Patient-reported outcome measures in patients with familial cerebral cavernous malformations: results from the Treat_CCM trial.

Background: The Phase 1/2 Treat_CCM randomized controlled trial for people with familial cerebral cavernous malformations (FCCMs) confirmed the safety of propranolol and suggested beneficial effects on intracerebral hemorrhage or new focal neurological deficits, but the effects on patient-reported outcome measures have not been reported. Methods: Participants completed self-reported questionnaires at baseline, 1 and 2 years. Depression was assessed with the Beck Depression Inventory-II (BDI-2); Anxiety with the State-Trait Anxiety Inventory X1 and X2 (STAI X-1 and STAI X-2); and Quality of Life with the Short Form 36 (SF-36), split into the physical and mental component scales (PCS and MCS). Differences between treatment groups and the general population were assessed. Change over time by treatment was assessed by means of mixed models. Results: In total, 71 participants (48 propranolol and 23 standard care) were enrolled, of whom 61 (73%) completed questionnaires at baseline and 2-year FU. At baseline, no differences between treatment groups for any of the questionnaires were present. Twenty (31.7%) patients were considered depressed at baseline, while this proportion was lower in the propranolol group after 2 years (28.6% vs. 55.5%, p = 0.047). The STAI X-1 and X-2 scores were stable over time. PCS was lower in FCCM patients as compared with the general Italian population, while the MCS was similar to the general population. No effect of propranolol was found for both PCS and MCS. Conclusion: Depression is common among patients with FCCM. Patients randomized to propranolol had a lower proportion of participants with depression after 2 years.Clinical trial registration: https://clinicaltrials.gov/, identifier (NCT03589014).

Consolidation Osimertinib Versus Durvalumab Versus Observation After Concurrent Chemoradiation in Unresectable EGFR-Mutant NSCLC: A Multicenter Retrospective Cohort Study.

INTRODUCTION: Durvalumab improves survival when used as consolidation therapy after chemoradiation (CRT) in patients with stage III NSCLC. The optimal consolidation therapy for patients with EGFR-mutant (EGFRmut) stage III NSCLC remains unknown. METHODS: In this multi-institutional, international retrospective analysis across 24 institutions, we evaluated outcomes in patients with stage III EGFRmut NSCLC treated with concurrent CRT followed by consolidation therapy with osimertinib, durvalumab, or observation between 2015 and 2022. Kaplan-Meier method was used to estimate real-world progression-free survival (rwPFS, primary end point) and overall survival (secondary end point). Treatment-related adverse events (trAEs) during consolidation treatment were defined using Common Terminology Criteria for Adverse Events version 5.0. Multivariable Cox regression analysis was used. RESULTS: Of 136 patients with stage III EGFRmut NSCLC treated with definitive concurrent CRT, 56 received consolidation durvalumab, 33 received consolidation osimertinib, and 47 was on observation alone. Baseline characteristics were similar across the three cohorts. With a median follow-up of 46 months for the entire cohort, the median duration of treatment was not reached (NR) for osimertinib (interquartile range: NR-NR) and was 5.5 (interquartile range: 2.4-10.8) months with durvalumab. After adjusting for nodal status, stage III A/B/C, and age, patients treated with consolidation osimertinib had significantly longer 24-month rwPFS compared to those treated with durvalumab or in the observation cohorts (osimertinib: 86%, durvalumab: 30%, observation: 27%, p < 0.001 for both comparisons). There was no difference in rwPFS between the durvalumab and the observation cohorts. No significant difference in overall survival across the three cohorts was detected, likely due to the limited follow-up. Any-grade trAE occurred in 52% (2 [6.1%] grade ≥3) and 48% (10 [18%] grade ≥3) of patients treated with osimertinib and durvalumab, respectively. Of 45 patients who progressed on consolidation durvalumab, 37 (82%) subsequently received EGFR tyrosine kinase inhibitors. Of these, 14 (38%) patients developed trAEs including five patients with pneumonitis (14%; 2 [5.4%] grade ≥3) and five patients with diarrhea (14%; 1 [2.7%] grade ≥3). CONCLUSIONS: This study suggests that among patients with stage III unresectable NSCLC with a sensitizing EGFR mutation, consolidation osimertinib was associated with a significantly longer rwPFS compared to durvalumab or observation. No unanticipated safety signals were observed with consolidation osimertinib.

Engineered cocultures of iPSC-derived atrial cardiomyocytes and atrial fibroblasts for modeling atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia treatable with antiarrhythmic drugs; however, patient responses remain highly variable. Human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are useful for discovering precision therapeutics, but current platforms yield phenotypically immature cells and are not easily scalable for high-throughput screening. Here, primary adult atrial, but not ventricular, fibroblasts induced greater functional iPSC-aCM maturation, partly through connexin-40 and ephrin-B1 signaling. We developed a protein patterning process within multiwell plates to engineer patterned iPSC-aCM and atrial fibroblast coculture (PC) that significantly enhanced iPSC-aCM structural, electrical, contractile, and metabolic maturation for 6+ weeks compared to conventional mono-/coculture. PC displayed greater sensitivity for detecting drug efficacy than monoculture and enabled the modeling and pharmacological or gene editing treatment of an AF-like electrophysiological phenotype due to a mutated sodium channel. Overall, PC is useful for elucidating cell signaling in the atria, drug screening, and modeling AF.

Evaluation of the Effects of Probiotics and Prebiotics on the Salmonella typhi Infections.

Typhoid fever is one of the most commonly disseminated diseases and is considered to be linked to poor sanitation. It is responsible for 2-5% of all deaths, and its causative agent is Salmonella typhi. The current study aimed to investigate the antibacterial activity of prebiotics (inulin and starch) and probiotics against multidrug resistance of S. typhi bacterial isolates. Determination of the inhibitory effect of probiotics and prebiotics against S. typhi isolates was performed by agar well diffusion method and minimal inhibitory concentration. Body samples of all eligible patients were collected and cultured. Finally, 50 (25%) out of the total cultured samples were S. Typhi bacteria isolated from different samples. The bacteria were mainly found in blood, followed by stool and fluid (74%, 24%, and 2%, respectively). On differential medium, xylose lysine deoxycholate agar, the colonies appear red with black centers, while on MacConkey agar, the colonies appear smooth, pale, transparent, colorless, and raised. Regarding the inhibition zone values of bacteriocins of Lactobacillus from Yogurt against S. typhi in plate, significant differences were identified between the ones with and without prebiotic addition. Accordingly, the value of the inhibition zone for those without prebiotic addition (13.18±7.403) was significantly lower than that of cutoff values of 20 with a significant difference of -6.820 (t= -6.514, df 49, P=0.000). Moreover, the inhibition effect of prebiotics (inulin and starch) against S. typhi at 37 °C for 24 h in part dish glucose as control, only the mean of inulin was found to be significantly lower than that of the cutoff value of 18 with the mean difference of -3.900 (t=-4.115, df 49, P=0.000). Other prebiotics of glucose and starch in 24 h showed negative inhibition. Probiotics are live microorganisms that have beneficial host effects by enhancing microbial balance in the intestine, whereas prebiotics are indigestible food components having beneficial effects by enhancing the activity and growth of one or more colonic bacteria. Lactobacillus filtrates had considerable effects against the test S. typhi isolates.

Evaluation of paediatric pulmonary vein stenosis by cardiac CT angiography: a comparative study with transthoracic echocardiography and catheter angiogram.

AIM: To compare prospective electrocardiogram (ECG)-gated cardiac computed tomographic angiography (CCTA) with transthoracic echocardiography (TTE) and cardiac catheter angiography (CCA) for paediatric pulmonary vein (PV) stenosis. MATERIALS AND METHODS: Retrospective chart review was undertaken of all patients who underwent CCTA for PV evaluation over a 4-year period. Patient demographics, findings of CCTA, TTE, and CCA, as well as interventions performed, were recorded for each PV. RESULTS: Thirty-five patients were included (23 male patients). All patients had a prior TTE with time interval between TTE and CCTA ranging from 0 to 90 days. CCTA detected 92 abnormalities in 32 patients. TTE missed 16 PV abnormalities (16/92, 17%), detected 37 abnormalities with certainty (37/92, 40%), and was suggestive in 39 abnormalities (39/92, 42%). CCTA was negative for PV abnormalities when TTE was positive or suspicious in three patients. Nineteen patients underwent CCA (18 patients with 52 abnormalities and one patient with normal PV), confirming CCTA findings. Thirty-nine were treated with angioplasty/stenting (39/52,75%). Failed recanalisation occurred in three PVs (3/52, 6%) and no intervention was attempted for the rest as the gradient was not significant (10/52,19%). Nine patients underwent surgical repair (26/92, 28%). Five patients (14/92, 15%) were managed with no intervention based on CCTA findings and poor clinical prognosis. CONCLUSIONS: CCTA plays an important role in detecting paediatric PV stenosis and identifies additional findings compared to TTE that have direct surgical/interventional implications. CCTA complements TTE in imaging these patients and helps guide management.

Influence of Co3O4-based catalysts on N2O catalytic decomposition and NO conversion.

This study investigated the effect of different Co3O4-based catalysts on the catalytic decomposition of nitrous oxide (N2O) and on nitric oxide (NO) conversion. The experiments were carried out using various reaction temperatures, alkaline solutions, pH, mixing conditions, aging times, space velocities, impregnation loads, and compounds. The results showed that Co3O4 catalysts prepared by precipitation methods have the highest catalytic activity and N2O conversion, even at low reaction temperatures, while the commercial nano and powder forms of Co3O4 (CS) have the lowest performance. The catalysts become inactive at temperatures below 400 °C, and their activity is strongly influenced by the mixing temperature. Samples without stirring during the aging process have higher catalytic activity than those with stirring, even at low reaction temperatures (200-300 °C). The catalytic activity of Co3O4 PM1 decreases with low W/F values and low reaction temperatures. Additionally, the catalyst's performance tends to increase with the reduction process. The study suggests that cobalt-oxide-based catalysts are effective in N2O catalytic decomposition and NO conversion. The findings may be useful in the design and optimization of catalytic systems for N2O and NO control. The results obtained provide important insights into the development of highly efficient, low-cost, and sustainable catalysts for environmental protection.

In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1 -mutant lung cancer.

LKB1/STK11 is a serine/threonine kinase that plays a major role in controlling cell metabolism, resulting in potential therapeutic vulnerabilities in LKB1-mutant cancers. Here, we identify the NAD + degrading ectoenzyme, CD38, as a new target in LKB1-mutant NSCLC. Metabolic profiling of genetically engineered mouse models (GEMMs) revealed that LKB1 mutant lung cancers have a striking increase in ADP-ribose, a breakdown product of the critical redox co-factor, NAD + . Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)-key downstream effectors of LKB1- induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCE: Loss-of-function mutations in the LKB1 tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B.

Familial hypercholesterolemia (FH) patients suffer from excessively high levels of Low Density Lipoprotein Cholesterol (LDL-C), which can cause severe cardiovascular disease. Statins, bile acid sequestrants, PCSK9 inhibitors, and cholesterol absorption inhibitors are all inefficient at treating FH patients with homozygous LDLR gene mutations (hoFH). Drugs approved for hoFH treatment control lipoprotein production by regulating steady-state Apolipoprotein B (apoB) levels. Unfortunately, these drugs have side effects including accumulation of liver triglycerides, hepatic steatosis, and elevated liver enzyme levels. To identify safer compounds, we used an iPSC-derived hepatocyte platform to screen a structurally representative set of 10,000 small molecules from a proprietary library of 130,000 compounds. The screen revealed molecules that could reduce the secretion of apoB from cultured hepatocytes and from humanized livers in mice. These small molecules are highly effective, do not cause abnormal lipid accumulation, and share a chemical structure that is distinct from any known cholesterol lowering drug.

Decellularized Liver Nanofibers Enhance and Stabilize the Long-Term Functions of Primary Human Hepatocytes In Vitro.

Owing to significant differences across species in liver functions, in vitro human liver models are used for screening the metabolism and toxicity of compounds, modeling diseases, and cell-based therapies. However, the extracellular matrix (ECM) scaffold used for such models often does not mimic either the complex composition or the nanofibrous topography of native liver ECM. Thus, here novel methods are developed to electrospin decellularized porcine liver ECM (PLECM) and collagen I into nano- and microfibers (≈200-1000 nm) without synthetic polymer blends. Primary human hepatocytes (PHHs) on nanofibers in monoculture or in coculture with nonparenchymal cells (3T3-J2 embryonic fibroblasts or primary human liver endothelial cells) display higher albumin secretion, urea synthesis, and cytochrome-P450 1A2, 2A6, 2C9, and 3A4 enzyme activities than on conventionally adsorbed ECM controls. PHH functions are highest on the collagen/PLECM blended nanofibers (up to 34-fold higher CYP3A4 activity relative to adsorbed ECM) for nearly 7 weeks in the presence of the fibroblasts. In conclusion, it is shown for the first time that ECM composition and topography synergize to enhance and stabilize PHH functions for several weeks in vitro. The nanofiber platform can prove useful for the above applications and to elucidate cell-ECM interactions in the human liver.

Engineered Platforms for Maturing Pluripotent Stem Cell-Derived Liver Cells for Disease Modeling.

Several liver diseases (eg, hepatitis B/C viruses, alcoholic/nonalcoholic fatty liver, malaria, monogenic diseases, and drug-induced liver injury) significantly impact global mortality and morbidity. Species-specific differences in liver functions limit the use of animals to fully elucidate/predict human outcomes; therefore, in vitro human liver models are used for basic and translational research to complement animal studies. However, primary human liver cells are in short supply and display donor-to-donor variability in viability/quality. In contrast, human hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells and embryonic stem cells are a near infinite cell resource that retains the patient/donor's genetic background; however, conventional protocols yield immature phenotypes. HLC maturation can be significantly improved using advanced techniques, such as protein micropatterning to precisely control cell-cell interactions, controlled sized spheroids, organoids with multiple cell types and layers, 3-dimensional bioprinting to spatially control cell populations, microfluidic devices for automated nutrient exchange and to induce liver zonation via soluble factor gradients, and synthetic biology to genetically modify the HLCs to accelerate and enhance maturation. Here, we present design features and characterization for representative advanced HLC maturation platforms and then discuss HLC use for modeling various liver diseases. Lastly, we discuss desirable advances to move this field forward. We anticipate that with continued advances in this space, pluripotent stem cell-derived liver models will provide human-relevant data much earlier in preclinical drug development and reduce animal usage, help elucidate liver disease mechanisms for the discovery of efficacious and safe therapeutics, and be useful as cell-based therapies for patients suffering from end-stage liver failure.

Sleep quality and its associations with disease activity and quality of life in older patients with rheumatoid arthritis.

PURPOSE: Sleep disturbance is among the most important geriatric syndromes, and its evaluation is part of the routine comprehensive geriatric assessment (CGA). Previous studies have demonstrated that older patients with rheumatoid arthritis (RA) have poorer sleep quality than younger control patients. However, there needs to be more data on the sleep quality of older patients with RA with age-matched controls. METHODS: Totally 100 participants, 50 older RA patients classified according to the ACR criteria, and 50 age- and gender-matched control patients without RA were included in the study. All patients underwent CGA, including assessing their functionality, depressive, cognitive, and nutritional status. In addition, sleep quality was assessed by the Pittsburg Sleep Quality Index (PSQI), and RA disease activity by a rheumatologist using the Disease Activity Score 28 (DAS28-CRP), and quality of life with the RA QoL questionnaire (RAQoL). RESULTS: The median age was 70 years (min-max: 65-86), and 62.5% were female. Co-morbidities and comprehensive geriatric assessment parameters were similar between the two groups. Median PSQI global score was higher in patients with RA than controls [9 (min-max: 1-20) vs. 5 (min-max: 1-13), p = 0.029). When the patients were categorized with respect to being 'poor sleepers' (PSQI score > 5), 62% of patients with RA and 38% of controls were poor sleepers (p = 0.016). The patients classified as poor sleepers were more likely to have a diagnosis of RA, higher DAS28-CRP and RAQoL scores, lower grip strength, and be a woman. PSQI global scores were significantly positively correlated with DAS28-CRP scores (r = 0.514, p < 0.001), RAQoL scores (r = 0.689, p < 0.001), number of medications used (r = 0.292, p = 0.003), and YDS scores (r = 0.407, p < 0.001), and significantly negatively correlated with handgrip strength (r = - 0.351, p = 0.001). CONCLUSION: The results suggest that older patients with RA might have poorer sleep quality compared to age- and gender-matched controls. Moreover, sleep quality correlated with RA disease activity and QoL in old age.

Diagnostic Accuracy of MRI for Detection of Meningitis in Infants.

PURPOSE: To determine the accuracy of MR imaging for diagnosis of meningitis in infants. MATERIALS AND METHODS: Retrospective review of infants less than 1 year of age who underwent a brain MR imaging for meningitis from 2010-2018. Gold standard for diagnosis of bacterial meningitis was a positive bacterial CSF culture or a positive blood culture with an elevated CSF WBC count, and diagnosis of viral meningitis was a positive CSF PCR result and elevated CSF WBC count. Sensitivity, specificity, PPV, NPV, and accuracy for MR imaging diagnosis of meningitis were calculated. RESULTS: Two hundred nine infants with mean age 80 days (range 0-347 days) were included. There were 178 true positives with the most common pathogens being: Group B Streptococcus (58), E. coli (50), Streptococcus pneumoniae (21), H. influenzae (4); Herpes simplex virus 1 or 2 (18); Enterovirus (4); and other (23). There were 31 true negatives. Range of sensitivity, specificity, PPV, NPV, and accuracy of MR imaging for detection of meningitis was 67.4-83.5%, 92.3-95.7%, 95.0-98.6%, 33.3-76.5%, and 71.3-86.5% respectively. MR imaging sensitivity decreased after 10 days from time of presentation while specificity remained stable. Among individual MR imaging findings, leptomeningeal enhancement was the most sensitive finding, while cerebritis, infarction, ventriculitis, abscess, and intraventricular purulent material were the most specific findings. CONCLUSIONS: MR imaging of the brain demonstrates high specificity and moderate sensitivity for diagnosis among infants presenting with signs and symptoms of meningitis. The results reflect current standard of care for imaging of infants with meningitis however a selection bias for imaging of more severe meningitis may affect these results.

Engineered matrix microenvironments reveal the heterogeneity of liver sinusoidal endothelial cell phenotypic responses.

Fibrosis is one of the hallmarks of chronic liver disease and is associated with aberrant wound healing. Changes in the composition of the liver microenvironment during fibrosis result in a complex crosstalk of extracellular cues that promote altered behaviors in the cell types that comprise the liver sinusoid, particularly liver sinusoidal endothelial cells (LSECs). Recently, it has been observed that LSECs may sustain injury before other fibrogenesis-associated cells of the sinusoid, implicating LSECs as key actors in the fibrotic cascade. A high-throughput cellular microarray platform was used to deconstruct the collective influences of defined combinations of extracellular matrix (ECM) proteins, substrate stiffness, and soluble factors on primary human LSEC phenotype in vitro. We observed remarkable heterogeneity in LSEC phenotype as a function of stiffness, ECM, and soluble factor context. LYVE-1 and CD-31 expressions were highest on 1 kPa substrates, and the VE-cadherin junction localization was highest on 25 kPa substrates. Also, LSECs formed distinct spatial patterns of LYVE-1 expression, with LYVE-1+ cells observed in the center of multicellular domains, and pattern size regulated by microenvironmental context. ECM composition also influenced a substantial dynamic range of expression levels for all markers, and the collagen type IV was observed to promote elevated expressions of LYVE-1, VE-cadherin, and CD-31. These studies highlight key microenvironmental regulators of LSEC phenotype and reveal unique spatial patterning of the sinusoidal marker LYVE-1. Furthermore, these data provide insight into understanding more precisely how LSECs respond to fibrotic microenvironments, which will aid drug development and identification of targets to treat liver fibrosis.

Polygodial, a Sesquiterpene Dialdehyde, Activates Apoptotic Signaling in Castration-Resistant Prostate Cancer Cell Lines by Inducing Oxidative Stress.

Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. Surgery, radiation therapy, chemotherapy, and androgen deprivation therapy are currently the standard treatment options for PCa. These have poor outcomes and result in the development of castration-resistant prostate cancer (CRPC), which is the foremost underlying cause of mortality associated with PCa. Taxanes, diterpene compounds approved to treat hormonal refractory PCa, show poor outcomes in CRPC. Polygodial (PG) is a natural sesquiterpene isolated from water pepper (Persicaria hydropiper), Dorrigo pepper (Tasmannia stipitata), and mountain pepper (Tasmannia lanceolata). Previous reports show that PG has an anticancer effect. Our results show that PG robustly inhibits the cell viability, colony formation, and migration of taxane-resistant CRPC cell lines and induces cell cycle arrest at the G0 phase. A toxicity investigation shows that PG is not toxic to primary human hepatocytes, 3T3-J2 fibroblast co-cultures, and non-cancerous BPH-1 cells, implicating that PG is innocuous to healthy cells. In addition, PG induces oxidative stress and activates apoptosis in drug-resistant PCa cell lines. Our mechanistic evaluation by a proteome profiler-human apoptotic array in PC3-TXR cells shows that PG induces upregulation of cytochrome c and caspase-3 and downregulation of antiapoptotic markers. Western blot analysis reveals that PG activates apoptotic and DNA damage markers in PCa cells. Our results suggest that PG exhibits its anticancer effect by promoting reactive oxygen species generation and induction of apoptosis in CRPC cells.

Reanalysis of Trichloroethylene and Tetrachloroethylene Metabolism to Glutathione Conjugates Using Human, Rat, and Mouse Liver in Vitro Models to Improve Precision in Risk Characterization.

BACKGROUND: Both trichloroethylene (TCE) and tetrachloroethylene (PCE) are high-priority chemicals subject to numerous human health risk evaluations by a range of agencies. Metabolism of TCE and PCE determines their ultimate toxicity; important uncertainties exist in quantitative characterization of metabolism to genotoxic moieties through glutathione (GSH) conjugation and species differences therein. OBJECTIVES: This study aimed to address these uncertainties using novel in vitro liver models, interspecies comparison, and a sensitive assay for quantification of GSH conjugates of TCE and PCE, S-(1,2-dichlorovinyl)glutathione (DCVG) and S-(1,2,2-trichlorovinyl) glutathione (TCVG), respectively. METHODS: Liver in vitro models used herein were suspension, 2-D culture, and micropatterned coculture (MPCC) with primary human, rat, and mouse hepatocytes, as well as human induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep). RESULTS: We found that, although efficiency of metabolism varied among models, consistent with known differences in their metabolic capacity, formation rates of DCVG and TCVG generally followed the patterns human≥rat≥mouse, and primary hepatocytes>iHep. Data derived from MPCC were most consistent with estimates from physiologically based pharmacokinetic models calibrated to in vivo data. DISCUSSION: For TCE, the new data provided additional empirical support for inclusion of GSH conjugation-mediated kidney effects as critical for the derivation of noncancer toxicity values. For PCE, the data reduced previous uncertainties regarding the extent of TCVG formation in humans; this information was used to update several candidate kidney-specific noncancer toxicity values. Overall, MPCC-derived data provided physiologically relevant estimates of GSH-mediated metabolism of TCE and PCE to reduce uncertainties in interspecies extrapolation that constrained previous risk evaluations, thereby increasing the precision of risk characterizations of these high-priority toxicants. https://doi.org/10.1289/EHP12006.

Modulation of human iPSC-derived hepatocyte phenotype via extracellular matrix microarrays.

In vitro human liver models are essential for drug screening, disease modeling, and cell-based therapies. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHeps) mitigate sourcing limitations of primary human hepatocytes (PHHs) and enable precision medicine; however, current protocols yield iHeps with very low differentiated functions. The composition and stiffness of liver's extracellular matrix (ECM) cooperatively regulate hepatic phenotype in vivo, but such effects on iHeps remain unelucidated. Here, we utilized ECM microarrays and high content imaging to assess human iHep attachment and functions on ten major liver ECM proteins in single and two-way combinations robotically spotted onto polyacrylamide gels of liver-like stiffnesses; microarray findings were validated using hydrogel-conjugated multiwell plates. Collagen-IV supported higher iHep attachment than collagen-I over 2 weeks on 1 kPa, while laminin and its combinations with collagen-III, fibronectin, tenascin C, or hyaluronic acid led to both high iHep attachment and differentiated functions; laminin and its combination with tenascin or fibronectin led to similar albumin expression in iHeps and PHHs. Additionally, several collagen-IV-, laminin-, fibronectin-, and collagen-V-containing combinations on 1 kPa led to similar or higher CYP3A4 staining in iHeps than PHHs. Lastly, collagen-I or -III mixed with laminin, collagen-IV mixed with lumican, and collagen-V mixed with fibronectin led to high and stable functional output (albumin/urea secretions; CYP1A2/2C9/3A4 activities) in iHep cultures versus declining PHH numbers/functions for 3 weeks within multiwell plates containing 1 kPa hydrogels. Ultimately, these platforms can help elucidate ECM's role in liver diseases and serve as building blocks of engineered tissues for applications. STATEMENT OF SIGNIFICANCE: We utilized high-throughput extracellular matrix (ECM) microarrays and high content imaging to assess the attachment and differentiated functions of iPSC-derived human hepatocyte-like cells (iHep) on major liver ECM protein combinations spotted onto polyacrylamide gels of liver-like stiffnesses. We observed that iHep responses are regulated in unexpected ways via the cooperation between ECM stiffness and protein composition. Using this approach, we induced mature functions in iHeps on substrates of physiological stiffness and select ECM coatings at higher levels over 3+ weeks than analogous primary human hepatocyte cultures, which is useful for building platforms for drug screening, disease modeling, and regenerative medicine.

Intratumoral Therapy to Make a "Cold" Tumor "Hot": The Jury Is Still Out.

Tilsotolimod, an oligodeoxynucleotide TLR9 agonist, administered intratumorally, has been clinically evaluated. This compound has demonstrated the ability to induce changes within the tumor microenvironment, to convert noninflamed cold tumors into inflamed hot tumors, with the hope that these tumors will be more responsive to immune checkpoint blockade. See related article by Babiker et al., p. 5079.

Immunotherapy in non-small cell lung cancer: Past, present, and future directions.

Many decades in the making, immunotherapy has demonstrated its ability to produce durable responses in several cancer types. In the last decade, immunotherapy has shown itself to be a viable therapeutic approach for non-small cell lung cancer (NSCLC). Several clinical trials have established the efficacy of immune checkpoint blockade (ICB), particularly in the form of anti-programmed death 1 (PD-1) antibodies, anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibodies and anti-programmed death 1 ligand (PD-L1) antibodies. Many trials have shown progression free survival (PFS) and overall survival (OS) benefit with either ICB alone or in combination with chemotherapy when compared to chemotherapy alone. The identification of biomarkers to predict response to immunotherapy continues to be evaluated. The future of immunotherapy in lung cancer continues to hold promise with the development of combination therapies, cytokine modulating therapies and cellular therapies. Lastly, we expect that innovative advances in technology, such as artificial intelligence (AI) and machine learning, will begin to play a role in the future care of patients with lung cancer.

The current state of the art and future trends in RAS-targeted cancer therapies.

Despite being the most frequently altered oncogenic protein in solid tumours, KRAS has historically been considered 'undruggable' owing to a lack of pharmacologically targetable pockets within the mutant isoforms. However, improvements in drug design have culminated in the development of inhibitors that are selective for mutant KRAS in its active or inactive state. Some of these inhibitors have proven efficacy in patients with KRASG12C-mutant cancers and have become practice changing. The excitement associated with these advances has been tempered by drug resistance, which limits the depth and/or duration of responses to these agents. Improvements in our understanding of RAS signalling in cancer cells and in the tumour microenvironment suggest the potential for several novel combination therapies, which are now being explored in clinical trials. Herein, we provide an overview of the RAS pathway and review the development and current status of therapeutic strategies for targeting oncogenic RAS, as well as their potential to improve outcomes in patients with RAS-mutant malignancies. We then discuss challenges presented by resistance mechanisms and strategies by which they could potentially be overcome.

PD-L1 Crosslinking as a New Strategy of 4-1BB Agonism Immunotherapy.

4-1BB has been considered a promising target in cancer immunotherapy for decades. Nevertheless, early 4-1BB-targeted agents demonstrated significant liver immuno-toxicity. A new wave of 4-1BB-based therapy is being developed to circumvent hepatotoxicity with a bispecific molecule that directs 4-1BB agonism to the tumor microenvironment by targeting tumor-associated immune checkpoint molecule PD-L1. See related article by Peper-Gabriel et al., p. 3387.