peptides. We propose this tissue-centric approach as the next generation of 3D hydrogel design for applications in tissue engineering and beyond.

Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases.

Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors.

Tumor cell-organized fibronectin maintenance of a dormant breast cancer population.

Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, nonproliferative state before reactivation and outgrowth. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created an in vitro cell culture system with carefully controlled ECM substrates to observe entrance into and exit from dormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle-arrested, and actively proliferating cells. Cell populations capable of entering dormancy formed an organized, fibrillar fibronectin matrix via αvß3 and α5ß1 integrin adhesion, ROCK-generated tension, and TGFß2 stimulation, and cancer cell outgrowth after dormancy required MMP-2-mediated fibronectin degradation. We propose this approach as a useful, in vitro method to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation.

Comparative Study of Multicellular Tumor Spheroid Formation Methods and Implications for Drug Screening.

Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and clinical application. Multicellular tumor spheroids (MCTS) are a popular method for three-dimensional (3D) cell culture, because they capture some aspects of the dimensionality, cell-cell contact, and cell-matrix interactions seen in vivo. Many approaches exist to create MCTS from cell lines, and they have been used to study tumor cell invasion, growth, and how cells respond to drugs in physiologically relevant 3D microenvironments. However, there are several discrepancies in the observations made of cell behaviors when comparing between MCTS formation methods. To resolve these inconsistencies, we created and compared the behavior of breast, prostate, and ovarian cancer cells across three MCTS formation methods: in polyNIPAAM gels, in microwells, or in suspension culture. These methods formed MCTS via proliferation from single cells or passive aggregation, and therefore showed differential reliance on genes important for cell-cell or cell-matrix interactions. We also found that the MCTS formation method dictated drug sensitivity, where MCTS formed over longer periods of time via clonal growth were more resistant to treatment. Toward clinical application, we compared an ovarian cancer cell line MCTS formed in polyNIPAAM with cells from patient-derived malignant ascites. The method that relied on clonal growth (PolyNIPAAM gel) was more time and cost intensive, but yielded MCTS that were uniformly spherical, and exhibited the most reproducible drug responses. Conversely, MCTS methods that relied on aggregation were faster, but yielded MCTS with grapelike, lobular structures. These three MCTS formation methods differed in culture time requirements and complexity, and had distinct drug response profiles, suggesting the choice of MCTS formation method should be carefully chosen based on the application required.

A biomaterial screening approach reveals microenvironmental mechanisms of drug resistance.

Traditional drug screening methods lack features of the tumor microenvironment that contribute to resistance. Most studies examine cell response in a single biomaterial platform in depth, leaving a gap in understanding how extracellular signals such as stiffness, dimensionality, and cell-cell contacts act independently or are integrated within a cell to affect either drug sensitivity or resistance. This is critically important, as adaptive resistance is mediated, at least in part, by the extracellular matrix (ECM) of the tumor microenvironment. We developed an approach to screen drug responses in cells cultured on 2D and in 3D biomaterial environments to explore how key features of ECM mediate drug response. This approach uncovered that cells on 2D hydrogels and spheroids encapsulated in 3D hydrogels were less responsive to receptor tyrosine kinase (RTK)-targeting drugs sorafenib and lapatinib, but not cytotoxic drugs, compared to single cells in hydrogels and cells on plastic. We found that transcriptomic differences between these in vitro models and tumor xenografts did not reveal mechanisms of ECM-mediated resistance to sorafenib. However, a systems biology analysis of phospho-kinome data uncovered that variation in MEK phosphorylation was associated with RTK-targeted drug resistance. Using sorafenib as a model drug, we found that co-administration with a MEK inhibitor decreased ECM-mediated resistance in vitro and reduced in vivo tumor burden compared to sorafenib alone. In sum, we provide a novel strategy for identifying and overcoming ECM-mediated resistance mechanisms by performing drug screening, phospho-kinome analysis, and systems biology across multiple biomaterial environments.

Osteocytes serve as a progenitor cell of osteosarcoma.

Osteosarcoma (OSA) is the most common primary bone tumor in humans. However, the cell of origin of OSA is not clearly defined although there is evidence that osteoblasts may serve as OSA progenitors. The role of osteocytes, terminally differentiated osteoblasts, as OSA progenitors has yet to be described. Analysis of patient cDNA from publicly available microarray data revealed that patients with OSA have increased expression of dentin matrix phosphoprotein 1 (DMP1), a marker of osteocytes. Analysis of multiple murine, human, and canine OSA cell lines revealed DMP1 expression. To test the tumorigenic potential of osteocytes, MLO-Y4, a SV-40 immortalized murine osteocyte cell line, was injected into subcutaneous and orthotopic (intratibial) sites of mice. Tumor growth occurred in both locations. Orthotopic MLO-Y4 tumors produced mixed osteoblastic/osteolytic radiographic lesions; a hallmark of OSA. Together, these data demonstrate for the first time that osteocytes can serve as OSA progenitors.

HER2 drives luminal breast cancer stem cells in the absence of HER2 amplification: implications for efficacy of adjuvant trastuzumab.

Although current breast cancer treatment guidelines limit the use of HER2-blocking agents to tumors with HER2 gene amplification, recent retrospective analyses suggest that a wider group of patients may benefit from this therapy. Using breast cancer cell lines, mouse xenograft models and matched human primary and metastatic tissues, we show that HER2 is selectively expressed in and regulates self-renewal of the cancer stem cell (CSC) population in estrogen receptor-positive (ER(+)), HER2(-) luminal breast cancers. Although trastuzumab had no effects on the growth of established luminal breast cancer mouse xenografts, administration after tumor inoculation blocked subsequent tumor growth. HER2 expression is increased in luminal tumors grown in mouse bone xenografts, as well as in bone metastases from patients with breast cancer as compared with matched primary tumors. Furthermore, this increase in HER2 protein expression was not due to gene amplification but rather was mediated by receptor activator of NF-κB (RANK)-ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore, these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Cancer Res; 73(5); 1635-46. ©2012 AACR.

Integrin alpha2beta 1 (α2ß1) promotes prostate cancer skeletal metastasis.

Men who die of prostate cancer (PCa) do so because of systemic metastases, the most frequent of which are within the skeleton. Recent data suggest that the colonization of the skeleton is mediated in part by collagen type I, the most abundant protein within the bone. We have shown that enhanced collagen I binding through increased expression of integrin α2ß1 stimulated in vitro invasion and promoted the growth of PCa cells within the bone. Accordingly, we sought to determine whether α2ß1 integrin is a potential mediator of skeletal metastasis. To examine whether α2ß1 integrin mediates PCa metastasis, α2 integrin was over-expressed in low-tumorigenic LNCaP PCa cells or selectively knocked-down in highly metastatic LNCaPcol PCa cells. We document that the over-expression of α2 cDNA stimulated whereas α2 shRNA inhibited the ability of transduced cells to bind to or migrate towards collagen in vitro. Correspondingly, α2 integrin knock-down reduced the tumor burden of intra-osseous tumors compared to control-transduced cells. To investigate the clinical significance of α2ß1 expression in PCa, α2ß1 protein was measured in prostatic tissues and in soft tissue and bone metastases. The data demonstrate that α2ß1 protein was elevated in PCa skeletal metastases compared to either PCa primary lesions or soft tissue metastases suggesting that α2ß1 contributes to the selective metastasis to the bone. Taken together, these data support that α2ß1 integrin is needed for the efficient metastasis of PCa cells to the skeleton.

Wnt and Wnt inhibitors in bone metastasis.

Bone metastasis is a clinically devastating development of progressive cancers including prostate carcinoma, breast carcinoma and multiple myeloma. Bone metastases are typically painful, lead to adverse skeletal-related events, such as fracture, and are highly resistant to therapy. A major contribution to the ability of cancers to successfully establish bone metastases is their ability to exploit mechanisms of normal bone remodeling. Wnts are a large family of morphogenic proteins that are critical for bone development and contribute to maintaining bone mass in the mature organism. Wnt function is balanced by the presence of a variety of endogenous inhibitors, such as the dickkopf family members, secreted frizzled related proteins and sclerostin. Together, these factors contribute to normal bone homeostasis, allowing for dynamic changes in bone to withstand alterations in physical forces and physiological needs. In this review, we describe the role that Wnts and their inhibitors have in normal bone biology and cancer-related bone pathology. An overview of Wnt signaling pathways is discussed and key bone microenvironment cellular players, as they pertain to Wnt biology, are examined. Finally, we describe clinical trials of several Wnt inhibitor antagonists for patients with tumor-related bone disease. As few options currently exist for the treatment of bone-metastatic disease, Wnt proteins and their inhibitors offer promise for the development of novel therapeutics.

p21CIP-1/WAF-1 induction is required to inhibit prostate cancer growth elicited by deficient expression of the Wnt inhibitor Dickkopf-1.

Osteoblastic bone metastases are the most common metastases produced by human prostate cancers (PCa). Deregulated activity of Wnt growth factors resulting from overexpression of the Wnt inhibitor Dickkopf-1 (DKK-1) is known to contribute to formation of the osteoblastic component of PCa skeletal bone metastases. In this study, we report that DKK-1 knockdown in osteolytic human PCa cells unexpectedly delays the development of both soft tissue and osseous lesions. PCa cells deficient in DKK-1 expression did not increase canonical Wnt signaling in target osteoblast cell lines; however, DKK-1 knockdown PCa cells exhibited increased expression of the CDK inhibitor p21(CIP1/WAF1) and a 32% increase in G(1) arrest compared with control cells. Ablating p21(CIP1/WAF1) in PCa cells deficient in DKK-1 was sufficient to rescue tumor growth. Collectively, our findings demonstrate that DKK-1 overexpression supports tumor growth in part by restricting expression of p21(CIP1/WAF1) through a mechanism independent of canonical Wnt signaling.

Expression of PGK1 by prostate cancer cells induces bone formation.

Prostate cancer (PCa) is one of the solid tumors that metastasize to the bone. Once there, the phenotype of the bone lesions is dependent upon the balance between osteoblastogenesis and osteoclastogenesis. We previously reported that overexpression of phosphoglycerate kinase 1 (PGK1) in PCa cell lines enhanced bone formation at the metastatic site in vivo. Here, the role of PGK1 in the bone formation was further explored. We show that PCa-derived PGK1 induces osteoblastic differentiation of bone marrow stromal cells. We also found that PGK1 secreted by PCa inhibits osteoclastogenesis. Finally, the expression levels of the bone-specific markers in PCa cells were higher in cells overexpressing PGK1 than controls. Together, these data suggest that PGK1 secreted by PCa regulates bone formation at the metastatic site by increasing osteoblastic activity, decreasing osteoclastic function, and expressing an osteoblastic phenotype by PCa cells.

A bioluminescent orthotopic mouse model of human osteosarcoma that allows sensitive and rapid evaluation of new therapeutic agents In vivo.

Osteosarcoma (OSA) is the most common primary malignant bone tumor in children, 30% of whom develop lung metastases despite aggressive treatment. Our objective was to develop a mouse model of OSA for preclinical studies that (i) incorporates the natural history of OSA including tumor growth in bone and development of lung metastasis and (ii) is amenable to non-invasive detection methods. A human OSA cell line that expresses high levels of luciferase was created. Following subcutaneous injection, nine out of ten mice showed tumor growth. Eight out of ten mice showed tumor growth following orthotopic injection into the proximal tibia. Thirty percent of mice showed pulmonary metastasis by bioluminescent imaging eight to 10 weeks following orthotopic injection. Animals receiving cisplatin treatment showed reduced tumor volume compared to animals treated with vehicle alone. This model allows real-time detection of tumors and can be used to study mechanisms of OSA metastasis and test new therapeutic agents.

Type I collagen receptor (alpha2beta1) signaling promotes prostate cancer invasion through RhoC GTPase.

The most frequent site of metastasis in human prostate cancer (PCa) is the bone. Preferential adhesion of PCa cells to bone-specific factors may facilitate the selective metastasis of the skeleton. The most abundant protein within the skeleton is type I collagen. We previously demonstrated that PCa cells selected in vitro for collagen I binding (LNCaP(col)) are highly motile and acquired the capacity to grow within the bone compared to nontumorigenic LNCaP parental cells. Treatment with alpha(2)beta(1)-neutralizing antibodies selectively blocked collagen-stimulated migration, suggesting that integrin signaling mediates PCa migration. To elucidate the mechanism of collagen-stimulated migration, we evaluated integrin-associated signaling pathways in non-collagen-binding LNCaP parental cells and in collagen-binding isogenic C4-2B and LNCaP(col) PCa cells. The expression and activity of RhoC guanosine triphosphatase was increased five- to eightfold in collagen-binding LNCaP(col) and C4-2B cells, respectively, compared to parental LNCaP cells. RhoC activation was selectively blocked with antibodies to alpha(2)beta(1) where treatment with a small hairpin RNA specific for RhoC suppressed collagen-mediated invasion without altering the PCa cells' affinity for collagen I. We conclude that the ligation of alpha(2)beta(1) by collagen I activates RhoC guanosine triphosphatase, which mediates PCa invasion, and suggests a mechanism for the preferential metastasis of PCa cells within the bone.

Prostate cancer induces bone metastasis through Wnt-induced bone morphogenetic protein-dependent and independent mechanisms.

Prostate cancer (PCa) is frequently accompanied by osteosclerotic (i.e., excessive bone production) bone metastases. Although bone morphogenetic proteins (BMP) and Wnts are mediators of PCa-induced osteoblastic activity, the relation between them in PCa bone metastases is unknown. The goal of this study was to define this relationship. Wnt3a and Wnt5a administration or knockdown of DKK-1, a Wnt inhibitor, induced BMP-4 and 6 expression and promoter activation in PCa cells. DKK-1 blocked Wnt activation of the BMP promoters. Transfection of C4-2B cells with axin, an inhibitor of canonical Wnt signaling, blocked Wnt3a but not Wnt5a induction of the BMP promoters. In contrast, Jnk inhibitor I blocked Wnt5a but not Wnt3a induction of the BMP promoters. Wnt3a, Wnt5a, and conditioned medium (CM) from C4-2B or LuCaP23.1 cells induced osteoblast differentiation in vitro. The addition of DKK-1 and Noggin, a BMP inhibitor, to CM diminished PCa CM-induced osteoblast differentiation in a synergistic fashion. However, pretreatment of PCa cells with DKK-1 before collecting CM blocked osteoblast differentiation, whereas pretreatment with Noggin only partially reduced osteoblast differentiation, and pretreatment with both DKK-1 and Noggin had no greater effect than pretreatment with DKK-1 alone. Additionally, knockdown of BMP expression in C4-2B cells inhibited Wnt-induced osteoblastic activity. These results show that PCa promotes osteoblast differentiation through canonical and noncanonical Wnt signaling pathways that stimulate both BMP-dependent and BMP-independent osteoblast differentiation. These results show a clear link between Wnts and BMPs in PCa-induced osteoblast differentiation and provide novel targets, including the noncanonical Wnt pathway, for therapy of PCa.

Dickkopf-1 expression increases early in prostate cancer development and decreases during progression from primary tumor to metastasis.

BACKGROUND: Prostate cancer (PCa) frequently metastasizes to the bone and induces osteoblastic lesions. We previously demonstrated through over-expression of the Wnt inhibitor dickkopf-1 (DKK-1) that Wnts contribute to the osteoblastic component of PCa osseous lesions in vivo. METHODS: To test the clinical significance of DKK-1 expression during PCa progression, tissue microarrays were stained for DKK-1 protein by immunohistochemistry. RESULTS: DKK-1 expression index (EI) was found to increase in PIN and primary lesions compared to non-neoplastic tissue (106 +/- 10 vs. 19 +/- 6, respectively, where the EI is the product of the percent expression and staining intensity). DKK-1 expression was also found to be higher in all PCa metastatic lesions (56 +/- 21 EI) compared to non-neoplastic tissues but was significantly decreased versus primary PCa lesions (P < 0.008). The decline in DKK-1 correlated with a shift of beta-catenin staining from the nucleus to the cytoplasm suggesting possible mechanism for the observed decrease in DKK-1 levels during PCa progression. Within metastatic lesions, DKK-1 expression was least abundant in PCa bone metastases relative to all soft tissue PCa metastatic lesions except lymph node metastases. High DKK-1 expression within PCa metastases was further associated with shorter over-all patient survival. CONCLUSIONS: Taken together, these data demonstrate that elevated DKK-1 expression is an early event in PCa and that as PCa progresses DKK-1 expression declines, particularly in advanced bone metastases. The decline of DKK-1 in bone metastases can unmask Wnts' osteoblastic activity. These data support a model in which DKK-1 is a molecular switch that transitions the phenotype of PCa osseous lesions from osteolytic to osteoblastic.

New trends in the treatment of bone metastasis.

Bone metastasis is often the penultimate harbinger of death for many cancer patients. Bone metastases are often associated with fractures and severe pain resulting in decreased quality of life. Accordingly, effective therapies to inhibit the development or progression of bone metastases will have important clinical benefits. To achieve this goal understanding the mechanisms through which bone metastases develop and progress may provide targets to inhibit the metastases. In the past few years, there have been advances in both understanding the mechanisms through which bone metastases develop and how they impact bone remodeling. Additionally, gains in promising clinical strategies to target bone metastases have been developed. In this prospectus, we will discuss some of these advances.

The role of Wnts in bone metastases.

Wnts are a large family of secreted glycoproteins that mediate bone development in the embryo and promote bone production in the adult. Autocrine Wnt signaling within tumor cells has been shown to promote tumorigenesis by enhancing tumor cell proliferation and survival. We recently demonstrated that prostate cancer cells (CaP) produce Wnts which act in a paracrine fashion to induce osteoblastic activity in CaP bone metastases. The ability of tumor-derived Wnts to influence bone development is regulated by multiple families of secreted antagonists including soluble frizzled related receptors (sFrp) and dickkopfs (DKK). CaP cells appear to produce DKK-1 early in the development of skeletal metastases, which masks osteogenic Wnts and thus favors an osteolytic environment at the metastatic site. As the metastases progresses, DKK-1 expression is lost allowing for a Wnt mediated osteoblastic response which predominates CaP boney lesions. Interestingly, blocking DKK-1 expression early in CaP metastasis prevents tumor establishment within the bone suggesting that osteolysis is a required first step in the development of CaP bone metastases. In this review, we discuss our data on the Wnt inhibitor DKK-1 in CaP bone metastasis in the context of current literature evidence that demonstrate that Wnt inhibitors can function as both tumor suppressors and tumor promoters. We provide a model that the affect of Wnt inhibitors on tumor development is dependent on the tumor micro-environment and suggest that DKK-1 is a switch which transitions CaP bone metastases from osteolytic to osteoblastic.

Type I collagen receptor (alpha 2 beta 1) signaling promotes the growth of human prostate cancer cells within the bone.

The most frequent site of prostate cancer metastasis is the bone. Adhesion to bone-specific factors may facilitate the selective metastasis of prostate cancer to the skeleton. Therefore, we tested whether prostate cancer bone metastasis is mediated by binding to type I collagen, the most abundant bone protein. We observed that only bone metastatic prostate cancer cells bound collagen I, whereas cells that form only visceral metastases failed to bind collagen. To confirm the relationship between collagen adhesion and bone metastatic potential, a collagen-binding variant of human LNCaP prostate cancer cells was derived through serial passage on type I collagen (LNCaP(col)). Fluorescence-activated cell sorting analysis showed that LNCaP(col) cells express increased levels of the integrin collagen I receptor alpha(2)beta(1) compared with LNCaP cells. Antibodies to the alpha(2)beta(1) complex inhibited LNCaP(col) binding to collagen, confirming that integrins mediated the attachment. Correspondingly, LNCaP(col) cells displayed enhanced chemotactic migration toward collagen I compared with LNCaP cells, an activity that could be blocked with alpha(2)beta(1) antibodies. To directly test the role of alpha(2)beta(1)-dependent collagen binding in bone metastasis, LNCaP and LNCaP(col) cells were injected into the tibia of nude mice. After 9 weeks, 7 of 13 (53%) mice injected with LNCaP(col) developed bone tumors, whereas 0 of 8 mice injected with LNCaP cells had evidence of boney lesions. LNCaP(col) cells were found to express increased levels of the metastasis-promoting RhoC GTPase compared with parental LNCaP. We conclude that collagen I attachment mediated by alpha(2)beta(1) initiates motility programs through RhoC and suggest a mechanism for prostate cancer metastasis to the bone.