Integrin αvß3 Is a Master Regulator of Resistance to TKI-Induced Ferroptosis in HER2-Positive Breast Cancer.

Human epidermal growth factor receptor-2 (HER2)-targeting therapies provide clinical benefits for patients with HER2-positive breast cancer. However, the resistance to monotherapies invariably develops and leads to disease relapse and treatment failure. Previous studies have demonstrated a link between the potency of HER2-targeting tyrosine kinase inhibitors (TKIs) and their ability to induce an iron-dependent form of cell death called ferroptosis. The aim of this study was to understand the mechanisms of resistance to TKI-induced ferroptosis and identify novel approaches to overcome treatment resistance. We used mouse and human HER2-positive models of acquired TKI resistance to demonstrate an intimate link between the resistance to TKIs and to ferroptosis and present the first evidence that the cell adhesion receptor αvß3 integrin is a critical mediator of resistance to TKI-induced ferroptosis. Our findings indicate that αvß3 integrin-mediated resistance is associated with the re-wiring of the iron/antioxidant metabolism and persistent activation of AKT signalling. Moreover, using gene manipulation approaches and pharmacological inhibitors, we show that this "αvß3 integrin addiction" can be targeted to reverse TKI resistance. Collectively, these findings provide critical insights into new therapeutic strategies to improve the treatment of advanced HER2-positive breast cancer patients.

The FDA modernisation act 2.0: Bringing non-animal technologies to the regulatory table.

The FDA modernisation Act 2.0 marks a game-changing legislation enabling drug registration without the absolute requirement for the use of animals in safety toxicology assessment. We discuss landmark developments in the legislation under which the FDA operates and consider the implications of this most recent chapter in the evolution of the drug regulation pathway, focussing on new opportunities to embed microphysiological systems.

Bifunctional Aptamer-Doxorubicin Conjugate Crosses the Blood-Brain Barrier and Selectively Delivers Its Payload to EpCAM-Positive Tumor Cells.

The prognosis for breast cancer patients diagnosed with brain metastases is poor, with survival time measured merely in months. This can largely be attributed to the limited treatment options capable of reaching the tumor as a result of the highly restrictive blood-brain barrier (BBB). While methods of overcoming this barrier have been developed and employed with current treatment options, the majority are highly invasive and nonspecific, leading to severe neurotoxic side effects. A novel approach to address these issues is the development of therapeutics targeting receptor-mediated transport mechanisms on the BBB endothelial cell membranes. Using this approach, we intercalated doxorubicin (DOX) into a bifunctional aptamer targeting the transferrin receptor on the BBB and epithelial cell adhesion molecule (EpCAM) on metastatic cancer cells. The ability of the DOX-loaded aptamer to transcytose the BBB and selectively deliver the payload to EpCAM-positive tumors was evaluated in an in vitro model and confirmed for the first time in vivo using the MDA-MB-231 breast cancer metastasis model (MDA-MB-231Br). We show that colocalized aptamer and DOX are clearly detectable within the brain lesions 75 min postadministration. Collectively, results from this study demonstrate that through intercalation of a cytotoxic drug into the bifunctional aptamer, a therapeutic delivery vehicle can be developed for specific targeting of EpCAM-positive brain metastases.

Neoadjuvant neratinib promotes ferroptosis and inhibits brain metastasis in a novel syngeneic model of spontaneous HER2+ve breast cancer metastasis.

BACKGROUND: Human epidermal growth factor receptor-2 (HER2)-targeted therapies prolong survival in HER2-positive breast cancer patients. Benefit stems primarily from improved control of systemic disease, but up to 50% of patients progress to incurable brain metastases due to acquired resistance and/or limited permeability of inhibitors across the blood-brain barrier. Neratinib, a potent irreversible pan-tyrosine kinase inhibitor, prolongs disease-free survival in the extended adjuvant setting, and several trials evaluating its efficacy alone or combination with other inhibitors in early and advanced HER2-positive breast cancer patients are ongoing. However, its efficacy as a first-line therapy against HER2-positive breast cancer brain metastasis has not been fully explored, in part due to the lack of relevant pre-clinical models that faithfully recapitulate this disease. Here, we describe the development and characterisation of a novel syngeneic model of spontaneous HER2-positive breast cancer brain metastasis (TBCP-1) and its use to evaluate the efficacy and mechanism of action of neratinib. METHODS: TBCP-1 cells were derived from a spontaneous BALB/C mouse mammary tumour and characterised for hormone receptors and HER2 expression by flow cytometry, immunoblotting and immunohistochemistry. Neratinib was evaluated in vitro and in vivo in the metastatic and neoadjuvant setting. Its mechanism of action was examined by transcriptomic profiling, function inhibition assays and immunoblotting. RESULTS: TBCP-1 cells naturally express high levels of HER2 but lack expression of hormone receptors. TBCP-1 tumours maintain a HER2-positive phenotype in vivo and give rise to a high incidence of spontaneous and experimental metastases in the brain and other organs. Cell proliferation/viability in vitro is inhibited by neratinib and by other HER2 inhibitors, but not by anti-oestrogens, indicating phenotypic and functional similarities to human HER2-positive breast cancer. Mechanistically, neratinib promotes a non-apoptotic form of cell death termed ferroptosis. Importantly, metastasis assays demonstrate that neratinib potently inhibits tumour growth and metastasis, including to the brain, and prolongs survival, particularly when used as a neoadjuvant therapy. CONCLUSIONS: The TBCP-1 model recapitulates the spontaneous spread of HER2-positive breast cancer to the brain seen in patients and provides a unique tool to identify novel therapeutics and biomarkers. Neratinib-induced ferroptosis provides new opportunities for therapeutic intervention. Further evaluation of neratinib neoadjuvant therapy is warranted.

Identification of brain metastasis genes and therapeutic evaluation of histone deacetylase inhibitors in a clinically relevant model of breast cancer brain metastasis.

Breast cancer brain metastases remain largely incurable. Although several mouse models have been developed to investigate the genes and mechanisms regulating breast cancer brain metastasis, these models often lack clinical relevance since they require the use of immunocompromised mice and/or are poorly metastatic to brain from the mammary gland. We describe the development and characterisation of an aggressive brain metastatic variant of the 4T1 syngeneic model (4T1Br4) that spontaneously metastasises to multiple organs, but is selectively more metastatic to the brain from the mammary gland than parental 4T1 tumours. As seen by immunohistochemistry, 4T1Br4 tumours and brain metastases display a triple-negative phenotype, consistent with the high propensity of this breast cancer subtype to spread to brain. In vitro assays indicate that 4T1Br4 cells have an enhanced ability to adhere to or migrate across a brain-derived endothelial monolayer and greater invasive response to brain-derived soluble factors compared to 4T1 cells. These properties are likely to contribute to the brain selectivity of 4T1Br4 tumours. Expression profiling and gene set enrichment analyses demonstrate the clinical relevance of the 4T1Br4 model at the transcriptomic level. Pathway analyses implicate tumour-intrinsic immune regulation and vascular interactions in successful brain colonisation, revealing potential therapeutic targets. Evaluation of two histone deacetylase inhibitors, SB939 and 1179.4b, shows partial efficacy against 4T1Br4 metastasis to brain and other sites in vivo, and potent radio-sensitising properties in vitro The 4T1Br4 model provides a clinically relevant tool for mechanistic studies and to evaluate novel therapies against brain metastasis.This article has an associated First Person interview with Soo-Hyun Kim, joint first author of the paper.

Copper accumulation in senescent cells: Interplay between copper transporters and impaired autophagy.

Cellular senescence is characterized by irreversible growth arrest incurred through either replicative exhaustion or by pro-oncogenic cellular stressors (radioactivity, oxidative stress, oncogenic activation). The enrichment of senescent cells in tissues with age has been associated with tissue dyshomeostasis and age-related pathologies including cancers, neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, etc.) and metabolic disorders (e.g. diabetes). We identified copper accumulation as being a universal feature of senescent cells [mouse embryonic fibroblasts (MEF), human prostate epithelial cells and human diploid fibroblasts] in vitro. Elevated copper in senescent MEFs was accompanied by elevated levels of high-affinity copper uptake protein 1 (Ctr1), diminished levels of copper-transporting ATPase 1 (Atp7a) (copper export) and enhanced antioxidant defence reflected by elevated levels of glutathione (GSH), superoxide dismutase 1 (SOD1) and glutaredoxin 1 (Grx1). The levels of intracellular copper were further increased in senescent MEFs cultured in copper supplemented medium and in senescent Mottled Brindled (Mobr) MEFs lacking functional Atp7a. Finally, we demonstrated that the restoration/preservation of autophagic-lysosomal degradation in senescent MEFs following rapamycin treatment correlated with attenuation of copper accumulation in these cells despite a further decrease in Atp7a levels. This study for the first time establishes a link between Atp7a and the autophagic-lysosomal pathway, and a requirement for both to effect efficient copper export. Such a connection between cellular autophagy and copper homeostasis is significant, as both have emerged as important facets of age-associated degenerative disease.

Copper Complexes in Cancer Therapy.

Copper homeostasis is tightly regulated in both prokaryotic and eukaryotic cells to ensure sufficient amounts for cuproprotein biosynthesis, while limiting oxidative stress production and toxicity. Over the last century, copper complexes have been developed as antimicrobials and for treating diseases involving copper dyshomeostasis (e.g., Wilson's disease). There now exists a repertoire of copper complexes that can regulate bodily copper through a myriad of mechanisms. Furthermore, many copper complexes are now being appraised for a variety of therapeutic indications (e.g., Alzheimer's disease and amyotrophic lateral sclerosis) that require a range of copper-related pharmacological affects. Cancer therapy is also drawing considerable attention since copper has been recognized as a limiting factor for multiple aspects of cancer progression including growth, angiogenesis, and metastasis. Consequently, 'old copper complexes' (e.g., tetrathiomolybdate and clioquinol) have been repurposed for cancer therapy and have demonstrated anticancer activity in vitro and in preclinical models. Likewise, new tailor-made copper complexes have been designed based on structural and biological features ideal for their anticancer activity. Human clinical trials continue to evaluate the therapeutic efficacy of copper complexes as anticancer agents and considerable progress has been made in understanding their pharmacological requirements. In this chapter, we present a historical perspective on the main copper complexes that are currently being repurposed for cancer therapy and detail several of the more recently developed compounds that have emerged as promising anticancer agents. We further provide an overview of the known mechanisms of action, including molecular targets and we discuss associated clinical trials.

Iron accumulation in senescent cells is coupled with impaired ferritinophagy and inhibition of ferroptosis.

Cellular senescence is characterised by the irreversible arrest of proliferation, a pro-inflammatory secretory phenotype and evasion of programmed cell death mechanisms. We report that senescence alters cellular iron acquisition and storage and also impedes iron-mediated cell death pathways. Senescent cells, regardless of stimuli (irradiation, replicative or oncogenic), accumulate vast amounts of intracellular iron (up to 30-fold) with concomitant changes in the levels of iron homeostasis proteins. For instance, ferritin (iron storage) levels provided a robust biomarker of cellular senescence, for associated iron accumulation and for resistance to iron-induced toxicity. Cellular senescence preceded iron accumulation and was not perturbed by sustained iron chelation (deferiprone). Iron accumulation in senescent cells was driven by impaired ferritinophagy, a lysosomal process that promotes ferritin degradation and ferroptosis. Lysosomal dysfunction in senescent cells was confirmed through several markers, including the build-up of microtubule-associated protein light chain 3 (LC3-II) in autophagosomes. Impaired ferritin degradation explains the iron accumulation phenotype of senescent cells, whereby iron is effectively trapped in ferritin creating a perceived cellular deficiency. Accordingly, senescent cells were highly resistant to ferroptosis. Promoting ferritin degradation by using the autophagy activator rapamycin averted the iron accumulation phenotype of senescent cells, preventing the increase of TfR1, ferritin and intracellular iron, but failed to re-sensitize these cells to ferroptosis. Finally, the enrichment of senescent cells in mouse ageing hepatic tissue was found to accompany iron accumulation, an elevation in ferritin and mirrored our observations using cultured senescent cells.

[10]-gingerol induces apoptosis and inhibits metastatic dissemination of triple negative breast cancer in vivo.

There is increasing interest in the use of non-toxic natural products for the treatment of various pathologies, including cancer. In particular, biologically active constituents of the ginger oleoresin (Zingiber officinale Roscoe) have been shown to mediate anti-tumour activity and to contribute to the anti-inflammatory, antioxidant, antimicrobial, and antiemetic properties of ginger. Here we report on the inhibitory properties of [10]-gingerol against metastatic triple negative breast cancer (TNBC) in vitro and in vivo. We show that [10]-gingerol concentration-dependently induces apoptotic death in mouse and human TNBC cell lines in vitro. In addition, [10]-gingerol is well tolerated in vivo, induces a marked increase in caspase-3 activation and inhibits orthotopic tumour growth in a syngeneic mouse model of spontaneous breast cancer metastasis. Importantly, using both spontaneous and experimental metastasis assays, we show for the first time that [10]-gingerol significantly inhibits metastasis to multiple organs including lung, bone and brain. Remarkably, inhibition of brain metastasis was observed even when treatment was initiated after surgical removal of the primary tumour. Taken together, these results indicate that [10]-gingerol may be a safe and useful complementary therapy for the treatment of metastatic breast cancer and warrant further investigation of its efficacy, either alone or in combination with standard systemic therapies, in pre-clinical models of metastatic breast cancer and in patients.

Sulfonation of Tyrosine as a Method To Improve Biodistribution of Peptide-Based Radiotracers: Novel 18F-Labeled Cyclic RGD Analogues.

Control of the biodistribution of radiolabeled peptides has proven to be a major challenge in their application as imaging agents for positron emission tomography (PET). Modification of peptide hydrophilicity in order to increase renal clearance has been a common endeavor to improve overall biodistribution. Herein, we examine the effect of site-specific sulfonation of tyrosine moieties in cyclic(RGDyK) peptides as a means to enhance their hydrophilicity and improve their biodistribution. The novel sulfonated cyclic(RGDyK) peptides were conjugated directly to 4-nitrophenyl 2-[18F]fluoropropionate, and the biodistribution of the radiolabeled peptides was compared with that of their nonsulfonated, clinically relevant counterparts, [18F]GalactoRGD and [18F]FPPRGD2. Site-specific sulfonation of the tyrosine residues was shown to increase hydrophilicity and improve biodistribution of the RGD peptides, despite contributing just 79 Da toward the MW, compared with 189 Da for both the "Galacto" and mini-PEG moieties, suggesting this may be a broadly applicable approach to enhancing biodistribution of radiolabeled peptides.

Ceruloplasmin is regulated by copper and lactational hormones in PMC42-LA mammary epithelial cell culture models.

Ceruloplasmin (Cp) is a multicopper ferroxidase that is considered to be an important source of copper in milk for normal neonatal development. We investigated the expression, subcellular localization and secretion of Cp in PMC42-LA cell culture models representative of resting, lactating and suckled human mammary epithelia. Both secreted Cp (sCp) and plasma membrane associated glycosylphosphatidylinositol-linked Cp (GPI-Cp) were expressed in PMC42-LA cells. In all three epithelial models (resting, lactating and suckled), the expression and secretion of copper-bound, ferroxidase active, Cp (holo-Cp) was dependent on media copper concentration. In low copper (bathocuproinedisulphonic acid/d-penicillamine treated models) there was greater than a 2-fold decrease in holo-Cp expression and secretion, which was mirrored by a 2-fold increase in the expression and secretion of copper-free Cp protein (apo-Cp). Cell surface biotinylation studies revealed that the state of PMC42-LA cell differentiation (functionality), and the level of extracellular copper, had no significant effect on the level of plasma membrane bound GPI-Cp. Quantitative real time PCR analyses determined that there was no significant (P > 0.05) difference in Cp mRNA levels across all copper conditions investigated (0, 5, 50 µM). However, there was a significant (P < 0.05) increase (∼2-fold) in Cp mRNA in both the lactating and suckled models in comparison to the resting model. Furthermore, the Cp mRNA increase in response to PMC42-LA differentiation corresponded with more secreted Cp protein, both apo and holo forms, indicating a link between function and Cp requirement. Our results provide significant insight on the regulation of Cp expression and secretion in lactation and copper incorporation into milk.

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution.

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed.

Targeting copper in cancer therapy: 'Copper That Cancer'.

Copper is an essential micronutrient involved in fundamental life processes that are conserved throughout all forms of life. The ability of copper to catalyze oxidation-reduction (redox) reactions, which can inadvertently lead to the production of reactive oxygen species (ROS), necessitates the tight homeostatic regulation of copper within the body. Many cancer types exhibit increased intratumoral copper and/or altered systemic copper distribution. The realization that copper serves as a limiting factor for multiple aspects of tumor progression, including growth, angiogenesis and metastasis, has prompted the development of copper-specific chelators as therapies to inhibit these processes. Another therapeutic approach utilizes specific ionophores that deliver copper to cells to increase intracellular copper levels. The therapeutic window between normal and cancerous cells when intracellular copper is forcibly increased, is the premise for the development of copper-ionophores endowed with anticancer properties. Also under investigation is the use of copper to replace platinum in coordination complexes currently used as mainstream chemotherapies. In comparison to platinum-based drugs, these promising copper coordination complexes may be more potent anticancer agents, with reduced toxicity toward normal cells and they may potentially circumvent the chemoresistance associated with recurrent platinum treatment. In addition, cancerous cells can adapt their copper homeostatic mechanisms to acquire resistance to conventional platinum-based drugs and certain copper coordination complexes can re-sensitize cancer cells to these drugs. This review will outline the biological importance of copper and copper homeostasis in mammalian cells, followed by a discussion of our current understanding of copper dysregulation in cancer, and the recent therapeutic advances using copper coordination complexes as anticancer agents.

Synthesis and in Vivo Evaluation of [123I]Melanin-Targeted Agents.

This study reports the synthesis, [(123)I]radiolabeling, and biological profile of a new series of iodinated compounds for potential translation to the corresponding [(131)I]radiolabeled compounds for radionuclide therapy of melanoma. Radiolabeling was achieved via standard electrophilic iododestannylation in 60-90% radiochemical yield. Preliminary SPECT imaging demonstrated high and distinct tumor uptake of all compounds, as well as high tumor-to-background ratios compared to the literature compound [(123)I]4 (ICF01012). The most favorable compounds ([(123)I]20, [(123)I]23, [(123)I]41, and [(123)I]53) were selected for further biological investigation. Biodistribution studies indicated that all four compounds bound to melanin containing tissue with low in vivo deiodination; [(123)I]20 and [(123)I]53 in particular displayed high and prolonged tumor uptake (13% ID/g at 48 h). [(123)I]53 had the most favorable overall profile of the cumulative uptake over time of radiosensitive organs. Metabolite analysis of the four radiotracers found [(123)I]41 and [(123)I]53 to be the most favorable, displaying high and prolonged amounts of intact tracer in melanin containing tissues, suggesting melanin specific binding. Results herein suggest that compound [(123)I]53 displays favorable in vivo pharmacokinetics and stability and hence is an ideal candidate to proceed with further preclinical [(131)I] therapeutic evaluation.

Heterogeneous copper concentrations in cancerous human prostate tissues.

BACKGROUND: Therapeutics that target copper for the treatment of prostate cancer are being evaluated in human clinical trials. Elevated intracellular copper is considered to sensitize prostate cancer cells to certain copper-coordination compounds, especially those with ionophoric properties. While there is compelling in vitro evidence that prostate cancer cells accumulate intracellular copper, a corresponding status for copper in patient tissues has not been corroborated. We therefore established whether copper concentrations increase in cancerous prostate tissues, and in sera, in patients throughout disease progression. METHODS: Human prostate tissue samples were obtained from patient prostatectomies (n = 28), and together with patient-matched sera, were analyzed for copper content by inductively coupled plasma mass spectrometry. RESULTS: When grouped together, cancerous prostate tissues exhibiting moderate disease severity (Gleason Score 7) (n = 10) had 1.6-fold more copper than age-matched normal tissues (n = 10) (P < 0.05). Those with more aggressive disease (Gleason Score 9) (n = 8) had 1.8-fold more copper (P < 0.05). In both disease stages however, the copper concentrations between individual samples were rather variable (0.55-3.02 µg/g), with many clearly within the normal range (0.52-1.28 µg/g). Additionally, we found that there was no change in serum copper concentrations in patients with either moderate or aggressive prostate cancer (Gleason Score 7 or 9), compared with reference intervals and to age-matched controls. CONCLUSIONS: The heterogeneous nature of copper concentrations in cancerous prostate tissues, suggest that a small subset of patients may respond to treatments that target elevated intratumoral copper. Therefore, such approaches would likely require personalized treatment strategies.

Analysis of 177Lu-DOTA-octreotate therapy-induced DNA damage in peripheral blood lymphocytes of patients with neuroendocrine tumors.

UNLABELLED: Ionizing radiation-induced DNA double-strand breaks (DSBs) can lead to cell death, genome instability, and carcinogenesis. Immunofluorescence detection of phosphorylated histone variant H2AX (γ-H2AX) is a reliable and sensitive technique to monitor external-beam ionizing radiation-induced DSBs in peripheral blood lymphocytes (PBLs). Here, we investigated whether γ-H2AX could be used as an in vivo marker to assess normal-tissue toxicity after extended internal irradiation with (177)Lu-DOTA-octreotate (LuTate) peptide receptor radionuclide therapy (PRRT) of neuroendocrine tumors. METHODS: We analyzed the kinetics of γ-H2AX foci in PBLs of 11 patients undergoing PRRT. The number of γ-H2AX foci was determined before and up to 72 h after treatment. These values were compared with the estimated absorbed dose to blood, spleen, bone marrow, and tumor and with subsequent PBL reduction. RESULTS: The decrease in (177)Lu activity in blood with time followed a biexponential kinetic pattern, with approximately 90% of circulating activity in blood cleared within 2 h. Absorbed dose to blood, but not to spleen or bone marrow, correlated with the administered (177)Lu activity. PRRT increased γ-H2AX foci in lymphocytes in all patients, relative to pretherapy values. The response varied significantly between patients, but the average number of foci indicated a general trend toward an increase at 0.5-4 h with a subsequent decrease by 24-72 h after treatment. The peak number of foci correlated with the absorbed dose to tumor and bone marrow and the extent of PBL reduction. CONCLUSION: γ-H2AX can be exploited in the LuTate PRRT as a biomarker of PBL cytotoxicity. Long-term follow-up studies investigating whether elevated residual γ-H2AX values are associated with acute myelotoxicity and secondary blood malignancy may be worthwhile.

Tumour but not stromal expression of ß3 integrin is essential, and is required early, for spontaneous dissemination of bone-metastatic breast cancer.

Although many preclinical studies have implicated ß3 integrin receptors (αvß3 and αIIbß3) in cancer progression, ß3 inhibitors have shown only modest efficacy in patients with advanced solid tumours. The limited efficacy of ß3 inhibitors in patients could arise from our incomplete understanding of the precise function of ß3 integrin and, consequently, inappropriate clinical application. Data from animal studies are conflicting and indicate heterogeneity with respect to the relative contributions of ß3-expressing tumour and stromal cell populations in different cancers. Here we aimed to clarify the function and relative contributions to metastasis of tumour versus stromal ß3 integrin in clinically relevant models of spontaneous breast cancer metastasis, with particular emphasis on bone metastasis. We show that stable down-regulation of tumour ß3 integrin dramatically impairs spontaneous (but not experimental) metastasis to bone and lung without affecting primary tumour growth in the mammary gland. Unexpectedly, and in contrast to subcutaneous tumours, orthotopic tumour vascularity, growth and spontaneous metastasis were not altered in mice null for ß3 integrin. Tumour ß3 integrin promoted migration, protease expression and trans-endothelial migration in vitro and increased vascular dissemination in vivo, but was not necessary for bone colonization in experimental metastasis assays. We conclude that tumour, rather than stromal, ß3 expression is essential and is required early for efficient spontaneous breast cancer metastasis to bone and soft tissues. Accordingly, differential gene expression analysis in cohorts of breast cancer patients showed a strong association between high ß3 expression, early metastasis and shorter disease-free survival in patients with oestrogen receptor-negative tumours. We propose that ß3 inhibitors may be more efficacious if used in a neoadjuvant setting, rather than after metastases are established.

Foxp3 expression in macrophages associated with RENCA tumors in mice.

The transcription factor Foxp3 represents the most specific functional marker of CD4+ regulatory T cells (TRegs). However, previous reports have described Foxp3 expression in other cell types including some subsets of macrophages, although there are conflicting reports and Foxp3 expression in cells other than Treg is not well characterized. We performed detailed investigations into Foxp3 expression in macrophages in the normal tissue and tumor settings. We detected Foxp3 protein in macrophages infiltrating mouse renal cancer tumors injected subcutaneously or in the kidney. Expression was demonstrated using flow cytometry and Western blot with two individual monoclonal antibodies. Further analyses confirmed Foxp3 expression in macrophages by RT PCR, and studies using ribonucleic acid-sequencing (RNAseq) demonstrated a previously unknown Foxp3 messenger (m)RNA transcript in tumor-associated macrophages. In addition, depletion of Foxp3+ cells using diphtheria toxin in Foxp3DTR mice reduced the frequency of type-2 macrophages (M2) in kidney tumors. Collectively, these results indicate that tumor-associated macrophages could express Foxp3.

Postchemotherapy and tumor-selective targeting with the La-specific DAB4 monoclonal antibody relates to apoptotic cell clearance.

UNLABELLED: Early identification of tumor responses to treatment is crucial for devising more effective and safer cancer treatments. No widely applicable, noninvasive method currently exists for specifically detecting tumor cell death after cytotoxic treatment and thus for predicting treatment outcomes. METHODS: We have further characterized the targeting of the murine monoclonal antibody DAB4 specifically to dead tumor cells in vitro, in vivo, and in clinical samples. We found that sustained DAB4 binding to treated cells was closely associated with markers of intrinsic apoptosis and DNA double-strand break formation. In a competition binding assay, DAB4 bound EL4 murine thymic lymphoma cells in preference to the normal counterpart of murine thymocytes. Defective in vivo clearance of apoptotic cells augmented in vivo accumulation of DAB4 in tumors particularly after chemotherapy but was unchanged in normal tissues. Tumor targeting of DAB4 was selective for syngeneic murine tumors and for human tumor xenografts of prostate cancer (PC-3) and pancreatic cancer (Panc-1) before and more so after chemotherapy. Furthermore, DAB4 was shown to bind to dead primary acute lymphoblastic leukemic blasts cultured with cytotoxic drugs and dead epithelial cancer cells isolated from peripheral blood of small cell lung carcinoma patients given chemotherapy. CONCLUSION: Collectively, these results further demonstrate the selectivity of DAB4 for chemotherapy-induced dead tumor cells. This postchemotherapy selectivity is related to a relative increase in the availability of DAB4-binding targets in tumor tissue rather than in normal tissues. The in vitro findings were translated in vivo to human xenograft models and to ex vivo analyses of clinical samples, providing further evidence of the potential of DAB4 as a marker of tumor cell death after DNA-damaging cytotoxic treatment that could be harnessed as a predictive marker of treatment responses.

Bone-derived soluble factors and laminin-511 cooperate to promote migration, invasion and survival of bone-metastatic breast tumor cells.

Tumor intrinsic and extrinsic factors are thought to contribute to bone metastasis but little is known about how they cooperate to promote breast cancer spread to bone. We used the bone-metastatic 4T1BM2 mammary carcinoma model to investigate the cooperative interactions between tumor LM-511 and bone-derived soluble factors in vitro. We show that bone conditioned medium cooperates with LM-511 to enhance 4T1BM2 cell migration and invasion and is sufficient alone to promote survival in the absence of serum. These responses were associated with increased secretion of MMP-9 and activation of ERK and AKT signaling pathways and were partially blocked by pharmacological inhibitors of MMP-9, AKT-1/2 or MEK. Importantly, pre-treatment of 4T1BM2 cells with an AKT-1/2 inhibitor significantly reduced experimental metastasis to bone in vivo. Promotion of survival and invasive responses by bone-derived soluble factors and tumor-derived LM-511 are likely to contribute to the metastatic spread of breast tumors to bone.