Residual Population Research Articles

Quantum properties of light propagating in a coherent-population-oscillation storage medium

We study the propagation and storage of a quantum field using ultranarrow coherent population oscillations (CPOs) in a $\mathrm{\ensuremath{\Lambda}}$-type atomic medium. The predictions for classical fields are checked experimentally in a vapor at room temperature. We derive the evolution of its squeezing spectrum in the presence of a large classical pump field which enables CPOs to exist. We show that the spontaneous emission of the residual population pumped into the excited state progressively destroys the quantum noise properties of the quantum field along propagation. The output quantum field therefore tends to be a coherent state, discarding the possibility to store quantum states of light with CPO.

Effect of microstructure and initial cell conditions on thermal inactivation kinetics and sublethal injury of Listeria monocytogenes in fish-based food model systems

The development of more accurate predictive models that describe the microbial kinetics of mild thermal treatments of foods requires knowledge concerning the influence of food microstructure and initial cell conditions on foodborne pathogens’ inactivation kinetics. The effect of food microstructure and initial cell conditions on thermal inactivation kinetics and sublethal injury (SI) of Listeria monocytogenes was investigated at 59, 64 and 69°C. Fish-based food model systems with different microstructures, possessing minimal compositional and physicochemical variations, were used. L. monocytogenes growth morphology had no significant influence on thermal inactivation kinetics. A gelled matrix resulted in a lower specific inactivation rate kmax and a higher residual cell population Nres, while the presence of fat droplets resulted in a higherkmaxand did not influenceNres. SI was higher in viscous than in gelled systems and more prominent for cells that were grown inside the matrix. Hence, predictive thermal inactivation models could benefit from the inclusion of factors related to the nature of the food matrix and fat properties. Starting inactivation from cells that were grown inside the matrix, resulted in lower (i.e., fail-safe)kmaxvalues and more uncertainty onNres as compared to starting from cells grown at optimal conditions.

Consistent Model of Ultrafast Energy Transfer in Peridinin Chlorophyll-a Protein Using Two-Dimensional Electronic Spectroscopy and Förster Theory.

Solar light harvesting begins with electronic energy transfer in structurally complex light-harvesting antennae such as the peridinin chlorophyll-a protein from dinoflagellate algae. Peridinin chlorophyll-a protein is composed of a unique combination of chlorophylls sensitized by carotenoids in a 4:1 ratio, and ultrafast spectroscopic methods have previously been utilized in elucidating their energy-transfer pathways and timescales. However, due to overlapping signals from various chromophores and competing pathways and timescales, a consistent model of intraprotein electronic energy transfer has been elusive. Here, we used a broad-band two-dimensional electronic spectroscopy, which alleviates the spectral congestion by dispersing excitation and detection wavelengths. Interchromophoric couplings appeared as cross peaks in two-dimensional electronic spectra, and these spectral features were observed between the peridinin S2 states and chlorophyll-a Qx and Qy states. In addition, the inherently high time and frequency resolutions of two-dimensional electronic spectroscopy enabled accurate determination of the ultrafast energy-transfer dynamics. Kinetic analysis near the peridinin S1 excited-state absorption, which forms in 24 fs after optical excitation, reveals an ultrafast energy-transfer pathway from the peridinin S2 state to the chlorophyll-a Qx state, a hitherto unconfirmed pathway critical for fast interchromophoric transfer. We propose a model of ultrafast peridinin chlorophyll-a protein photophysics that includes (1) a conical intersection between peridinin S2 and S1 states to explain both the ultrafast peridinin S1 formation and the residual peridinin S2 population for energy transfer to chlorophyll-a, and (2) computationally and experimentally derived peridinin S2 site energies that support the observed ultrafast peridinin S2 to chlorophyll-a Qx energy transfer.

Efficient demultiplexed single-photon source with a quantum dot coupled to a nanophotonic waveguide

Planar nanostructures allow near-ideal extraction of emission from a quantum emitter embedded within, thereby realizing deterministic single-photon sources. Such a source can be transformed into M single-photon sources by implementing active temporal-to-spatial mode demultiplexing. We report on the realization of such a demultiplexed source based on a quantum dot embedded in a nanophotonic waveguide. Efficient outcoupling (>60%) from the waveguide into a single mode optical fiber is obtained with high-efficiency grating couplers. As a proof-of-concept, active demultiplexing into M = 4 spatial channels is demonstrated by the use of electro-optic modulators with an end-to-end efficiency of >81% into single-mode fibers. Overall, we demonstrate four-photon coincidence rates of >1 Hz even under nonresonant excitation of the quantum dot. The main limitation of the current source is the residual population of other exciton transitions, which corresponds to a finite preparation efficiency of the desired transition. We quantitatively extract a preparation efficiency of 15% using the second-order correlation function measurements. The experiment highlights the applicability of planar nanostructures as efficient multiphoton sources through temporal-to-spatial demultiplexing and lays out a clear path way of how to scale up toward demonstrating quantum advantages with the quantum dot sources.

Abstract 4486: Androgen-deprivation therapy promotes immune suppression in a murine model of prostate cancer

Abstract Most prostate cancer (PCa) deaths are due to castration resistant PCa (CRPC), following failure of androgen deprivation therapy (ADT). ADT is the standard of care for patients with advanced PCa but nearly universal progression to CRPC occurs 2-3 years after ADT is initiated. Immunotherapy with checkpoint inhibitors has not been effective in most prostate cancers, perhaps because such cancers lack functional CD8 T-cells. This may be caused by infiltration of myeloid cell populations into the tumor immune cell microenvironment (TIME). Recently, we found that in a PTEN-deficient mouse PCa model, castration induces an immunosuppressive state within the tumor that is concurrent with tumor recurrence. The response to castration/ADT is tri-phasic: a pro-apoptotic regression phase when tumor shrinks, followed by selection for a residual population of resistant tumor cells and finally recurrent growth as CRPC. Using PCa cell lines to model the first two phases of the response to ADT, we have shown that ADT induces apoptosis, thereby enriching for an ADT-resistant stem/progenitor population that we propose is the in vivo source of TNF. Mechanistically, in our model system the response to ADT is driven by the soluble mediators TNF and CCL2, which facilitate communication within the TIME. Specifically, a TNF-CCL2-CCR2 paracrine loop is induced between prostate cancer cells and non-tumor cells in the microenvironment: TNF produced by tumor cells acts on myofibroblasts to induce CCL2 production, which in turn recruits CCR2+ tumor-associated macrophages (TAMs). To investigate the ADT response within the TIME in an in vivo model of prostate cancer, we employed a prostate-specific PTEN-deficient mouse model (PbCre4 x PTENf/f). Castration caused the tumors to regress, consistent with initial phase of the response that is seen in the human disease. At late times post-castration (5-6 weeks), corresponding to the selection phase, we observed a coordinate increase in the stem/progenitor tumor cell population, as well as TNF and CCL2, within the TIME. Immunohistochemical staining of tumors 5 weeks post-castration revealed an increase in TAMs, and a decrease in CD8 T cells, consistent with an immuno-suppressive or immuno-evasive state. This phenotype was reversed by a soluble receptor that binds TNF (etanercept). Thus, following ADT, TNF derived from an ADT-resistant stem/progenitor epithelial tumor cell population promotes an immunosuppressive state via CCL2 in the TIME. Analysis of public human PCa data sets show the transcripts for TNF, as well as gene signatures for stem/progenitor tumor cells and M2 TAMs are increased in CRPC, consistent with our hypothesis that ADT drives the development of myeloid immuno-suppressive state via a TNF-CCL2-CCR2 axis. Our results set the stage for the future development of immunotherapies that could improve the efficacy of ADT. Citation Format: John J. Krolewski, Kai Sha, Kent L. Nastiuk. Androgen-deprivation therapy promotes immune suppression in a murine model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4486.

PB2019 MOLECULAR ANALYSIS OF A COMPOSITE B‐CELL TUMOR – HAIRY CELL LEUKEMIA AND MANTLE CELL LYMPHOMA COMBINATION

Background:Composite lymphomas are unique models to study the clonal transformation process according stages of lymphoid cell differentiation. Clonal relationship is a key point in the research. At the moment, molecular analysis of V(D)J‐recombination is the most accurate method to evaluate related lymphoid populations.Aims:To study clonal rearrangements of IGH genes in isolated tumor populations in order to determine the mechanism of their occurrence during pathogenesis of composite lymphoma – hairy cell leukemia (HCL) and mantle cell lymphoma (MCL) combinationMethods:Patient was diagnosed and treated at the National Research Center for Hematology. Informed consent was obtained. Isolation of MCL and HCL cells was carried out by magnetic selection of bone marrow leukocyte fraction using anti‐CD103 and anti‐CD5 magnetic particles according to the standard Miltenyi Biotec protocol (www.miltenyibiotec.com). B‐cell clonality testing and IGHV sequencing was performed according to BIOMED‐2 protocol. Fragment analysis and Sanger sequencing was performed on ABI PRISM 3100 genetic analyzer, followed by comparison with the germinal sequence databases www.imgt.org.Results:Patient was performed with leukopenia and lymphocytosis, splenomegaly, lymphadenopathy in May, 2018 and was diagnosed with HCL. The diagnosis was confirmed by the BRAF mutation detection and peripheral blood lymphocytes immunophenotyping (table 1). Patient was consistently treated with interferon α2b and cladribine with good partial response. Within 4 months after therapy was completed spleen repeatedly enlarged. Diagnostic splenectomy was performed and MCL was revealed. The diagnosis was confirmed by the t(11;14) detection. Immunophenotyping of spleen suspension allowed to identify not only MCL cells but minimal residual HCL population (table 1). Analysis of clonally rearranged IGH genes is isolated CD103+ and CD5+ population revealed two clonal products corresponding to components of composite lymphoma (figure 1). The sequence analysis identified the dissimilar repertoire of gene segments involved in V(D)J‐recombination in MCL (IGHV3‐9/IGHD2/IGHJ4) and HCL (IGHV1‐69/IGHD3/IGHJ4). This distinction indicated the independence of two clones’ origin, at least at the stage of B‐lymphocyte precursors as we do not have other molecular markers for progenitor lymphoid cells. Nucleotide sequence was 100% germinal in MCL and only 90% matches with the germinal sequence were detected in HCL reflecting the postgerminal phenotype. Thus not only the existence of separate tumor precursors was strictly supposed but also the different way of parallel lymphoid cell differentiation was proven. Patient was treated with R‐BAC cycles for MCL. Complete remission was achieved and has been maintained for 6 months.Summary/Conclusion:At the moment, there is no known description of the synchronous development of MCL and HCL. The presented clinical case is the first observation of the clinical course of such the composite lymphoma. We demonstrated independent origin of two tumor components and proved the different way of their differentiation. The proposed algorithm for the analysis of isolated cell populations and the obtained results make an additional contribution to the lymphomagenesis understanding, which is necessary for the development of effective methods of diagnosis and therapy.image

Diverse MR1-restricted T cells in mice and humans

Mucosal-associated invariant T (MAIT) cells express an invariant TRAV1/TRAJ33 TCR-α chain and are restricted to the MHC-I-like molecule, MR1. Whether MAIT cell development depends on this invariant TCR-α chain is unclear. Here we generate Traj33-deficient mice and show that they are highly depleted of MAIT cells; however, a residual population remains and can respond to exogenous antigen in vitro or pulmonary Legionella challenge in vivo. These residual cells include some that express Trav1+ TCRs with conservative Traj-gene substitutions, and others that express Trav1- TCRs with a broad range of Traj genes. We further report that human TRAV1-2- MR1-restricted T cells contain both MAIT-like and non-MAIT-like cells, as judged by their TCR repertoire, antigen reactivity and phenotypic features. These include a MAIT-like population that expresses a public, canonical TRAV36+ TRBV28+ TCR. Our findings highlight the TCR diversity and the resulting potential impact on antigen recognition by MR1-restricted T cells.

Impact of Diabetic Retinopathy on Sleep, Mood, and Quality of Life.

PurposeDiabetic retinopathy (DR) is associated with retinal neuronal and vascular damage. DR has previously been shown to affect the photosensitive retinal ganglion cells (pRGCs). PRGCs are essential for the entrainment of circadian rhythms; thus, DR progression could lead to worsening sleep quality and mood. We investigate the relationship between increasing DR severity, and its impact on sleep quality and mood.MethodsA total of 430 participants with DR, and 303 healthy controls with no ocular disease or preexisting sleep disorders were recruited. DR severity was grouped as follows: 1, mild nonproliferative (NPDR); 2, moderate/severe NPDR; and 3, proliferative diabetic retinopathy (PDR). Sleep, mood, and quality of life were assessed using the Pittsburgh Sleep Quality Index (PSQI), quality of life (SF-36), and Hospital Anxiety and Depression Score (HADS) questionnaires. Data were analyzed by severity of DR, and correlated with sleep, QOL, and mood and compared to controls.ResultsNo significant difference between PSQI scores in the DR group or the control group was identified despite severity of DR. Mean anxiety and depression scores were within the normal range for both groups. Despite a lower general health and physical function, the DR group had lower anxiety scores than controls.ConclusionsThese data show that even in severe DR, sleep quality is similar to controls. However, this could be explained by the majority of individuals in this study having good visual acuities in the better eye with a residual population of pRGCs remaining unaffected by DR.

145 An in vitro model of immunotherapy-tolerant melanoma persister cells

Cancer persister cells survive cytotoxic therapies by entering a drug-tolerant, pro-survival cell state governed by poorly understood nongenetic mechanisms. Persister cells have been observed in a wide range of tumor types and are known to exhibit tolerance to targeted therapies and chemotherapies. Key characteristics of persister cells include reversible drug-tolerance, quiescence/slow proliferation, low genome-wide H3K4me3 marks, cancer stem cell and EMT gene expression patterns, and cross resistance to other cytotoxic drugs. Persister cells constitute a surviving cancer cell reservoir from which drug resistant tumors may emerge. Therefore, targeting persister cells is a promising therapeutic strategy to prevent tumor relapse. Immune checkpoint inhibitors have been proven to extend the survival of melanoma patients. However, the majority of patients who respond experience partial responses followed by relapse. Relatively little is known about residual melanoma cells that survive during initial immunotherapy responses. To explore this residual cell population, we developed an in vitro model system of melanoma persister cells which survive CD8 T cell-mediated killing. We have modeled this process by treating human A375 BRAFV600E melanoma cells with the components of T cell lytic granules that serve as the primary mechanism of T cell-mediated killing, perforin and granzyme B. Upon co-treatment with purified perforin and granzyme B, >80% of A375 melanoma cells undergo apoptosis. No melanoma cell death is observed from treatment with perforin or granzyme B alone, consistent with the synergistic mechanism of these lytic granule components. Reminiscent of chemical drug-derived persister cells, the residual melanoma cells exhibit reversible resistance to perforin and granzyme B indicating a nongenetic mechanism of resistance. This in vitro model of immunotherapy-tolerant melanoma persister cells will be useful for exploring this important melanoma cell population.

Mammalian circannual pacemakers

Circannual clocks drive rhythms in reproduction and many other seasonal characteristics but the underlying control of these long-term oscillators remains a mystery. Now, we propose that circannual timing involves mechanisms that are integral to the ontogenetic life-history programme where annual transitions are generated by cell birth, death and tissue regeneration throughout the life cycle ߝ the histogenesis hypothesis. The intrinsic cycle is then timed by cues from the environment. The concept is that in specific sites in the brain, pituitary and peripheral tissues, residual populations of progenitor cells (adult stem cells) synchronously initiate a phase of cell division to begin a cycle. The progeny cells then proliferate, migrate and differentiate, providing the substrate that drives physiological change over long time-spans (e.g. summer/winter); cell death may be required to trigger the next cycle. We have begun to characterise such a tissue-based timer in our Soay sheep model focusing on the pars tuberalis (PT) of the pituitary gland and the sub-ventricular zone of the mediobasal hypothalamus (MBH) as potential circannual pacemakers. The PT is of special interest because it is a melatonin-responsive tissue containing undifferentiated cells, strategically located at the gateway between the brain and pituitary gland. The PT also governs long-photoperiod activation of thyroid hormone dependant processes in the MBH required for neurogenesis. In sheep, exposure to long photoperiod markedly activates BrDU-labelled cell proliferation in the PT and MBH, and acts to entrain the circannual reproductive cycle. Variation in expression and co-ordination of multiple tissue timers may explain species differences in circannual rhythmicity. This paper is dedicated to the memory of Ebo Gwinner3.

Seasonal malaria vector and transmission dynamics in western Burkina Faso

BackgroundIn the context of widespread mosquito resistance to currently available pesticides, novel, precise genetic vector control methods aimed at population suppression or trait replacement are a potentially powerful approach that could complement existing malaria elimination interventions. Such methods require knowledge of vector population composition, dynamics, behaviour and role in transmission. Here were characterized these parameters in three representative villages, Bana, Pala and Souroukoudingan, of the Sudano-Sahelian belt of Burkina Faso, a region where bed net campaigns have recently intensified.MethodsFrom July 2012 to November 2015, adult mosquitoes were collected monthly using pyrethroid spray catches (PSC) and human landing catches (HLC) in each village. Larval habitat prospections assessed breeding sites abundance at each site. Mosquitoes collected by PSC were identified morphologically, and then by molecular technique to species where required, to reveal the seasonal dynamics of local vectors. Monthly entomological inoculation rates (EIR) that reflect malaria transmission dynamics were estimated by combining the HLC data with mosquito sporozoite infection rates (SIR) identified through ELISA-CSP. Finally, population and EIR fluctuations were fit to locally-collected rainfall data to highlight the strong seasonal determinants of mosquito abundance and malaria transmission in this region.ResultsThe principal malaria vectors found were in the Anopheles gambiae complex. Mosquito abundance peaked during the rainy season, but there was variation in vector species composition between villages. Mean survey HLC and SIR were similar across villages and ranged from 18 to 48 mosquitoes/person/night and from 3.1 to 6.6% prevalence. The resulting monthly EIRs were extremely high during the rainy season (0.91–2.35 infectious bites/person/day) but decreased substantially in the dry season (0.03–0.22). Vector and malaria transmission dynamics generally tracked seasonal rainfall variations, and the highest mosquito abundances and EIRs occurred in the rainy season. However, despite low residual mosquito populations, mosquitoes infected with malaria parasites remained present in the dry season.ConclusionThese results highlight the important vector control challenge facing countries with high EIR despite the recent campaigns of bed net distribution. As demonstrated in these villages, malaria transmission is sustained for large parts of the year by a very high vector abundance and high sporozoite prevalence, resulting in seasonal patterns of hyper and hypo-endemicity. There is, therefore, an urgent need for additional vector control tools that can target endo and exophillic mosquito populations.

Long-Term Vegetation Recovery and Invasive Annual Suppression in Native and Introduced Postfire Seeding Treatments

Seed mixes used for postfire seeding in the Great Basin are often selected on the basis of short-term rehabilitation objectives, such as ability to rapidly establish and suppress invasive exotic annuals (e.g., cheatgrass, Bromus tectorum L.). Longer-term considerations are also important, including whether seeded plants persist, continue to suppress invasives, and promote recovery of desired vegetation. To better understand long-term effects of postfire seed mixes, we revisited study sites in Tintic Valley, Utah, where seeding experiments had been initiated after the 1999 Railroad wildfire. Four different mixes, including two comprised entirely of native species, had been applied using rangeland drills at a shrubland site and aerial seeding followed by one-way Ely chaining at a woodland site. New vegetation data collected 16 years post fire revealed changes relative to 3 years post fire. We found significant increases in total cover of seed-mix species in all treatments, including the unseeded control where these species were present as residual populations or had spread from seeded treatments. Significant increases of seed-mix species cover and density were observed in blocks where seeding treatments had previously been considered unsuccessful. Some seed-mix species, particularly rhizomatous grasses, increased while others declined. Exotic annual forb cover decreased in all treatments while cheatgrass increased in the unseeded control and to a lesser extent in the native-only seeded treatments. Recruitment of non-seed-mix native perennials was highest in the unseeded control. Results indicate that postfire seeding has lasting effects on vegetation composition and structure, implying that seed mixes should be carefully formulated to promote long-term management objectives. Seed mixes containing large amounts of competitive introduced species may be especially effective for long-term cheatgrass suppression, but native-only mixes can also serve this purpose to a lesser degree while avoiding drawbacks of non-native species introductions.

Abstract P5-04-08: Timing provides context for the paradoxical effects of AMPK activation in ER+ breast cancer: Suppressing growing tumors, but promoting dormant tumor cell survival and recurrence

Abstract Precision oncology requires delivering the right drug to the right patient at the right time, but “time” is rarely studied preclinically before a drug enters a particular clinical setting. Despite showing efficacy against recurrent/metastatic solid tumors, drugs sometimes fail to prevent tumor recurrence when given (neo)adjuvantly. The biology and microenvironments of overt tumors likely differ substantially from dormant cancer cells. The development of preclinical models to investigate clinically dormant disease will increase understanding of residual tumor cell biology and enable the development of therapeutics for rational adjuvant trials. Despite treatment with adjuvant anti-estrogen therapies, ˜30% of patients with ER+ breast cancer (BC) experience recurrence. In contrast to other BC subtypes, ER+ relapses occur late, often appearing years to decades after initial diagnosis and treatment. This delay suggests that ER+ BC cells can undergo extended periods of clinical dormancy. We developed novel, clinically relevant xenograft models of dormancy in ER+ BC. Low systemic levels of estrogens in mice can be further suppressed by ovariectomy, mimicking the effects of aromatase inhibitor (AI)-induced estrogen deprivation (ED) therapy. In ovariectomized mice, luciferase-labeled MCF-7, HCC-1428, HCC-1500, and MDA-MB-415 cells, as well as the HCI-017 PDX model, formed palpable orthotopic tumors upon 17b-estradiol supplementation. ED induced rapid tumor regression and decreased bioluminescent signal. However, a small proportion of ER+ BC cells survived ED for >1 year in a clinically dormant, growth-suppressed state. This residual cell population stabilized after ˜90 days of ED, as evidenced by stabilization of bioluminescent signal. Transcriptional and immunohistochemical analyses revealed significant upregulation of AMP-activated protein kinase (AMPK)-alpha-2 levels and activity in clinically dormant tumor cells compared to estrogen-driven or acutely ED xenografts. Dormant tumor cells were dependent upon AMPK activity for survival, as short-term pharmacologic inhibition of AMPK reduced residual bioluminescent signal. Metformin is an AMPK-activating drug approved for the treatment of diabetes. Metformin is currently being tested as an anti-cancer agent in many clinical trials at various points in the disease progression of diverse cancer types. In our models of clinically dormant ER+ BC, AMPK activation via metformin slowed ED-induced tumor regression, promoted residual tumor cell survival, and caused earlier tumor regrowth. In vitro studies indicated that metformin promotes cell survival during ED by enhancing fatty acid β-oxidation. Conversely, metformin treatment slowed estrogen-driven tumor growth, in agreement with prior observations that metformin slows growth of various tumor subtypes. These findings suggest that AMPK activation may be efficacious against growing tumors, but deleterious when used in combination with drugs that suppress tumor growth and induce regression. More broadly, this work highlights the issue that the time in a disease course needs to be considered when testing potential anti-cancer agents such as AMPK modulators. Citation Format: Miller TW, Hampsch RA, McCleery CF, Wells JD, Fields JL, Dillon LM, Shee K. Timing provides context for the paradoxical effects of AMPK activation in ER+ breast cancer: Suppressing growing tumors, but promoting dormant tumor cell survival and recurrence [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-08.

Abstract A084: Towards combining androgen deprivation and immunotherapy to prevent progression to castration-resistant prostate cancer

Abstract Most prostate cancer (PCa) deaths are due to castration-resistant PCa (CRPC), following failure of androgen-deprivation therapy (ADT). ADT is the standard of care for patients with advanced PCa. However, nearly universal progression to castration-resistant prostate cancer (CRPC) occurs 2-3 years after ADT is initiated. Although there have been recent improvements in the treatment of CRPC, even the most promising therapies are still not curative. One approach to this problem is to improve the initial treatment of advanced prostate cancers, by combining complementary therapies with ADT, to prevent progression of such advanced cancers to CRPC. Immunotherapy with checkpoint inhibitors (CPIs) has not been effective in prostate cancers, perhaps because such cancers are “cold” (lacking cytolytic CD8 T-cells). Some cold tumors may be caused by infiltration of myeloid cell populations (tumor associated macrophages and myeloid-derived suppressor cells) into the tumor immune cell microenvironment (TIME). Recently, we found that in a PTEN-deficient mouse PCa model, castration induces an immunosuppressive state within the tumor that is concurrent with tumor recurrence. The response to castration/ADT is tri-phasic: a pro-apoptotic regression phase when tumor shrinks, followed by selection for a residual population of resistant tumor cells and finally recurrent growth as CRPC. Using PCa cell lines to model the first two phases of the response to ADT, we have shown that ADT induces apoptosis, thereby enriching for an ADT-resistant stem/progenitor population that we propose is the in vivo source of TNF. Mechanistically, in our model system the response to ADT is driven by the soluble mediators TNF and CCL2, which facilitate communication within the TIME. Specifically, a TNF-CCL2-CCR2 paracrine loop is induced between prostate cancer cells and non-tumor cells in the microenvironment: TNF produced by tumor cells acts on myofibroblasts to induce CCL2 production, which in turn recruits CCR2+ tumor-associated macrophages (TAMs). To investigate the ADT response within the TIME in an in vivo model of prostate cancer, we employed a prostate-specific PTEN-deficient mouse model (PbCre4 x PTENf/f). Castration caused the tumors to regress, consistent with initial phase of the response that is seen in the human disease. At late times post-castration (5-6 weeks), corresponding to the selection phase, we observed a coordinate increase in the stem/progenitor tumor cell population, as well as TNF and CCL2, within the TIME. Immunohistochemical staining of tumors 5 weeks post-castration revealed an increase in TAMs, and a decrease in CD8 T-cells, consistent with an immunosuppressive or immunoevasive state. This phenotype was reversed by a soluble receptor that binds TNF (etanercept). We also observed increased myeloid-derived suppressor cells (MDSC). Thus, following ADT, TNF derived from an ADT-resistant stem/progenitor epithelial tumor cell population promotes an immunosuppressive state via CCL2 in the TIME. Analysis of public human PCa data sets shows TNF and stem/progenitor marker expression are both increased in CRPC, consistent with our hypothesis that ADT drives the development of an immunosuppressive state via a TNF-CCL2-CCR2 axis. Our results set the stage for the future development of immunotherapies that could improve the efficacy of ADT. Citation Format: John J. Krolewski, Kai Sha, Michalis Mastri, Dean Tang, Kevin Eng, Kent L. Nastiuk. Towards combining androgen deprivation and immunotherapy to prevent progression to castration-resistant prostate cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A084.

Cavity Attenuators for Superconducting Qubits

Dephasing induced by residual thermal photons in the readout resonator is a leading factor limiting the coherence times of qubits in the circuit QED architecture. This residual thermal population, of the order of $10^{-1}$--$10^{-3}$, is suspected to arise from noise impinging on the resonator from its input and output ports. To address this problem, we designed and tested a new type of band-pass microwave attenuator that consists of a dissipative cavity well thermalized to the mixing chamber stage of a dilution refrigerator. By adding such a cavity attenuator inline with a 3D superconducting cavity housing a transmon qubit, we have reproducibly measured increased qubit coherence times. At base temperature, through Hahn echo experiment, we measured $T_{2\mathrm{e}}/2T_1 = 1.0\,({+0.0}/{-0.1})$ for two qubits over multiple cooldowns. Through noise-induced dephasing measurement, we obtained an upper bound $2\times 10^{-4}$ on the residual photon population in the fundamental mode of the readout cavity, which to our knowledge is the lowest value reported so far. These results validate an effective method for protecting qubits against photon noise, which can be developed into a standard technology for quantum circuit experiments.

'Superbull' males: what role do they play and what drives their appearance within the Doryteuthis gahi Patagonian Shelf population?

Cephalopod populations exhibit high variability in life history characteristics, such as longevity and size-at-age. The aim of this study was to understand how characteristics of a newly described ‘superbull’ male morph in Doryteuthis gahi populations (Patagonian Shelf) arise and whether there is a selective advantage. At the population level, it is speculated that superbulls provide temporal and spatial connectivity, but individual benefit is less obvious. Age structure and reproductive potential of males was investigated to determine whether superbulls could provide connectivity. Environmental variables affecting size-at-age were explored to ascertain whether morphological differences were primarily phenotypically driven. Superbulls from the autumn spawning cohort were significantly older than the residual population, with added longevity potentially leading to spawning with the following cohort. A reduction in relative testis weight was apparent in superbulls, but spermatophore production remained high. Generalised additive mixed models indicated temperature, location and hatch year had significant effects on size-at-age. Weak correlations between warm El Niño–Southern Oscillation phases and superbull abundance were found. The results suggest that superbulls provide temporal connectivity and arise through phenotypic plasticity, likely providing connectivity as a side effect of body shape and size rather than a genetically selected advantage.

Genetic identification of native populations of Mediterranean brown trout Salmo trutta L. complex (Osteichthyes: Salmonidae) in central Italy

Italian native populations of Mediterranean brown trout belong to the Salmo trutta complex. This species complex includes many mitochondrial lineages and phenotypes that have caused taxonomic controversies over time. The spatial distribution and the genetic diversity of these fishes are threatened by habitat destruction, global warming and, mainly, by the introduction of domestic trout of Atlantic origin. Indeed allochthonous trouts were massively restocked in Italian rivers for a century and they admixed with native populations. In order to identify residual native populations of Mediterranean brown trout, a genetic analysis of specimens collected within Latium region, on the Tyrrhenian slope of central Italy, was undertaken. To this purpose, 210 trout specimens were collected from six different rivers and analyzed for the identification of their nuclear (LDH-C1* RFLP) and mitochondrial (Control Region sequences) genotypes. Genetic characterization with these molecular markers allowed a quantitative estimate of allochthonous genotypes, which are present in all brown trout populations of the six sites, even if not equally distributed across the sampling area. At least three populations, inhabiting diverse lotic ecosystems (mountain, hilly and coastal streams respectively), are characterized by a high percentage of native nuclear allele *100 at locus LDH-C1* and typical Mediterranean haplotypes (of AD and ME lineages), which can be considered as different management units (MUs). This finding highlighted the aquatic ecosystems of the Latium Region as an important hotspot of salmonid biodiversity within the Italian peninsula, with important implications from a conservation perspective.

Harnessing stem cells and biomaterials to promote neural repair.

With the limited capacity for self-repair in the adult CNS, efforts to stimulate quiescent stem cell populations within discrete brain regions, as well as harness the potential of stem cell transplants, offer significant hope for neural repair. These new cells are capable of providing trophic cues to support residual host populations and/or replace those cells lost to the primary insult. However, issues with low-level adult neurogenesis, cell survival, directed differentiation and inadequate reinnervation of host tissue have impeded the full potential of these therapeutic approaches and their clinical advancement. Biomaterials offer novel approaches to stimulate endogenous neurogenesis, as well as for the delivery and support of neural progenitor transplants, providing a tissue-appropriate physical and trophic milieu for the newly integrating cells. In this review, we will discuss the various approaches by which bioengineered scaffolds may improve stem cell-based therapies for repair of the CNS.

Image-guided study of inter-fraction and intra-fraction set-up variability and margins in reverse semi-decubitus breast radiotherapy

BackgroundThis study aimed to evaluate the inter-fraction set-up error and intra-fraction motion during reverse semi-decubitus (RSD) breast radiotherapy, and to determine a planning target volume (PTV) margin.Material and methodsPre- and post-treatment cone-beam computed tomography (CBCT) scans were prospectively acquired at fractions 1, 4, 7, 8, 11, and 14 for 30 patients who underwent RSD breast radiotherapy. Online correction for initial set-up error greater than 5 mm or 2° was performed and post-correction CBCT was acquired. An off-line analysis was performed to quantify initial and residual inter-fraction set-up errors and intra-fraction motion in three-dimensions. Patient inter-fraction errors were analysed for time trends during the course of radiotherapy. PTV margins were calculated from the systematic and random errors.ResultsThe initial inter-fraction population systematic errors were 1.8–3.3 mm (translation) and 0.5° (rotation); random errors were 1.8–2.1 mm (translation) and 0.3–0.5° (rotation). After online correction, the residual inter-fraction population systematic errors were 1.2–1.8 mm (translation) and 0.3–0.4° (rotation); random errors were 1.4–1.6 mm (translation) and 0.3–0.4° (rotation). Intra-fraction population systematic and random errors were ≤ 1.3 mm (translation) and ≤ 0.2° (rotation). The magnitude of inter-fraction set-up errors in the anterior-posterior direction, roll, and yaw were significantly correlated with higher body weight and body mass index (BMI). The inter-fraction set-up error did not change significantly as a function of time during the course of radiotherapy. The magnitude of intra-fraction motion was not correlated with patient characteristics and treatment time. The total PTV margins accounting for pre-correction and intra-fraction errors were 6.5–10.2 mm; those accounting for post-correction and intra-fraction errors were 4.7–6.3 mm.ConclusionsCBCT is an effective modality to evaluate and improve the inter-fraction set-up reproducibility in RSD breast radiotherapy, particularly for patients with higher BMI. Intra-fraction motion was minimal during RSD breast radiotherapy.

Adipogeneis in the Bone Marrow Microenvironment Confers a Proliferative Advantage in Residual Leukaemic Cell Populations in Acute Myeloid Leukaemia after Allogeneic Stem Cell Transplantation

Up to 40% of poor risk Acute Myeloid Leukaemia (AML) patients receiving an allogeneic bone marrow transplant (BMT) from a donor will suffer disease recurrence/relapse within a year, significantly shortening their overall survival. Drug development to date has focused on clearing the bulk of leukaemic cells and how to target residual ‘Leukaemic stem cells,‘ which may give rise to relapse, however little work has been undertaken to discover the mechanisms by which the bone marrow niche may be unable to support normal haematopoietic stem cell growth and this may have a significant impact on the re-emergence of the patient's leukaemia. With the rise in the use of reduced intensity conditioning (RIC) regimes prior to transplant, the role of the patient microenvironment into which donor HSCs are engrafted warrants closer investigation.Stromal layers (AML-MSC) were derived from a cohort of 45 serial diagnostic, early (1-3 month), mid (6-9 month) and late (12 month+) post-transplant AML patients. Uniformity of stromal immunophenotyping markers (CD29, CD73, CD90, CD105, CD166, CD44, CD146) was present from passage 2 with concomitant loss of CD45 and other myeloid lineage markers (CD14, CD13, CD33, CD34). Diagnostic AML-MSCs showed a significant increase in stromal multipotency (CFU-F colony assays and CD146 expression, p<0.001) compared to both NBM and post-transplant MSCs. In comparison, early post-transplant colony formation was significantly reduced compared to other timepoints.AML-MSC supportive capacity for matched allogeneic donor (normal HSC) or autologous (malignant) blast populations was investigated using 14 day co-culture assays. Flow cytometric analysis of suspension and adherent co-culture fractions revealed AML blast cell numbers were consistently higher than comparative donor cell cultures at all post-transplant stages (p<0.001), suggesting patient AML-MSCs retain a preference for supporting their own blasts throughout different treatment stages. This observation was also confirmed in long-term LTC-IC assays in the stroma-adherent cobblestone fraction. Throughout serial samples, supportive capacity increased in later transplant AML-MSCs samples, confirming previous multipotency observations that stromal fitness to support stem cell growth may be compromised early post-transplant.Cytokine induced differentiation of AML-MSCs to osteoblast and adipocyte lineages resulted in a significant enhancement of adhesion and supportive capacity in the adipocyte lineage for AML blasts at all timepoints (p<0.002), which was absent in donor cell cultures. Osteoblast cultures gave results similar to that of the undifferentiated stromal layers for both AML and donor blast support. Parallel experiments using donor NBM-MSC layers revealed reduced support for AML blasts compared to that of matched AML-MSC stroma, however the same predisposition for adipogenic support of AML blasts was also observed.Luminex secretome profiling of serial AML-MSC supernatants using a panel of 105 pro-survival, inflammatory, migratory and immunomodulation cytokines revealed significant alterations in a number of targets associated with the diagnostic and post-transplant setting. Of the 80/105 targets detectable (>100pg/ml), high expression levels were seen in adhesion and ECM remodelling targets highlighting the ability of stromal secretory molecules to modify the surrounding environment. Hierarchical clustering analysis revealed post-transplant AML profiles correlated more closely with that of NBM-MSC at later time points, however significant increases in inflammatory/metabolic stress signalling molecules including IGFBP-3, Protein S, DKK1, VEGF, MMP1,MMP3, TPO (p,0.001), which have all been implicated HSC quiescence were associated with diagnostic and early post-transplant secretory profiles.In conclusion we identified several changes in the molecular and functional behaviour of the patient bone marrow microenvironment that may promote pro-leukaemic cell survival in post-transplant AML. Alterations in stromal fitness, adipogenic preference and metabolic stress signalling may contribute to a failing ability to support normal donor blood cells. Further exploration of therapies that target these alterations and prime the niche in favour of donor cells may provide a critical window for early clinical intervention and improved patient outcome. DisclosuresOttmann:Novartis: Consultancy; Celgene: Consultancy, Research Funding; Pfizer: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Fusion Pharma: Consultancy, Research Funding; Incyte: Consultancy, Research Funding.