Signaling events at TMEM doorways provide potential targets for inhibiting breast cancer dissemination.

Tumor cell intravasation is essential for metastatic dissemination, but its exact mechanism is incompletely understood. We have previously shown that in breast cancer, the direct and stable association of a tumor cell expressing Mena, a Tie2hi/VEGFhi macrophage, and a vascular endothelial cell, creates an intravasation portal, called a "tumor microenvironment of metastasis" (TMEM) doorway, for tumor cell intravasation, leading to dissemination to distant sites. The density of TMEM doorways, also called TMEM doorway score, is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that tumor cells utilize TMEM doorway-associated transient vascular openings to intravasate, the precise signaling mechanisms involved in TMEM doorway function are only partially understood. Using two mouse models of breast cancer and an in vitro assay of intravasation, we report that CSF-1 secreted by the TMEM doorway tumor cell stimulates local secretion of VEGF-A from the Tie2hi TMEM doorway macrophage, leading to the dissociation of endothelial junctions between TMEM doorway associated endothelial cells, supporting tumor cell intravasation. Acute blockade of CSF-1R signaling decreases macrophage VEGF-A secretion as well as TMEM doorway-associated vascular opening, tumor cell trans-endothelial migration, and dissemination. These new insights into signaling events regulating TMEM doorway function should be explored further as treatment strategies for metastatic disease.

SHP2 Inhibition Sensitizes Diverse Oncogene-Addicted Solid Tumors to Re-treatment with Targeted Therapy.

Rationally targeted therapies have transformed cancer treatment, but many patients develop resistance through bypass signaling pathway activation. PF-07284892 (ARRY-558) is an allosteric SHP2 inhibitor designed to overcome bypass-signaling-mediated resistance when combined with inhibitors of various oncogenic drivers. Activity in this setting was confirmed in diverse tumor models. Patients with ALK fusion-positive lung cancer, BRAFV600E-mutant colorectal cancer, KRASG12D-mutant ovarian cancer, and ROS1 fusion-positive pancreatic cancer who previously developed targeted therapy resistance were treated with PF-07284892 on the first dose level of a first-in-human clinical trial. After progression on PF-07284892 monotherapy, a novel study design allowed the addition of oncogene-directed targeted therapy that had previously failed. Combination therapy led to rapid tumor and circulating tumor DNA (ctDNA) responses and extended the duration of overall clinical benefit. SIGNIFICANCE: PF-07284892-targeted therapy combinations overcame bypass-signaling-mediated resistance in a clinical setting in which neither component was active on its own. This provides proof of concept of the utility of SHP2 inhibitors in overcoming resistance to diverse targeted therapies and provides a paradigm for accelerated testing of novel drug combinations early in clinical development. See related commentary by Hernando-Calvo and Garralda, p. 1762. This article is highlighted in the In This Issue feature, p. 1749.

Assessment of MRI to estimate metastatic dissemination risk and prometastatic effects of chemotherapy.

Metastatic dissemination in breast cancer is regulated by specialized intravasation sites called "tumor microenvironment of metastasis" (TMEM) doorways, composed of a tumor cell expressing the actin-regulatory protein Mena, a perivascular macrophage, and an endothelial cell, all in stable physical contact. High TMEM doorway number is associated with an increased risk of distant metastasis in human breast cancer and mouse models of breast carcinoma. Here, we developed a novel magnetic resonance imaging (MRI) methodology, called TMEM Activity-MRI, to detect TMEM-associated vascular openings that serve as the portal of entry for cancer cell intravasation and metastatic dissemination. We demonstrate that TMEM Activity-MRI correlates with primary tumor TMEM doorway counts in both breast cancer patients and mouse models, including MMTV-PyMT and patient-derived xenograft models. In addition, TMEM Activity-MRI is reduced in mouse models upon treatment with rebastinib, a specific and potent TMEM doorway inhibitor. TMEM Activity-MRI is an assay that specifically measures TMEM-associated vascular opening (TAVO) events in the tumor microenvironment, and as such, can be utilized in mechanistic studies investigating molecular pathways of cancer cell dissemination and metastasis. Finally, we demonstrate that TMEM Activity-MRI increases upon treatment with paclitaxel in mouse models, consistent with prior observations that chemotherapy enhances TMEM doorway assembly and activity in human breast cancer. Our findings suggest that TMEM Activity-MRI is a promising precision medicine tool for localized breast cancer that could be used as a non-invasive test to determine metastatic risk and serve as an intermediate pharmacodynamic biomarker to monitor therapeutic response to agents that block TMEM doorway-mediated dissemination.

Phase Ib/II Trial of Ribociclib in Combination with Binimetinib in Patients with NRAS-mutant Melanoma.

PURPOSE: Enhanced MAPK pathway signaling and cell-cycle checkpoint dysregulation are frequent in NRAS-mutant melanoma and, as such, the regimen of the MEK inhibitor binimetinib and the selective CDK4/6 inhibitor ribociclib is a rational combination. PATIENTS AND METHODS: This is a phase Ib/II, open-label study of ribociclib + binimetinib in patients with NRAS-mutant melanoma (NCT01781572). Primary objectives were to estimate the MTD/recommended phase II dose (RP2D) of the combination (phase Ib) and to characterize combination antitumor activity at the RP2D (phase II). Tumor genomic characterization and pharmacokinetics/pharmacodynamics were also evaluated. RESULTS: Ten patients (16.4%) experienced dose-limiting toxicities in cycle 1 of phase Ib. Overall response rate in the phase II cohort (n = 41) for the selected RP2D (binimetinib 45 mg twice daily + ribociclib 200 mg once daily, 21 days on/7 days off) was 19.5% [8/41; 95% confidence interval (CI), 8.8-34.9]. The response rate was 32.5% (13/40; 95% CI, 20.1-48.0) in patients with NRAS mutation with concurrent alterations of CDKN2A, CDK4, or CCND1. Median progression-free survival was 3.7 months (95% CI, 3.5-5.6) and median overall survival was 11.3 months (95% CI, 9.3-14.2) for all patients. Common treatment-related toxicities included creatine phosphokinase elevation, rash, edema, anemia, nausea, diarrhea, and fatigue. Pharmacokinetics and safety were consistent with single-agent data, supporting a lack of drug-drug interaction. CONCLUSIONS: Ribociclib + binimetinib can be safely administered and is clinically active in patients with NRAS-mutant melanoma. Co-mutations of cell-cycle genes may define a population with greater likelihood of treatment benefit. See related commentary by Moschos, p. 2977.

Homophilic CD44 Interactions Mediate Tumor Cell Aggregation and Polyclonal Metastasis in Patient-Derived Breast Cancer Models.

Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. The intercellular CD44-CD44 homophilic interactions directed multicellular aggregation, requiring its N-terminal domain, and initiated CD44-PAK2 interactions for further activation of FAK signaling. Our studies highlight that CD44+ CTC clusters, whose presence is correlated with a poor prognosis of patients with breast cancer, can serve as novel therapeutic targets of polyclonal metastasis. SIGNIFICANCE: CTCs not only serve as important biomarkers for liquid biopsies, but also mediate devastating metastases. CD44 homophilic interactions and subsequent CD44-PAK2 interactions mediate tumor cluster aggregation. This will lead to innovative biomarker applications to predict prognosis, facilitate development of new targeting strategies to block polyclonal metastasis, and improve clinical outcomes.See related commentary by Rodrigues and Vanharanta, p. 22.This article is highlighted in the In This Issue feature, p. 1.

A Unidirectional Transition from Migratory to Perivascular Macrophage Is Required for Tumor Cell Intravasation.

Tumor-associated macrophages (TAMs) are critical for tumor metastasis. Two TAM subsets support cancer cell intravasation: migratory macrophages guide cancer cells toward blood vessels, where sessile perivascular macrophages assist their entry into the blood. However, little is known about the inter-relationship between these functionally distinct TAMs or their possible inter-conversion. We show that motile, streaming TAMs are newly arrived monocytes, recruited via CCR2 signaling, that then differentiate into the sessile perivascular macrophages. This unidirectional process is regulated by CXCL12 and CXCR4. Cancer cells induce TGF-ß-dependent upregulation of CXCR4 in monocytes, while CXCL12 expressed by perivascular fibroblasts attracts these motile TAMs toward the blood vessels, bringing motile cancer cells with them. Once on the blood vessel, the migratory TAMs differentiate into perivascular macrophages, promoting vascular leakiness and intravasation.

The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2Hi Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors.

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2+ myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2Hi/Vegf-AHi macrophages in the tumor microenvironment of metastasis (TMEM). The antitumor effects of rebastinib enhance the efficacy of microtubule inhibiting chemotherapeutic agents, either eribulin or paclitaxel, by reducing tumor volume, metastasis, and improving overall survival. Rebastinib inhibition of angiopoietin/Tie2 signaling impairs multiple pathways in tumor progression mediated by protumoral Tie2+ macrophages, including TMEM-dependent dissemination and angiopoietin/Tie2-dependent angiogenesis. Rebastinib is a promising therapy for achieving Tie2 inhibition in cancer patients. Mol Cancer Ther; 16(11); 2486-501. ©2017 AACR.

Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism.

Breast cancer cells disseminate through TIE2/MENACalc/MENAINV-dependent cancer cell intravasation sites, called tumor microenvironment of metastasis (TMEM), which are clinically validated as prognostic markers of metastasis in breast cancer patients. Using fixed tissue and intravital imaging of a PyMT murine model and patient-derived xenografts, we show that chemotherapy increases the density and activity of TMEM sites and Mena expression and promotes distant metastasis. Moreover, in the residual breast cancers of patients treated with neoadjuvant paclitaxel after doxorubicin plus cyclophosphamide, TMEM score and its mechanistically connected MENAINV isoform expression pattern were both increased, suggesting that chemotherapy, despite decreasing tumor size, increases the risk of metastatic dissemination. Chemotherapy-induced TMEM activity and cancer cell dissemination were reversed by either administration of the TIE2 inhibitor rebastinib or knockdown of the MENA gene. Our results indicate that TMEM score increases and MENA isoform expression pattern changes with chemotherapy and can be used in predicting prometastatic changes in response to chemotherapy. Furthermore, inhibitors of TMEM function may improve clinical benefits of chemotherapy in the neoadjuvant setting or in metastatic disease.

The Multifaceted Role of Perivascular Macrophages in Tumors.

Evidence has emerged for macrophages in the perivascular niche of tumors regulating important processes like angiogenesis, various steps in the metastatic cascade, the recruitment and activity of other tumor-promoting leukocytes, and tumor responses to frontline therapies like irradiation and chemotherapy. Understanding the mechanisms controlling the recruitment, retention, and function of these cells could identify important targets for anti-cancer therapeutics.

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

Brightness-equalized quantum dots.

As molecular labels for cells and tissues, fluorescent probes have shaped our understanding of biological structures and processes. However, their capacity for quantitative analysis is limited because photon emission rates from multicolour fluorophores are dissimilar, unstable and often unpredictable, which obscures correlations between measured fluorescence and molecular concentration. Here we introduce a new class of light-emitting quantum dots with tunable and equalized fluorescence brightness across a broad range of colours. The key feature is independent tunability of emission wavelength, extinction coefficient and quantum yield through distinct structural domains in the nanocrystal. Precise tuning eliminates a 100-fold red-to-green brightness mismatch of size-tuned quantum dots at the ensemble and single-particle levels, which substantially improves quantitative imaging accuracy in biological tissue. We anticipate that these materials engineering principles will vastly expand the optical engineering landscape of fluorescent probes, facilitate quantitative multicolour imaging in living tissue and improve colour tuning in light-emitting devices.

Tuning cobalt(III) Schiff base complexes as activated protein inhibitors.

Cobalt(III) Schiff base complexes ([Co(acacen)(L)2](+), where L = NH3) inhibit histidine-containing proteins through dissociative exchange of the labile axial ligands (L). This work investigates axial ligand exchange dynamics of [Co(acacen)(L)2](+) complexes toward the development of protein inhibitors that are activated by external triggers such as light irradiation. We sought to investigate ligand exchange dynamics to design a Co(III) complex that is substitutionally inert under normal physiological conditions for selective activation. Fluorescent imidazoles (C3Im) were prepared as axial ligands in [Co(acacen)(L)2](+) to produce complexes (CoC3Im) that could report on ligand exchange and, thus, complex stability. These fluorescent imidazole reporters guided the design of a new dinuclear Co(III) Schiff base complex containing bridging diimidazole ligands, which exhibits enhanced stability to ligand exchange with competing imidazoles and to hydrolysis within a biologically relevant pH range. These studies inform the design of biocompatible Co(III) Schiff base complexes that can be selectively activated for protein inhibition with spatial and temporal specificity.

Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA.

UNLABELLED: Dissemination of tumor cells is an essential step in metastasis. Direct contact between a macrophage, mammalian-enabled (MENA)-overexpressing tumor cell, and endothelial cell [Tumor MicroEnvironment of Metastasis (TMEM)] correlates with metastasis in breast cancer patients. Here we show, using intravital high-resolution two-photon microscopy, that transient vascular permeability and tumor cell intravasation occur simultaneously and exclusively at TMEM. The hyperpermeable nature of tumor vasculature is described as spatially and temporally heterogeneous. Using real-time imaging, we observed that vascular permeability is transient, restricted to the TMEM, and required for tumor cell dissemination. VEGFA signaling from TIE2(hi) TMEM macrophages causes local loss of vascular junctions, transient vascular permeability, and tumor cell intravasation, demonstrating a role for the TMEM within the primary mammary tumor. These data provide insight into the mechanism of tumor cell intravasation and vascular permeability in breast cancer, explaining the value of TMEM density as a predictor of distant metastatic recurrence in patients. SIGNIFICANCE: Tumor vasculature is abnormal with increased permeability. Here, we show that VEGFA signaling from TIE2(hi) TMEM macrophages results in local, transient vascular permeability and tumor cell intravasation. These data provide evidence for the mechanism underlying the association of TMEM with distant metastatic recurrence, offering a rationale for therapies targeting TMEM.

Axial ligand exchange of N-heterocyclic cobalt(III) Schiff base complexes: molecular structure and NMR solution dynamics.

The kinetic and thermodynamic ligand exchange dynamics are important considerations in the rational design of metal-based therapeutics and therefore, require detailed investigation. Co(III) Schiff base complex derivatives of bis(acetylacetone)ethylenediimine [acacen] have been found to be potent enzyme and transcription factor inhibitors. These complexes undergo solution exchange of labile axial ligands. Upon dissociation, Co(III) irreversibly interacts with specific histidine residues of a protein, and consequently alters structure and causes inhibition. To guide the rational design of next generation agents, understanding the mechanism and dynamics of the ligand exchange process is essential. To investigate the lability, pH stability, and axial ligand exchange of these complexes in the absence of proteins, the pD- and temperature-dependent axial ligand substitution dynamics of a series of N-heterocyclic [Co(acacen)(X)(2)](+) complexes [where X = 2-methylimidazole (2MeIm), 4-methylimidazole (4MeIm), ammine (NH(3)), N-methylimidazole (NMeIm), and pyridine (Py)] were characterized by NMR spectroscopy. The pD stability was shown to be closely related to the nature of the axial ligand with the following trend toward aquation: 2MeIm > NH(3) ≫ 4MeIm > Py > Im > NMeIm. Reaction of each [Co(III)(acacen)(X)(2)](+) derivative with 4MeIm showed formation of a mixed ligand Co(III) intermediate via a dissociative ligand exchange mechanism. The stability of the mixed ligand adduct was directly correlated to the pD-dependent stability of the starting Co(III) Schiff base with respect to [Co(acacen)(4MeIm)(2)](+). Crystal structure analysis of the [Co(acacen)(X)(2)](+) derivatives confirmed the trends in stability observed by NMR spectroscopy. Bond distances between the Co(III) and the axial nitrogen atoms were longest in the 2MeIm derivative as a result of distortion in the planar tetradentate ligand, and this was directly correlated to axial ligand lability and propensity toward exchange.

Kinetics and thermodynamics of irreversible inhibition of matrix metalloproteinase 2 by a Co(III) Schiff base complex.

Cobalt(III) Schiff base complexes have been used as potent inhibitors of protein function through the coordination to histidine residues essential for activity. The kinetics and thermodynamics of the binding mechanism of Co(acacen)(NH(3))(2)Cl [Co(acacen); where H(2)acacen is bis(acetylacetone)ethylenediimine] enzyme inhibition has been examined through the inactivation of matrix metalloproteinase 2 (MMP-2) protease activity. Co(acacen) is an irreversible inhibitor that exhibits time- and concentration-dependent inactivation of MMP-2. Co(acacen) inhibition of MMP-2 is temperature-dependent, with the inactivation increasing with temperature. Examination of the formation of the transition state for the MMP-2/Co(acacen) complex was determined to have a positive entropy component indicative of greater disorder in the MMP-2/Co(acacen) complex than in the reactants. With further insight into the mechanism of Co(acacen) complexes, Co(III) Schiff base complex protein inactivators can be designed to include features regulating activity and protein specificity. This approach is widely applicable to protein targets that have been identified to have clinical significance, including matrix metalloproteinases. The mechanistic information elucidated here further emphasizes the versatility and utility of Co(III) Schiff base complexes as customizable protein inhibitors.

Targeted inactivation of Snail family EMT regulatory factors by a Co(III)-Ebox conjugate.

Snail family proteins are core EMT (epithelial-mesenchymal transition) regulatory factors that play essential roles in both development and disease processes and have been associated with metastasis in carcinomas. Snail factors are required for the formation of neural crest stem cells in most vertebrate embryos, as well as for the migratory invasive behavior of these cells. Snail factors have recently been linked to the formation of cancer stem cells, and expression of Snail proteins may be associated with tumor recurrence and resistance to chemotherapy and radiotherapy. We report that Co(III)-Ebox is a potent inhibitor of Snail-mediated transcriptional repression in breast cancer cells and in the neural crest of Xenopus. We further show that the activity of Co(III)-Ebox can be modulated by temperature, increasing the utility of this conjugate as a Snail inhibitor in model organisms. We exploit this feature to further delineate the requirements for Snail function during neural crest development, showing that in addition to the roles that Snail factors play in neural crest precursor formation and neural crest EMT/migration, inhibition of Snail function after the onset of neural crest migration leads to a loss of neural crest derived melanocytes. Co(III)-Ebox-mediated inhibition therefore provides a powerful tool for analysing the function of these core EMT factors with unparalleled temporal resolution. Moreover, the potency of Co(III)-Ebox as a Snail inhibitor in breast cancer cells suggests its potential as a therapeutic inhibitor of tumor progression and metastasis.

Specific inhibition of the transcription factor Ci by a cobalt(III) Schiff base-DNA conjugate.

We describe the use of Co(III) Schiff base-DNA conjugates, a versatile class of research tools that target C2H2 transcription factors, to inhibit the Hedgehog (Hh) pathway. In developing mammalian embryos, Hh signaling is critical for the formation and development of many tissues and organs. Inappropriate activation of the Hedgehog (Hh) pathway has been implicated in a variety of cancers including medulloblastomas and basal cell carcinomas. It is well-known that Hh regulates the activity of the Gli family of C2H2 zinc finger transcription factors in mammals. In Drosophila the function of the Gli proteins is performed by a single transcription factor with an identical DNA binding consensus sequence, Cubitus Interruptus (Ci). We have demonstrated previously that conjugation of a specific 17 base-pair oligonucleotide to a Co(III) Schiff base complex results in a targeted inhibitor of the Snail family C2H2 zinc finger transcription factors. Modification of the oligonucleotide sequence in the Co(III) Schiff base-DNA conjugate to that of Ci's consensus sequence (Co(III)-Ci) generates an equally selective inhibitor of Ci. Co(III)-Ci irreversibly binds the Ci zinc finger domain and prevents it from binding DNA in vitro. In a Ci responsive tissue culture reporter gene assay, Co(III)-Ci reduces the transcriptional activity of Ci in a concentration dependent manner. In addition, injection of wild-type Drosophila embryos with Co(III)-Ci phenocopies a Ci loss of function phenotype, demonstrating effectiveness in vivo. This study provides evidence that Co(III) Schiff base-DNA conjugates are a versatile class of specific and potent tools for studying zinc finger domain proteins and have potential applications as customizable anticancer therapeutics.

Synthesis, Characterization, and in vitro Testing of a Bacteria-Targeted MR Contrast Agent.

A bacteria-targeted MR contrast agent, Zn-1, consisting of two Zn-dipicolylamine (Zn-dpa) groups conjugated to a GdIII chelate has been synthesized and characterized. In vitro studies with S. aureus and E. coli show that Zn-1 exhibits a significant improvement in bacteria labeling efficiency vs. control. Studies with a structural analogue, Zn-2, indicate that removal of one Zn-dpa moiety dramatically reduces the agent's affinity for bacteria. The ability of Zn-1 to significantly reduce the T1 of labeled vs. unlabeled bacteria, resulting in enhanced MR image contrast, demonstrates its potential for visualizing bacterial infections in vivo.

Chiral porphyrazine near-IR optical imaging agent exhibiting preferential tumor accumulation.

A chiral porphyrazine (pz), H(2)[pz(trans-A(2)B(2))] (247), has been prepared that exhibits preferential in vivo accumulation in the cells of tumors. Pz 247 exhibits near-infrared (NIR) emission with lambda > 700 nm in the required wavelength range for maximum tissue penetration. When MDA-MB-231 breast tumor cells are treated with 247, the agent shows strong intracellular fluorescence with an emission maximum, 704 nm, which indicates that it localizes within a hydrophobic microenvironment. Pz 247 is shown to associate with the lipophilic core of LDL and undergo cellular entry primarily through receptor-mediated endocytosis accumulating in lysosomes. Preliminary in vivo studies show that 247 exhibits preferential accumulation and retention in the cells of MDA-MB-231 tumors subcutaneously implanted in mice, thereby enabling NIR optical imaging with excellent contrast between tumor and surrounding tissue. The intensity of fluorescence from 247 within the tumor increases over time up to 48 h after injection presumably due to the sequestration of circulating 247/LDL complex by the tumor tissue. As the need for cholesterol, and thus LDL, is elevated in highly proliferative tumor cells over nontumorigenic cells, 247 has potential application for all such tumors.