Insights into Disease Progression of Translational Preclinical Rat Model of Interstitial Pulmonary Fibrosis through Endpoint Analysis.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease characterized by the relentless deposition of extracellular matrix (ECM), causing lung distortions and dysfunction. Animal models of human IPF can provide great insight into the mechanistic pathways underlying disease progression and a means for evaluating novel therapeutic approaches. In this study, we describe the effect of bleomycin concentration on disease progression in the classical rat bleomycin model. In a dose-response study (1.5, 2, 2.5 U/kg i.t), we characterized lung fibrosis at day 14 after bleomycin challenge using endpoints including clinical signs, inflammatory cell infiltration, collagen content, and bronchoalveolar lavage fluid-soluble profibrotic mediators. Furthermore, we investigated fibrotic disease progression after 2 U/kg i.t. bleomycin administration at days 3, 7, and 14 by quantifying the expression of clinically relevant signaling molecules and pathways, epithelial mesenchymal transition (EMT) biomarkers, ECM components, and histopathology of the lung. A single bleomycin challenge resulted in a progressive fibrotic response in rat lung tissue over 14 days based on lung collagen content, histopathological changes, and modified Ashcroft score. The early fibrogenesis phase (days 3 to 7) is associated with an increase in profibrotic mediators including TGFß1, IL6, TNFα, IL1ß, CINC1, WISP1, VEGF, and TIMP1. In the mid and late fibrotic stages, the TGFß/Smad and PDGF/AKT signaling pathways are involved, and clinically relevant proteins targeting galectin-3, LPA1, transglutaminase-2, and lysyl oxidase 2 are upregulated on days 7 and 14. Between days 7 and 14, the expressions of vimentin and α-SMA proteins increase, which is a sign of EMT activation. We confirmed ECM formation by increased expressions of procollagen-1Aα, procollagen-3Aα, fibronectin, and CTGF in the lung on days 7 and 14. Our data provide insights on a complex network of several soluble mediators, clinically relevant signaling pathways, and target proteins that contribute to drive the progressive fibrotic phenotype from the early to late phase (active) in the rat bleomycin model. The framework of endpoints of our study highlights the translational value for pharmacological interventions and mechanistic studies using this model.

Characterization of Large Immune Complexes with Size Exclusion Chromatography and Native Mass Spectrometry Supplemented with Gas Phase Ion Chemistry.

Large immune complexes formed by the cross-linking of antibodies with polyvalent antigens play critical roles in modulating cell-mediated immunity. While both the size and the shape of immune complexes are important determinants in Fc receptor-mediated signaling responsible for phagocytosis, degranulation, and, in some instances, autoimmune pathologies, their characterization remains extremely challenging due to their large size and structural heterogeneity. We use native mass spectrometry (MS) supplemented with limited charge reduction in the gas phase to determine the stoichiometry of immune complexes formed by a bivalent (homodimeric) antigen, a 163 kDa aminopeptidase P2 (APP2), and a monoclonal antibody (mAb) to APP2. The observed (APP2·mAb)n complexes populate a wide range of stoichiometries (n = 1-4) with the largest detected species exceeding 1 MDa, although the gas-phase dissociation products are also evident in the mass spectra. While frequently considering a nuisance that complicates interpretation of native MS data, limited dissociation provides an additional dimension for characterization of the immune complex quaternary structure. APP2/mAb associations with identical composition but slightly different elution times in size exclusion chromatography exhibit notable differences in their spontaneous fragmentation profiles. The latter indicates the presence of both extended linear and cyclized (APP2·mAb)n configurations. The unique ability of MS to distinguish between such isomeric structures will be invaluable for a variety of applications where the biological effects of immune complexes are determined by their ability to assemble Fc receptor clusters of certain density on cell surfaces, such as platelet activation by clustering the low-affinity receptors FcγRIIa on their surface.

Progression of Acute Lung Injury in Intratracheal LPS Rat Model: Efficacy of Fluticasone, Dexamethasone, and Pirfenidone.

INTRODUCTION: We investigated the potential of LPS (10-300 µg/rat) administered intratracheally (i.t.) to induce reproducible features of acute lung injury (ALI) and compared the pharmacological efficacy of anti-inflammatory glucocorticoids and antifibrotic drugs to reduce the disease. Additionally, we studied the time-dependent progression of ALI in this LPS rat model. METHODS: We conducted (1) dose effect studies of LPS administered i.t. at 10, 30, 100, and 300 µg/rat on ALI at 4 h timepoint; (2) pharmacological interventions using i.t. fluticasone (100 and 300 µg/rat), i.t. pirfenidone (4,000 µg/rat), and peroral dexamethasone (1 mg/kg) at 4 h timepoint; (3) kinetic studies at 0, 2, 4, 6, 8, 10, and 24 h post-LPS challenge. Phenotype or pharmacological efficacy was assessed using predetermined ALI features such as pulmonary inflammation, edema, and inflammatory mediators. RESULTS: All LPS doses induced a similar increase of inflammation, edema, and inflammatory mediators, e.g., IL6, IL1ß, TNFα, and CINC-1. In pharmacological intervention studies, we showed fluticasone and dexamethasone ameliorated ALI by inhibiting inflammation (>60-80%), edema (>70-100%), and the increase of cytokines IL6, IL1ß, and TNFα (≥70-90%). We also noticed some inhibition of CINC-1 (25-35%) and TIMP1 (57%) increase with fluticasone and dexamethasone. Conversely, pirfenidone failed to inhibit inflammation, edema, and mediators of inflammation. Last, in ALI kinetic studies, we observed progressive pulmonary inflammation and TIMP1 levels, which peaked at 6 h and remained elevated up to 24 h. Progressive pulmonary edema started between 2 and 4 h and was sustained at later timepoints. On average, levels of IL6 (peak at 6-8 h), IL1ß (peak at 2-10 h), TNFα (peak at 2 h), CINC-1 (peak at 2-6 h), and TGFß1 (peak at 8 h) were elevated between 2 and 10 h and declined toward 24 h post-LPS challenge. CONCLUSION: Our data show that 10 µg/rat LPS achieved a robust, profound, and reproducible experimental ALI phenotype. Glucocorticoids ameliorated key ALI features at the 4-h timepoint, but the antifibrotic pirfenidone failed. Progressive inflammation and sustained pulmonary edema were present up to 24 h, whereas levels of inflammatory mediators were dynamic during ALI progression. This study's data might be helpful in designing appropriate experiments to test the potential of new therapeutics to cure ALI.

Worldwide Regional Differences in Obesity, Elderly, and COVID-19 Mortality: Do the Exceptions Prove the Rule?

Objectives: Obesity and old age are commonly assumed to be risk factors for COVID-19 mortality. On a worldwide basis, we examine quantitative measures of obesity and elderly in the populations of individual countries and territories, and investigate whether these measures are predictive of COVID-19 mortality in those countries. In particular, we highlight regional differences relative to obesity and elderly metrics, and how these relate to COVID-19 mortality. Methods: In this retrospective, population-based study, we obtained data relating to percentages of obese or elderly individuals in 199 countries, as well as COVID-19 mortality rates in these countries. We used negative binomial regression analyses to assess associations between COVID-19 mortality rates and the putative risk factors, in six regions - Africa, Asia, Europe, North America, Oceania, and South America. Results: We found significant differences between regions relative to COVID-19 mortality, as well as obesity and elderly population proportions. There were also substantial differences between countries within regions relative to proportions of obesity and elderly individuals, and COVID-19 mortality. Conclusions: There are significant differences both between regions and within regions relative to COVID-19 mortality rates, as well as proportions of obese or elderly individuals. A global pronouncement that obesity and elderly constitute definitive risk factors for COVID-19 mortality masks the subtleties engendered by these intra- and inter-regional differences.

State Government Policy Responses to the COVID-19 Pandemic in the United States 2020-2022: Concordant Heterogeneity?

Objectives: We investigate governmental responses to the COVID-19 pandemic on a statewide basis between January 2020 and June 2022, together with mortality rates attributable to COVID-19 over the same period. Our aim is to demarcate the states' responses, and examine whether these differential responses are associated with COVID-19 mortality. Methods: Our study is based on individual state data from the Oxford COVID-19 Government Response Tracker, OxCGRT. We focus on the Government Response Index, the most comprehensive index tracked in the OxCGRT dataset. We use multivariate techniques to group the states into clusters relative to their similarities on the Government Response Index, and determine mortality rates attributable to COVID-19 in the individual groups. Results: We find that the Government Response Index was sustained at relatively constant levels in the states, with two major transition periods: a rapid rise in stringency during April through June of 2020, and a gradual decline in May and June of 2021. Heterogeneity in the Government Response Index dramatically increased in 2022. No consistent patterns emerge when relating government stringency measures with COVID-19 mortality rates. Conclusions: There is inconsistent evidence that increased governmental stringency is associated with lower COVID-19 mortality; judicious selection of time frames can lead to contrasting inferences. Political trends and motivations appear to have an outsized influence on governmental responses to the COVID-19 public health crisis, to the detriment of the populace.

Characterization of acute lung injury in the bleomycin rat model.

The aim of this study was to describe and characterize the pathophysiological changes occurring during the early inflammatory phase (first 3 days) in the rat bleomycin model of lung injury preceding the development of fibrosis. Further, we wanted to understand the kinetics and factors contributing to bleomycin-induced acute lung injury (ALI) and provide a robust, reliable and reproducible framework of features of ALI readouts to assess effects of therapeutics on bleomycin-induced ALI in rats. We induced ALI in rats with intratracheal (i.t.) installation of bleomycin. The animals were sacrificed on predetermined time points, that is, Day 0, 1, 2, and 3 post the bleomycin challenge. We analyzed bronchoalveolar lavage fluid (BALF) and lung tissue to establish and assess relevant experimental features of ALI. We demonstrated that bleomycin induced key features of experimental ALI including a profound increase in neutrophils in BALF (50-60%), pulmonary edema, and lung pathology on Day 3 after challenge. Furthermore, we showed that TGF-ß1, IL-1ß, TNF-α, IL-6, CINC-1, TIMP-1, and WISP-1 were induced by studying their kinetic profile during the first 3 days after bleomycin injury consistent with their known role ALI. We also confirmed that detectable fibrogenesis occurs at the earliest on Day 3 after injury based on collagen content, along with changes in the TGF-ß/Smad signaling pathway and increased expression of Galectin-3, Vimentin, and Fibronectin in lung homogenate. Our report presents robust features and contributing mediators/factors to the pathology of bleomycin-induced ALI in rats on Day 3. The kinetic data provide insights on the progression of ALI and a detailed understanding of early events before actual fibrosis development. This set of experimental endpoints is very appropriate and invaluable for efficacy testing of potential novel therapeutic treatments (single or combined) in ALI and understanding their mechanism of action.

Targeting caveolae to pump bispecific antibody to TGF-ß into diseased lungs enables ultra-low dose therapeutic efficacy.

The long-sought-after "magic bullet" in systemic therapy remains unrealized for disease targets existing inside most tissues, theoretically because vascular endothelium impedes passive tissue entry and full target engagement. We engineered the first "dual precision" bispecific antibody with one arm pair to precisely bind to lung endothelium and drive active delivery and the other to precisely block TGF-ß effector function inside lung tissue. Targeting caveolae for transendothelial pumping proved essential for delivering most of the injected intravenous dose precisely into lungs within one hour and for enhancing therapeutic potency by >1000-fold in a rat pneumonitis model. Ultra-low doses (µg/kg) inhibited inflammatory cell infiltration, edema, lung tissue damage, disease biomarker expression and TGF-ß signaling. The prodigious benefit of active vs passive transvascular delivery of a precision therapeutic unveils a new promising drug design, delivery and therapy paradigm ripe for expansion and clinical testing.

Déjà vu all over again: racial, ethnic and age disparities in mortality from influenza 1918-19 and COVID-19 in the United States.

Background: Examination of the mortality patterns in the United States among racial, ethnic, and age groups attributed to the 1918-19 influenza pandemic revealed stark disparities, causes for which could have been addressed and rectified this past century. However, these disparities have been amplified during the current COVID-19 pandemic.We have ignored the lessons of the past, and were destined to repeat its failings. Objectives: Compare and contrast mortality patterns by age, race, and ethnicity attributable to the 1918-19 influenza pandemic in the United States with corresponding patterns during the COVID-19 pandemic. Methods: This is a retrospective study, establishing mortality rates according to age, race and ethnicity attributable to the 1918-19 influenza pandemic in the United States and to the current COVID-19 pandemic, using mortality data published by the U.S. Public Health Service and the Centers for Disease Control and Prevention. Negative binomial regression models were used to establish rate ratios, that is, ratios of mortality rates across the various racial/ethnic groups, and associated 95% confidence intervals. Results: Mortality patterns by age differ significantly between the 1918-19 influenza pandemic and the COVID-19 pandemic: with infant and young adult (25-40 years old) mortality substantially higher in the former. Disparities in mortality between racial and ethnic groups are amplified in the COVID-19 pandemic compared to the 1918-19 experience. Conclusions: As we evaluate our nation's response to COVID-19 and design public policy to prepare better for coming pandemics, we cannot ignore the stark disparities in mortality rates experienced by different racial and ethnic groups. This will require a sustained resolve by society and government to delineate and remedy the causative factors, through science devoid of political interpretation and exploitation.

Where Do We Go From Here? A Framework for Using Susceptible-Infectious-Recovered Models for Policy Making in Emerging Infectious Diseases.

OBJECTIVES: Throughout the coronavirus disease 2019 pandemic, susceptible-infectious-recovered (SIR) modeling has been the preeminent modeling method to inform policy making worldwide. Nevertheless, the usefulness of such models has been subject to controversy. An evolution in the epidemiological modeling field is urgently needed, beginning with an agreed-upon set of modeling standards for policy recommendations. The objective of this article is to propose a set of modeling standards to support policy decision making. METHODS: We identify and describe 5 broad standards: transparency, heterogeneity, calibration and validation, cost-benefit analysis, and model obsolescence and recalibration. We give methodological recommendations and provide examples in the literature that employ these standards well. We also develop and demonstrate a modeling practices checklist using existing coronavirus disease 2019 literature that can be employed by readers, authors, and reviewers to evaluate and compare policy modeling literature along our formulated standards. RESULTS: We graded 16 articles using our checklist. On average, the articles met 6.81 of our 19 categories (36.7%). No articles contained any cost-benefit analyses and few were adequately transparent. CONCLUSIONS: There is significant room for improvement in modeling pandemic policy. Issues often arise from a lack of transparency, poor modeling assumptions, lack of a system-wide perspective in modeling, and lack of flexibility in the academic system to rapidly iterate modeling as new information becomes available. In anticipation of future challenges, we encourage the modeling community at large to contribute toward the refinement and consensus of a shared set of standards for infectious disease policy modeling.

Lessons from the Past: Methodological Issues Arising from Comparison of the Disease Burden of the Influenza A (H1N1) Pandemic 2009-10 and Seasonal Influenza 2010-2019 in the United States.

BACKGROUND: Annual influenza outbreaks constitute a major public health concern, both in the United States and worldwide. Comparisons of the health burdens of outbreaks might lead to the identification of specific at-risk populations, for whom public health resources should be marshaled appropriately and equitably. METHODS: We examined the disease burden of the 2009-10 influenza A (H1N1) pandemic relating to illnesses, medical visits, hospitalizations, and mortality, compared to influenza seasons 2010 to 2019, in the United States, as compiled by the Centers for Disease Control. RESULTS: With regard to seasonal influenza, rates of illnesses and medical visits were highest in infants aged 0-4 years, followed by adults aged 50-64 years. Rates of hospitalizations and deaths evinced a starkly different pattern, both dominated by elderly adults aged 65 and over. Youths aged 0 to 17 years were especially adversely affected by the H1N1 pandemic relative to hospitalizations and mortality compared to seasonal influenza; but curiously the opposite pattern was observed in elderly adults (aged 65 and older). CONCLUSIONS: Determination of a baseline influenza mortality profile in the United States over the 2010-19 decade is not straightforward. The disease burden of the 2009-10 influenza A pandemic among the elderly was strikingly unlike that observed in the subsequent influenza seasons 2010 to 2019: the past did not predict the future.

A Note on Excess Mortality Attributable to COVID-19 in the United States.

Background: Annual influenza outbreaks constitute a major public health concern in the United States. But this health burden appears dwarfed by the impact of COVID-19. Our aim is to quantify the excess mortality attributable to COVID-19, compared to previous influenza seasons. Methods: We retrospectively compare weekly mortality figures attributable to influenza and pneumonia in the United States from 2013 to 2019 with corresponding figures attributable to influenza, pneumonia, and COVID-19 from 2019 to 2021. We utilize a difference in differences regression methodology to estimate excess mortality observed in 2019-21 compared to 2013-2019. Results: Mortality patterns attributable to influenza, pneumonia, and COVID-19 differ significantly from the 2013-19 experience. Notably, distinct, aperiodic mortality waves occur in the 2019-2021 window, and mortality is well in excess of what is observed in typical influenza seasons. Conclusions: The COVID-19 pandemic has led to considerable excess mortality in the United States, and has strained public health resources. One might expect that the mortality waves observed during the pandemic will be damped by increasing levels of vaccination, and prior infections.

Impact of Cisplatin Dosing Regimens on Mammary Tumor Growth in an Animal Model.

BACKGROUND: We have recently introduced a modification of the seminal Simeoni model for tumor growth, the modification entailing the incorporation of delay differential equations into its formulation. We found that the modification was competitive with the Simeoni construct in modeling mammary tumor growth under cisplatin treatment in an animal model. METHODS: In our original study, we had two cohorts of animals: untreated, and treatment with bolus injection of cisplatin on day 0. We here explore how modifications in the cisplatin dosing scheme affect tumor growth in our model. RESULTS: We found that modest fractionation dosing schemes have little ultimate impact on tumor growth. In contrast, metronomic dosing schemes seem quite efficacious, and might yield effective control over tumor progression. CONCLUSIONS: With regard to cisplatin as single agent chemotherapy, a minimum level of drug for a prolonged period of time seems more critical than rapid achievement of a very high dose for a shorter time frame for deterring tumor growth or progression. Exploration of tumor dose schedules with mathematical models can provide valuable insights into potentially effective therapeutic regimens.

Different ODE models of tumor growth can deliver similar results.

BACKGROUND: Simeoni and colleagues introduced a compartmental model for tumor growth that has proved quite successful in modeling experimental therapeutic regimens in oncology. The model is based on a system of ordinary differential equations (ODEs), and accommodates a lag in therapeutic action through delay compartments. There is some ambiguity in the appropriate number of delay compartments, which we examine in this note. METHODS: We devised an explicit delay differential equation model that reflects the main features of the Simeoni ODE model. We evaluated the original Simeoni model and this adaptation with a sample data set of mammary tumor growth in the FVB/N-Tg(MMTVneu)202Mul/J mouse model. RESULTS: The experimental data evinced tumor growth heterogeneity and inter-individual diversity in response, which could be accommodated statistically through mixed models. We found little difference in goodness of fit between the original Simeoni model and the delay differential equation model relative to the sample data set. CONCLUSIONS: One should exercise caution if asserting a particular mathematical model uniquely characterizes tumor growth curve data. The Simeoni ODE model of tumor growth is not unique in that alternative models can provide equivalent representations of tumor growth.

Known Knowns, Known Unknowns, and Unknown Unknowns: Coverage in MS Experiments.

A mathematical model from ecology, namely, the capture-recapture model with a closed population and time-varying and heterogeneous individual probabilities of capture, is implemented to model the number of protein identifications across the various cycles of a mass spectroscopy experiment. Rcapture, a package available in the R computing environment, can easily provide estimates of the cardinality of the proteome from such experiments. Alternatively, model fitting can be undertaken in other software platforms, such as Matlab, that can accommodate general linear models. It has not escaped our notice that capture-recapture models can be more broadly applied to other settings, so as to estimate the number of missing observations in an experiment.

Overcoming key biological barriers to cancer drug delivery and efficacy.

Poor delivery efficiency continues to hamper the effectiveness of cancer therapeutics engineered to destroy solid tumors using different strategies such as nanocarriers, targeting agents, and matching treatments to specific genetic mutations. All contemporary systemic anti-cancer agents are dependent upon passive transvascular mechanisms for their delivery into solid tumors. The therapeutic efficacies of our current drug arsenal could be significantly improved with an active delivery strategy. Here, we discuss how drug delivery and therapeutic efficacy are greatly hindered by barriers presented by the vascular endothelial cell layer and by the aberrant nature of tumor blood vessels in general. We describe mechanisms by which molecules cross endothelial cell (EC) barriers in normal tissues and in solid tumors, including paracellular and transcellular pathways that enable passive or active transport. We also discuss specific obstacles to drug delivery that make solid tumors difficult to treat, as well strategies to overcome them and enhance drug penetration. Finally, we describe the caveolae pumping system, a promising active transport alternative to passive drug delivery across the endothelial cell barrier. Each strategy requires further testing to define its therapeutic applicability and clinical utilities.

Tumor targeting, trifunctional dendritic wedge.

We report in vitro and in vivo evaluation of a newly designed trifunctional theranostic agent for targeting solid tumors. This agent combines a dendritic wedge with high boron content for boron neutron capture therapy or boron MRI, a monomethine cyanine dye for visible-light fluorescent imaging, and an integrin ligand for efficient tumor targeting. We report photophysical properties of the new agent, its cellular uptake and in vitro targeting properties. Using live animal imaging and intravital microscopy (IVM) techniques, we observed a rapid accumulation of the agent and its retention for a prolonged period of time (up to 7 days) in fully established animal models of human melanoma and murine mammary adenocarcinoma. This macromolecular theranostic agent can be used for targeted delivery of high boron load into solid tumors for future applications in boron neutron capture therapy.

In vivo proteomic imaging analysis of caveolae reveals pumping system to penetrate solid tumors.

Technologies are needed to map and image biological barriers in vivo that limit solid tumor delivery and, ultimately, the effectiveness of imaging and therapeutic agents. Here we integrate proteomic and imaging analyses of caveolae at the blood-tumor interface to discover an active transendothelial portal to infiltrate tumors. A post-translationally modified form of annexin A1 (AnnA1) is selectively concentrated in human and rodent tumor caveolae. To follow trafficking, we generated a specific AnnA1 antibody that targets caveolae in the tumor endothelium. Intravital microscopy of caveolae-immunotargeted fluorophores even at low intravenous doses showed rapid and robust pumping across the endothelium to enter mammary, prostate and lung tumors. Within 1 h, the fluorescence signal concentrated throughout tumors to exceed the peak levels in blood. This transvascular pumping required the expression of caveolin 1 and annexin A1. Tumor uptake with other antibodies were >100-fold less. This proteomic imaging strategy reveals a unique target, antibody and caveolae pumping system for solid tumor penetration.

Experimental model of transthoracic, vascular-targeted, photodynamically induced myocardial infarction.

We describe a novel model of myocardial infarction (MI) in rats induced by percutaneous transthoracic low-energy laser-targeted photodynamic irradiation. The procedure does not require thoracotomy and represents a minimally invasive alternative to existing surgical models. Target cardiac area to be photodynamically irradiated was triangulated from the thoracic X-ray scans. The acute phase of MI was histopathologically characterized by the presence of extensive vascular occlusion, hemorrhage, loss of transversal striations, neutrophilic infiltration, and necrotic changes of cardiomyocytes. Consequently, damaged myocardium was replaced with fibrovascular and granulation tissue. The fibrotic scar in the infarcted area was detected by computer tomography imaging. Cardiac troponin I (cTnI), a specific marker of myocardial injury, was significantly elevated at 6 h (41 ± 6 ng/ml, n = 4, P < 0.05 vs. baseline) and returned to baseline after 72 h. Triphenyltetrazolium chloride staining revealed transmural anterolateral infarcts targeting 25 ± 3% of the left ventricle at day 1 with a decrease to 20 ± 3% at day 40 (n = 6 for each group, P < 0.01 vs. day 1). Electrocardiography (ECG) showed significant ST-segment elevation in the acute phase with subsequent development of a pathological Q wave and premature ventricular contractions in the chronic phase of MI. Vectorcardiogram analysis of spatiotemporal electrical signal transduction revealed changes in inscription direction, QRS loop morphology, and redistribution in quadrant areas. The photodynamically induced MI in n = 51 rats was associated with 12% total mortality. Histological findings, ECG abnormalities, and elevated cTnI levels confirmed the photosensitizer-dependent induction of MI after laser irradiation. This novel rodent model of MI might provide a platform to evaluate new diagnostic or therapeutic interventions.

Assessing the barriers to image-guided drug delivery.

Imaging has become a cornerstone for medical diagnosis and the guidance of patient management. A new field called image-guided drug delivery (IGDD) now combines the vast potential of the radiological sciences with the delivery of treatment and promises to fulfill the vision of personalized medicine. Whether imaging is used to deliver focused energy to drug-laden particles for enhanced, local drug release around tumors, or it is invoked in the context of nanoparticle-based agents to quantify distinctive biomarkers that could risk stratify patients for improved targeted drug delivery efficiency, the overarching goal of IGDD is to use imaging to maximize effective therapy in diseased tissues and to minimize systemic drug exposure in order to reduce toxicities. Over the last several years, innumerable reports and reviews covering the gamut of IGDD technologies have been published, but inadequate attention has been directed toward identifying and addressing the barriers limiting clinical translation. In this consensus opinion, the opportunities and challenges impacting the clinical realization of IGDD-based personalized medicine were discussed as a panel and recommendations were proffered to accelerate the field forward.

III. Cellular ultrastructures in situ as key to understanding tumor energy metabolism: biological significance of the Warburg effect.

Despite the universality of metabolic pathways, malignant cells were found to have their metabolism reprogrammed to generate energy by glycolysis even under normal oxygen concentrations (the Warburg effect). Therefore, the pathway energetically 18 times less efficient than oxidative phosphorylation was implicated to match increased energy requirements of growing tumors. The paradox was explained by an abnormally high rate of glucose uptake, assuming unlimited availability of substrates for tumor growth in vivo. However, ultrastructural analysis of tumor vasculature morphogenesis showed that the growing tissue regions did not have continuous blood supply and intermittently depended on autophagy for survival. Erythrogenic autophagy, and resulting ATP generation by glycolysis, appeared critical to initiating vasculature formation where it was missing. This study focused on ultrastructural features that reflected metabolic switch from aerobic to anaerobic. Morphological differences between and within different types of cells were evident in tissue sections. In cells undergoing nucleo-cytoplasmic conversion into erythrosomes (erythrogenesis), gradual changes led to replacing mitochondria with peroxisomes, through an intermediate form connected to endoplasmic reticulum. Those findings related to the issue of peroxisome biogenesis and to the phenomenon of hemogenic endothelium. Mitochondria were compacted also during mitosis. In vivo, cells that lost and others that retained capability to use oxygen coexisted side-by-side; both types were important for vasculature morphogenesis and tissue growth. Once passable, the new vasculature segment could deliver external oxygen and nutrients. Nutritional and redox status of microenvironment had similar effect on metabolism of malignant and non-malignant cells demonstrating the necessity to maintain structure-energy equivalence in all living cells. The role of glycolysis in initiating vasculature formation, and in progression of cell cycle through mitosis, indicated that Warburg effect had a fundamental biological significance extending to non-malignant tissues. The approach used here could facilitate integration of accumulated cyber knowledge on cancer metabolism into predictive science.