Joint single-cell measurements of nuclear proteins and RNA in vivo.

Identifying gene-regulatory targets of nuclear proteins in tissues is a challenge. Here we describe intranuclear cellular indexing of transcriptomes and epitopes (inCITE-seq), a scalable method that measures multiplexed intranuclear protein levels and the transcriptome in parallel across thousands of nuclei, enabling joint analysis of transcription factor (TF) levels and gene expression in vivo. We apply inCITE-seq to characterize cell state-related changes upon pharmacological induction of neuronal activity in the mouse brain. Modeling gene expression as a linear combination of quantitative protein levels revealed genome-wide associations of each TF and recovered known gene targets. TF-associated genes were coexpressed as distinct modules that each reflected positive or negative TF levels, showing that our approach can disentangle relative putative contributions of TFs to gene expression and add interpretability to inferred gene networks. inCITE-seq can illuminate how combinations of nuclear proteins shape gene expression in native tissue contexts, with direct applications to solid or frozen tissues and clinical specimens.

SCITO-seq: single-cell combinatorial indexed cytometry sequencing.

The development of DNA-barcoded antibodies to tag cell surface molecules has enabled the use of droplet-based single-cell sequencing (dsc-seq) to profile protein abundances from thousands of cells simultaneously. As compared to flow and mass cytometry, the high per cell cost of current dsc-seq-based workflows precludes their use in clinical applications and large-scale pooled screens. Here, we introduce SCITO-seq, a workflow that uses splint oligonucleotides (oligos) to enable combinatorially indexed dsc-seq of DNA-barcoded antibodies from over 105 cells per reaction using commercial microfluidics. By encoding sample barcodes into splint oligos, we demonstrate that multiplexed SCITO-seq produces reproducible estimates of cellular composition and surface protein expression comparable to those from mass cytometry. We further demonstrate two modified splint oligo designs that extend SCITO-seq to achieve compatibility with commercial DNA-barcoded antibodies and simultaneous expression profiling of the transcriptome and surface proteins from the same cell. These results demonstrate SCITO-seq as a flexible and ultra-high-throughput platform for sequencing-based single-cell protein and multimodal profiling.

A Quantitative Pharmacology Model of Exosome-Mediated Drug Efflux and Perturbation-Induced Synergy.

Exosomes, naturally occurring vesicles secreted by cells, are undergoing development as drug carriers. We used experimental and computational studies to investigate the kinetics of intracellular exosome processing and exosome-mediated drug efflux and the effects of exosome inhibition. The experiments used four human-breast or ovarian cancer cells, a cytotoxic drug paclitaxel (PTX), two exosome inhibitors (omeprazole (OME), which inhibits exosome release, and GW4869 (GW), which inhibits synthesis of sphingolipid ceramide required for exosome formation), LC-MS/MS analysis of PTX levels in exosomes, and confocal microscopic study of endocytic transport (monitored using fluorescent nanoparticles and endocytic organelle markers). In all four cells, exosome production was enhanced by PTX but diminished by OME or GW (p < 0.05); the PTX enhancement was completely reversed by OME or GW. Co-treatment with OME or GW simultaneously reduced PTX amount in exosomes and increased PTX amount and cytotoxicity in exosome-donor cells (corresponding to >2-fold synergy as indicated by curve shift and uncertainty envelope analyses). This synergy is consistent with the previous reports that OME co-administration significantly enhances the taxane activity in tumor-bearing mice and in patients with triple negative metastatic breast cancer. The experimental results were used to develop a quantitative pharmacology model; model simulations revealed the different effects of the two exosome inhibitors on intracellular PTX processing and subcellular distribution.

Integrated analysis of multimodal single-cell data.

The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce "weighted-nearest neighbor" analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.

Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells.

Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.

Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis.

BACKGROUND: High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. METHODS: Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. RESULTS: Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. CONCLUSIONS: We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

Characterizing the molecular regulation of inhibitory immune checkpoints with multimodal single-cell screens.

The expression of inhibitory immune checkpoint molecules, such as programmed death-ligand (PD-L)1, is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here, we apply expanded CRISPR-compatible (EC)CITE-seq, a technology that combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1 and leverage our multimodal data to identify both transcriptional and post-transcriptional modes of regulation. Specifically, we discover that the Kelch-like protein KEAP1 and the transcriptional activator NRF2 mediate the upregulation of PD-L1 after interferon (IFN)-γ stimulation. Our results identify a new mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multimodal single-cell perturbation screens.

Author Correction: Nuclei multiplexing with barcoded antibodies for single-nucleus genomics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

T cell-inducing vaccine durably prevents mucosal SHIV infection even with lower neutralizing antibody titers.

Recent efforts toward an HIV vaccine focus on inducing broadly neutralizing antibodies, but eliciting both neutralizing antibodies (nAbs) and cellular responses may be superior. Here, we immunized macaques with an HIV envelope trimer, either alone to induce nAbs, or together with a heterologous viral vector regimen to elicit nAbs and cellular immunity, including CD8+ tissue-resident memory T cells. After ten vaginal challenges with autologous virus, protection was observed in both vaccine groups at 53.3% and 66.7%, respectively. A nAb titer >300 was generally associated with protection but in the heterologous viral vector + nAb group, titers <300 were sufficient. In this group, protection was durable as the animals resisted six more challenges 5 months later. Antigen stimulation of T cells in ex vivo vaginal tissue cultures triggered antiviral responses in myeloid and CD4+ T cells. We propose that cellular immune responses reduce the threshold of nAbs required to confer superior and durable protection.

Nuclei multiplexing with barcoded antibodies for single-nucleus genomics.

Single-nucleus RNA-seq (snRNA-seq) enables the interrogation of cellular states in complex tissues that are challenging to dissociate or are frozen, and opens the way to human genetics studies, clinical trials, and precise cell atlases of large organs. However, such applications are currently limited by batch effects, processing, and costs. Here, we present an approach for multiplexing snRNA-seq, using sample-barcoded antibodies to uniquely label nuclei from distinct samples. Comparing human brain cortex samples profiled with or without hashing antibodies, we demonstrate that nucleus hashing does not significantly alter recovered profiles. We develop DemuxEM, a computational tool that detects inter-sample multiplets and assigns singlets to their sample of origin, and validate its accuracy using sex-specific gene expression, species-mixing and natural genetic variation. Our approach will facilitate tissue atlases of isogenic model organisms or from multiple biopsies or longitudinal samples of one donor, and large-scale perturbation screens.

Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics.

Despite rapid developments in single cell sequencing, sample-specific batch effects, detection of cell multiplets, and experimental costs remain outstanding challenges. Here, we introduce Cell Hashing, where oligo-tagged antibodies against ubiquitously expressed surface proteins uniquely label cells from distinct samples, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its original sample, robustly identify cross-sample multiplets, and "super-load" commercial droplet-based systems for significant cost reduction. We validate our approach using a complementary genetic approach and demonstrate how hashing can generalize the benefits of single cell multiplexing to diverse samples and experimental designs.

Quantitative contributions of processes by which polyanion drugs reduce intracellular bioavailability and transfection efficiency of cationic siRNA lipoplex.

RNA Interference (RNAi) is a potentially useful tool to correct the detrimental effects of faulty genes; several RNAi are undergoing clinical evaluation in various diseases. The present study identified the relative contributions of three mechanisms by which polyanion drugs reduced the gene silencing activity of Lipoplex, a complex of small interfering RNA (siRNA) and cationic liposomes. The study used a siRNA against the chemoresistance gene survivin and two model polyanion drugs (suramin, heparin). Products of Lipoplex destabilization were separated, identified, and/or quantified using ultrafiltration, gel electrophoresis, and RT-qPCR (quantitative reverse transcription polymerase chain reaction). Cell binding and endocytosis of fluorescence-labeled Lipoplex and the amount of siRNA at its site of action RISC (RNA-induced silencing complex) were evaluated using endocytosis markers, confocal microscopy, quantitative image analysis, immunoprecipitation, and RT-qPCR. The results show suramin and heparin exerted multiple concentration-dependent effects. First, these agents altered several Lipoplex properties (i.e., reduced particle size, changed surface charge, modified composition of protein biocorona). Second, both caused Lipoplex destabilization to release double- and single-strand siRNA and/or smaller siRNA-lipid complexes with reduced siRNA cargo. Third, both prevented the cell surface binding and internalization of Lipoplex, diminished the siRNA concentration in RISC, and retarded the mRNA knockdown. Suramin and heparin yielded qualitatively and quantitatively different results. Analysis of the experimental results of suramin using quantitative pharmacology (QP) modeling indicated the major cause of gene silencing activity loss depended on drug concentration, changing from inhibition of endocytosis at lower concentration (accounting for 60% loss at ~9µM) to inhibition of cell surface binding and loss of siRNA cargo at higher concentrations (accounting for 64% and 27%, respectively, at 70µM). In summary, the present study demonstrates the complex and dynamic interactions between polyanions and Lipoplex, and the use of QP modeling to delineate the contributions of three mechanisms to the eventual loss of gene silencing activity.

Exosome is a mechanism of intercellular drug transfer: Application of quantitative pharmacology.

PURPOSE: Exosomes are small membrane vesicles (30-100nm in diameter) secreted by cells into extracellular space. The present study evaluated the effect of chemotherapeutic agents on exosome production and/or release, and quantified the contribution of exosomes to intercellular drug transfer and pharmacodynamics. METHODS: Human cancer cells (breast MCF7, breast-to-lung metastatic LM2, ovarian A2780 and OVCAR4) were treated with paclitaxel (PTX, 2-1000nM) or doxorubicin (DOX, 20-1000nM) for 24-48h. Exosomes were isolated from the culture medium of drug-treated donor cells (Donor cells) using ultra-centrifugation, and analyzed for acetylcholinesterase activity, total proteins, drug concentrations, and biological effects (cytotoxicity and anti-migration) on drug-naïve recipient cells (Recipient cells). These results were used to develop computational predictive quantitative pharmacology models. RESULTS: Cells in exponential growth phase released ~220 exosomes/cell in culture medium. PTX and DOX significantly promoted exosome production and/or release in a dose- and time-dependent manner, with greater effects in ovarian cancer cells than in breast cancer cells. Exosomes isolated from Donor cells contained appreciable drug levels (2-7pmole/106 cells after 24h treatment with 100-1000nM PTX), and caused cytotoxicity and inhibited migration of Recipient cells. Quantitative pharmacology models that integrated cellular PTX pharmacokinetics with PTX pharmacodynamics successfully predicted effects of exosomes on intercellular drug transfer, cytotoxicity of PTX on Donor cells and cytotoxicity of PTX-containing exosomes on Recipient cells. Additional model simulations indicate that within clinically achievable PTX concentrations, the contribution of exosomes to active drug efflux increased with drug concentration and exceeded the p-glycoprotein efflux when the latter was saturated. CONCLUSIONS: Our results indicate (a) chemotherapeutic agents stimulate exosome production or release, and (b) exosome is a mechanism of intercellular drug transfer that contributes to pharmacodynamics of neighboring cells.

Delivery of cancer therapeutics to extracellular and intracellular targets: Determinants, barriers, challenges and opportunities.

Advances in molecular medicine have led to identification of worthy cellular and molecular targets located in extracellular and intracellular compartments. Effectiveness of cancer therapeutics is limited in part by inadequate delivery and transport in tumor interstitium. Parts I and II of this report give an overview on the kinetic processes in delivering therapeutics to their intended targets, the transport barriers in tumor microenvironment and extracellular matrix (TME/ECM), and the experimental approaches to overcome such barriers. Part III discusses new concepts and findings concerning nanoparticle-biocorona complex, including the effects of TME/ECM. Part IV outlines the challenges in animal-to-human translation of cancer nanotherapeutics. Part V provides an overview of the background, current status, and the roles of TME/ECM in immune checkpoint inhibition therapy, the newest cancer treatment modality. Part VI outlines the development and use of multiscale computational modeling to capture the unavoidable tumor heterogeneities, the multiple nonlinear kinetic processes including interstitial and transvascular transport and interactions between cancer therapeutics and TME/ECM, in order to predict the in vivo tumor spatiokinetics of a therapeutic based on experimental in vitro biointerfacial interaction data. Part VII provides perspectives on translational research using quantitative systems pharmacology approaches.

High Sensitivity RT-qPCR Assay of Nonlabeled siRNA in Small Blood Volume for Pharmacokinetic Studies: Application to Survivin siRNA.

RNAi therapeutics provide an opportunity to correct faulty genes, and several RNAi have entered clinical evaluation. The existing quantification methods typically use radioactivity- or fluorescence-labeled RNAi, require large blood volumes, and/or have a limited dynamic detection range. We established a quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assay to measure RNAi; the model analyte was survivin siRNA (siSurvivin). A second siRNA was used as the internal standard. The three major steps were (a) extraction of the two siRNAs from blood or water, (b) synthesis of their cDNA by poly-A extension, and (c) qPCR of cDNA. Standard curves were established. Utility of the assay was demonstrated in a pharmacokinetic study where all 12 samples for the blood concentration-time profile were obtained from a single mouse given an intravenous dose of 1 nmole siSurvivin (prepared as lipoplex with pegylated cationic liposomes). The RT-qPCR assay was sensitive (lower detection limit of 100 fM) and had a 5 × 107-fold dynamic range and low sample volume requirement (10 µL). The 16-point standard curves constructed using whole blood samples were linear (R (2) > 0.98). The intraday and interday variations for the slopes were ≤6%, although the variations for accuracy and precision at individual concentrations were substantially higher (58-145%). Standard curves prepared with water in place of blood showed similar results (<6% difference), indicating water may be used when blood is not available. The current RT-qPCR assay enabled the measurement of nonlabeled siRNA in small volume of blood samples.

Paclitaxel tumor priming promotes delivery and transfection of intravenous lipid-siRNA in pancreatic tumors.

The major barrier for using small interfering RNA (siRNA) as cancer therapeutics is the inadequate delivery and transfection in solid tumors. We have previously shown that paclitaxel tumor priming, by inducing apoptosis, expands the tumor interstitial space, improves the penetration and dispersion of nanoparticles and siRNA-lipoplexes in 3-dimensional tumor histocultures, and promotes the delivery and transfection efficiency of siRNA-lipoplexes under the locoregional setting in vivo (i.e., intraperitoneal treatment of intraperitoneal tumors). The current study evaluated whether tumor priming is functional for systemically delivered siRNA via intravenous injection, which would subject siRNA to several additional delivery barriers and elimination processes. We used the same pegylated cationic (PCat)-siRNA lipoplexes as in the intraperitoneal study to treat mice bearing subcutaneous human pancreatic Hs766T xenograft tumors. The target gene was survivin, an inducible chemoresistance gene. The results show single agent paclitaxel delayed tumor growth but also significantly induced the survivin protein level in residual tumors, whereas addition of PCat-siSurvivin completely reversed the paclitaxel-induced survivin and enhanced the paclitaxel activity (p<0.05). In comparison, PCat-siSurvivin alone did not yield survivin knockdown or antitumor activity, indicating the in vivo effectiveness of intravenous siRNA-mediated gene silencing requires paclitaxel cotreatment. Additional in vitro studies showed that paclitaxel promoted the cytoplasmic release of siGLO, a 22 nucleotide double-stranded RNA that has no mRNA targets, from its PCat lipoplex and/or endosomes/lysosomes. Taken together, our earlier and current data show paclitaxel tumor priming, by promoting the interstitial transport and cytoplasmic release, is critical to promote the delivery and transfection of siRNA in vivo. In addition, because paclitaxel has broad spectrum activity and is used to treat multiple types of solid tumors including the hard-to-treat pancreatic cancer, the synergistic paclitaxel+siSurvivin combination represents a potentially useful chemo-gene therapy.

Pharmacodynamics of telomerase inhibition and telomere shortening by noncytotoxic suramin.

We reported that suramin is an effective chemosensitizer at noncytotoxic concentrations (<50 µM); this effect was observed in multiple types of human xenograft tumors in vitro and in vivo. Clinical evaluation of noncytotoxic suramin is ongoing. Because (a) suramin inhibits reverse transcriptase, (b) telomerase is a reverse transcriptase, and (c) inhibition of telomerase enhances tumor chemosensitivity, we studied the pharmacodynamics of noncytotoxic suramin on telomerase activity and telomere length in cultured cells and tumors grown in animals. In three human cancer cells that depend on telomerase for telomere maintenance (pharynx FaDu, prostate PC3, breast MCF7), suramin inhibited telomerase activity in cell extracts and intact cells at concentrations that exhibited no cytotoxicity (IC50 of telomerase was between 1 and 3 µM vs. >60 µM for cytotoxicity), and continuous treatment at 10-25 µM for 6 weeks resulted in gradual telomere shortening (maximum of 30%) and cell senescence (measured by ß-galactosidase activity and elevation of mRNA levels of two senescence markers p16 and p21). In contrast, noncytotoxic suramin did not shorten the telomere in telomerase-independent human osteosarcoma Saos-2 cells. In mice bearing FaDu tumors, treatment with noncytotoxic suramin for 6 weeks resulted in telomere erosion in >95% of the tumor cells with an average telomere shortening of >40%. These results indicate noncytotoxic suramin inhibits telomerase, shortens telomere and induces cell senescence, and suggest telomerase inhibition as a potential mechanism of its chemosensitization.

Predicting diffusive transport of cationic liposomes in 3-dimensional tumor spheroids.

Nanotechnology is widely used in cancer research. Models that predict nanoparticle transport and delivery in tumors (including subcellular compartments) would be useful tools. This study tested the hypothesis that diffusive transport of cationic liposomes in 3-dimensional (3D) systems can be predicted based on liposome-cell biointerface parameters (binding, uptake, retention) and liposome diffusivity. Liposomes comprising different amounts of cationic and fusogenic lipids (10-30mol% DOTAP or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-20mol% DOPE or 1,2-dioleoyl-3-trimethylammonium-propane, +25 to +44mV zeta potential) were studied. We (a) measured liposome-cell biointerface parameters in monolayer cultures, and (b) calculated effective diffusivity based on liposome size and spheroid composition. The resulting parameters were used to simulate the liposome concentration-depth profiles in 3D spheroids. The simulated results agreed with the experimental results for liposomes comprising 10-30mol% DOTAP and ≤10mol% DOPE, but not for liposomes with higher DOPE content. For the latter, model modifications to account for time-dependent extracellular concentration decrease and liposome size increase did not improve the predictions. The difference among low- and high-DOPE liposomes suggests concentration-dependent DOPE properties in 3D systems that were not captured in monolayers. Taken together, our earlier and present studies indicate the diffusive transport of neutral, anionic and cationic nanoparticles (polystyrene beads and liposomes, 20-135nm diameter, -49 to +44mV) in 3D spheroids, with the exception of liposomes comprising >10mol% DOPE, can be predicted based on the nanoparticle-cell biointerface and nanoparticle diffusivity. Applying the model to low-DOPE liposomes showed that changes in surface charge affected the liposome localization in intratumoral subcompartments within spheroids.

Tumor priming enhances siRNA delivery and transfection in intraperitoneal tumors.

Cancers originating from the digestive system account for 290,000 or ~20% of all new cancer cases annually in the US. We previously developed paclitaxel-loaded tumor-penetrating microparticles (TPM) for intraperitoneal (IP) treatment of peritoneal tumors (Lu et al., 2008; Tsai et al., 2007; Tsai et al., 2013). TPM is undergoing NIH-supported IND-enabling studies for clinical evaluation. The present study evaluated the hypothesis that TPM, via inducing apoptosis and expanding the interstitial space, promotes the delivery and transfection of lipid vectors containing siRNA. The in vivo model was the metastatic human Hs766T pancreatic tumor that, upon IP injection, produced widely distributed solid tumors and ascites in the peritoneal cavity in 100% of animals. The target gene was survivin, an anti-apoptotic protein induced by chemotherapy and associated with metastases and poor prognosis of patients with gastric and colorectal cancers. The siRNA carrier was pegylated liposomes comprising cationic and neutral lipids plus a fusogenic lipid (PCat). PCat-loaded with survivin siRNA (PCat-siSurvivin) was active in cultured cells (decreased survivin mRNA and protein levels, reduced cell clonogenicity, enhanced paclitaxel activity), but lost its activity in vivo; this difference is consistent with the well-known problem of inadequate delivery and transfection of siRNA in vivo. In comparison, single agent TPM prolonged animal survival and, as expected, induced survivin expression in tumors. Addition of PCat-siSurvivin reversed the TPM-induced survivin expression and enhanced the antitumor activity of TPM. The finding that in vivo survivin knockdown by PCat-siSurvivin was successful only when it was given in combination with TPM provides the proof-of-concept that tumor priming promotes the delivery and transfection of liposomal siRNA. The data further suggest the TPM/PCat-siSurvivin combination as a potentially useful chemo-gene therapy for peritoneal cancer.

Vulnerability of dentate granule cells to disruption of arc expression in human amyloid precursor protein transgenic mice.

Activity-induced expression of Arc is necessary for maintenance of long-term potentiation and for memory consolidation. In transgenic (TG) mice with neuronal production of human amyloid precursor protein (hAPP) and hAPP-derived amyloid-beta (Abeta) peptides, basal Arc expression was reduced primarily in granule cells of the dentate gyrus. After exploration of a novel environment, Arc expression in these neurons was unaltered in hAPP mice but increased markedly in nontransgenic controls. Other TG neuronal populations showed no or only minor deficits in Arc expression, indicating a special vulnerability of dentate granule cells. The phosphorylation states of NR2B and ERK1/2 were reduced in the dentate gyrus of hAPP mice, suggesting attenuated activity in NMDA-dependent signaling pathways that regulate synaptic plasticity as well as Arc expression. Arc reductions in hAPP mice correlated with reductions in the actin-binding protein alpha-actinin-2, which is located in dendritic spines and, like Arc, fulfills important functions in excitatory synaptic activity. Reductions in Arc and alpha-actinin-2 correlated tightly with reductions in Fos and calbindin, shown previously to reflect learning deficits in hAPP mice. None of these alterations correlated with the extent of plaque formation, suggesting a plaque-independent mechanism of hAPP/Abeta-induced neuronal deficits. The brain region-specific depletion of factors that participate in activity-dependent modification of synapses may critically contribute to cognitive deficits in hAPP mice and possibly in humans with Alzheimer's disease.