Interface-guided phenotyping of coding variants in the transcription factor RUNX1 with SEUSS.

Understanding the consequences of single amino acid substitutions in cancer driver genes remains an unmet need. Perturb-seq provides a tool to investigate the effects of individual mutations on cellular programs. Here we deploy SEUSS, a Perturb-seq like approach, to generate and assay mutations at physical interfaces of the RUNX1 Runt domain. We measured the impact of 115 mutations on RNA profiles in single myelogenous leukemia cells and used the profiles to categorize mutations into three functionally distinct groups: wild-type (WT)-like, loss-of-function (LOF)-like and hypomorphic. Notably, the largest concentration of functional mutations (non-WT-like) clustered at the DNA binding site and contained many of the more frequently observed mutations in human cancers. Hypomorphic variants shared characteristics with loss of function variants but had gene expression profiles indicative of response to neural growth factor and cytokine recruitment of neutrophils. Additionally, DNA accessibility changes upon perturbations were enriched for RUNX1 binding motifs, particularly near differentially expressed genes. Overall, our work demonstrates the potential of targeting protein interaction interfaces to better define the landscape of prospective phenotypes reachable by amino acid substitutions.

Effects of Particle Geometry for PLGA-Based Nanoparticles: Preparation and In Vitro/In Vivo Evaluation.

The physicochemical properties (size, shape, zeta potential, porosity, elasticity, etc.) of nanocarriers influence their biological behavior directly, which may result in alterations of the therapeutic outcome. Understanding the effect of shape on the cellular interaction and biodistribution of intravenously injected particles could have fundamental importance for the rational design of drug delivery systems. In the present study, spherical, rod and elliptical disk-shaped PLGA nanoparticles were developed for examining systematically their behavior in vitro and in vivo. An important finding is that the release of the encapsulated human serum albumin (HSA) was significantly higher in spherical particles compared to rod and elliptical disks, indicating that the shape can make a difference. Safety studies showed that the toxicity of PLGA nanoparticles is not shape dependent in the studied concentration range. This study has pioneering findings on comparing spherical, rod and elliptical disk-shaped PLGA nanoparticles in terms of particle size, particle size distribution, colloidal stability, morphology, drug encapsulation, drug release, safety of nanoparticles, cellular uptake and biodistribution. Nude mice bearing non-small cell lung cancer were treated with 3 differently shaped nanoparticles, and the accumulation of nanoparticles in tumor tissue and other organs was not statistically different (p > 0.05). It was found that PLGA nanoparticles with 1.00, 4.0 ± 0.5, 7.5 ± 0.5 aspect ratios did not differ on total tumor accumulation in non-small cell lung cancer.

A novel combination for the treatment of small cell lung cancer: Active targeted irinotecan and stattic co-loaded PLGA nanoparticles.

Polymeric nanoparticles are widely used drug delivery systems for cancer treatment due to their properties such as ease of passing through biological membranes, opportunity to modify drug release, specifically targeting drugs to diseased areas, and potential of reducing side effects. Here, we formulated irinotecan and Stattic co-loaded PLGA nanoparticles targeted to small cell lung cancer. Nanoparticles were successfully conjugated with CD56 antibody with a conjugation efficiency of 84.39 ± 1.01%, and characterization of formulated nanoparticles was conducted with in-vitro and in-vivo studies. Formulated particles had sizes in the range of 130-180 nm with PDI values smaller than 0.3. Encapsulation and active targeting of irinotecan and Stattic resulted in increased cytotoxicity and anti-cancer efficiency in-vitro. Furthermore, it was shown with ex-vivo biodistribution studies that conjugated nanoparticles were successfully targeted to CD56-expressing SCLC cells and distributed mainly to tumor tissue and lungs. Compliant with our hypothesis and literature, the STAT3 pathway was successfully inhibited with Stattic solution and Stattic loaded nanoparticles. Additionally, intravenous injection of conjugated co-loaded nanoparticles resulted in decreased side effects and better anti-tumor activity than individual solutions of drugs in SCLC tumor-bearing mice. These results may indicate a new treatment option for clinically aggressive small cell lung cancer.

Mixed micelles formulation for carvedilol delivery: In-vitro characterization and in-vivo evaluation.

Carvedilol (CAR) is a widely studied, beta and alpha-1 blocker, antihypertensive drug due to its poor water solubility and low oral bioavailability (25-35%). The aim of this work is to improve poor water solubility and the pharmacokinetic parameters of carvedilol by using an optimized and self-assembly prepared micelle formulation. Optimized micelle formulation composed of Pluronic® F127, D-α-tocopheryl polyethylene glycol 1000 succinate, L-cysteine HCl in a ratio of 4:3:3. Micellar size, polydispersity index, zeta potential, morphology, critical micelle concentration, thermal behaviors, in-vitro dissolution of micelles and pharmacokinetic parameters in rats were characterized in this study. Carvedilol aqueous solubility increased (up to 271-fold) as a result of its encapsulation within a mixed micelle formulation. The measured micellar sizes of blank and carvedilol loaded mixed micelles are lower than 30 nm with size distributions of 26.69 ±â€¯2.93 nm and 24.16 ±â€¯4.89 nm, respectively. Transmission electron microscopy revealed that the micelles were spherically shaped. There is a significant enhancement of carvedilol dissolution compared to commercially available tablet formulation (f2 < 50). The in-vivo test demonstrated that the t1/2 and AUC0-∞ values of micelles were approximately 10.89- and 2.65-fold greater than that of the commercial tablets, respectively. Based on our study, bring such applications into being may provide effective new drugs for treatment armamentarium of cardiovascular diseases and hypertension in near future.

In silico analysis suggests less effective MHC-II presentation of SARS-CoV-2 RBM peptides: Implication for neutralizing antibody responses.

SARS-CoV-2 antibodies develop within two weeks of infection, but wane relatively rapidly post-infection, raising concerns about whether antibody responses will provide protection upon re-exposure. Here we revisit T-B cooperation as a prerequisite for effective and durable neutralizing antibody responses centered on a mutationally constrained RBM B cell epitope. T-B cooperation requires co-processing of B and T cell epitopes by the same B cell and is subject to MHC-II restriction. We evaluated MHC-II constraints relevant to the neutralizing antibody response to a mutationally-constrained B cell epitope in the receptor binding motif (RBM) of the spike protein. Examining common MHC-II alleles, we found that peptides surrounding this key B cell epitope are predicted to bind poorly, suggesting a lack MHC-II support in T-B cooperation, impacting generation of high-potency neutralizing antibodies in the general population. Additionally, we found that multiple microbial peptides had potential for RBM cross-reactivity, supporting previous exposures as a possible source of T cell memory.

Phenotypically supervised single-cell sequencing parses within-cell-type heterogeneity.

To better understand cellular communication driving diverse behaviors, we need to uncover the molecular mechanisms of within-cell-type functional heterogeneity. While single-cell RNA sequencing (scRNAseq) has advanced our understanding of cell heterogeneity, linking individual cell phenotypes to transcriptomic data remains challenging. Here, we used a phenotypic cell sorting technique to ask whether phenotypically supervised scRNAseq analysis (pheno-scRNAseq) can provide more insight into heterogeneous cell behaviors than unsupervised scRNAseq. Using a simple 3D in vitro breast cancer (BRCA) model, we conducted pheno-scRNAseq on invasive and non-invasive cells and compared the results to phenotype-agnostic scRNAseq analysis. Pheno-scRNAseq identified unique and more selective differentially expressed genes than unsupervised scRNAseq analysis. Functional studies validated the utility of pheno-scRNAseq in understanding within-cell-type functional heterogeneity and revealed that migration phenotypes were coordinated with specific metabolic, proliferation, stress, and immune phenotypes. This approach lends new insight into the molecular systems underlying BRCA cell phenotypic heterogeneity.

Aggregation of chitosan nanoparticles in cell culture: Reasons and resolutions.

Nanoparticles are promising drug delivery systems which are flexible for targeting specific tissues to reduce therapeutic doses and minimize side effects. Nanoparticles should be maintained with high stability and uniformity; however, aggregation is a major challenge which commonly impairs stability and efficacy of nanocarriers. In this study, we revisited the factors that influence the stability of chitosan (Protasan™ UP CL113) nanoparticles prepared with ionotropic gelation, widely recognized to be prone to aggregation, and proposed a model to overcome the negative influence of aggregation while testing in vitro efficacy. Decrease in pH due to cell proliferation, 37 °C cell culture temperature, serum in culture media, and incubation time were considered as factors causing chitosan nanoparticles' aggregation which deteriorates cell culture assay readouts, increases optical density values and leads to false-positive results. Size and stability studies were not sufficient to avoid misleading results in cell culture. The chitosan nanoparticle aggregation was almost inevitable under standard culture conditions; nevertheless, the removal of nanoparticles before aggregation but after an incubation period long enough for efficient cellular uptake was determined as a feasible and inexpensive method for testing the in vitro efficacy of polymeric nanoformulations. This approach was used with blank and gemcitabine-loaded chitosan nanoparticles on pancreatic cancer cells and proved to be useful for reliable cytotoxicity results.

Elevated neoantigen levels in tumors with somatic mutations in the HLA-A, HLA-B, HLA-C and B2M genes.

BACKGROUND: The major histocompatibility complex class I (MHC-I) molecule is a protein complex that displays intracellular peptides to T cells, allowing the immune system to recognize and destroy infected or cancerous cells. MHC-I is composed of a highly polymorphic HLA-encoded alpha chain that binds the peptide and a Beta-2-microglobulin (B2M) protein that acts as a stabilizing scaffold. HLA mutations have been implicated as a mechanism of immune evasion during tumorigenesis, and B2M is considered a tumor suppressor gene. However, the implications of somatic HLA and B2M mutations have not been fully explored in the context of antigen presentation via the MHC-I molecule during tumor development. To understand the effect that B2M and HLA MHC-I molecule mutations have on mutagenesis, we analyzed the accumulation of mutations in patients from The Cancer Genome Atlas according to their MHC-I molecule mutation status. RESULTS: Somatic B2M and HLA mutations in microsatellite stable tumors were associated with higher overall mutation burden and a larger fraction of HLA-binding neoantigens when compared to B2M and HLA wild type tumors. B2M and HLA mutations were highly enriched in patients with microsatellite instability. B2M mutations tended to occur relatively early during patients' respective tumor development, whereas HLA mutations were either early or late events. In addition, B2M and HLA mutated patients had higher levels of immune infiltration by natural killer and CD8+ T cells and higher levels of cytotoxicity. CONCLUSIONS: Our findings add to a growing body of evidence that somatic B2M and HLA mutations are a mechanism of immune evasion by demonstrating that such mutations are associated with a higher load of neoantigens that should be presented via MHC-I.

Identifying Driver Interfaces Enriched for Somatic Missense Mutations in Tumors.

Human cancers often harbor large numbers of somatic mutations. However, only a small proportion of these mutations are expected to contribute to tumor growth and progression. Therefore, determining causal driver mutations and the genes they target is becoming an important challenge in cancer genomics. Here we describe an approach for mapping somatic mutations onto 3D structures of human proteins in complex to identify "driver interfaces." Our strategy relies on identifying protein-interaction interfaces that are unexpectedly biased toward nonsynonymous mutations, which suggests that these interfaces are subject to positive selection during tumorigenesis, implicating the interacting proteins as candidate drivers.

The Emerging Potential for Network Analysis to Inform Precision Cancer Medicine.

Precision cancer medicine promises to tailor clinical decisions to patients using genomic information. Indeed, successes of drugs targeting genetic alterations in tumors, such as imatinib that targets BCR-ABL in chronic myelogenous leukemia, have demonstrated the power of this approach. However, biological systems are complex, and patients may differ not only by the specific genetic alterations in their tumor, but also by more subtle interactions among such alterations. Systems biology and more specifically, network analysis, provides a framework for advancing precision medicine beyond clinical actionability of individual mutations. Here we discuss applications of network analysis to study tumor biology, early methods for N-of-1 tumor genome analysis, and the path for such tools to the clinic.

Tumor-Induced Myeloid Cells Are Reduced by Gemcitabine-Loaded PAMAM Dendrimers Decorated with Anti-Flt1 Antibody.

While reshaping their microenvironment, tumors are also capable of influencing systemic processes including myeloid cell production. Therefore, the tumor-induced myeloid cells, such as myeloid-derived suppressor cells (MDSCs), which are characterized with pro-cancer properties, became another target in order to increase the success of the therapy. This study evaluated the capacity of a novel dendrimeric drug delivery platform to eliminate tumor-induced myeloid cells. As described in a previous study by our research group, the anti-Flt1 antibody-conjugated polyethylene glycol (PEG)-cored poly(amidoamine) (PAMAM) dendrimers improved the efficacy of gemcitabine against pancreatic cancer. Here, the biodistribution studies showed that these dendrimeric structures accumulated in the compartments that became rich in myeloid cells in the pancreatic tumor-bearing mice. When gemcitabine was loaded into the dendrimer complexes, the number of myeloid cells was significantly reduced while the percentage distribution of granulocytic and monocytic myeloid cells was not always significantly altered. The CD11b+Ly6G-Ly6C+ monocytes were more severely affected by the treatments than CD11b+Ly6G+Ly6C+ granulocytes. Immune infiltration levels in the tumor tissue were also altered. Myeloid cells in the spleen and F4/80+ macrophages of the liver were protected. The compartments, such as the liver and the bone marrow, which are known to have high vascular endothelial growth factor (VEGF)-Flt1 pathway activity, were particularly targeted by gemcitabine when delivered through anti-Flt1 antibody-conjugated PAMAM dendrimers. In conclusion, chemotherapeutic agents complexed with dendrimers not only improve anticancer efficacy, but they also assist in the elimination of the tumor-induced myeloid cells.

Effective targeting of gemcitabine to pancreatic cancer through PEG-cored Flt-1 antibody-conjugated dendrimers.

Tumor-targeted delivery of anticancer drugs using dendrimers has been recognized as a promising strategy to increase efficiency and reduce adverse effects of chemotherapy. Herein, we developed a dendrimer-based drug delivery system targeting Flt-1 (a receptor for vascular endothelial growth factors (VEGF)) receptor to improve therapeutic efficacy of gemcitabine in pancreatic cancer. Synthesized polyethylene glycol (PEG)-cored PAMAM dendrimers, which bear anionic carboxylic acid groups on the surface were modified with PEG chains, which were then conjugated with Flt-1 antibody. Following structural and chemical characterization studies, gemcitabine HCl-dendrimer inclusion complexes were successfully prepared. These complexes were efficiently engulfed by Flt-1 expressing pancreatic cancer cells, which enhanced the cytotoxicity of gemcitabine. Moreover, pancreatic tumors established in mice were highly targeted by PEG-cored Flt-1 antibody-conjugated dendrimers and increased accumulation of these gemcitabine-loaded complexes exhibited satisfactory in vivo anti-cancer efficacy. In conclusion, dendrimer-based targeted delivery of chemotherapeutics may serve as a promising approach for the treatment of malignancies such as pancreatic cancer that do not benefit from conventional chemotherapy.

Preparation and in vitro evaluation of 5-fluorouracil-loaded PCL nanoparticles for colon cancer treatment.

Nanoparticles loaded with 5-fluorouracil (5-FU) for colon cancer therapies were prepared using the solvent evaporation technique, which involved lyophilization by freeze-drying. Formulations produced a substantially high encapsulation efficiency of approximately 93%. A positive correlation was seen when increasing polycaprolactone (PCL) and/or PVA concentrations and the size of nanoparticles produced. Increasing PCL concentration had a considerable influence on PDI while increasing PVA concentration had a lesser effect. All nanoparticles possessed a negative zeta potential, particularly in formulations with low polymer and polymer emulsifier concentrations. The formulation with the lowest PCL and PVA concentration was characterized by the most optimal properties; which accounts for the desirable delayed release profile of the active drug in dissolution testing indicating an improved targeting capability and enhanced bioavailability at the action site. Cytotoxicity studies showed that 5-FU loaded PCL nanoparticles had higher antiproliferative effect than free 5-FU on Caco-2 cell line (p < 0.05). The encouraging results obtained offer reasons for optimism regarding the future of 5-FU nanoparticles as a promising drug delivery system which could be further improved by including either enteric coating or encapsulating the nanoparticles onto microparticles to overcome unanticipated degradation.