Wiskott-Aldrich syndrome protein is involved in alphaIIb beta3-mediated cell adhesion

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked immunodeficiency disorder. The most common symptom seen in WAS patients is bleeding. One of the main causes of bleeding is defective platelet aggregation. The causative gene of WAS encodes WAS protein (WASP). Here, we show that WASP binds to the calcium- and integrin-binding protein (CIB) in platelets. CIB was originally identified as a protein binding to the alphaIIb cytoplasmic tail of platelet integrin alphaIIb beta3, which has a primary role in platelet aggregation. We also show that the WASP-CIB complex is important in alphaIIb beta3-mediated cell adhesion, and that in patients mutant forms of WASP are expressed at reduced levels or show lower affinities for CIB than wild-type WASP. Our results indicate that impaired complex formation between mutant WASPs and CIB reduces alphaIIb beta3-mediated cell adhesion and causes defective platelet aggregation, resulting in bleeding.     

Predominant inhibition of ganodermic acid S on the thromboxane A2-dependent pathway in human platelets response to collagen

Ganodermic acid S (GAS), a membrane acting agent, exerts multiple effects on human platelet function (C.N. Wang et al. (1991) Biochem. J. 277, 189-197). The study reported how GAS affected the response of human gel-filtered platelets (GFP) to collagen. The agent inhibited cell aggregation by prolonging lag and shape change periods and decreasing the initial cell aggregation rate. However, the inhibitory efficiency was less than its inhibition on GFP response to U46619, a thromboxane (TX) A2 mimetic. In the agent-effect on biochemical events, GAS effectively inhibited Ca2+ mobilization, phosphorylation of myosin light chain, dense granule secretion and TXB2 generation. The inhibitions might originate from blocking Ca2+ mobilization of the TXA2-dependent pathway. GAS partially decreased the phosphorylation of most phosphotyrosine proteins from early activation to the integrin alphaIIbbeta3-regulated steps. The agent did not affect the phosphorylation of three proteins at the steps regulated by integrin alphaIIbbeta3. The results suggest that GAS inhibits the collagen response predominantly on the TXA2-dependent signaling, and the tyrosine kinase-dependent pathway in collagen response plays a major role in aggregation.     

Anti-transforming growth factor ß antibody treatment rescues bone loss and prevents breast cancer metastasis to bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p?=?0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p?=?0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.     

Pharmacological evaluation of the novel thromboxane modulator BM-567 (II/II). Effects of BM-567 on osteogenic sarcoma-cell-induced platelet aggregation

Evidence exists that a large number of tumor cells such as osteosarcoma cells stimulate platelet aggregation, which can be an early step in the metastatic processes of these tumors. Thromboxane A(2) (TXA(2)) is released during platelet aggregation, and it has been suggested that this release may be pathogenic for tumor metastasis for several reasons:Some tumors release large amounts of TXA(2) compared to normal tissue.TXA(2) potentiates tumor growth in culture and increases metastasis in animals.TXA(2) is a potent stimulant of platelet aggregation and causes vascular injuries that may promote implantation of tumor cell-platelet aggregates.If TXA(2) participates in tumor metastasis, it may be hypothesized that TXA(2) inhibitors should decrease tumor metastasis. So, we have evaluated the effects of the original TXA(2) synthase inhibitor and TXA(2) receptor antagonist BM-567 on platelet aggregation induced by osteosarcoma cells using MG-63 tumor cells. Results obtained showed that this drug inhibited both MG-63 tumor-cell-induced platelet aggregation and platelet TXA(2) release following the tumor cell stimulation with IC(50) values of 3.04x10(-7) and 2.51x10(-8)M, respectively.     

Cancer Cell Expression of Autotaxin Controls Bone Metastasis Formation in Mouse through Lysophosphatidic Acid-Dependent Activation of Osteoclasts

Background

Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorbtive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2) is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD) activity, ATX controls the level of lysophosphatidic acid (LPA) in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models.

Methodology/Principal Findings

Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX) to inmmunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis.

Conclusion/Significance

Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a factor that stimulates directly cancer growth and metastasis, and osteoclast differentiation. Therefore, targeting the autotaxin/LPA track emerges as a potential new therapeutic approach to improve the outcome of patients with bone metastases.     

Characterization of mice lacking the tetraspanin superfamily member CD151

The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially alpha3beta1, alpha6beta1, alpha7beta1, and alpha6beta4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of alpha3 and alpha6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.     

The a3 isoform vacuolar type H⁺-ATPase promotes distant metastasis in the mouse B16 melanoma cells

Accumulating evidence indicates that the acidic microenvironments critically influence malignant behaviors of cancer including invasiveness, metastasis, and chemoresistance. Because the vacuolar-type H(+)-ATPase (V-ATPase) has been shown to cause extracellular acidification by pumping protons, we studied the role of V-ATPase in distant metastasis. Real-time PCR analysis revealed that the high-metastatic B16-F10 melanoma cells strongly expressed the a3 isoform V-ATPase compared to the low-metastatic B16 parental cells. Consistent with this, B16-F10 cells created acidic environments in lung metastases by acridine orange staining and strong a3 V-ATPase expression in bone metastases by immunohistochemistry. Immunocytochemical analysis showed B16-F10 cells expressed a3 V-ATPase not only in cytoplasm but also plasma membrane, whereas B16 parental cells exhibited its expression only in cytoplasm. Of note, knockdown of a3 V-ATPase suppressed invasiveness and migration with reduced MMP-2 and MMP-9 expression in B16-F10 cells and significantly decreased lung and bone metastases, despite that tumor growth was not altered. Importantly, administration of a specific V-ATPase a3 inhibitor FR167356 reduced bone metastasis of B16-F10 cells. These results suggest that a3 V-ATPase promotes distant metastasis of B16-F10 cells by creating acidic environments via proton secretion. Our results also suggest that inhibition of the development of cancer-associated acidic environments by suppressing a3 V-ATPase could be a novel therapeutic approach for the treatment of cancer metastasis.     

Targeted overexpression of BSP in osteoclasts promotes bone metastasis of breast cancer cells

Bone is one of the most common sites of breast cancer metastasis while bone sialoprotein (BSP) is thought to play an important role in bone metastasis of malignant tumors. The objective of this study is to determine the role of BSP overexpression in osteolytic metastasis using two homozygous transgenic mouse lines in which BSP expression is elevated either in all the tissues (CMV-BSP mice) or only in the osteoclasts (CtpsK-BSP mice). The results showed that skeletal as well as systemic metastases of 4T1 murine breast cancer cells were dramatically increased in CMV-BSP mice. In CtpsK-BSP mice, it was found that targeted BSP overexpression in osteoclasts promoted in vitro osteoclastogenesis and activated osteoclastic differentiation markers such as Cathepsin K, TRAP and NFAT2. MicroCT scan demonstrated that CtpsK/BSP mice had reduced trabecular bone volume and bone mineral density (BMD). The real-time IVIS Imaging System showed that targeted BSP overexpression in osteoclasts promoted bone metastasis of breast cancer cells. The osteolytic lesion area was significantly larger in CtpsK/BSP mice than in the controls as demonstrated by both radiographic and histomorphometric analyses. TRAP staining demonstrated a twofold increase in the number of osteoclasts in the bone lesion area from CtpsK/BSP mice compared with that from wild type mice. We conclude that host tissue-derived BSP also plays important roles in breast cancer metastasis through inducing tumor cell seeding into the remote host tissues. Furthermore, osteoclast-derived BSP promotes osteoclast differentiation in an autocrine manner and consequently promotes osteolytic bone metastasis of breast cancer.     

The role of the BMP signaling antagonist noggin in the development of prostate cancer osteolytic bone metastasis

Members of the BMP and Wnt protein families play a relevant role in physiologic and pathologic bone turnover. Extracellular antagonists are crucial for the modulation of their activity. Lack of expression of the BMP antagonist noggin by osteoinductive, carcinoma-derived cell lines is a determinant of the osteoblast response induced by their bone metastases. In contrast, osteolytic, carcinoma-derived cell lines express noggin constitutively. We hypothesized that cancer cell-derived noggin may contribute to the pathogenesis of osteolytic bone metastasis of solid cancers by repressing bone formation. Intra-osseous xenografts of PC-3 prostate cancer cells induced osteolytic lesions characterized not only by enhanced osteoclast-mediated bone resorption, but also by decreased osteoblast-mediated bone formation. Therefore, in this model, uncoupling of the bone remodeling process contributes to osteolysis. Bone formation was preserved in the osteolytic lesions induced by noggin-silenced PC-3 cells, suggesting that cancer cell-derived noggin interferes with physiologic bone coupling. Furthermore, intra-osseous tumor growth of noggin-silenced PC-3 cells was limited, most probably as a result of the persisting osteoblast activity. This investigation provides new evidence for a model of osteolytic bone metastasis where constitutive secretion of noggin by cancer cells mediates inhibition of bone formation, thereby preventing repair of osteolytic lesions generated by an excess of osteoclast-mediated bone resorption. Therefore, noggin suppression may be a novel strategy for the treatment of osteolytic bone metastases.     

The TLR3, PI3K,survin, FasL,and Fas genes as major risk factors of occurrence and development of cervical cancer disease

To investigate the role of TLR3/PI3K signals in the occurrence and development of cervical cancer disease, TLR3–siRNA was used to block key signaling pathways involved in cervical cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Results show that tumor U14 cell growth, migration and invasion in TLR3–siRNA treatment group were significantly decreased. Through LY294002 suppressing targeted gene, the LY294002 treatment specifically and significantly knocked down the expressions of tumor TLR3 and PI3K proteins in cervical cancer mice. Furthermore, expressions of tumor Survivin and FasL proteins were markedly suppressed, whereas expressions of Fas protein were upregulated in LY294002 treatment group mice. LY294002 treatment suppressed tumor growth and increased the thymus and spleen indeces and survival days of cervical cancer mice. This study demonstrates that TLR3–siRNA and LY294002 treatments can markedly suppress cervical cancer cell invasion and tumor growth and increase survival life by silencing targeted genes.     

Anti-Transforming Growth Factor ? Antibody Treatment Rescues Bone Loss and Prevents Breast Cancer Metastasis to Bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.     

Activation of the RalGEF/Ral pathway promotes prostate cancer metastasis to bone

A hallmark of metastasis is organ specificity; however, little is known about the underlying signaling pathways responsible for the colonization and growth of tumor cells in target organs. Since tyrosine kinase receptor activation is frequently associated with prostate cancer progression, we have investigated the role of a common signaling intermediary, activated Ras, in prostate cancer metastasis. Three effector pathways downstream of Ras, Raf/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase, and Ral guanine nucleotide exchange factors (RalGEFs), were assayed for their ability to promote the metastasis of a tumorigenic, nonmetastatic human prostate cancer cell line, DU145. Oncogenic Ras promoted the metastasis of DU145 to multiple organs, including bone and brain. Activation of the Raf/ERK pathway stimulated metastatic colonization of the brain, while activation of the RalGEF pathway led to bone metastases, the most common organ site for prostate cancer metastasis. In addition, loss of RalA in the metastatic PC3 cell line inhibited bone metastasis but did not affect subcutaneous tumor growth. Loss of Ral appeared to suppress expansive growth of prostate cancer cells in bone, whereas homing and initial colonization were less affected. These data extend our understanding of the functional roles of the Ral pathway and begin to identify signaling pathways relevant for organ-specific metastasis.     

Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein.

We have used recombinant or synthetic alphaIIb and beta3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha.beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha.beta complex by decreasing the dissociation rate. alpha.beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bound to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding of CIB to intact alphaIIbbeta3 occurred preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alphaIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevented activated alphaIIbbeta3 occupancy by PAC-1, we conclude that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor occupancy event.     

TNF inhibitor suppresses bone metastasis in a breast cancer cell line

In the evolution of cancer, tumor necrosis factor-alpha (TNF-α) plays a paradoxical role. High doses induce significant anticancer effects, but conversely, physiologic and pathologic levels of TNF-α may be involved in cancer promotion, tumor growth, and metastasis.Infliximab is a chimeric murine monoclonal antibody that binds with high affinity to soluble and membrane TNF-α and inhibits binding of TNF-α to its receptors. In the present study, we investigated the effect of infliximab, a TNF-α antagonist, on breast cancer aggressiveness and bone metastases.Infliximab greatly reduced cell motility and bone metastases in a metastatic breast cancer cell line, MDA-MB-231. The mechanism of bone metastasis inhibition involved decreased expression of CXC chemokine receptor 4 (CXCR4) and increased expression of decorin, which is the prototype of an expanding family of small leucine-rich proteoglycans. These results suggest a novel role for TNF-α inhibition in the reduction or prevention of bone metastases in this breast cancer model. Our study suggests that inhibition of TNF-α using infliximab may become a preventive therapeutic option for breast cancer.     

Fibrin but not adsorbed fibrinogen supports fibronectin assembly by spread platelets. Effects of the interaction of alphaIIb beta3 with the C terminus of the fibrinogen gamma-chain

We investigated the assembly of soluble fibronectin by lysophosphatidic acid-activated platelets adherent to fibrinogen or fibrin. More fibronectin was assembled by activated platelets spread on fibrin matrices than by platelets spread on adsorbed fibrinogen. The difference between platelets adherent to fibrinogen and fibrin occurred under both static and flow conditions. Similar differences were seen in binding of the 70-kDa N-terminal fragment of fibronectin that recognizes fibronectin assembly sites on adherent cells. Antibody and peptide blocking studies demonstrated that alphaIIb beta3 integrin mediates platelet adhesion to fibrinogen, whereas both alphav beta3 and alphaIIb beta3 mediate platelet adhesion to fibrin. The hypothesis that engagement of the C-terminal QAGDV sequence of the fibrinogen gamma-chain by alphaIIb beta3 inhibits the ability of the platelet to assemble fibronectin was tested by several experiments. Activated platelets adherent to adsorbed mutant fibrinogen lacking the QAGDV sequence (gammadelta5FG) were assembly-competent, as were platelets adherent to adsorbed normal fibrinogen that had been pretreated with the 7E9 antibody to the C terminus of the gamma-chain. Moreover, adsorbed normal fibrinogen but not gammadelta5FG suppressed the ability of co-adsorbed fibronectin to direct assembly of soluble fibronectin by spread platelets. The suppressive effect was lost when a surface of co-adsorbed fibronectin and fibrinogen was pretreated with 7E9. These results support a model in which the engagement of alphaIIb beta3 by the C-terminal sequence of the fibrinogen gamma-chain initiates signals that suppress subsequent fibronectin assembly by spread platelets. This interaction is less dominant when platelets adhere to fibrin, resulting in enhanced fibronectin assembly.     

Osteoprotegrin and the bone homing and colonization potential of breast cancer cells

Breast cancer cells preferentially metastasize to bone, leading to the formation of primarily osteolytic lesions. Osteoprotegerin (OPG) plays multifactorial roles in the development of osteolytic bone metastases. An increase in the ratio of receptor activator of nuclear factor kappaB ligand (RANKL) to OPG increases osteoclastogenesis within the bone microenvironment. OPG also acts as a survival factor for cancer cells by protecting them from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apoptosis. This study compares OPG production in vitro in a number of breast cancer cell lines exhibiting both differences in metastatic capacity and in preferential metastasis to bone. Our studies demonstrated that OPG expression by MDA-231, MDA-MET, and MDA-231/K cancer cells was directly correlated with bone specific homing and colonization potential but not with metastasis of cancer cells to other organs; both in IL-1 beta stimulated and control cells. We also demonstrated expression of other bone-related markers including type I collagen, osteocalcin, osteopontin, and Runx2 in these cells. However, the generally lower expression of these markers in the bone selective cell line MDA-MET suggested that increased OPG expression in the bone specific variant was not merely a consequence of enhanced osteomimicry by these cells but that it has a significant role in the metastatic process. Co-culture of breast cancer cells with osteoblastic cells (hFOB 1.19) led to an overall downregulation in OPG production, which was not affected by the bone homing and colonization potential of the cell lines, suggesting that OPG alone is not indicative of osteolytic bone activity by breast cancer cells.     

Alpha-TEA plus cisplatin reduces human cisplatin-resistant ovarian cancer cell tumor burden and metastasis

A novel nonhydrolyzable ether-linked acetic acid analog of vitamin E, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (alpha-TEA) in combination with cisplatin, reduces tumor burden of A2780/cp70 (cp70) cisplatin-resistant human ovarian cancer cells xenografted into immune compromised nude mice. Two xenograft studies were conducted using cp70 cells stably expressing green fluorescent protein (cp70-GFP) subcutaneously transplanted into NU/NU mice. For studies 1 and 2, alpha-TEA was formulated in liposomes and delivered by aerosol such that approximately 36 microg and 72 microg of alpha-TEA were deposited in the respiratory tract of each mouse each day, respectively. Cisplatin at 5 mg/kg was administered by intraperitoneal injections once weekly for the first 3 weeks in Study 1 and on the third and 10th days following treatment initiation in Study 2. The combination alpha-TEA + cisplatin treatment reduced tumor burden and metastasis of cp70-GFP cells in comparison to control mice or mice treated with alpha-TEA or cisplatin singly. A significant reduction (P < 0.001) in growth of subcutaneous transplanted tumors was obtained with alpha-TEA + cisplatin for both studies. Visible metastases were observed in the lungs of animals from control and cisplatin-treated groups but not in animals from the alpha-TEA- or alpha-TEA + cisplatin-treated groups. The alpha-TEA + cisplatin significantly reduced the total number of lung and axillary lymph node micrometastasis (P < 0.03 and P < 0.0001, respectively). Analyses of tumor sections showed the alpha-TEA + cisplatin treatment group, in comparison to control, to have a significantly lower level of cell proliferation (Ki-67 staining; P < 0.0001) and a significantly higher level of apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling [TUNEL]; P < 0.0001). In summary, combinations of alpha-TEA + cisplatin significantly reduced tumor burden and metastases in a xenograft model of cisplatin-resistant human ovarian cancer cells. These data show promise for combination alpha-TEA + cisplatin chemotherapy for ovarian cancer.     

Comparison of Immunity in Mice Cured of Primary/Metastatic Growth of EMT6 or 4THM Breast Cancer by Chemotherapy or Immunotherapy

Purpose

We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy.

Methods

We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel.

Results

In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment.

Conclusion

Immunotherapy, but not chemotherapy, enhances CD4⁺ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.     

Disseminated Breast Cancer Cells Acquire a Highly Malignant and Aggressive Metastatic Phenotype during Metastatic Latency in the Bone

Background

Disseminated tumor cells (DTCs) in the bone marrow may exist in a dormant state for extended periods of time, maintaining the ability to proliferate upon activation, engraft at new sites, and form detectable metastases. However, understanding of the behavior and biology of dormant breast cancer cells in the bone marrow niche remains limited, as well as their potential involvement in tumor recurrence and metastasis. Therefore, the purpose of this study was to investigate the tumorigenicity and metastatic potential of dormant disseminated breast cancer cells (prior to activation) in the bone marrow.

Methodology/Principal Findings

Total bone marrow, isolated from mice previously injected with tumorspheres into the mammary fat pad, was injected into the mammary fat pad of NUDE mice. As a negative control, bone marrow isolated from non-injected mice was injected into the mammary fat pad of NUDE mice. The resultant tumors were analyzed by immunohistochemistry for expression of epithelial and mesenchymal markers. Mouse lungs, livers, and kidneys were analyzed by H+E staining to detect metastases. The injection of bone marrow isolated from mice previously injected with tumorspheres into the mammary fat pad, resulted in large tumor formation in the mammary fat pad 2 months post-injection. However, the injection of bone marrow isolated from non-injected mice did not result in tumor formation in the mammary fat pad. The DTC-derived tumors exhibited accelerated development of metastatic lesions within the lung, liver and kidney. The resultant tumors and the majority of metastatic lesions within the lung and liver exhibited a mesenchymal-like phenotype.

Conclusions/Significance

Dormant DTCs within the bone marrow are highly malignant upon injection into the mammary fat pad, with the accelerated development of metastatic lesions within the lung, liver and kidney. These results suggest the acquisition of a more aggressive phenotype of DTCs during metastatic latency within the bone marrow microenvironment.     

Feasibility and limits of an orthotopic human colon cancer model in nude mice

We sought to develop an accurate colorectal cancer model in nude mice with stable local growth, tumor cell dissemination, and reproducible metastatic capacity. To this end, we orthotopically transplanted histologically intact human colorectal cancer tissue from 10 human patients into nude mice. After successful local tumor growth, tumor tissues were retransplanted as many as 9 times in serial passage. All specimens were transplanted using microsurgical techniques. Histologic, immunohistochemical, and polymerase chain reaction techniques were used to determine tumor growth rates and kinetics, development of regional lymph node and distant hepatic metastases, and the induction of minimal residual disease (MRD). Stable local tumor growth rates with variable growth kinetics were detected in 73.4% of all mice. The lymph node and hepatic metastasis rates were low, at 18.4% and 4.9%, respectively. MRD, as reflected by CK20 positivity of the bone marrow in animals with lymph node and hepatic metastases, was present in 22.2%. The orthotopic colorectal cancer model described here is feasible for the induction of reproducible local tumor growth but is limited by variable growth kinetics and the low rate of lymph node and hepatic metastases. Cytokeratin-positive cells indicative of MRD could be detected in the bone marrow of approximately 25% of the nude mice with metastases. The observed induction of MRD after orthotopic implantation of intact human colon cancer in animals with lymph node and hepatic metastases might be improved if established colon cancer cell lines were used.