Search

Adipocyte iron overload is a maladaptation associated with obesity and insulin resistance. The objective of the current study was to determine whether and how adipose tissue macrophages (ATMs) regulate adipocyte iron concentrations and whether this is impacted by obesity. Using bone marrow-derived macrophages (BMDMs) polarized to M0, M1, M2, or metabolically activated (MMe) phenotypes, we showed that MMe BMDMs and ATMs from obese mice have reduced expression of several iron-related proteins. Furthermore, the bioenergetic response to iron in obese ATMs was hampered. ATMs from iron-injected lean mice increased their glycolytic and respiratory capacities, thus maintaining metabolic flexibility, while ATMs from obese mice did not. Using an isotope-based system, we found that iron exchange between BMDMs and adipocytes was regulated by macrophage phenotype. At the end of the co-culture, MMe macrophages transferred and received more iron from adipocytes than M0, M1, and M2 macrophages. This culminated in a decrease in total iron in MMe macrophages and an increase in total iron in adipocytes compared with M2 macrophages. Taken together, in the MMe condition, the redistribution of iron is biased toward macrophage iron deficiency and simultaneous adipocyte iron overload. These data suggest that obesity changes the communication of iron between adipocytes and macrophages and that rectifying this iron communication channel may be a novel therapeutic target to alleviate insulin resistance.

INTRODUCTION: Cancer is a disease driven by variable genetic mutations. Overcoming this variability while sparing normal cells has stymied broad-acting therapy development. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity, raising the possibility that it can produce similar effects in cancer. Previous studies showed that neutrophil elastase (ELANE) - a neutrophil-derived serine protease - killed a wide range of cancer cells without harming non-cancer cells by cleaving CD95, the FAS receptor (Cui et al.,Cell, 2021). ELANE attenuated primary tumor growth and produced a CD8+T cell-mediated abscopal effect to attack metastases. Here we leveraged this ELANE-mediated pathway to produce an optimized N17350 biologic and tested its effects on tumor development, both as a monotherapy and in combination with checkpoint inhibitors. Our findings underscore the viability of N17350 as a new therapeutic modality leveraging innate immunity. METHODS: Anti-cancer effects of N17350 were examined in vitro and in vivo and compared with standard of care (SoC) agents. For in vitro studies, cells were treated with N17350 and viability was quantified by calcein-AM and immunogenic cell death (ICD) markers. Cancer/non-cancer cells included human/murine cell lines and primary cells isolated from healthy donors and ovarian cancer patients. We further tested the ability of cancer cells to develop resistance to N17350 and FAS-L. For in vivo studies, a single dose of N17350 was delivered intratumorally into CT26 (colon) and 4T1 (metastatic breast) tumors. Effects on primary and metastatic tumor growth, immunology, and survival were assessed in comparison to SoC agents (oxaliplatin, cyclophosphamide) or in combination with a checkpoint inhibitor (anti-CTLA4). RESULTS: N17350 killed and induced ICD markers in all cancer cell types tested without harming non-cancer cells, while SoC agents were similarly toxic to both cell types. Repeatedly killing E0771 cancer cells with N17350 did not produce resistance, which was observed with a FAS-L that targets CD95 via a distinct mechanism. A single intra-tumoral dose of N17350 produced durable effects in the 4T1 and CT26 models (CT26:75%-100% tumor-free). N17350 induced a robust innate/adaptive immune profile, abscopal effect to limit 4T1 lung metastasis, and synergized with anti-CTLA4 in cold (4T1) and hot (CT26) tumors. Finally, N17350 showed markedly improved efficacy over SoC agents in both models. CONCLUSIONS: Taken together, our data suggest that N17350 selectively kills cancer cells, produces complete responses in a subset of mice, induces favorable innate and adaptive immunology, and combines with checkpoint inhibitors in cold and hot tumors. Its ability to escape resistance, produce abscopal effects, and outperform SoC chemotherapies warrants further studies of this unique therapeutic modality in a clinical setting. Citation Format: Ravindra Gujar, Chang Cui, Lilibet Valdovinos, Christine Lee, Arezoo Arjmand, Nicole Grigaitis, Asna Khalid, Catherine A. Reardon, Kelly Q. Schoenfelt, Sonia Feau, Chris Twitty, Lev Becker. N17350 is an emerging therapeutic modality that selectively kills cancer cells and stimulates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6390.

Summary Monocytes and neutrophils are widely distributed throughout the body and play essential roles in health and disease. Here, we present a detailed protocol to isolate polymorphonuclear neutrophils and monocytes from a single sample of human peripheral blood. We have optimized several aspects of the procedure, including the density gradient, timing of each cell processing step, and the buffer/media conditions to preserve cell viability for subsequent functional assays. This protocol is reproducible and can be scaled as required for downstream applications. For complete details on the use and execution of this protocol, please refer to Cui et al. (2021)., Graphical abstract, Highlights • A protocol for isolating neutrophils and monocytes from a single human blood sample • Optimizes key processing steps to ensure high purity, yield, and viability of cells • Scalable for a wide range of downstream applications, Monocytes and neutrophils are widely distributed throughout the body and play essential roles in health and disease. Here, we present a detailed protocol to isolate polymorphonuclear neutrophils and monocytes from a single sample of human peripheral blood. We have optimized several aspects of the procedure, including the density gradient, timing of each cell processing step, and the buffer/media conditions to preserve cell viability for subsequent functional assays. This protocol is reproducible and can be scaled as required for downstream applications.

Tiwari et al. identify metabolically activated macrophages in obese mammary adipose tissue as an important source of IL-6, which fuels triple-negative breast cancer stemness and tumorigenesis through GP130 signaling. These mechanistic insights provide potential targets for treating obesity-associated triple-negative breast cancer., Obesity is associated with increased incidence and severity of triple-negative breast cancer (TNBC); however, mechanisms underlying this relationship are incompletely understood. Here, we show that obesity reprograms mammary adipose tissue macrophages to a pro-inflammatory metabolically activated phenotype (MMe) that alters the niche to support tumor formation. Unlike pro-inflammatory M1 macrophages that antagonize tumorigenesis, MMe macrophages are pro-tumorigenic and represent the dominant macrophage phenotype in mammary adipose tissue of obese humans and mice. MMe macrophages release IL-6 in an NADPH oxidase 2 (NOX2)–dependent manner, which signals through glycoprotein 130 (GP130) on TNBC cells to promote stem-like properties including tumor formation. Deleting Nox2 in myeloid cells or depleting GP130 in TNBC cells attenuates obesity-augmented TNBC stemness. Moreover, weight loss reverses the effects of obesity on MMe macrophage inflammation and TNBC tumor formation. Our studies implicate MMe macrophage accumulation in mammary adipose tissue as a mechanism for promoting TNBC stemness and tumorigenesis during obesity., Graphical Abstract

Activating CD8+ T cells by antigen cross-presentation is remarkably effective at eliminating tumours. Although this function is traditionally attributed to dendritic cells, tumour-associated macrophages (TAMs) can also cross-present antigens. TAMs are the most abundant tumour-infiltrating leukocyte. Yet, TAMs have not been leveraged to activate CD8+ T cells because mechanisms that modulate their ability to cross-present antigens are incompletely understood. Here we show that TAMs harbour hyperactive cysteine protease activity in their lysosomes, which impedes antigen cross-presentation, thereby preventing CD8+ T cell activation. We developed a DNA nanodevice (E64-DNA) that targets the lysosomes of TAMs in mice. E64-DNA inhibits the population of cysteine proteases that is present specifically inside the lysosomes of TAMs, improves their ability to cross-present antigens and attenuates tumour growth via CD8+ T cells. When combined with cyclophosphamide, E64-DNA showed sustained tumour regression in a triple-negative-breast-cancer model. Our studies demonstrate that DNA nanodevices can be targeted with organelle-level precision to reprogram macrophages and achieve immunomodulation in vivo.

Background CFTR modulators decrease some etiologies of CF airway inflammation; however, data indicate that non-resolving airway infection and inflammation persist in individuals with CF and chronic bacterial infections. Thus, identification of therapies that diminish airway inflammation without allowing unrestrained bacterial growth remains a critical research goal. Novel strategies for combatting deleterious airway inflammation in the CFTR modulator era require better understanding of cellular contributions to chronic CF airway disease, and how inflammatory cells change after initiation of CFTR modulator therapy. Peripheral blood monocytes, which traffic to the CF airway, can develop both pro-inflammatory and inflammation-resolving phenotypes, represent intriguing cellular targets for focused therapies. This therapeutic approach, however, requires a more detailed knowledge of CF monocyte cellular programming and phenotypes. Material and methods In order to characterize the inflammatory phenotype of CF monocytes, and how these cells change after initiation of CFTR modulator therapy, we studied adults (n=10) with CF, chronic airway infections, and the CFTR-R117H mutations before and 7 days after initiation of ivacaftor. Transcriptomes of freshly isolated blood monocytes were interrogated by RNA-sequencing (RNA-seq) followed by pathway-based analyses. Plasma concentrations of cytokines and chemokines were evaluated by multiplex ELISA. Results RNAseq identified approximately 50 monocyte genes for which basal expression was significantly changed in all 10 subjects after 7 days of ivacaftor. Of these, the majority were increased in expression post ivacaftor, including many genes traditionally associated with enhanced inflammation and immune responses. Pathway analyses confirmed that transcriptional programs were overwhelmingly up-regulated in monocytes after 7 days of ivacaftor, including biological modules associated with immunity, cell cycle, oxidative phosphorylation, and the unfolded protein response. Ivacaftor increased plasma concentrations of CXCL2, a neutrophil chemokine secreted by monocytes and macrophages, and CCL2, a monocyte chemokine. Conclusions Our results demonstrate that ivacaftor causes acute changes in blood monocyte transcriptional profiles and plasma chemokines, and suggest that increased monocyte inflammatory signals and changes in myeloid cell trafficking may contribute to changes in airway inflammation in people taking CFTR modulators. To our knowledge, this is the first report investigating the transcriptomic response of circulating blood monocytes in CF subjects treated with a CFTR modulator.

Management of cystic fibrosis has been revolutionised by the introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators. These compounds treat the underlying molecular basis of the disease by increasing activity of defective CFTR channels, which improves many clinical parameters and enhances patient quality of life [1]. Next-generation modulators, also known as triple combination therapy, promise to be highly efficacious in up to 90% of patients [2] and will likely dramatically change the landscape of cystic fibrosis disease. Studies examining individuals before and after initiation of CFTR modulators have revealed novel functions of CFTR and shown that CFTR modulators do not reverse all disease manifestations [3–5]. Thus, knowledge of the post-modulator cystic fibrosis disease state is crucial for understanding what continued therapies will be needed for people with cystic fibrosis and what new challenges may arise., This study demonstrates that initiation of the CFTR modulator ivacaftor in people with cystic fibrosis and susceptible CFTR mutations causes an acute reduction in blood monocyte sensitivity to the key proinflammatory cytokine IFN-γ http://bit.ly/2TeI6LG

SUMMARY Type 2 diabetes (T2D) is associated with increased risk for atherosclerosis; however, the mechanisms underlying this relationship are poorly understood. Macrophages, which are activated in T2D and causatively linked to atherogenesis, are an attractive mechanistic link. Here, we use proteomics to show that diet-induced obesity and insulin resistance (obesity/IR) modulate a pro-atherogenic “macrophage-sterol-responsive-network” (MSRN), which, in turn, predisposes macrophages to cholesterol accumulation. We identify IFNγ as the mediator of obesity/IR-induced MSRN dysregulation and increased macrophage cholesterol accumulation and show that obesity/IR primes T cells to increase IFNγ production. Accordingly, myeloid cell-specific deletion of the IFNγ receptor (Ifngr1−/−) restores MSRN proteins, attenuates macrophage cholesterol accumulation and atherogenesis, and uncouples the strong relationship between hyperinsulinemia and aortic root lesion size in hypercholesterolemic Ldlr−/− mice with obesity/IR, but does not affect these parameters in Ldlr−/− mice without obesity/IR. Collectively, our findings identify an IFNγ-macrophage pathway as a mechanistic link between obesity/IR and accelerated atherogenesis., Graphical Abstract In Brief: Obesity and insulin resistance are major risk factors for cardiovascular disease, but the underlying mechanisms are poorly understood. Reardon et al. show that obesity and insulin resistance induce an IFNγ-macrophage pathway that exacerbates foam cell formation and aortic lesion size in atherosclerotic mice.

The Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) is a nuclear corepressor, regulating the transcriptional activity of many transcription factors critical for metabolic processes. While the importance of the role of SMRT in the adipocyte has been well-established, our comprehensive understanding of its in vivo function in the context of homeostatic maintenance is limited due to contradictory phenotypes yielded by prior generalized knockout mouse models. Multiple such models agree that SMRT deficiency leads to increased adiposity, although the effects of SMRT loss on glucose tolerance and insulin sensitivity have been variable. We therefore generated an adipocyte-specific SMRT knockout (adSMRT-/-) mouse to more clearly define the metabolic contributions of SMRT. In doing so, we found that SMRT deletion in the adipocyte does not cause obesity—even when mice are challenged with a high-fat diet. This suggests that adiposity phenotypes of previously described models were due to effects of SMRT loss beyond the adipocyte. However, an adipocyte-specific SMRT deficiency still led to dramatic effects on systemic glucose tolerance and adipocyte insulin sensitivity, impairing both. This metabolically deleterious outcome was coupled with a surprising immune phenotype, wherein most genes differentially expressed in the adipose tissue of adSMRT-/- mice were upregulated in pro-inflammatory pathways. Flow cytometry and conditioned media experiments demonstrated that secreted factors from knockout adipose tissue strongly informed resident macrophages to develop a pro-inflammatory, MMe (metabolically activated) phenotype. Together, these studies suggest a novel role for SMRT as an integrator of metabolic and inflammatory signals to maintain physiological homeostasis.

Tumor-associated macrophages (TAMs) are the most prevalent immune cell in the tumor microenvironment (TME). They mainly adopt an M2-like phenotype that supports angiogenesis, attenuates anti-tumor immunity, and promotes metastasis. Blocking M2 activation of TAMs in pre-clinical models attenuates tumor growth and metastasis, and high abundance of M2-like TAMs is associated with poor patient survival across many cancer types. Despite of TAMs as a promising therapeutic target, the mechanisms producing their M2-like phenotype is poorly understood. This understanding is required to develop effective TAM-targeting therapeutics and identify patients that might benefit from them. One potential pathway to influence macrophage polarization is via metabolic reprogramming. The traditional view is that glycolysis supports a pro-inflammatory M1 phenotype in macrophages, while oxidative phosphorylation is required for their M2 phenotypes. Our recent work challenged this paradigm. We showed that treating macrophages with LPS or bacteria (conditions that support M1 activation) induces lactate production, which in turn drives a late phase switch to an M2-like phenotypes. The mechanism underlying this surprising observation involves a novel lactate-induced epigenetic modification termed histone lysine lactylation (Kla) that marks promoters of M2-like genes and directly promote transcription. In tumors, hypoxia is a key environmental stimulus that induces lactate production. The goal of this study was to examine if hypoxia-induced lactate production by TAMs metabolically reprograms them to promote Kla-M2 pathway to influence tumorigenesis. Here, we showed that hypoxia-induced lactate production by macrophages elevates the expression of M2-like genes, marked by Kla at their promotes. We further demonstrated the spatial and quantitative relationship between hypoxia, Kla, and M2-like phenotype within a single tumor (GEM model & human tumors) and across tumors with variable hypoxia (syngeneic models). Moreover, inhibiting endogenous lactate production by TAMs (via Ldha deletion) reduces tumor growth, attenuates M2-like phenotype of TAMs, and increases CD8+ T cells in tumor with high hypoxia, but not low hypoxia. Importantly, lactate level in TME is independent of tumor hypoxia or LDHA status, suggesting that this epigenetic pathway is primarily driven by endogenous lactate production by TAM rather than exogenous lactate in TME. Collectively, our studies demonstrated an important role for a “hypoxia-induced lactate-Kla-M2 pathway” in TAMs to promote tumorigenesis. Citation Format: Chang Cui, kasturi Chakraborty, Kelly Schoenfelt, Guolin Zhou, Xu Anna Tang, Catherine Reardon, Di Zhang, Alexander Muir, Yingming Zhao, Lev Becker. Hypoxia-induced lactate production by tumor-associated macrophages promote tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2766.

Cancer cell genetic variability and similarity to host cells have stymied development of broad anti-cancer therapeutics. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity; however, whether/how innate immunity can produce similar effects in cancer is unknown. Here, we show that human, but not murine, neutrophils release catalytically active neutrophil elastase (ELANE) to kill many cancer cell types while sparing non-cancer cells. ELANE proteolytically liberates the CD95 death domain, which interacts with histone H1 isoforms to selectively eradicate cancer cells. ELANE attenuates primary tumor growth and produces a CD8+T cell-mediated abscopal effect to attack distant metastases. Porcine pancreatic elastase (ELANE homolog) resists tumor-derived protease inhibitors and exhibits markedly improved therapeutic efficacy. Altogether, our studies suggest that ELANE kills genetically diverse cancer cells with minimal toxicity to non-cancer cells, raising the possibility of developing it as a broad anti-cancer therapy.

SUMMARYObesity is associated with increased incidence and severity of triple-negative breast cancer (TNBC); however, mechanisms underlying this relationship are incompletely understood. Macrophages, which accumulate in adipose tissue and are activated during obesity, are an attractive mechanistic link. Here, we show that, during obesity, murine and human mammary adipose tissue macrophages adopt a pro-inflammatory, metabolically- activated (MMe) macrophage phenotype that promotes TNBC stem-like markers and functions, including increased tumorsphere growthin vitroand tumor-initiating potentialin vivo. We demonstrate that MMe macrophages release cytokines in an NADPH oxidase 2 (NOX2)-dependent manner that signal through glycoprotein 130 (GP130) on TNBC cells to promote their stem-like properties. Accordingly, deletingNox2in myeloid cells or depleting GP130 in TNBC cells attenuates the ability of obesity to drive TNBC tumor formation. Our studies implicate MMe macrophage accumulation in mammary adipose tissue during obesity as a mechanism for promoting TNBC stemness and tumorigenesis.HIGHLIGHTS⁘Obesity promotes TNBC tumor formation and stemness.⁘Mammary adipose tissue macrophages are metabolically activated (MMe) in obese mice and humans.⁘MMe macrophages in mammary adipose tissue contribute to obesity-induced stemness.⁘MMe macrophages promote TNBC stemness through GP130 signaling.

Length-dependent activation (LDA) is a prominent feature of cardiac muscle characterized by decreases in the Ca(2+) levels required to generate force (i.e., increases in Ca(2+) sensitivity) when muscle is stretched. Previous studies have concluded that LDA originates from the increased ability of (strong) cross-bridges to attach when muscle is lengthened, which in turn enhances Ca(2+) binding to the troponin C (TnC) subunit of the troponin complex. However, our results demonstrate that inhibition of strong cross-bridge attachment with blebbistatin had no effect on the length-dependent modulation of Ca(2+) sensitivity (i.e., EC(50)) or Ca(2+) cooperativity, suggesting that LDA originates upstream of cross-bridge attachment. To test whether LDA arises from length dependence of thin-filament activation, we replaced native cTnC with a mutant cTnC (DM-TnC) that is incapable of binding Ca(2+). Although progressive replacement of native cTnC with DM-TnC caused an expected monotonic decrease in the maximal force (F(max)), DM-TnC incorporation induced much larger increases in EC(50) and decreases in Ca(2+) cooperativity at short lengths than at long lengths. These findings support the conclusion that LDA arises primarily from the influence of length on the modulation of the Ca(2+) cooperativity arising from interaction between adjacent troponin-tropomyosin complexes on the thin filament.

Triple-negative breast cancer (TNBC) patients have the poor prognosis due to their high metastatic potential and lack of targeted therapies. Emerging epidemiological data suggest that obesity is associated with increased incidence of more aggressive triple-negative breast cancer; however, mechanisms are unclear. During obesity, adipocytes within breast tissues recruit macrophages. M2-like macrophages can exacerbate TNBC progression; however, obese breast tissue is reported to be enriched with M1-like adipose tissue macrophages (ATMs), which are anti-tumorigenic. These results raise a paradox: how does obesity promote tumor progression if it activates an anti-tumorigenic macrophage phenotype? Here we show that obesity promotes a distinct pro-inflammatory phenotype (metabolically activated macrophages; MMe) in breast ATMs, which promotes TNBC progression. We showed that ATMs isolated from obese women express cell surface markers of MMe (CD36, ABCA1) but not M1 (CD38, CD319) macrophages. We further found that pretreating TNBC cells with MMe (but not M1) derived conditioned media increase tumorsphere growth, and frequency of tumor-incidence when injected into mice in limited dilutions. Remarkably, we saw the same effect with obesity on tumor-incidence in both spontaneous and syngeneic TNBC mouse models. Moreover, blocking cytokine expression specifically in MMe using myeloid specific genetic knockout mice diminishes the effect of obesity on tumor-incidence. Interestingly, a subset of these MMe-secreted cytokines shared a common signal transducer, gp130, which led us to investigate the role of gp130 in tumors. Knocking down gp130 in cancer cells inhibits the MMe-induced stat-3 phosphorylation and tumorsphere growth in vitro, and diminishes the MMe-mediated effect of obesity on tumor-incidence in mice. Together these findings suggest that obesity-induced metabolically activated macrophages (MMe) in breast adipose tissue secrete inflammatory cytokines that promotes the TNBC tumor-progression by activating gp130 in cancer cells. A comprehensive understanding of the signaling mechanism(s) involved in metabolic activation of mammary ATMs would enable development of targeted therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact. Citation Format: Payal Tiwari, Ariane Blank, Chang Cui, Kelly Schoenfelt, Seema A. Khan, Marsha R. Rosner, Lev Becker. Metabolically activated macrophages mediate obesity-driven TNBC progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4964.

Triple negative breast cancer (TNBC) patients have an extremely poor prognosis due to their high metastatic potential and lack of targeted drug therapies. Emerging epidemiological data suggest that obesity is strongly linked to the incidence and severity of TNBC. However, mechanisms by which obesity potentiates TNBC progression are unclear. Adipose tissue macrophages (ATMs) are an attractive mechanistic link between obesity and TNBC because they are the predominant type of macrophage in the breast during early tumorigenesis and obesity causes accumulation of ATMs. However, during obesity, the macrophage phenotype was reported to switch from an apparent pro-tumorigenic M2-like phenotype to an apparent anti-tumorigenic M1-like phenotype. This result raises a paradox: how does obesity promote tumor progression if it activates an anti-tumorigenic macrophage phenotype? Here we show that metabolic dysfunction promotes a mechanistically distinct pro-inflammatory phenotype (metabolic activation; MMe) in breast adipose tissue macrophages isolated from obese women. These ATMs express cell surface markers of MMe (CD36, ABCA1) but not M1 (CD38, CD319, CD274) macrophages. We further demonstrate that pretreating TNBC cells with conditioned media derived from MMe but not M1 macrophages promotes mammosphere formation as well as invasion in vitro, and intravasation of cancer cells into the blood in vivo. Moreover, pre-treated cancer cells showed a two-fold increase in the expression of stem-like cell markers (OCT4, SOX2 and NANOG). Remarkably, we saw the same effect on ‘stemness’ of tumor cells in a spontaneous TNBC mouse model when fed a 60% high fat diet and a significant increase in tumor incidence in obese mice compared to lean mice in a syngeneic mouse model. We further show that blocking inflammatory cytokines expression in MMe(s) using genetic knockouts inhibits the ability of MMe(s) to promote both mammosphere formation and invasion. These findings suggest that obesity-induced changes to mammary adipose tissue reprogram macrophages to an MMe phenotype that potentiates TNBC initiation and metastasis via inflammatory cytokines. A comprehensive understanding of the signaling mechanism(s) involved in metabolic activation of mammary ATMs would enable development of directed therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact. Citation Format: Payal Tiwari, Kelly Schoenfelt, Ariane Blank, Chang Cui, Marsha Rich Rosner, Lev Becker. Metabolically active macrophages: A novel link between obesity and TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4229. doi:10.1158/1538-7445.AM2017-4229

The cellular mechanism underlying the Frank-Starling law of the heart is myofilament length-dependent activation. The mechanism(s) whereby sarcomeres detect changes in length and translate this into increased sensitivity to activating calcium has been elusive. Small-angle X-ray diffraction studies have revealed that the intact myofilament lattice undergoes numerous structural changes upon an increase in sarcomere length (SL): lattice spacing and the I1,1/I1,0 intensity ratio decreases, whereas the M3 meridional reflection intensity (IM3) increases, concomitant with increases in diastolic and systolic force. Using a short (∼10 ms) X-ray exposure just before electrical stimulation, we were able to obtain detailed structural information regarding the effects of external osmotic compression (with mannitol) and obtain SL on thin intact electrically stimulated isolated rat right ventricular trabeculae. We show that over the same incremental increases in SL, the relative changes in systolic force track more closely to the relative changes in myosin head orientation (as reported by IM3) than to the relative changes in lattice spacing. We conclude that myosin head orientation before activation determines myocardial sarcomere activation levels and that this may be the dominant mechanism for length-dependent activation.

Changes interfilament lattice spacing is a major determinant of force production in striated muscle with reductions in force generation being observed with both expansion and compression of the lattice spacing relative to the normal physiological values. Previous studies have concluded that lattice spacing depends complexly on the balance of outward repulsive forces and inward attractive and compressive forces between thick and thin filaments. Since lattice spacing has important implications on force generation, we examined the effects of alterations in filament charge, induced by changes in intracellular pH (by the rapid application and withdrawal of 30 mM NH4Cl), on lattice spacing in intact twitching cardiac trabeculae. Since we observed changes in sarcomere length in response to changes in intracellular pH and since changes in sarcomere length induce changes in lattice spacing as a result of the isovolumic volume behavior of intact sarcomeres, sarcomere length was maintained at a fixed value (∼2.2 μm) during the pH interventions. Lattice spacing increased (p < 0.05) following NH4Cl wash in (measured after 5 minutes) when the pH is estimated to increase to ∼7.8 (Balnave 2000 J. Physiol.) to 36.9 +/-0.2. Following the washout of NH4Cl (which is estimated to decrease pH to ∼6.5 (Swietach 2005 J. Physiol.)) lattice spacing increased to 37.6+/−0.4 elevated above (P < 0.05) the space observed at control pH. Since the isoelectric point for myofilaments is ∼5 (Naylor, Biophys J, 1985), our findings suggest that, in addition to electrostatic, van der Waals' and osmotic forces, other pH-sensitive forces are also critical determinants of the lattice spacing, possibly the M-protein, myomesin or other M-line proteins which are postulated to hold thick filaments together in the M-band of striated muscle.

It is becoming clear that upregulated protein kinase C (PKC) signaling plays a role in reduced ventricular myofilament contractility observed in congestive heart failure. However, data are scant regarding which PKC isozymes are involved. There is evidence that PKC-α may be of particular importance. Here, we examined PKC-α quantity, activity, and signaling to myofilaments in chronically remodeled myocytes obtained from rats in either early heart failure or end-stage congestive heart failure. Immunoblotting revealed that PKC-α expression and activation was unaltered in early heart failure but increased in end-stage congestive heart failure. Left ventricular myocytes were isolated by mechanical homogenization, Triton-skinned, and attached to micropipettes that projected from a force transducer and motor. Myofilament function was characterized by an active force–[Ca 2+ ] relation to obtain Ca 2+ -saturated maximal force (F max ) and myofilament Ca 2+ sensitivity (indexed by EC 50 ) before and after incubation with PKC-α, protein phosphatase type 1 (PP1), or PP2a. PKC-α treatment induced a 30% decline in F max and 55% increase in the EC 50 in control cells but had no impact on myofilament function in failing cells. PP1-mediated dephosphorylation increased F max (15%) and decreased EC 50 (≈20%) in failing myofilaments but had no effect in control cells. PP2a-dependent dephosphorylation had no effect on myofilament function in either group. Lastly, PP1 dephosphorylation restored myofilament function in control cells hyperphosphorylated with PKC-α. Collectively, our results suggest that in end-stage congestive heart failure, the myofilament proteins exist in a hyperphosphorylated state attributable, in part, to increased activity and signaling of PKC-α.

Triple negative breast cancer (TNBC) accounts for about 20% of all breast cancers. TNBC patients have an extremely poor prognosis due to their high metastatic potential and lack of targeted drug therapies. Emerging epidemiological data suggest that obesity is strongly linked to the incidence and severity of TNBC; obese women have a 35% higher risk of developing TNBC and 46% higher risk of developing distant metastases. Thus, understanding the biological processes that link obesity and TNBC has important clinical applications for prognosis and treatment. Mechanisms by which obesity worsens TNBC prognosis are incompletely understood. One clue to its action is that obesity causes chronic inflammation, and macrophage infiltration into adipose tissue is a key mediator of this inflammation. Recent studies demonstrated that macrophages are enriched in breast adipose tissue of obese humans and mice. Moreover, depletion of macrophages in mice diminished the effects of obesity on TNBC growth and metastasis. Although anti-inflammatory ‘M2-like’ TAMs are key effector cells that promote tumorigenesis, it is well accepted that obesity stimulates macrophages to adopt a pro-inflammatory ‘M1-like’ state. However, M1 macrophages exhibit potent anti-tumor functions. From these discordant observations an important paradox emerges: How can obesity promote breast cancer if it elicits an M1, anti-tumor macrophage phenotype? Using a combination of proteomics, immunology, and cell biology, we recently identified a novel metabolically activated (MMe) macrophage phenotype produced by exposure to high levels of insulin, glucose, and palmitate, conditions characteristic of obese and diabetic patients. Here we show that gene expression patterns in MMe (but not M1) macrophages are strongly associated with pathways involved in breast cancer. Moreover, pre-treating BM1 cells with conditioned media derived from MMe macrophages promotes TNBC cell invasion by 2.3 fold, suggesting that MMe macrophages potentiate metastasis. We further show that breast adipose tissue macrophages isolated from obese women express cell surface markers of MMe (CD36, ABCA1), but not M1 (CD38, CD319, CD274), macrophages. Furthermore, treating naïve macrophages with media conditioned by human mammary adipose tissue from an obese subject (BMI = 37), but not a lean subject (BMI = 19), induced genes diagnostic of the MMe phenotype. Thus, mammary adipose tissue from obese women supports metabolic activation of macrophages. Together, these observations suggest that obesity-induced changes to adipose tissue reprogram macrophages to an MMe phenotype that potentiates TNBC. A comprehensive understanding of signaling mechanisms involved in metabolic activation would enable development of directed therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact. Citation Format: Payal Tiwari, Kelly Schoenfelt, Swati Kulkarni, Marsha Rosner, Lev Becker. Metabolically activated macrophages in obesity associated TNBC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2888. doi:10.1158/1538-7445.AM2015-2888

The main cellular mechanism that underlies the so-called “Frank -Starling Law of the Heart” is an increase in the responsiveness of cardiac myofilaments to activating Ca2+ ions at longer sarcomere lengths (SL). The fundamental mechanism responsible for this increase in responsiveness has been elusive, despite considerable experimental scrutiny. Here we tested the hypothesis that the increase in calcium sensitivity upon increasing SL is correlated with a radially outward movement of the myosin heads during diastole. 2D x-ray diffraction patterns were obtained from electrically stimulated intact, twitching papillary muscle isolated from rat hearts during a 10 ms time window in diastole just prior to electrical stimulation. A range of sarcomere lengths was compared either at Lmax (SL= ∼2.3 µm) or following a quick release to slack length (SL=∼1.9µm). The relative position of myosin heads was first assessed by the I11/I10 equatorial intensity ratio. To our surprise, I 11/I10 was negatively correlated with SL, i.e. I11/I10 was less at Lmax vs. slack length. A more direct measure of the radial position of the myosin heads can be estimated from the position of the first maxima on the unsampled myosin layer lines, which are prominent in diastole. The intensity maxima, when examined pair-wise, moved outwards to a maximum of 5-6% for a 0.4µm change in SL indicating that the heads must be moving radially outward at slack length. Our data suggest that myofilament length dependent activation does not derive from a radial extension of the myosin heads at the long SL and must, therefore, involve some other mechanism. Supported by NIH HL75494 and RR08630.

A complete understanding of excitation /contraction coupling in cardiac muscle requires knowledge of the sequence of structural changes in the myofilaments in response to the release of calcium from internal stores. We used isolated, membrane intact, electrically stimulated, cardiac trabeculae to obtain improved 2-dimensional X-ray patterns under three conditions: 1) diastolic conditions (no Calcium), 2) at peak calcium response but with 5 mM EGTA to inhibit calcium response and 3) at peak calcium response but where force was inhibited using the myosin ATPase inhibitor Blebbistatin which prevents strong binding of myosin heads to the thin filament. The resulting 2 dimensional X-ray diffraction patterns indicated that with the release of calcium from internal stores, the myosin heads, without generating active force, move towards the thin filaments as evidenced by an inward shift of the first maximum on the unsampled 4th myosin layer line. Surprisingly, the diffraction patterns, in the presence of Blebbistatin and calcium, indicated a more ordered structure, than in its absence, suggesting that the attachment of myosin heads and force development involves transient increases in cross-bridge ordering prior to tension generation. This is in contrast to previous results, from skeletal muscle preparations, that have been interpreted as the process activation inevitably involves a rapid disordering of the thick filament.