A Microfluidic Platform for Evaluating the Internalization of Liposome Drug Carriers in Tumor Spheroids.

The development of in vitro models recapitulating nanoparticle transport under physiological flow conditions is of great importance for predicting the efficacy of nanoparticle drug carriers. Liposomes are extensively used for drug delivery owing to their biocompatibility and biodegradability and the ability to carry both hydrophilic and hydrophobic compounds. Here, we used a library of liposomes with various dimensions and a microfluidic platform comprising a large array of uniformly sized breast cancer spheroids to explore size-dependent liposome internalization and retention in the spheroids under close-to-physiological interstitial conditions. Such a platform showed promising applications in the preclinical screening of small molecule drugs; however, the capability to deliver nanoparticles in the spheroid interior under close-to-physiological flow conditions was not explored. For the liposomes with diameters in the range of 45-200 nm, we show experimentally and by simulations that in comparison with liposome delivery solely by diffusion, flow significantly enhances liposome internalization in the microgels and mitigates the size-dependent spheroid penetration by the liposomes. The utility of the microfluidic platform was validated by evaluating the efficacy of clinically approved doxorubicin-loaded liposomes (Doxil), which exhibited superior retention in the spheroids under flow conditions, in comparison with free doxorubicin. This MF platform can serve as an in vitro model for screening the efficacy of drugs encapsulated in liposomes and find applications for screening other types of nanoparticle carriers for vaccine delivery, diagnostics, and skincare.

Elimination of Cancer Cells in Co-Culture: Role of Different Nanocarriers in Regulation of CD47 and Calreticulin-Induced Phagocytosis.

Under healthy conditions, pro- and anti-phagocytic signals are balanced. Cluster of Differentiation 47 (CD47) is believed to act as an anti-phagocytic marker that is highly expressed on multiple types of human cancer cells including acute myeloid leukemia (AML) and lung and liver carcinomas, allowing them to escape phagocytosis by macrophages. Downregulating CD47 on cancer cells discloses calreticulin (CRT) to macrophages and recovers their phagocytic activity. Herein, we postulate that using a modified graphene oxide (GO) carrier to deliver small interfering RNA (siRNA) CD47 (CD47_siRNA) in AML, A549 lung, and HepG2 liver cancer cells in co-culture in vitro will silence CD47 and flag cancer cells for CRT-mediated phagocytosis. Results showed a high knockdown efficiency of CD47 and a significant increase in CRT levels simultaneously by using GO formulation as carriers in all used cancer cell lines. The presence of CRT on cancer cells was significantly higher than levels before knockdown of CD47 and was required to achieve phagocytosis in co-culture with human macrophages. Lipid nanoparticles (LNPs) and modified boron nitride nanotubes (BNPs) were used to carry CD47_siRNA, and the knockdown efficiency values of CD47 were compared in three cancer cells in co-culture, with an achieved knockdown efficiency of >95% using LNPs as carriers. Interestingly, the high efficiency of CD47 knockdown was obtained by using the LNPs and BNP carriers; however, an increase in CRT levels on cancer cells was not required for phagocytosis to happen in co-culture with human macrophages, indicating other pathways' involvement in the phagocytosis process. These findings highlight the roles of 2D (graphene oxide), 1D (boron nitride nanotube), and "0D" (lipid nanoparticle) carriers for the delivery of siRNA to eliminate cancer cells in co-culture, likely through different phagocytosis pathways in multiple types of human cancer cells. Moreover, these results provide an explanation of immune therapies that target CD47 and the potential use of these carriers in screening drugs for such therapies in vitro.

Upregulated integrin α11 in the stroma of cutaneous squamous cell carcinoma promotes skin carcinogenesis.

Integrin α11ß1 is a collagen-binding integrin that is needed to induce and maintain the myofibroblast phenotype in fibrotic tissues and during wound healing. The expression of the α11 is upregulated in cancer-associated fibroblasts (CAFs) in various human neoplasms. We investigated α11 expression in human cutaneous squamous cell carcinoma (cSCC) and in benign and premalignant human skin lesions and monitored its effects on cSCC development by subjecting α11-knockout (Itga11-/- ) mice to the DMBA/TPA skin carcinogenesis protocol. α11-deficient mice showed significantly decreased tumor cell proliferation, leading to delayed tumor development and reduced tumor burden. Integrin α11 expression was significantly upregulated in the desmoplastic tumor stroma of human and mouse cSCCs, and the highest α11 expression was detected in high-grade tumors. Our results point to a reduced ability of α11-deficient stromal cells to differentiate into matrix-producing and tumor-promoting CAFs and suggest that this is one causative mechanism underlying the observed decreased tumor growth. An unexpected finding in our study was that, despite reduced CAF activation, the α11-deficient skin tumors were characterized by the presence of thick and regularly aligned collagen bundles. This finding was attributed to a higher expression of TGFß1 and collagen crosslinking lysyl oxidases in the Itga11-/- tumor stroma. In summary, our data suggest that α11ß1 operates in a complex interactive tumor environment to regulate ECM synthesis and collagen organization and thus foster cSCC growth. Further studies with advanced experimental models are still needed to define the exact roles and molecular mechanisms of stromal α11ß1 in skin tumorigenesis.

Stimulus-Responsive Nanoconjugates Derived from Phytoglycogen Nanoparticles.

Plant-derived phytoglycogen nanoparticles (PhG NPs) have the advantages of size uniformity, dispersibility in water, excellent lubrication properties, and lack of cytotoxicity; however, their chemical functionalization may lead to loss of NP structural integrity. Here, we report a straightforward approach to the generation of PhG NP conjugates with biologically active molecules. Hydrogen bonding of bovine serum albumin with electroneutral PhG NPs endows them with additional ligand binding affinity and enables the electrostatically governed attachment of methotrexate (MTX), a therapeutic agent commonly used in the treatment of cancer and arthritis diseases, to the protein-capped NPs. We showed stimuli-responsive release of MTX from the PhG-based nanoconjugates under physiological cues such as temperature and ionic strength. The results of this study stimulate future exploration of biomedical applications of nanoconjugates of PhG NPs.

Clinical utility of targeted next-generation sequencing assay in IDH-wildtype glioblastoma for therapy decision-making.

BACKGROUND: Targeted gene NGS testing is available through many academic institutions and commercial entities and is increasingly incorporated in practice guidelines for glioblastoma (GBM). This single-center retrospective study aimed to evaluate the clinical utility of incorporating NGS results in the management of GBM patients at a clinical trials-focused academic center. METHODS: We identified 1011 consecutive adult patients with pathologically confirmed GBM (IDHwt or IDHmut) who had somatic tumor sequencing (Oncopanel, ~500 cancer gene panel) at DFCI from 2013-2019. Clinical records of all IDHwt GBM patients were reviewed to capture clinical trial enrollment and off-label targeted therapy use based on NGS results. RESULTS: Of the 557 IDHwt GBM patients with sequencing, 182 entered clinical trials at diagnosis (32.7%) and 213 (38.2%) entered after recurrence. Sequencing results for 130 patients (23.3%) were utilized for clinical trial enrollment for either targeted therapy indications (6.9 % upfront and 27.7% at recurrent clinical trials and 3.1% for off-label targeted therapy) or exploratory studies (55.4% upfront and 6.9% recurrent clinical trials). Median overall survival was 20.1 months with no survival difference seen between patients enrolled in clinical trials compared to those who were not, in a posthoc analysis. CONCLUSIONS: While NGS testing has become essential for improved molecular diagnostics, our study illustrates that targeted gene panels remain underutilized for selecting therapy in GBM-IDHwt. Targeted therapy and clinical trial design remain to be improved to help leverage the potential of NGS in clinical care.

Neighborhood Contexts and Breast Cancer Among Asian American Women.

BACKGROUND: This study examines how neighborhood socioeconomic status (nSES) and ethnic composition are associated with breast cancer risk for Asian American women. METHODS: We linked individual level data from a population-based case-control study of breast cancer among Asian American women with neighborhood level data in the Greater San Francisco Bay Area (cases: n = 118, controls: n = 390). Multivariable logistic regression models examined the association between nSES, ethnic composition, and odds of having breast cancer. RESULTS: Asian American women living in neighborhoods with high nSES and high ethnic composition had the highest odds of breast cancer, compared to those living in neighborhoods with high nSES and low ethnic composition (OR = 0.34, 95% CI [0.16-0.75]) or in neighborhoods with low nSES and high ethnic composition (OR = 0.37, 95% CI [0.17-0.83]). DISCUSSION: Neighborhood socioeconomic and ethnic contexts are associated with breast cancer for Asian American women. We discuss explanations and avenues for future research.

May Measurement Month 2019: an analysis of blood pressure screening results in the Philippines.

The Philippine Society of Hypertension (PSH) took part again in the annual May Measurement Month 2019 (MMM19) blood pressure (BP) measurement campaign to raise awareness of hypertension especially in those who are not aware of their condition. The MMM19 standard protocol designed by the International Society of Hypertension was used during screening. These included the collection of basic data on demography, lifestyle, and environmental factors. Standardized sitting BP measurements were taken two to three times, using an automated BP apparatus and were inputted either in the MMM19 app or data were recorded in paper form and manually transferred to Excel spreadsheets by encoders supervised by the PSH. A total of 89 941 participated through opportunistic convenience sampling. After multiple imputation, a total of 47 925 (53.3%) participants had hypertension (≥140/90 mmHg or on antihypertensive medication). Of this number, 31 151 (65%) were aware that they had high BP and 30 120 (62.8%) were on antihypertensive medications. Of the 30 120 participants on antihypertensive medications, only 18 373 (61.1%) had controlled BP (<140/90 mmHg). Being overweight or obese were significant predictors of high BP. Other predictors of high systolic BP and diastolic BP were alcohol intake, smoking, and a previous history of hypertension in pregnancy, while pregnant participants had significantly lower BP. The MMM19 campaign succeeded in raising awareness of high BP in our country, and the opportunistic sampling enhanced a sense of people empowerment by their knowing how easy it is to detect high BP and thereby enabling the prevention of long-term health complications. The higher BP control in the MMM19 hypertensive individuals possibly attests to the success of the previous MMM17 and MMM18 campaigns.

Anti-leukemia effect associated with down-regulated CD47 and up-regulated calreticulin by stimulated macrophages in co-culture.

CD47 is over-expressed in Acute Myeloid Leukemia (AML) and functions as an inhibitory signal, suppressing phagocytosis by binding to signal regulatory protein α (SIRPα) on the surface of macrophages. Inhibition of CD47 restores the immune surveillance of AML cells. However, the inhibition of CD47 in AML by activated macrophages and the subsequent effects on different immune response parameters are not fully understood. Here, we demonstrate the use of a distinct co-culture method to inhibit CD47 and therefore eliminate AML cells by macrophages in vitro. Human chemically induced THP-1 macrophages were activated using different concentrations of lipopolysaccharide (LPS) and co-culturing with three AML cancer cell lines (HL-60, NB4, and THP-1), respectively, as well as normal human peripheral blood mononuclear cells (PBMC). CD47 inhibition was observed in and selective to AML but not observed in normal PBMC. Additionally, calreticulin (CRT) levels were elevated in the same cell lines simultaneously, after co-culturing with activated human macrophages, but not elevated in normal cells. We also show that the activated macrophages secreted high levels of cytokines, including IL-12p70, IL-6, and TNF-α, consistent with the elimination of AML by macrophages. Our study reveals the potential of this model for screening new drugs against AML and the possibility of using human macrophages in AML treatment in the future.

Structure basis of the FERM domain of kindlin-3 in supporting integrin αIIbß3 activation in platelets.

Kindlin-3, a protein 4.1, ezrin, radixin, and moesin (FERM) domain-containing adaptor in hematopoietic cells, is essentially required for supporting the bidirectional integrin αIIbß3 signaling in platelets by binding to the integrin ß3 cytoplasmic tail. However, the structural details of kindlin-3's FERM domain remain unknown. In this study, we crystalized the kindlin-3's FERM domain protein and successfully solved its 3-dimensional structure. The structure shows that the 3 kindlin-3's FERM subdomains (F1, F2, and F3) compact together and form a cloverleaf-shaped conformation, which is stabilized by the binding interface between the F1 and F3 subdomains. Interestingly, the FERM domain of kindlin-3 exists as a monomer in both crystal and solution, which is different from its counterpart in kindlin-2 that is able to form a F2 subdomain-swapped dimer; nonetheless, dimerization is required for kindlin-3 to support integrin αIIbß3 activation, indicating that kindlin-3 may use alternative mechanisms for formation of a functional dimer in cells. To evaluate the functional importance of the cloverleaf-like FERM structure in kindlin-3, structure-based mutations were introduced into kindlin-3 to disrupt the F1/F3 interface. The results show that integrin αIIbß3 activation is significantly suppressed in platelets expressing the kindlin-3 mutant compared with those expressing wild-type kindlin-3. In addition, introduction of equivalent mutations into kindlin-1 and kindlin-2 also significantly compromises their ability to support integrin αIIbß3 activation in CHO cells. Together, our findings suggest that the cloverleaf-like FERM domain in kindlins is structurally important for supporting integrin αIIbß3 activation.

The Australian and New Zealand Society of Cardiac and Thoracic Surgeons Database Program - Two Decades of Quality Assurance Data.

Over two decades, the Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) cardiac surgery database program has evolved from a single state-based database to a national clinical quality registry program and is now the most comprehensive cardiac surgical registry in Australia. We report the current structure and governance of the program and its key activities.

Higher Breast Cancer Risk Among Immigrant Asian American Women Than Among US-Born Asian American Women.

INTRODUCTION: Given rising rates of breast cancer in parts of Asia, immigrant Asian American women in the United States may have higher rates of breast cancer than previously anticipated. This study examined breast cancer risk among Asian American women by nativity and percentage of life lived in the United States, accounting for established breast cancer risk factors. METHODS: We analyzed a breast cancer case-control data set of Asian American women living in the San Francisco Bay Area; this data set included 132 cases of women with breast cancer selected from a Surveillance, Epidemiology, and End Results cancer registry and 438 Asian American women without diagnosed breast cancer matched to cases by age and country of origin. We used logistic regression to compare 3 Asian American groups: US-born, immigrants who lived 50% or more of their life in the United States, and immigrants who lived less than 50% of their life in the United States. RESULTS: In the minimally adjusted and fully adjusted models, both groups of immigrant Asian American women had higher risk of breast cancer than US-born Asian American women. In the fully adjusted model, compared with US-born Asian American women, immigrant Asian American women who lived more than 50% of their life in United States were on average 3 times as likely (odds ratio = 3.00; 95% confidence interval, 1.56-5.75) and immigrants who lived less than 50% of their life in United States were on average 2.46 times as likely (odds ratio = 2.46; 95% confidence interval, 1.21-4.99) to have breast cancer. We found no difference in fully adjusted odds ratios of having breast cancer between the 2 immigrant groups. CONCLUSION: This study provides preliminary evidence that breast cancer risk among immigrant Asian American women may be higher than among their US-born counterparts.

Structured Feedback: Acceptability and Feasibility of a Strategy to Enhance the Role of a Clinical Quality Registry to Drive Change in Cardiac Surgical Practice.

BACKGROUND: The Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) Database collects and monitors preoperative, operative, and 30-day outcome data on patients undergoing cardiac surgery, and delivers regular performance feedback reports to key personnel with intent to drive quality improvement. The current feedback approach appears to be ineffective in driving change to minimise Unit performance variation. We sought to determine the acceptability and feasibility of providing structured feedback in addition. METHODS: Cardiac surgeons were surveyed to assess their evaluation of the current feedback reports and assist in developing the content of structured feedback. We then assessed acceptability and performance outcomes of control Units receiving current feedback reports via email, versus intervention Units that in addition received structured feedback. RESULTS: Survey respondents assessing the current feedback report agreed that the content is relevant (95%), key performance indicators (KPIs) are useful (85%), and that it would be beneficial to compare surgeons' KPIs (75%). Survey respondents rating method of feedback, requested structured feedback sessions one to two times annually (67%; control Units), and future structured feedback (83%; intervention Units). With combined report and structured feedback, improved performance was noted for an under-performing Unit. Limitations of feedback in driving quality improvement was high performance of Units at baseline, low surgeon participation, and scheduling challenges for structured feedback. CONCLUSIONS: In this pilot study, compared to the control method, structured feedback did not significantly improve communication. To maximise quality improvement efforts, a collaborative feedback approach that fosters a climate of continuous performance improvement, is recommended.

Dual-Mode Dark Field and Surface-Enhanced Raman Scattering Liposomes for Lymphoma and Leukemia Cell Imaging.

Multifunctional probes are needed to characterize individual cells simultaneously by different techniques to provide complementary information. A preparative method and an in vitro demonstration of function are presented for a dual-function dark field microscopy/surface-enhanced Raman scattering (SERS) liposome probe for cancer. Liposomes composed of zwitterionic lipids are valuable both to limit biofouling and to serve as a modular matrix to incorporate a variety of functional molecules and hence are used here as vehicles for SERS-active materials. Dark field microscopy and SERS represent new combined functionalities for targeted liposomal probes. Two methods of antibody conjugation to SERS liposomes are demonstrated: (i) direct conjugation to functional groups on the SERS liposome surface and (ii) postinsertion of lipid-functionalized antibody fragments (Fabs) into preformed SERS liposomes. In vitro experiments targeting both lymphoma cell line LY10 and primary human chronic lymphocytic leukemia (CLL) cells demonstrate the usefulness of these probes as optical contrast agents in both dark field and Raman microscopy.

Black-white mental status trajectories: What ages do differences emerge?

OBJECTIVES: Studies of older U.S. adults have consistently found that African Americans perform worse on cognitive measures than whites, but there are inconsistencies as to whether these findings hold over time. Moreover, studies have focused on adults 51 and older, without considering younger ages; thus it is unclear the age at which these disparities surface. The present study examines black-white disparities in mental status trajectories among adults as young as 25 years over a 25-year period. METHOD: Data come from the Americans' Changing Lives Study (ACL) (n = 3,617). Participants, ranging from ages 25-100 years old at baseline, were followed from 1986 to 2011 over 5 waves. Mental status was assessed at each wave using a 5-item Short Portable Mental Status Questionnaire. Growth models were used to estimate the associations between age, race, baseline status, and longitudinal changes in mental status, controlling for sociodemographic (e.g., education, income) and other health risk factors (diabetes, stroke, tobacco use, depression). RESULTS: Racial disparities were seen beginning in midlife and this relationship was curvilinear. Specifically, blacks had a steeper rate of mental status decline than whites and these disparities persisted after accounting for social and health risk factors (b = 0.0090, p < 0.0001). DISCUSSION: Study findings demonstrate disparities emerge at middles ages and worsen as age increases. This finding highlights the importance of addressing racial disparities in cognition across a larger part of the adult life course. By doing so, we may better be able to capture early-life exposures that influence later-life cognitive outcomes and ultimately lead to disparities.

Kindlin-3 negatively regulates the release of neutrophil extracellular traps.

Neutrophils fight infections by generating reactive oxygen species (ROS) and extracellular traps (NETs). However, how neutrophils modulate ROS/NET generation is mechanistically unclear. Kindlin-3, an essential integrin activator expressed in hematopoietic cells, is required to support integrin-mediated neutrophil recruitment during inflammation. Here, we report a novel role of kindlin-3 in regulating ROS/NET generation in neutrophils. When overexpressing kindlin-3 in neutrophil-like differentiated HL-60 cells (HL-60N), ROS/NET generation from these cells were significantly suppressed. Interestingly, overexpression of a kindlin-3 mutant that is defective for interacting with integrins in HL-60N cells still inhibited ROS/NET generation, suggesting that the role of kindlin-3 in inhibiting ROS/NET signaling may be independent of its binding to integrins. Consistently, knockdown of kindlin-3 in HL-60N cells led to enhanced ROS/NET generation. In addition, bone marrow neutrophils isolated from kindlin-3-deficient mice showed elevated ROS/NET generation when compared with WT counterparts. As expected, overexpression of exogenous kindlin-3 in mouse neutrophils could suppress NET release ex vivo and in vivo. Collectively, these results demonstrate that kindlin-3 in neutrophils is involved in modulating the ROS/NET signaling, providing a novel mechanism for fine-tuning neutrophil behaviors during inflammation.

Closed incision negative pressure therapy decreases complications after periprosthetic fracture surgery around the hip and knee.

INTRODUCTION: Periprosthetic fractures (PPFXs) are becoming increasingly common following total hip arthroplasty (THA) and total knee arthroplasty (TKA). Patients sustaining PPFXs face considerable perioperative morbidity, with relatively increased rates of surgical site infection. We sought to evaluate the efficacy of closed-incision negative-pressure wound therapy (ciNPT) in decreasing perioperative wound complications following lower extremity periprosthetic fracture surgery. METHODS: We performed a retrospective review of 69 consecutive patients who underwent surgery to address lower extremity periprosthetic fractures around hip or knee implants performed over a 6.5-year period. The population was divided into two groups based on the surgical dressing used at the conclusion of the procedure: (1) a sterile, antimicrobial hydrofiber dressing, or (2) ciNPT. There were no baseline demographic differences between the two groups. Rates of wound complications, surgical site infection, and reoperation related to the surgical site were compared between groups. Continuous variables were analyzed using a student's t-test, and categorical variables using either chi-square or fisher's exact test. RESULTS: Patients treated with ciNPT developed fewer wound complications (4% vs. 35%; p=0.002), fewer deep infections (0% vs. 25%; p=0.004), and underwent fewer reoperations related to the surgical site (4% vs. 25%; p=0.021) compared to patients treated with standard of care. CONCLUSIONS: Our findings suggest that ciNPT may reduce wound complications, SSIs, and reoperations in patients undergoing lower extremity periprosthetic fracture surgery. This is the first study to investigate ciNPT as a treatment for periprosthetic fracture surgery, and has the potential to change the postoperative management of these patients.

Kindlin supports platelet integrin αIIbß3 activation by interacting with paxillin.

Kindlins play an important role in supporting integrin activation by cooperating with talin; however, the mechanistic details remain unclear. Here, we show that kindlins interacted directly with paxillin and that this interaction could support integrin αIIbß3 activation. An exposed loop in the N-terminal F0 subdomain of kindlins was involved in mediating the interaction. Disruption of kindlin binding to paxillin by structure-based mutations significantly impaired the function of kindlins in supporting integrin αIIbß3 activation. Both kindlin and talin were required for paxillin to enhance integrin activation. Interestingly, a direct interaction between paxillin and the talin head domain was also detectable. Mechanistically, paxillin, together with kindlin, was able to promote the binding of the talin head domain to integrin, suggesting that paxillin complexes with kindlin and talin to strengthen integrin activation. Specifically, we observed that crosstalk between kindlin-3 and the paxillin family in mouse platelets was involved in supporting integrin αIIbß3 activation and in vivo platelet thrombus formation. Taken together, our findings uncover a novel mechanism by which kindlin supports integrin αIIbß3 activation, which might be beneficial for developing safer anti-thrombotic therapies.

Thrombosis in Cancer: Research Priorities Identified by a National Cancer Institute/National Heart, Lung, and Blood Institute Strategic Working Group.

The risk for venous thromboembolism (VTE) is increased in cancer and particularly with chemotherapy, and it portends poorer survival among patients with cancer. However, many fundamental questions about cancer-associated VTE, or Trousseau syndrome, remain unanswered. This report summarizes the proceedings of a working group assembled by the NCI and NHLBI in August 2014 to explore the state of the science in cancer-associated VTE, identify clinically important research gaps, and develop consensus on priorities for future research. Representing a convergence of research priorities between the two NIH Institutes, the workshop addressed epidemiologic, basic science, clinical, and translational issues in cancer-associated VTE. Cancer Res; 76(13); 3671-5. ©2016 AACR.