Computing the frequency-dependent NMR relaxation of 1H nuclei in liquid water.

We present a computational framework for reliably determining the frequency-dependent intermolecular and intramolecular nuclear magnetic resonance (NMR) dipole-dipole relaxation rates of spin 1/2 nuclei from Molecular Dynamics (MD) simulations. This approach avoids the alterations caused by the well-known finite-size effects of translational diffusion. Moreover, a procedure is derived to control and correct for effects caused by fixed distance-sampling cutoffs and periodic boundary conditions. By construction, this approach is capable of accurately predicting the correct low-frequency scaling behavior of the intermolecular NMR dipole-dipole relaxation rate and thus allows for the reliable calculation of the frequency-dependent relaxation rate over many orders of magnitude. Our approach is based on the utilization of the theory of Hwang and Freed for the intermolecular dipole-dipole correlation function and its corresponding spectral density [L.-P. Hwang and J. H. Freed, J. Chem. Phys. 63, 4017-4025 (1975)] and its combination with data from MD simulations. The deviations from the Hwang and Freed theory caused by periodic boundary conditions and sampling distance cutoffs are quantified by means of random walker Monte Carlo simulations. An expression based on the Hwang and Freed theory is also suggested for correcting those effects. As a proof of principle, our approach is demonstrated by computing the frequency-dependent intermolecular and intramolecular dipolar NMR relaxation rates of 1H nuclei in liquid water at 273 and 298 K based on the simulations of the TIP4P/2005 model. Our calculations are suggesting that the intermolecular contribution to the 1H NMR relaxation rate of the TIP4P/2005 model in the extreme narrowing limit has previously been substantially underestimated.

Computing Accurate True Self-Diffusion Coefficients and Shear Viscosities Using the OrthoBoXY Approach.

In a recent paper [Busch, J.; Paschek, D. J. Phys. Chem. B 2023, 127, 7983-7987], we have shown that for molecular dynamics (MD) simulations using orthorhombic periodic boundary conditions with "magic" box length ratios of Lz/Lx = Lz/Ly = 2.7933596497, the self-diffusion coefficients Dx and Dy in x- and y-directions are independent of the system size. They both represent the true self-diffusion coefficient D0 = (Dx + Dy)/2, while the shear viscosity can be calculated from diffusion coefficients in x-, y-, and z-directions, using η = kBT·8.1711245653/[3πLz(Dx + Dy - 2Dz)]. In this contribution, we test this "OrthoBoXY" approach by its application to a variety of different systems: liquid water, dimethyl ether, methanol, triglyme, water/methanol mixtures, water/triglyme mixtures, and imidazolium-based ionic liquids. The chosen systems range from small-sized molecular liquids to complex mixtures and ionic liquids, while spanning a viscosity range of almost 3 orders of magnitude. We assess the efficiency of the method for computing true self-diffusion and viscosity data and provide simple formulas for estimating the required MD simulation lengths and sizes for delivering reliable data with targeted uncertainty levels. Our analysis of the system size dependence of statistical uncertainties for both the viscosity and the self-diffusion coefficient leads us to the conclusion that it is preferable to extend the simulation length instead of increasing the system size. MD simulations consisting of 768 molecules or ion pairs seem to be perfectly adequate.

An OrthoBoXY-method for various alternative box geometries.

We have shown in a recent contribution [Busch and Paschek, J. Phys. Chem. B, 2023 127, 7983-7987] that for molecular dynamics (MD) simulations of isotropic fluids based on orthorhombic periodic boundary conditions with "magical" box length ratios of Lz/Lx = Lz/Ly = 2.7933596497, the computed self-diffusion coefficients Dx and Dy in x- and y-direction become system size independent. They thus represent the true self-diffusion coefficient D0 = (Dx + Dy)/2, while the shear viscosity can be determined from diffusion coefficients in x-, y-, and z-direction, using the expression η = kBT·8.1711245653/[3πLz(Dx + Dy - 2Dz)]. Here we present a more generalized version of this "OrthoBoXY"-approach, which can be applied to any orthorhombic MD box of any shape. In particular, we would like to test, how the efficiency is affected by using a shape more akin to the cubic form, albeit with different box length ratios Lx/Lz ≠ Ly/Lz and Lx < Ly < Lz. We use NVT and NpT simulations of systems of 1536 TIP4P/2005 water molecules as a benchmark and explore different box geometries to determine the influence of the box shape on the computed statistical uncertainties for D0 and η. Moreover, another "magical" set of box length ratios is discovered with Ly/Lz = 0.57804765578 and Lx/Lz = 0.33413909235, where the self-diffusion coefficient in x-direction becomes system size independent, such that D0 = Dx.

Dissecting Clinical Features of COVID-19 in a Cohort of 21,312 Acute Care Patients.

COVID-19 has resulted in over 645 million hospitalization and 7 million deaths globally. However, many questions still remain about clinical complications in COVID-19 and if these complications changed with different circulating SARS-CoV-2 strains. We analyzed a 2.5-year retrospective cohort of 47,063 encounters for 21,312 acute care patients at five Central Texas hospitals and define distinct trajectory groups (TGs) with latent class mixed modeling, based on the World Health Organization COVID-19 Ordinal Scale. Using this TG framework, we evaluated the association of demographics, diagnoses, vitals, labs, imaging, consultations, and medications with COVID-19 severity and broad clinical outcomes. Patients within 6 distinct TGs differed in manifestations of multi-organ disease and multiple clinical factors. The proportion of mild patients increased over time, particularly during Omicron waves. Age separated mild and fatal patients, though did not distinguish patients with severe versus critical disease. Male and Hispanic/Latino demographics were associated with more severe/critical TGs. More severe patients had a higher rate of neuropsychiatric diagnoses, consultations, and brain imaging, which did not change significantly in severe patients across SARS-CoV-2 variant waves. More severely affected patients also demonstrated an immunological signature of high neutrophils and immature granulocytes, and low lymphocytes and monocytes. Interestingly, low albumin was one of the best lab predictors of COVID-19 severity in association with higher malnutrition in severe/critical patients, raising concern of nutritional insufficiency influencing COVID-19 outcomes. Despite this, only a small fraction of severe/critical patients had nutritional labs checked (pre-albumin, thiamine, Vitamin D, B vitamins) or received targeted interventions to address nutritional deficiencies such as vitamin replacement. Our findings underscore the significant link between COVID-19 severity, neuropsychiatric complications, and nutritional insufficiency as key risk factors of COVID-19 outcomes and raise the question of the need for more widespread early assessment of patients' neurological, psychiatric, and nutritional status in acute care settings to help identify those at risk of severe disease outcomes.

OrthoBoXY: A Simple Way to Compute True Self-Diffusion Coefficients from MD Simulations with Periodic Boundary Conditions without Prior Knowledge of the Viscosity.

Recently, an analytical expression for the system size dependence and direction-dependence of self-diffusion coefficients for neat liquids due to hydrodynamic interactions has been derived for molecular dynamics (MD) simulations using orthorhombic unit cells. Based on this description, we show that for systems with a "magic" box length ratio of Lz/Lx = Lz/Ly = 2.7933596497 the computed self-diffusion coefficients Dx and Dy in the x- and y-direction become system-size independent and represent the true self-diffusion coefficient D0 = (Dx + Dy)/2. Moreover, by using this particular box geometry, the viscosity can be determined with a reasonable degree of accuracy from the difference of components of the diffusion coefficients in x-, y-, and z-directions using the simple expression η = kBT × 8.1711245653/[3πLz(Dx + Dy - 2Dz)], where kB denotes Boltzmann's constant and T represents the temperature. MD simulations of TIP4P/2005 water for various system sizes using both orthorhombic and cubic box geometries are used to test the approach.

Exploring the Impact of COVID-19 on Women Hospitalists: A Mixed-Gender Qualitative Analysis.

BACKGROUND: Women physicians have faced persistent challenges, including gender bias, salary inequities, a disproportionate share of caregiving and domestic responsibilities, and limited representation in leadership. Data indicate the COVID-19 pandemic further highlighted and exacerbated these inequities. OBJECTIVE: To understand the pandemic's impact on women physicians and to brainstorm solutions to better support women physicians. DESIGN: Mixed-gender semi-structured focus groups. PARTICIPANTS: Hospitalists in the Hospital Medicine Reengineering Network (HOMERuN). APPROACH: Six semi-structured virtual focus groups were held with 22 individuals from 13 institutions comprised primarily of academic hospitalist physicians. Rapid qualitative methods including templated summaries and matrix analysis were applied to identify major themes and subthemes. KEY RESULTS: Four key themes emerged: (1) the pandemic exacerbated perceived gender inequities, (2) women's academic productivity and career development were negatively impacted, (3) women held disproportionate roles as caregivers and household managers, and (4) institutional pandemic responses were often misaligned with workforce needs, especially those of women hospitalists. Multiple interventions were proposed including: creating targeted workforce solutions and benefits to address the disproportionate caregiving burden placed on women, addressing hospitalist scheduling and leave practices, ensuring promotion pathways value clinical and COVID-19 contributions, creating transparency around salary and non-clinical time allocation, and ensuring women are better represented in leadership roles. CONCLUSIONS: Hospitalists perceived and experienced that women physicians faced negative impacts from the pandemic in multiple domains including leadership opportunities and scholarship, while also shouldering larger caregiving duties than men. There are many opportunities to improve workplace conditions for women; however, current institutional efforts were perceived as misaligned to actual needs. Thus, policy and programmatic changes, such as those proposed by this cohort of hospitalists, are needed to advance equity in the workplace.

Role of Hydrogen Bond Defects for Cluster Formation and Distribution in Ionic Liquids by Means of Neutron Diffraction and Molecular Dynamics Simulations.

Defects fundamentally govern the properties of all real materials. Correlating molecular defects to macroscopic quantities remains a challenge, particularly in the liquid phase. Herein, we report the influence of hydrogen bonds (HB) acting as defects in mixtures of non-hydroxyl-functionalized ionic liquids (ILs) with an increasing concentration of hydroxyl-functionalized ILs. We observed two types of HB defects: The conventional HBs between cation and anion (c-a), and the elusive HBs between cations (c-c) despite the repulsive Coulomb forces. We use neutron diffraction with isotopic substitution in combination with molecular dynamics simulations for measuring the geometry, strength, and distribution of mobile OH defects in the IL mixtures. In principle, this procedure allows relating the number and stability of defects to macroscopic properties such as diffusion, viscosity, and conductivity, which are of utmost importance for the performance of electrolytes in batteries and other electrical devices.

Quantification and Distribution of Three Types of Hydrogen Bonds in Mixtures of an Ionic Liquid with the Hydrogen-Bond-Accepting Molecular Solvent DMSO Explored by Neutron Diffraction and Molecular Dynamics Simulations.

The concept of hydrogen bonding is celebrating its 100th birthday. Hydrogen bonds (H-bonds) play a key role in the structure and function of biological molecules, the strength of materials, and molecular binding. Herein, we study H-bonding in mixtures of a hydroxyl-functionalized ionic liquid with the neutral, H-bond-accepting molecular liquid dimethylsulfoxide (DMSO) using neutron diffraction experiments and molecular dynamics simulations. We report the geometry, strength, and distribution of three different types of H-bond OH···O, formed between the hydroxyl group of the cation and either the oxygen atom of another cation, the counteranion, or the neutral molecule. Such a variety of different strengths and distributions of H-bonds in one single mixture could hold the promise of providing solvents with potential applications in H-bond-related chemistry, for example, to alter the natural selectivity patterns of catalytic reactions or the conformation of catalysts.

Practical Applications of Rapid Qualitative Analysis for Operations, Quality Improvement, and Research in Dynamically Changing Hospital Environments.

BACKGROUND: Health care systems are in a constant state of change. As such, methods to quickly acquire and analyze data are essential to effectively evaluate current processes and improvement projects. Rapid qualitative analysis offers an expeditious approach to evaluate complex, dynamic, and time-sensitive issues. METHODS: We used rapid data acquisition and qualitative methods to assess six real-world problems the hospitalist field faced during the COVID-19 pandemic. We iteratively modified and applied a six-step framework for conducting rapid qualitative analysis, including determining if rapid methods are appropriate, creating a team, selecting a data collection approach, data analysis, and synthesis and dissemination. Virtual platforms were used for focus groups and interviews; templated summaries and matrix analyses were then applied to allow for rapid qualitative analyses. RESULTS: We conducted six projects using rapid data acquisition and rapid qualitative analysis from December 4, 2020, to January 14, 2022, each of which included 23 to 33 participants. One project involved participants from a single institution; the remainder included participants from 15 to 24 institutions. These projects led to the refinement of an adapted rapid qualitative method for evaluation of hospitalist-driven operational, research, and quality improvement efforts. We describe how we used these methods and disseminated our results. We also discuss situations for which rapid qualitative methods are well-suited and strengths and weaknesses of the methods. CONCLUSION: Rapid qualitative methods paired with rapid data acquisition can be employed for prompt turnaround assessments of quality, operational, and research projects in complex health care environments. Although rapid qualitative analysis is not meant to replace more traditional qualitative methods, it may be appropriate in certain situations. Application of a framework to guide projects using a rapid qualitative approach can help provide structure to the analysis and instill confidence in the findings.

An exact a posteriori correction for hydrogen bond population correlation functions and other reversible geminate recombinations obtained from simulations with periodic boundary conditions. Liquid water as a test case.

The kinetics of breaking and re-formation of hydrogen bonds (HBs) in liquid water is a prototype of reversible geminate recombination. HB population correlation functions (HBPCFs) are a means to study the HB kinetics. The long-time limiting behavior of HBPCFs is controlled by translatoric diffusion and shows a t-3/2 time-dependence, which can be described by analytical expressions based on the HB acceptor density and the donor-acceptor inter-diffusion coefficient. If the trajectories are not properly "unwrapped," the presence of periodic boundary conditions (PBCs) can perturb this long-time limiting behavior. Keeping the trajectories "wrapped," however, allows for a more efficient calculation of HBPCFs. We discuss the consequences of PBCs in combination with "wrapped" trajectories following from the approximations according to Luzar-Chandler and according to Starr, each deviating in a different fashion from the true long-time limiting behavior, but enveloping the unperturbed function. A simple expression is given for estimating the maximum time up to which the computed HBPCFs reliably describe the long-time limiting behavior. In addition, an exact a posteriori correction for systems with PBCs for "wrapped" trajectories is derived, which can be easily computed and which is able to fully recover the true t-3/2 long-time behavior. For comparison, HBPCFs are computed from MD simulations of TIP4P/2005 model water for varying system sizes and temperatures of 273 and 298 K using this newly introduced correction. Implications for the computations of HB lifetimes and the effect of the system-size are discussed.

Is the effect of precipitation on acute gastrointestinal illness in southwestern Uganda different between Indigenous and non-Indigenous communities?

Acute gastrointestinal illness (AGI) is a global public health priority that often disproportionately effects Indigenous populations. While previous research examines the association between meteorological conditions and AGI, little is known about how socio-cultural factors may modify this relationship. This present study seeks to address this research gap by comparing AGI prevalence and determinants between an Indigenous and non-Indigenous population in Uganda. We estimate the 14-day self-reported prevalence of AGI among adults in an Indigenous Batwa population and their non-Indigenous neighbours using cross-sectional panel data collected over four periods spanning typically rainy and dry seasons (January 2013 to April 2014). The independent associations between Indigenous status, precipitation, and AGI are examined with multivariable multi-level logistic regression models, controlling for relative wealth status and clustering at the community level. Estimated prevalence of AGI among the Indigenous Batwa was greater than among the non-Indigenous Bakiga. Our models indicate that both Indigenous identity and decreased levels of precipitation in the weeks preceding the survey period were significantly associated with increased AGI, after adjusting for confounders. Multivariable models stratified by Indigenous identity suggest that Indigenous identity may not modify the association between precipitation and AGI in this context. Our results suggest that short-term changes in precipitation affect both Indigenous and non-Indigenous populations similarly, though from different baseline AGI prevalences, maintaining rather than exacerbating this socially patterned health disparity. In the context of climate change, these results may challenge the assumption that changing weather patterns will necessarily exacerbate existing socially patterned health disparities.

Visual Awareness in Binocular Rivalry Modulates Induced Pupil Fluctuations.

When a visual stimulus oscillates in luminance, pupil size follows this oscillation. Recently, it has been demonstrated that such induced pupil oscillations can be used to tag which stimulus is covertly attended. Here we ask whether this "pupil frequency tagging" approach can be extended to visual awareness, specifically to inferring perceptual dominance in Binocular Rivalry between complex stimuli. We presented two distinct stimuli, a face and a house, to each eye and modulated their luminance at 1.7 Hz either in counter-phase (180° phase shift), with a 90° phase shift or in phase (0° control). In some conditions, we additionally asked observers to attend either of the stimuli. The luminance modulation was sufficiently subtle that rivalry dynamics did not differ among these conditions, and was also indistinguishable from unmodulated presentation of the stimuli. For the 180° and the 90° phase-shifted stimuli, we found that the phase of the pupil response relative to the stimuli was modulated by perceptual dominance; that is, the relative phase depended on the stimulus the observer was aware of. In turn, this perceptually dominant stimulus could be decoded from the phase of the pupil response significantly above chance. Neither percept dependence of the phase nor significant decoding was found for the 0° control condition. Our results show that visual awareness modulates pupil responses and provide proof of principle that dominance in rivalry for complex stimuli can be inferred from induced pupil fluctuations.

Increased expression of the frontotemporal dementia risk factor TMEM106B causes C9orf72-dependent alterations in lysosomes.

Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) is an important cause of dementia in individuals under age 65. Common variants in the TMEM106B gene were previously discovered by genome-wide association to confer genetic risk for FTLD-TDP (p = 1 × 10-11, OR = 1.6). Furthermore, TMEM106B may act as a genetic modifier affecting age at onset and age at death in the Mendelian subgoup of FTLD-TDP due to expansions of the C9orf72 gene. Evidence suggests that TMEM106B variants increase risk for developing FTLD-TDP by increasing expression of Transmembrane Protein 106B (TMEM106B), a lysosomal protein. To further understand the functional role of TMEM106B in disease pathogenesis, we investigated the cell biological effects of increased TMEM106B expression. Here, we report that increased TMEM106B expression results in the appearance of a vacuolar phenotype in multiple cell types, including neurons. Concomitant with the development of this vacuolar phenotype, cells over-expressing TMEM106B exhibit impaired lysosomal acidification and degradative function, as well as increased cytotoxicity. We further identify a potential lysosomal sorting motif for TMEM106B and demonstrate that abrogation of sorting to lysosomes rescues TMEM106B-induced defects. Finally, we show that TMEM106B-induced defects are dependent on the presence of C9orf72, as knockdown of C9orf72 also rescues these defects. In sum, our results suggest that TMEM106B exerts its effects on FTLD-TDP disease risk through alterations in lysosomal pathways. Furthermore, TMEM106B and C9orf72 may interact in FTLD-TDP pathophysiology.

Expression of TMEM106B, the frontotemporal lobar degeneration-associated protein, in normal and diseased human brain.

BACKGROUND: Frontotemporal lobar degeneration (FTLD) is the second most common cause of dementia in individuals under 65 years old and manifests as alterations in behavior, personality, or language secondary to degeneration of the frontal and/or temporal lobes. FTLD-TDP, the largest neuropathological subset of FTLD, is characterized by hyperphosphorylated, ubiquitinated TAR DNA-binding protein 43 (TDP-43) inclusions. Mutations in progranulin (GRN), a neuroprotective growth factor, are one of the most common Mendelian genetic causes of FTLD-TDP. Moreover, a recent genome-wide association study (GWAS) identified multiple SNPs within the uncharacterized gene TMEM106B that significantly associated with FTLD-TDP, suggesting that TMEM106B genotype confers risk for FTLD-TDP. Indeed, TMEM106B expression levels, which correlate with TMEM106B genotype, may play a role in the pathogenesis of disease. RESULTS: Since little is known about TMEM106B and its expression in human brain, we performed immunohistochemical studies of TMEM106B in postmortem human brain samples from normal individuals, FTLD-TDP individuals with and without GRN mutations, and individuals with other neurodegenerative diseases. We find that TMEM106B protein is cytoplasmically expressed in both histopathologically affected and unaffected areas of the brain by neurons, glia, and endothelial cells/pericytes. Furthermore, we demonstrate that TMEM106B expression may differ among neuronal subtypes. Finally, we show that TMEM106B neuronal expression is significantly more disorganized in FTLD-TDP cases with GRN mutations, compared to normal and disease controls, including FTLD-TDP cases without GRN mutations. CONCLUSIONS: Our data provide an initial neuropathological characterization of the newly discovered FTLD-TDP-associated protein TMEM106B. In addition, we demonstrate that FTLD-TDP cases with GRN mutations exhibit a loss of neuronal TMEM106B subcellular localization, adding to evidence that TMEM106B and progranulin may be pathophysiologically linked in FTLD-TDP.

SDF-1α mediates wound-promoted tumor growth in a syngeneic orthotopic mouse model of breast cancer.

Increased growth of residual tumors in the proximity of acute surgical wounds has been reported; however, the mechanisms of wound-promoted tumor growth remain unknown. Here, we used a syngeneic, orthotopic mouse model of breast cancer to study mechanisms of wound-promoted tumor growth. Our results demonstrate that exposure of metastatic mouse breast cancer cells (4T1) to SDF-1α, which is increased in wound fluid, results in increased tumor growth. Both, wounding and exposure of 4T1 cells to SDF-1α not only increased tumor growth, but also tumor cell proliferation rate and stromal collagen deposition. Conversely, systemic inhibition of SDF-1α signaling with the small molecule AMD 3100 abolished the effect of wounding, and decreased cell proliferation, collagen deposition, and neoangiogenesis to the levels observed in control animals. Furthermore, using different mouse strains we could demonstrate that the effect of wounding on tumor growth and SDF-1α levels is host dependent and varies between mouse strains. Our results show that wound-promoted tumor growth is mediated by elevated SDF-1α levels and indicate that the effect of acute wounds on tumor growth depends on the predetermined wound response of the host background and its predetermined wound response.

TMEM106B, the risk gene for frontotemporal dementia, is regulated by the microRNA-132/212 cluster and affects progranulin pathways.

Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) is a fatal neurodegenerative disease with no available treatments. Mutations in the progranulin gene (GRN) causing impaired production or secretion of progranulin are a common Mendelian cause of FTLD-TDP; additionally, common variants at chromosome 7p21 in the uncharacterized gene TMEM106B were recently linked by genome-wide association to FTLD-TDP with and without GRN mutations. Here we show that TMEM106B is neuronally expressed in postmortem human brain tissue, and that expression levels are increased in FTLD-TDP brain. Furthermore, using an unbiased, microarray-based screen of >800 microRNAs (miRs), we identify microRNA-132 as the top microRNA differentiating FTLD-TDP and control brains, with <50% normal expression levels of three members of the microRNA-132 cluster (microRNA-132, microRNA-132*, and microRNA-212) in disease. Computational analyses, corroborated empirically, demonstrate that the top mRNA target of both microRNA-132 and microRNA-212 is TMEM106B; both microRNAs repress TMEM106B expression through shared microRNA-132/212 binding sites in the TMEM106B 3'UTR. Increasing TMEM106B expression to model disease results in enlargement and poor acidification of endo-lysosomes, as well as impairment of mannose-6-phosphate-receptor trafficking. Finally, endogenous neuronal TMEM106B colocalizes with progranulin in late endo-lysosomes, and TMEM106B overexpression increases intracellular levels of progranulin. Thus, TMEM106B is an FTLD-TDP risk gene, with microRNA-132/212 depression as an event which can lead to aberrant overexpression of TMEM106B, which in turn alters progranulin pathways. Evidence for this pathogenic cascade includes the striking convergence of two independent, genomic-scale screens on a microRNA:mRNA regulatory pair. Our findings open novel directions for elucidating miR-based therapies in FTLD-TDP.

Angiogenesis and the tumor vasculature as antitumor immune modulators: the role of vascular endothelial growth factor and endothelin.

Cancer immunotherapies have yielded promising results in recent years, but new approaches must be utilized if more patients are to experience the benefits of these therapies. Angiogenesis and the tumor endothelium confer unique immune privilege to a growing tumor, with significant effects on diverse immunological processes such as hematopoietic cell maturation, antigen presentation, effector T cell differentiation, cytokine production, adhesion, and T cell homing and extravasation. Here, we review the role of angiogenesis and the tumor endothelium on regulation of the antitumor immune response. We place particular emphasis on the role of vascular endothelial growth factor (VEGF) in the suppression of numerous immunological processes that control tumor progression. Further, we describe the unique crosstalk between the VEGF and endothelin systems, and how their interactions may shape the antitumor immune response. These insights establish new targets for combinatorial approaches to modify existing cancer immunotherapies.

Transient tumor-fibroblast interactions increase tumor cell malignancy by a TGF-Beta mediated mechanism in a mouse xenograft model of breast cancer.

Carcinoma are complex societies of mutually interacting cells in which there is a progressive failure of normal homeostatic mechanisms, causing the parenchymal component to expand inappropriately and ultimately to disseminate to distant sites. When a cancer cell metastasizes, it first will be exposed to cancer associated fibroblasts in the immediate tumor microenvironment and then to normal fibroblasts as it traverses the underlying connective tissue towards the bloodstream. The interaction of tumor cells with stromal fibroblasts influences tumor biology by mechanisms that are not yet fully understood. Here, we report a role for normal stroma fibroblasts in the progression of invasive tumors to metastatic tumors. Using a coculture system of human metastatic breast cancer cells (MCF10CA1a) and normal murine dermal fibroblasts, we found that medium conditioned by cocultures of the two cell types (CoCM) increased migration and scattering of MCF10CA1a cells in vitro, whereas medium conditioned by homotypic cultures had little effect. Transient treatment of MCF10CA1a cells with CoCM in vitro accelerated tumor growth at orthotopic sites in vivo, and resulted in an expanded pattern of metastatic engraftment. The effects of CoCM on MCF10CA1a cells were dependent on small amounts of active TGF-beta1 secreted by fibroblasts under the influence of the tumor cells, and required intact ALK5-, p38-, and JNK signaling in the tumor cells. In conclusion, these results demonstrate that transient interactions between tumor cells and normal fibroblasts can modify the acellular component of the local microenvironment such that it induces long-lasting increases in tumorigenicity and alters the metastatic pattern of the cancer cells in vivo. TGF-beta appears to be a key player in this process, providing further rationale for the development of anti-cancer therapeutics that target the TGF-beta pathway.

Complex display of putative tumor stem cell markers in the NCI60 tumor cell line panel.

Tumor stem cells or cancer initiating cells (CICs) are single tumor cells that can regenerate a tumor or a metastasis. The identification and isolation of CICs remain challenging, and a variety of putative CIC markers have been described. We hypothesized that cell lines of the NCI60 panel contain CICs and express putative CIC markers. We investigated expression of putative CIC surface markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP) and the activity of aldehyde dehydrogenase in the NCI60 panel singly and in combination by six-color fluorescence-activated cell sorting analysis. All investigated markers were expressed in cell lines of the NCI60 panel. Expression levels of individual markers varied widely across the 60 cell lines, and neither single marker expression nor simple combinations nor co-expression patterns correlated with the colony-formation capacity of cell lines. Rather, marker expression patterns correlated with tumor types in multidimensional analysis. Whereas some expression patterns correlated with tumor entities such as basal breast cancer, other expression patterns occurred across different tumor types and largely related to expression of a more mesenchymal phenotype in individual breast, lung, renal, and melanoma cell lines. Our data for the first time demonstrate that tumor cell lines display CIC markers in a complex pattern that relates to the tumor type. The complexity and tumor type specificity of marker display creates challenges for the application of cell sorting and other approaches to isolation of putative tumor stem cell populations and suggests that therapeutic targeting strategies will need to take this into account.

Acute wounds accelerate tumorigenesis by a T cell-dependent mechanism.

We investigated the influence of acute wounding on tumor growth in a syngeneic mouse breast cancer model. Metastatic mouse breast cancer cells (4T1) were orthotopically injected into the mammary fat pads of BALB/c mice, and animals were wounded locally by full thickness dermal incisions above the mammary fat pads or remotely above the scapula 9 days later. Local, but not remote, wounding increased tumor size when compared with sham treatment. Injection of wound fluid close to the tumor site increased tumor growth, whereas in vitro wound fluid compared with serum increased the proliferation rate of 4T1 cells. Our results show that wound stroma can unfavorably influence growth of nearby tumors. This effect is T cell-dependent, as local wounding had no effect on tumor growth in nu/nu mice. The effect of wounding on tumor growth can be mimicked by acellular wound fluid, suggesting that T cells secrete or mediate secretion of cytokines or growth factors that then accelerate tumor growth. Here, we define an experimental model of wound-promoted tumor growth that will enable us to identify mechanisms and therapeutic targets to reduce the negative effect of tissue repair on residual tumors.