Association of the Telomerase Reverse Transcriptase rs10069690 Polymorphism with the Risk, Age at Onset and Prognosis of Triple Negative Breast Cancer.

Telomerase reverse transcriptase (TERT) plays a key role in the maintenance of telomere DNA length. The rs10069690 single nucleotide variant, located in intron 4 of TERT, was found to be associated with telomere length and the risk of estrogen receptor-negative but not-positive breast cancer. This study aimed at analysis of the association of rs10069690 genotype and TERT expression with the risk, age at onset, prognosis, and clinically and molecularly relevant subtypes of breast cancer. Accordingly, rs10069690 was genotyped in a hospital-based case-control study of 403 female breast cancer patients and 246 female controls of a Central European (Austrian) study population, and the mRNA levels of TERT were quantified in 106 primary breast tumors using qRT-PCR. We found that in triple-negative breast cancer patients, the minor rs10069690 TT genotype tended to be associated with an increased breast cancer risk (OR, 1.87; 95% CI, 0.75-4.71; p = 0.155) and was significantly associated with 11.7 years younger age at breast cancer onset (p = 0.0002), whereas the CC genotype was associated with a poor brain metastasis-free survival (p = 0.009). Overall, our data show that the rs10069690 CC genotype and a high TERT expression tended to be associated with each other and with a poor prognosis. Our findings indicate a key role of rs10069690 in triple-negative breast cancer.

Tidal Volume-Dependent Activation of the Renin-Angiotensin System in Experimental Ventilator-Induced Lung Injury.

OBJECTIVES: Ventilator-induced lung injury (VILI) is a major contributor to morbidity and mortality in critically ill patients. Mechanical damage to the lungs is potentially aggravated by the activation of the renin-angiotensin system (RAS). This article describes RAS activation profiles in VILI and discusses the effects of angiotensin (Ang) 1-7 supplementation or angiotensin-converting enzyme (ACE) inhibition with captopril as protective strategies. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: C57BL/6 mice. INTERVENTIONS: Anesthetized mice ( n = 12-18 per group) were mechanically ventilated with low tidal volume (LV T , 6 mL/kg), high tidal volume (HV T , 15 mL/kg), or very high tidal volume (VHV T , 30 mL/kg) for 4 hours, or killed after 3 minutes (sham). Additional VHV T groups received infusions of 60 µg/kg/hr Ang 1-7 or a single dose of 100 mg/kg captopril. MEASUREMENTS AND MAIN RESULTS: VILI was characterized by increased bronchoalveolar lavage fluid levels of interleukin (IL)-6, keratinocyte-derived cytokine, and macrophage inflammatory protein-2 (MIP2). The Ang metabolites in plasma measured with liquid chromatography tandem mass spectrometry showed a strong activation of the classical (Ang I, Ang II) and alternative RAS (Ang 1-7, Ang 1-5), with highest concentrations found in the HV T group. Although the lung-tissue ACE messenger RNA expression was unchanged, its protein expression showed a dose-dependent increase under mechanical ventilation. The ACE2 messenger RNA expression decreased in all ventilated groups, whereas ACE2 protein levels remained unchanged. Both captopril and Ang 1-7 led to markedly increased Ang 1-7 plasma levels, decreased Ang II levels, and ACE activity (Ang II/Ang I ratio), and effectively prevented VILI. CONCLUSIONS: VILI is accompanied by a strong activation of the RAS. Based on circulating Ang metabolite levels and tissue expression of RAS enzymes, classical ACE-dependent and alternative RAS cascades were activated in the HV T group, whereas classical RAS activation prevailed with VHV T ventilation. Ang 1-7 or captopril protected from VILI primarily by modifying the systemic RAS profile.

Sympathetic nerve innervation and metabolism in ischemic myocardium in response to remote ischemic perconditioning.

Sympathetic nerve denervation after myocardial infarction (MI) predicts risk of sudden cardiac death. Therefore, therapeutic approaches limit infarct size, improving adverse remodeling and restores sympathetic innervation have a great clinical potential. Remote ischemic perconditioning (RIPerc) could markedly attenuate MI-reperfusion (MIR) injury. In this study, we aimed to assess its effects on cardiac sympathetic innervation and metabolism. Transient myocardial ischemia is induced by ligature of the left anterior descending coronary artery (LAD) in male Sprague-Dawley rats, and in vivo cardiac 2-[18F]FDG and [11C]mHED PET scans were performed at 14-15 days after ischemia. RIPerc was induced by three cycles of 5-min-long unilateral hind limb ischemia and intermittent 5 min of reperfusion during LAD occlusion period. The PET quantitative parameters were quantified in parametric polar maps. This standardized format facilitates the regional radioactive quantification in deficit regions to remote areas. The ex vivo radionuclide distribution was additionally identified using autoradiography. Myocardial neuron density (tyrosine hydroxylase positive staining) and chondroitin sulfate proteoglycans (CSPG, inhibiting neuron regeneration) expression were assessed by immunohistochemistry. There was no significant difference in the mean hypometabolism 2-[18F]FDG uptake ratio (44.6 ± 4.8% vs. 45.4 ± 4.4%) between MIR rats and MIR + RIPerc rats (P > 0.05). However, the mean [11C]mHED nervous activity of denervated myocardium was significantly elevated in MIR + RIPerc rats compared to the MIR rats (35.9 ± 7.1% vs. 28.9 ± 2.3%, P < 0.05), coupled with reduced denervated myocardium area (19.5 ± 5.3% vs. 27.8 ± 6.6%, P < 0.05), which were associated with preserved left-ventricular systolic function, a less reduction in neuron density, and a significant reduction in CSPG and CD68 expression in the myocardium. RIPerc presented a positive effect on cardiac sympathetic-nerve innervation following ischemia, but showed no significant effect on myocardial metabolism.

Alterations in ACE and ACE2 Activities and Cardiomyocyte Signaling Underlie Improved Myocardial Function in a Rat Model of Repeated Remote Ischemic Conditioning.

Post-ischemic left ventricular (LV) remodeling and its hypothetical prevention by repeated remote ischemic conditioning (rRIC) in male Sprague-Dawley rats were studied. Myocardial infarction (MI) was evoked by permanent ligation of the left anterior descending coronary artery (LAD), and myocardial characteristics were tested in the infarcted anterior and non-infarcted inferior LV regions four and/or six weeks later. rRIC was induced by three cycles of five-minute-long unilateral hind limb ischemia and five minutes of reperfusion on a daily basis for a period of two weeks starting four weeks after LAD occlusion. Sham operated animals served as controls. Echocardiographic examinations and invasive hemodynamic measurements revealed distinct changes in LV systolic function between four and six weeks after MI induction in the absence of rRIC (i.e., LV ejection fraction (LVEF) decreased from 52.8 ± 2.1% to 50 ± 1.6%, mean ± SEM, p < 0.05) and in the presence of rRIC (i.e., LVEF increased from 48.2 ± 4.8% to 55.2 ± 4.1%, p < 0.05). Angiotensin-converting enzyme (ACE) activity was about five times higher in the anterior LV wall at six weeks than that in sham animals. Angiotensin-converting enzyme 2 (ACE2) activity roughly doubled in post-ischemic LVs. These increases in ACE and ACE2 activities were effectively mitigated by rRIC. Ca2+-sensitivities of force production (pCa50) of LV permeabilized cardiomyocytes were increased at six weeks after MI induction together with hypophosphorylation of 1) cardiac troponin I (cTnI) in both LV regions, and 2) cardiac myosin-binding protein C (cMyBP-C) in the anterior wall. rRIC normalized pCa50, cTnI and cMyBP-C phosphorylations. Taken together, post-ischemic LV remodeling involves region-specific alterations in ACE and ACE2 activities together with changes in cardiomyocyte myofilament protein phosphorylation and function. rRIC has the potential to prevent these alterations and to improve LV performance following MI.

Tenascin C promotes valvular remodeling in two large animal models of ischemic mitral regurgitation.

Ischemic mitral regurgitation (MR) is a frequent complication of myocardial infarction (MI) characterized by adverse remodeling both at the myocardial and valvular levels. Persistent activation of valvular endothelial cells leads to leaflet fibrosis through endothelial-to-mesenchymal transition (EMT). Tenascin C (TNC), an extracellular matrix glycoprotein involved in cardiovascular remodeling and fibrosis, was also identified in inducing epithelial-to-mesenchymal transition. In this study, we hypothesized that TNC also plays a role in the valvular remodeling observed in ischemic MR by contributing to valvular excess EMT. Moderate ischemic MR was induced by creating a posterior papillary muscle infarct (7 pigs and 7 sheep). Additional animals (7 pigs and 4 sheep) served as controls. Pigs and sheep were sacrificed after 6 weeks and 6 months, respectively. TNC expression was upregulated in the pig and sheep experiments at 6 weeks and 6 months, respectively, and correlated well with leaflet thickness (R = 0.68; p < 0.001 at 6 weeks, R = 0.84; p < 0.001 at 6 months). To confirm the translational potential of our findings, we obtained mitral valves from patients with ischemic cardiomyopathy presenting MR (n = 5). Indeed, TNC was also expressed in the mitral leaflets of these. Furthermore, TNC induced EMT in isolated porcine mitral valve endothelial cells (MVEC). Interestingly, Toll-like receptor 4 (TLR4) inhibition prevented TNC-mediated EMT in MVEC. We identified here for the first time a new contributor to valvular remodeling in ischemic MR, namely TNC, which induced EMT through TLR4. Our findings might set the path for novel therapeutic targets for preventing or limiting ischemic MR.

PO2 oscillations induce lung injury and inflammation.

BACKGROUND: Mechanical ventilation can lead to ventilator-induced lung injury (VILI). In addition to the well-known mechanical forces of volutrauma, barotrauma, and atelectrauma, non-mechanical mechanisms have recently been discussed as contributing to the pathogenesis of VILI. One such mechanism is oscillations in partial pressure of oxygen (PO2) which originate in lung tissue in the presence of within-breath recruitment and derecruitment of alveoli. The purpose of this study was to investigate this mechanism's possible independent effects on lung tissue and inflammation in a porcine model. METHODS: To separately study the impact of PO2 oscillations on the lungs, an in vivo model was set up that allowed for generating mixed-venous PO2 oscillations by the use of veno-venous extracorporeal membrane oxygenation (vvECMO) in a state of minimal mechanical stress. While applying the identical minimal-invasive ventilator settings, 16 healthy female piglets (weight 50 ± 4 kg) were either exposed for 6 h to a constant mixed-venous hemoglobin saturation (SmvO2) of 65% (which equals a PmvO2 of 41 Torr) (control group), or an oscillating SmvO2 (intervention group) of 40-90% (which equals PmvO2 oscillations of 30-68 Torr)-while systemic normoxia in both groups was maintained. The primary endpoint of histologic lung damage was assessed by ex vivo histologic lung injury scoring (LIS), the secondary endpoint of pulmonary inflammation by qRT-PCR of lung tissue. Cytokine concentration of plasma was carried out by ELISA. A bioinformatic microarray analysis of lung samples was performed to generate hypotheses about underlying pathomechanisms. RESULTS: The LIS showed significantly more severe damage of lung tissue after exposure to PO2 oscillations compared to controls (0.53 [0.51; 0.58] vs. 0.27 [0.23; 0.28]; P = 0.0025). Likewise, a higher expression of TNF-α (P = 0.0127), IL-1ß (P = 0.0013), IL-6 (P = 0.0007), and iNOS (P = 0.0013) in lung tissue was determined after exposure to PO2 oscillations. Cytokines in plasma showed a similar trend between the groups, however, without significant differences. Results of the microarray analysis suggest that inflammatory (IL-6) and oxidative stress (NO/ROS) signaling pathways are involved in the pathology linked to PO2 oscillations. CONCLUSIONS: Artificial mixed-venous PO2 oscillations induced lung damage and pulmonary inflammation in healthy animals during lung protective ventilation. These findings suggest that PO2 oscillations represent an independent mechanism of VILI.

PlGF and VEGF-A Regulate Growth of High-Risk MYCN-Single Copy Neuroblastoma Xenografts via Different Mechanisms.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and is a rapidly growing, highly-vascularized cancer. NBs frequently express angiogenic factors and high tumor angiogenesis has been associated with poor outcomes. Placental growth factor (PlGF) is an angiogenic protein belonging to the vascular endothelial growth factor (VEGF) family and is up-regulated mainly in pathologic conditions. Recently, PlGF was identified as a member of a gene expression signature characterizing highly malignant NB stem cells drawing attention as a potential therapeutic target in NB. In the present study, we sought to investigate the expression of PlGF in NB patients and the effect of PlGF inhibition on high-risk MYCN-non-amplified SK-N-AS NB xenografts. Human SK-N-AS cells, which are poorly differentiated and express PlGF and VEGF-A, were implanted subcutaneously in athymic nude mice. Treatment was done by intratumoral injection of replication-incompetent adenoviruses (Ad) expressing PlGF- or VEGF-specific short hairpin (sh)RNA, or soluble (s)VEGF receptor 2 (VEGFR2). The effect on tumor growth and angiogenesis was analyzed. High PlGF expression levels were observed in human advanced-stage NBs. Down-regulating PlGF significantly reduced NB growth in established NB xenografts by reducing cancer cell proliferation but did not suppress angiogenesis. In contrast, blocking VEGF by administration of Ad(sh)VEGF and Ad(s)VEGFR2 reduced tumor growth associated with decreased tumor vasculature. These findings suggest that PlGF and VEGF-A modulate MYCN-non-amplified NB tumors by different mechanisms and support a role for PlGF in NB biology.

Association of the rs1346044 Polymorphism of the Werner Syndrome Gene RECQL2 with Increased Risk and Premature Onset of Breast Cancer.

Like other RECQ helicases, WRN/RECQL2 plays a crucial role in DNA replication and the maintenance of genome stability. Inactivating mutations in RECQL2 lead to Werner syndrome, a rare autosomal disease associated with premature aging and an increased susceptibility to multiple cancer types. We analyzed the association of two coding single-nucleotide polymorphisms in WRN, Cys1367Arg (rs1346044), and Arg834Cys (rs3087425), with the risk, age at onset, and clinical subclasses of breast cancer in a hospital-based case-control study of an Austrian population of 272 breast cancer patients and 254 controls. Here we report that the rare homozygous CC genotype of rs1346044 was associated with an approximately two-fold elevated breast cancer risk. Moreover, patients with the CC genotype exhibited a significantly increased risk of developing breast cancer under the age of 55 in both recessive and log-additive genetic models. CC patients developed breast cancer at a mean age of 55.2 ± 13.3 years and TT patients at 60.2 ± 14.7 years. Consistently, the risk of breast cancer was increased in pre-menopausal patients in the recessive model. These findings suggest that the CC genotype of WRN rs1346044 may contribute to an increased risk and a premature onset of breast cancer.

Analysis of the rs10046 polymorphism of aromatase (CYP19) in premenopausal onset of human breast cancer.

The CYP19 gene encodes aromatase, an enzyme catalyzing the conversion of androgens to estrogens. Studies analyzing associations between single nucleotide polymorphisms in CYP19 and breast cancer risk have shown inconsistent results. The rs10046 polymorphism is located in the 3' untranslated region of the CYP19 gene, but the influence of this polymorphism on breast cancer risk is unclear. In this study, we investigated the impact of rs10046 SNP on breast cancer risk, age at onset and association with clinical characteristics in an Austrian population of 274 breast cancer patients and 253 controls. The results show that a significantly increased fraction of patients with the TT genotype of rs10046 develop breast cancer under the age of 50 (41.8% of TT patients, compared to 26.6% of C carriers; p = 0.018, Chi-square test). No rs10046 genotypes were significantly associated with increased breast cancer risk or patient characteristics other than age at onset. These results suggest that the rs10046 polymorphism in the CYP19 gene may have an effect on breast cancer susceptibility at an age under 50 in the investigated population.

Egr-1 upregulates Siva-1 expression and induces cardiac fibroblast apoptosis.

The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.

Targeting Cdc42 with the small molecule drug AZA197 suppresses primary colon cancer growth and prolongs survival in a preclinical mouse xenograft model by downregulation of PAK1 activity.

BACKGROUND: Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential. METHODS: We report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using a xenograft mouse model of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197. RESULTS: In SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo. CONCLUSIONS: These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.

Inhibition of stromal PlGF suppresses the growth of prostate cancer xenografts.

The growth and vascularization of prostate cancer is dependent on interactions between cancer cells and supporting stromal cells. The primary stromal cell type found in prostate tumors is the carcinoma-associated fibroblast, which produces placental growth factor (PlGF). PlGF is a member of the vascular endothelial growth factor (VEGF) family of angiogenic molecules and PlGF mRNA levels increase after androgen deprivation therapy in prostate cancer. In this study, we show that PlGF has a direct dose-dependent proliferative effect on human PC-3 prostate cancer cells in vitro and fibroblast-derived PlGF increases PC-3 proliferation in co-culture. In xenograft tumor models, intratumoral administration of murine PlGF siRNA reduced stromal-derived PlGF expression, reduced tumor burden and decreased the number of Ki-67 positive proliferating cells associated with reduced vascular density. These data show that targeting stromal PlGF expression may represent a therapeutic target for the treatment of prostate cancer.

Combined angiotensin-converting enzyme and aminopeptidase inhibition for treatment of experimental ventilator-induced lung injury in mice.

Introduction: Ventilator-induced lung injury (VILI) may aggravate critical illness. Although angiotensin-converting enzyme (ACE) inhibition has beneficial effects in ventilator-induced lung injury, its clinical application is impeded by concomitant hypotension. We hypothesized that the aminopeptidase inhibitor ALT-00 may oppose the hypotension induced by an angiotensin-converting enzyme inhibitor, and that this combination would activate the alternative renin-angiotensin system (RAS) axis to counteract ventilator-induced lung injury. Methods: In separate experiments, C57BL/6 mice were mechanically ventilated with low (LVT, 6 mL/kg) and high tidal volumes (HVT, 30 mL/kg) for 4 h or remained unventilated (sham). High tidal volume-ventilated mice were treated with lisinopril (0.15 µg/kg/min) ± ALT-00 at 2.7, 10 or 100 µg/kg/min. Blood pressure was recorded at baseline and after 4 h. Lung histology was evaluated for ventilator-induced lung injury and the angiotensin (Ang) metabolite profile in plasma (equilibrium levels of Ang I, Ang II, Ang III, Ang IV, Ang 1-7, and Ang 1-5) was measured with liquid chromatography tandem mass spectrometry at the end of the experiment. Angiotensin concentration-based markers for renin, angiotensin-converting enzyme and alternative renin-angiotensin system activities were calculated. Results: High tidal volume-ventilated mice treated with lisinopril showed a significant drop in the mean arterial pressure at 4 h compared to baseline, which was prevented by adding ALT-00 at 10 and 100 µg/kg/min. Ang I, Ang II and Ang 1-7 plasma equilibrium levels were elevated in the high tidal volumes group versus the sham group. Lisinopril reduced Ang II and slightly increased Ang I and Ang 1-7 levels versus the untreated high tidal volumes group. Adding ALT-00 at 10 and 100 µg/kg/min increased Ang I and Ang 1-7 levels versus the high tidal volume group, and partly prevented the downregulation of Ang II levels caused by lisinopril. The histological lung injury score was higher in the high tidal volume group versus the sham and low tidal volume groups, and was attenuated by lisinopril ± ALT-00 at all dose levels. Conclusion: Combined angiotensin-converting enzyme plus aminopeptidase inhibition prevented systemic hypotension and maintained the protective effect of lisinopril. In this study, a combination of lisinopril and ALT-00 at 10 µg/kg/min appeared to be the optimal approach, which may represent a promising strategy to counteract ventilator-induced lung injury that merits further exploration.

Neuregulin-1ß Improves Uremic Cardiomyopathy and Renal Dysfunction in Rats.

Chronic kidney disease is a global health problem affecting 10% to 12% of the population. Uremic cardiomyopathy is often characterized by left ventricular hypertrophy, fibrosis, and diastolic dysfunction. Dysregulation of neuregulin-1ß signaling in the heart is a known contributor to heart failure. The systemically administered recombinant human neuregulin-1ß for 10 days in our 5/6 nephrectomy-induced model of chronic kidney disease alleviated the progression of uremic cardiomyopathy and kidney dysfunction in type 4 cardiorenal syndrome. The currently presented positive preclinical data warrant clinical studies to confirm the beneficial effects of recombinant human neuregulin-1ß in patients with chronic kidney disease.

Cardiovascular phenotype of the Dmdmdx rat - a suitable animal model for Duchenne muscular dystrophy.

Besides skeletal muscle abnormalities, Duchenne muscular dystrophy (DMD) patients present with dilated cardiomyopathy development, which considerably contributes to morbidity and mortality. Because the mechanisms responsible for the cardiac complications in the context of DMD are largely unknown, evidence-based therapy approaches are still lacking. This has increased the need for basic research efforts into animal models for DMD. Here, we characterized in detail the cardiovascular abnormalities of Dmdmdx rats, with the aim of determining the suitability of this recently established dystrophin-deficient small animal as a model for DMD.Various methods were applied to compare cardiovascular properties between wild-type and Dmdmdx rats, and to characterize the Dmdmdx cardiomyopathy. These methods comprised echocardiography, invasive assessment of left ventricular hemodynamics, examination of adverse remodeling and endothelial cell inflammation, and evaluation of vascular function, employing wire myography. Finally, intracellular Ca2+ transient measurements, and recordings of currents through L-type Ca2+ channels were performed in isolated single ventricular cardiomyocytes. We found that, similar to respective observations in DMD patients, the hearts of Dmdmdx rats show significantly impaired cardiac function, fibrosis and inflammation, consistent with the development of a dilated cardiomyopathy. Moreover, in Dmdmdx rats, vascular endothelial function is impaired, which may relate to inflammation and oxidative stress, and Ca2+ handling in Dmdmdx cardiomyocytes is abnormal.These findings indicate that Dmdmdx rats represent a promising small-animal model to elucidate mechanisms of cardiomyopathy development in the dystrophic heart, and to test mechanism-based therapies aiming to combat cardiovascular complications in DMD.

Stromal cell-derived CSF-1 blockade prolongs xenograft survival of CSF-1-negative neuroblastoma.

The molecular mechanisms of tumor-host interactions that render neuroblastoma (NB) cells highly invasive are unclear. Cancer cells upregulate host stromal cell colony-stimulating factor-1 (CSF-1) production to recruit tumor-associated macrophages (TAMs) and accelerate tumor growth by affecting extracellular matrix remodeling and angiogenesis. By coculturing NB with stromal cells in vitro, we showed the importance of host CSF-1 expression for macrophage recruitment to NB cells. To examine this interaction in NB in vivo, mice bearing human CSF-1-expressing SK-N-AS and CSF-1-negative SK-N-DZ NB xenografts were treated with intratumoral injections of small interfering RNAs directed against mouse CSF-1. Significant suppression of both SK-N-AS and SK-N-DZ NB growth by these treatments was associated with decreased TAM infiltration, matrix metalloprotease (MMP)-12 levels and angiogenesis compared to controls, while expression of tissue inhibitors of MMPs increased following mouse CSF-1 blockade. Furthermore, Tie-2-positive and -negative TAMs recruited by host CSF-1 were identified in NB tumor tissue by confocal microscopy and flow cytometry. However, host-CSF-1 blockade prolonged survival only in CSF-1-negative SK-N-DZ NB. These studies demonstrated that increased CSF-1 production by host cells enhances TAM recruitment and NB growth and that the CSF-1 phenotype of NB tumor cells adversely affects survival.

Adenoviral-mediated endothelial precursor cell delivery of soluble CD115 suppresses human prostate cancer xenograft growth in mice.

Prostate cancer tumor growth and neovascularization is promoted by an interplay between migratory tumor stromal cells such as specialized tumor-associated macrophages (TAMs) and circulating endothelial precursor cells (CEPs). As vehicles for tumor therapy, human CEPs are relatively easy to isolate from peripheral blood, are able to proliferate long-term in vitro, are amenable to viral manipulation, and preferentially home to regions of ischemia found in growing tumors. We show here that human peripheral blood CEPs expanded ex vivo migrate to prostate cancer cells in vitro and efficiently home to human prostate tumor xenografts in vivo. Infection of precursors ex vivo with an adenovirus constructed to secrete a soluble form of the colony-stimulating factor-1 receptor CD115 that inhibits macrophage viability and migration in vitro significantly decreases the number of TAMs in xenografts (p < .05), reduces proliferation (p < .01) and vascular density (p < .03), and suppresses the growth of xenografts (p < .03). These data show for the first time that targeting stromal cell processes with cellular therapy has the potential to retard prostate tumor growth.

Cancer Immunotherapy: Targeting Tumor-Associated Macrophages by Gene Silencing.

Tumor-associated macrophages (TAMs) are representing a major leukocyte population in solid tumors. Macrophages are very heterogeneous and plastic cells and can acquire distinct functional phenotypes ranging from antitumorigenic to immunosuppressive tumor-promoting M2-like TAMs, depending on the local tissue microenvironment (TME). TAMs express cytokines, chemokines, growth factors, and extracellular matrix (ECM) modifying factors, and the cross talk with the TME regulates pathways involved in the recruitment, polarization, and metabolism of TAMs during tumor progression. Due to their crucial role in tumor growth and metastasis, selective targeting of TAM for the treatment of cancer with therapeutic agents that promote phagocytosis or suppress survival, proliferation, trafficking, or polarization of TAMs may prove to be beneficial in cancer therapy. In this chapter, we will discuss TAM biology and current strategies for the targeting of TAMs using small interfering RNA (siRNA)-based drugs. In the past few years, advances in the field of nanomedicine pave the way for the development of siRNA-based drugs as an additional class of personalized cancer immuno-nanomedicines. Fundamental challenges associated with this group of therapeutics include the development process, delivery system, and clinical translation for siRNA-based drugs.

Comment on：Kadomoto, S. et al. "Tumor-Associated Macrophages Induce Migration of Renal Cell Carcinoma Cells via Activation of the CCL20-CCR6 Axis" Cancers 2020, 12, 89.

Macrophages form a major component of the leukocyte infiltrate in solid tumors and it has become increasingly clear that tumor-associated macrophages (TAMs) have tumor-promoting effects within the stroma [1]. Renal cell carcinoma (RCC) solid tumors are comprised of a heterogeneous microenvironment of both malignant and normal stromal cells containing large numbers of macrophages [2].We read with interest the paper by Suguru Kadomoto et al. entitled "Tumor-associated macrophages induce migration of renal cell carcinoma cells via activation of the CCL20-CCR6 axis", published in Cancers [3], in which they report that the CCL20-CCR6 axis induces migration and epithelial-mesenchymal transition (EMT) of ACHN and Caki-1 RCC cells in co-cultures with THP-1/U937-derived tumor conditioned macrophages.[...].

Targeting stromal-cancer cell interactions with siRNAs.

Tumors are composed of both malignant and normal cells, including fibroblasts, endothelial cells, mesenchymal stem cells, and inflammatory immune cells such as macrophages. These various stromal components interact with cancer cells to promote growth and metastasis. For example, macrophages, attracted by colony-stimulating factor-1 (CSF-1) produced by tumor cells, in turn produce various growth factors such as vascular endothelial growth factor, which supports the growth of tumor cells and their interaction with blood vessels leading to enhanced tumor cell spreading. The activation of autocrine and paracrine oncogenic signaling pathways by stroma-derived growth factors and cytokines has been implicated in promoting tumor cell proliferation and metastasis. Furthermore, matrix metalloproteinases (MMPs) derived from both tumor cells and the stromal compartment are regarded as major players assisting tumor cells during metastasis. Collectively, these recent findings indicate that targeting tumor-stroma interactions is a promising strategy in the search for novel treatment modalities in human cancer. This chapter summarizes our current understanding of the tumor microenvironment and highlights some potential targets for therapeutic intervention with small interfering RNAs.