Molecularly defined and spatially resolved cell atlas of the whole mouse brain.

In mammalian brains, millions to billions of cells form complex interaction networks to enable a wide range of functions. The enormous diversity and intricate organization of cells have impeded our understanding of the molecular and cellular basis of brain function. Recent advances in spatially resolved single-cell transcriptomics have enabled systematic mapping of the spatial organization of molecularly defined cell types in complex tissues1-3, including several brain regions (for example, refs. 1-11). However, a comprehensive cell atlas of the whole brain is still missing. Here we imaged a panel of more than 1,100 genes in approximately 10 million cells across the entire adult mouse brains using multiplexed error-robust fluorescence in situ hybridization12 and performed spatially resolved, single-cell expression profiling at the whole-transcriptome scale by integrating multiplexed error-robust fluorescence in situ hybridization and single-cell RNA sequencing data. Using this approach, we generated a comprehensive cell atlas of more than 5,000 transcriptionally distinct cell clusters, belonging to more than 300 major cell types, in the whole mouse brain with high molecular and spatial resolution. Registration of this atlas to the mouse brain common coordinate framework allowed systematic quantifications of the cell-type composition and organization in individual brain regions. We further identified spatial modules characterized by distinct cell-type compositions and spatial gradients featuring gradual changes of cells. Finally, this high-resolution spatial map of cells, each with a transcriptome-wide expression profile, allowed us to infer cell-type-specific interactions between hundreds of cell-type pairs and predict molecular (ligand-receptor) basis and functional implications of these cell-cell interactions. These results provide rich insights into the molecular and cellular architecture of the brain and a foundation for functional investigations of neural circuits and their dysfunction in health and disease.

A novel pan-PI3K inhibitor KTC1101 synergizes with anti-PD-1 therapy by targeting tumor suppression and immune activation.

BACKGROUND: Phosphoinositide 3-kinases (PI3Ks) are critical regulators of diverse cellular functions and have emerged as promising targets in cancer therapy. Despite significant progress, existing PI3K inhibitors encounter various challenges such as suboptimal bioavailability, potential off-target effects, restricted therapeutic indices, and cancer-acquired resistance. Hence, novel inhibitors that overcome some of these challenges are needed. Here, we describe the characterization of KTC1101, a novel pan-PI3K inhibitor that simultaneously targets tumor cell proliferation and the tumor microenvironment. Our studies demonstrate that KTC1101 significantly increases the anti-PD-1 efficacy in multiple pre-clinical mouse models. METHODS: KTC1101 was synthesized and characterized employing chemical synthesis, molecular modeling, Nuclear Magnetic Resonance (NMR), and mass spectrometry. Its target specificity was confirmed through the kinase assay, JFCR39 COMPARE analysis, and RNA-Seq analysis. Metabolic stability was verified via liver microsome and plasma assays, pharmacokinetics determined by LC-MS/MS, and safety profile established through acute toxicity assays to determine the LD50. The antiproliferative effects of KTC1101 were evaluated in a panel of cancer cell lines and further validated in diverse BALB/c nude mouse xenograft, NSG mouse xenograft and syngeneic mouse models. The KTC1101 treatment effect on the immune response was assessed through comprehensive RNA-Seq, flow cytometry, and immunohistochemistry, with molecular pathways investigated via Western blot, ELISA, and qRT-PCR. RESULTS: KTC1101 demonstrated strong inhibition of cancer cell growth in vitro and significantly impeded tumor progression in vivo. It effectively modulated the Tumor Microenvironment (TME), characterized by increased infiltration of CD8+ T cells and innate immune cells. An intermittent dosing regimen of KTC1101 enhanced these effects. Notably, KTC1101 synergized with anti-PD-1 therapy, significantly boosting antitumor immunity and extending survival in preclinical models. CONCLUSION: KTC1101's dual mechanism of action-directly inhibiting tumor cell growth and dynamically enhancing the immune response- represents a significant advancement in cancer treatment strategies. These findings support incorporating KTC1101 into future oncologic regimens to improve the efficacy of immunotherapy combinations.

Elucidating siRNA Cellular Delivery Mechanism Mediated by Quaternized Starch Nanoparticles.

Starch-based nanoparticles are highly utilized in the realm of drug delivery taking advantage of their biocompatibility and biodegradability. Studies have utilized Quaternized starch (Q-starch) for small interfering RNA (siRNA) delivery, in which quaternary amines enable interaction with negatively charged siRNA, resulting in self-assembly complexation. Although reports present numerous applications, the demonstrated efficacy is nonetheless limited due to undiscovered cellular mechanistic delivery. In this study, a deep dive into Q-starch/siRNA complexes' cellular mechanism and kinetics at the cellular level is revealed using single-particle tracking and cell population level using imaging flow cytometry. Uptake studies depict the efficient cellular internalization via endocytosis while a significant fraction of complexes' intracellular fate is lysosome. Utilizing single-particle tracking, it is found that an average of 15% of cellular detected complexes escape the endosome which holds the potential for the integration in the cytoplasmatic gene silencing mechanism. Additional experimental manipulations (overcoming endosomal escape) demonstrate that the complex's disassembly is the rate-limiting step, correlating Q-starch's structure-function properties as siRNA carrier. Structure-function properties accentuating the high affinity of the interaction between Q-starch's quaternary groups and siRNA's phosphate groups that results in low release efficiency. However, low-frequency ultrasound (20 kHz) application may have induced siRNA release resulting in faster gene silencing kinetics.

Highly Sensitive and Multiplexed Protein Measurements.

Proteins are involved in many biological processes. Misfolded, truncated, or mutated proteins as well as over- or underexpressed proteins have been implicated in many diseases. Therefore, detection and quantification of proteins is extremely important. Conventional techniques such as the enzyme-linked immunosorbent assay, Western Blot, and mass spectrometry have enabled discovery and study of proteins in biological samples. However, many important proteins are present at low concentrations, rendering them undetectable using conventional techniques. Furthermore, limited ability to simultaneously measure multiple proteins in a sample has constrained our ability to fully study the proteome. In this review, we comprehensively discuss approaches for protein detection. We first discuss the fundamentals of proteins and protein assays, including affinity reagents, surface functionalization, assay formats, signal detection, and multiplexing. We then discuss the challenges with these methods and review existing methods for highly sensitive and multiplexed protein detection. Finally, we review recent advances in protein detection from the literature and discuss challenges and future directions.

A SARS-CoV-2 Neutralization Assay Using Single Molecule Arrays.

Coronavirus disease 2019 (COVID-19) manifests with high clinical variability and warrants sensitive and specific assays to analyze immune responses in infected and vaccinated individuals. Using Single Molecule Arrays (Simoa), we developed an assay to assess antibody neutralization with high sensitivity and multiplexing capabilities based on antibody-mediated blockage of the ACE2-spike interaction. The assay does not require live viruses or cells and can be performed in a biosafety level 2 laboratory within two hours. We used this assay to assess neutralization and antibody levels in patients who died of COVID-19 and patients hospitalized for a short period of time and show that neutralization and antibody levels increase over time. We also adapted the assay for SARS-CoV-2 variants and measured neutralization capacity in pre-pandemic healthy, COVID-19 infected, and vaccinated individuals. This assay is highly adaptable for clinical applications, such as vaccine development and epidemiological studies.

MET activation confers resistance to cetuximab, and prevents HER2 and HER3 upregulation in head and neck cancer.

An understanding of the mechanisms underlying acquired resistance to cetuximab is urgently needed to improve cetuximab efficacy in patients with head and neck squamous cell carcinoma (HNSCC). Here, we present a clinical observation that MET pathway activation constitutes the mechanism of acquired resistance to cetuximab in a patient with HNSCC. Specifically, RNA sequencing and mass spectrometry analysis of cetuximab-sensitive (CetuxSen ) and cetuximab-resistant (CetuxRes ) tumors indicated MET amplification and overexpression in the CetuxRes tumor compared to the CetuxSen lesion. Stimulation of MET in HNSCC cell lines was sufficient to reactivate the MAPK pathway and to confer resistance to cetuximab in vitro and in vivo. In addition to the direct role of MET in reactivation of the MAPK pathway, MET stimulation abrogates the well-known cetuximab-induced compensatory feedback loop of HER2/HER3 expression. Mechanistically, we showed that the overexpression of HER2 and HER3 following cetuximab treatment is mediated by the ETS homologous transcription factor (EHF), and is suppressed by MET/MAPK pathway activation. Collectively, our findings indicate that evaluation of MET and HER2/HER3 in response to cetuximab in HNSCC patients can provide the rationale of successive line of treatment.

Digital direct detection of microRNAs using single molecule arrays.

MicroRNAs (miRNAs) are involved in many biological pathways, and detecting miRNAs accurately is critical for diagnosing a variety of diseases including cancer. However, most current methods for miRNA detection require lengthy sample preparation and amplification steps that can bias the results. In addition, lack of specificity and reproducibility give rise to various challenges in detection of circulating miRNAs in biological samples. In this work, we applied the Single Molecule Array (Simoa) technique to develop an ultra-sensitive sandwich assay for direct detection of multiple miRNAs without pre-amplification. We successfully detected miRNAs at femtomolar concentrations (with limits of detection [LODs] ranging from 1 to 30 fM) and high specificity (distinguishing miRNAs with a single nucleotide mismatch). This method was effective against a range of diverse target sequences, suggesting a general approach for miRNA detection. To demonstrate the practical application of this technique, we detected miRNAs in a variety of sample types including human serum and total RNA. The high sensitivity and simple workflow of the Simoa method represent excellent advantages for miRNA-based diagnostics of human diseases.

Competitive Immunoassays for the Detection of Small Molecules Using Single Molecule Arrays.

Small-molecule detection is important for many applications including clinical diagnostics, drug discovery, and measurements of environmental samples and agricultural products. Current techniques for small-molecule detection suffer from various limitations including low analytical sensitivity and complex sample processing. Furthermore, as a result of their small size, small molecules are difficult to detect using an antibody pair in a traditional sandwich assay format. To overcome these limitations, we developed an ultrasensitive competitive immunoassay for small-molecule detection using Single Molecule Arrays (Simoa). We show that the competitive Simoa assay is approximately 50-fold more sensitive than the conventional ELISA. We performed theoretical calculations to determine the factors that influence the sensitivity of competitive Simoa assays and used them to achieve maximal sensitivity. We also demonstrate detection of small molecules in complex biological samples. We show that the competitive Simoa assay is a simple, fast, and highly sensitive approach for ultrasensitive detection of small molecules.

Evaluation of Antibody Biotinylation Approaches for Enhanced Sensitivity of Single Molecule Array (Simoa) Immunoassays.

In this study, we evaluated the performance of Single Molecule Array (Simoa) immunoassays based on various detection antibody biotinylation approaches. Simoa immunoassays, like other sandwich ELISAs, are highly dependent on the interaction of a biotinylated detection antibody with an enzyme conjugated to streptavidin. Thus, we sought to assess whether different biotinylation reagents can improve the performance and sensitivity of Simoa assays. We selected three proteins, GM-CSF, IFNγ, and IL-2, that are present at ultralow levels in many biological samples. We compared the performance of these Simoa assays by using five different biotinylation reagents and varying the amount of molar fold excess biotin during the biotinylation process. We found that the choice of biotinylation reagent and the molar fold excess biotin can highly affect the performance of the Simoa assays, with differences of up to an order of magnitude in sensitivity. We also tested the performance of bulk ELISAs using the different biotinylated detection antibodies and observe differences greater than an order of magnitude in sensitivity. We show that evaluating different strategies for detection antibody biotinylation is a simple approach for optimizing immunoassay performance for enhanced sensitivity.

Regulation of natural cytotoxicity receptors by heparan sulfate proteoglycans in -cis: A lesson from NKp44.

NKp44 (NCR2) is a distinct member of natural cytotoxicity receptors (NCRs) family that can induce cytokine production and cytolytic activity in human NK cells. Heparan sulfate proteoglycans (HSPGs) are differentially expressed in various normal and cancerous tissues. HSPGs were reported to serve as ligands/co-ligands for NKp44 and other NCRs. However, HSPG expression is not restricted to either group and can be found also in NK cells. Our current study reveals that NKp44 function can be modulated through interactions with HSPGs on NK cells themselves in -cis rather than on target cells in -trans. The intimate interaction of NKp44 and the NK cell-associated HSPG syndecan-4 (SDC4) in -cis can directly regulate membrane distribution of NKp44 and constitutively dampens the triggering of the receptor. We further demonstrate, that the disruption of NKp44 and SDC4 interaction releases the receptor to engage with its ligands in -trans and therefore enhances NKp44 activation potential and NK cell functional response.

Extracellular pH regulates zinc signaling via an Asp residue of the zinc-sensing receptor (ZnR/GPR39).

Zinc activates a specific Zn(2+)-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca(2+) responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na(+)/H(+) exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn(2+) binding site, His(17) or His(19), or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp(313) with alanine resulted in similar Ca(2+) responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na(+)/H(+) exchange at pH 7.4 and pH 6.5. Substitution of Asp(313) to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp(313), which was shown to modulate Zn(2+) binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity.

A molecularly defined and spatially resolved cell atlas of the whole mouse brain.

In mammalian brains, tens of millions to billions of cells form complex interaction networks to enable a wide range of functions. The enormous diversity and intricate organization of cells in the brain have so far hindered our understanding of the molecular and cellular basis of its functions. Recent advances in spatially resolved single-cell transcriptomics have allowed systematic mapping of the spatial organization of molecularly defined cell types in complex tissues1-3. However, these approaches have only been applied to a few brain regions1-11 and a comprehensive cell atlas of the whole brain is still missing. Here, we imaged a panel of >1,100 genes in ~8 million cells across the entire adult mouse brain using multiplexed error-robust fluorescence in situ hybridization (MERFISH)12 and performed spatially resolved, single-cell expression profiling at the whole-transcriptome scale by integrating MERFISH and single-cell RNA-sequencing (scRNA-seq) data. Using this approach, we generated a comprehensive cell atlas of >5,000 transcriptionally distinct cell clusters, belonging to ~300 major cell types, in the whole mouse brain with high molecular and spatial resolution. Registration of the MERFISH images to the common coordinate framework (CCF) of the mouse brain further allowed systematic quantifications of the cell composition and organization in individual brain regions defined in the CCF. We further identified spatial modules characterized by distinct cell-type compositions and spatial gradients featuring gradual changes in the gene-expression profiles of cells. Finally, this high-resolution spatial map of cells, with a transcriptome-wide expression profile associated with each cell, allowed us to infer cell-type-specific interactions between several hundred pairs of molecularly defined cell types and predict potential molecular (ligand-receptor) basis and functional implications of these cell-cell interactions. These results provide rich insights into the molecular and cellular architecture of the brain and a valuable resource for future functional investigations of neural circuits and their dysfunction in diseases.

Ultrasensitive detection of circulating LINE-1 ORF1p as a specific multi-cancer biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. While proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1, L1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible detectable expression in corresponding normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore the potential of ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 M) ORF1p concentrations in patient plasma samples across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multi-analyte panel, and provides early therapeutic response monitoring in gastric and esophageal cancers. Together, these observations nominate ORF1p as a multi-cancer biomarker with potential utility for disease detection and monitoring.

Ultrasensitive Detection of Circulating LINE-1 ORF1p as a Specific Multicancer Biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. Although proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible expression in normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 mol/L) ORF1p concentrations in plasma across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multianalyte panel, provides early therapeutic response monitoring in gastroesophageal cancers, and is prognostic for overall survival in gastroesophageal and colorectal cancers. Together, these observations nominate ORF1p as a multicancer biomarker with potential utility for disease detection and monitoring. SIGNIFICANCE: The LINE-1 ORF1p transposon protein is pervasively expressed in many cancers and is a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 µL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring. See related commentary by Doucet and Cristofari, p. 2502. This article is featured in Selected Articles from This Issue, p. 2489.

Ectopic Lymphoid Follicle Formation and Human Seasonal Influenza Vaccination Responses Recapitulated in an Organ-on-a-Chip.

Lymphoid follicles (LFs) are responsible for generation of adaptive immune responses in secondary lymphoid organs and form ectopically during chronic inflammation. A human model of ectopic LF formation will provide a tool to understand LF development and an alternative to non-human primates for preclinical evaluation of vaccines. Here, it is shown that primary human blood B- and T-lymphocytes autonomously assemble into ectopic LFs when cultured in a 3D extracellular matrix gel within one channel of a two-channel organ-on-a-chip microfluidic device. Superfusion via a parallel channel separated by a microporous membrane is required for LF formation and prevents lymphocyte autoactivation. These germinal center-like LFs contain B cells expressing Activation-Induced Cytidine Deaminase and exhibit plasma cell differentiation upon activation. To explore their utility for seasonal vaccine testing, autologous monocyte-derived dendritic cells are integrated into LF Chips. The human LF chips demonstrate improved antibody responses to split virion influenza vaccination compared to 2D cultures, which are enhanced by a squalene-in-water emulsion adjuvant, and this is accompanied by increases in LF size and number. When inoculated with commercial influenza vaccine, plasma cell formation and production of anti-hemagglutinin IgG are observed, as well as secretion of cytokines similar to vaccinated humans over clinically relevant timescales.

MEK1/2 inhibition transiently alters the tumor immune microenvironment to enhance immunotherapy efficacy against head and neck cancer.

BACKGROUND: Although the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Therefore, we aimed to characterize the effect of MEK1/2 inhibition on the tumor microenvironment (TME) of MAPK-driven HNC, elucidate tumor-host interaction mechanisms facilitating immune escape on treatment, and apply rationale-based therapy combination immunotherapy and MEK1/2 inhibitor to induce tumor clearance. METHODS: Mouse syngeneic tumors and xenografts experiments were used to analyze tumor growth in vivo. Single-cell cytometry by time of flight, flow cytometry, and tissue stainings were used to profile the TME in response to trametinib (MEK1/2 inhibitor). Co-culture of myeloid-derived suppressor cells (MDSC) with CD8+ T cells was used to measure immune suppression. Overexpression of colony-stimulating factor-1 (CSF-1) in tumor cells was used to show the effect of tumor-derived CSF-1 on sensitivity to trametinib and anti-programmed death- 1 (αPD-1) in mice. In HNC patients, the ratio between CSF-1 and CD8A was measured to test the association with clinical benefit to αPD-1 and αPD-L1 treatment. RESULTS: Using preclinical HNC models, we demonstrated that treatment with trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the antitumor immunity of CD8+ T cells. Activation of CD8+ T cells by supplementation with αPD-1 antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to αPD-1-supplementation by attenuating the expression of tumor-derived CSF-1, which reduced the abundance of two CSF-1R+CD11c+ MDSC populations in the TME. In contrast, prolonged treatment with trametinib abolished the antitumor activity of αPD-1, because tumor cells undergoing the epithelial to mesenchymal transition in response to trametinib restored CSF-1 expression and recreated an immune-suppressive TME. CONCLUSION: Our findings provide the rationale for testing the trametinib/αPD-1 combination in HNC and highlight the importance of sensitizing tumors to αPD-1 by using MEK1/2 to interfere with the tumor-host interaction. Moreover, we describe the concept that treatment of cancer with a targeted therapy transiently induces an immune-active microenvironment, and supplementation of immunotherapy during this time further activates the antitumor machinery to cause tumor elimination.

Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements. SIGNIFICANCE: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397.

IGF2 Mediates Resistance to Isoform-Selective-Inhibitors of the PI3K in HPV Positive Head and Neck Cancer.

Over 50% of human papilloma positive head-and-neck cancer (HNCHPV+) patients harbor genomic-alterations in PIK3CA, leading to hyperactivation of the phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) pathway. Nevertheless, despite PI3K pathway activation in HNCHPV+ tumors, the anti-tumor activities of PI3K pathway inhibitors are moderate, mostly due to the emergence of resistance. Thus, for potent and long-term tumor management, drugs blocking resistance mechanisms should be combined with PI3K inhibitors. Here, we delineate the molecular mechanisms of the acquisition of resistance to two isoform-selective inhibitors of PI3K (isiPI3K), alpelisib (BYL719) and taselisib (GDC0032), in HNCHPV+ cell lines. By comparing the transcriptional landscape of isiPI3K-sensitive tumor cells with that of their corresponding isiPI3K-acquired-resistant tumor cells, we found upregulation of insulin growth factor 2 (IGF2) in the resistant cells. Mechanistically, we show that upon isiPI3K treatment, isiPI3K-sensitive tumor cells upregulate the expression of IGF2 to induce cell proliferation via the activation of the IGF1 receptor (IGF1R). Stimulating tumor cells with recombinant IGF2 limited isiPI3K efficacy and released treated cells from S phase arrest. Knocking-down IGF2 with siRNA, or blocking IGF1R with AEW541, resulted in superior anti-tumor activity of isiPI3K in vitro and ex vivo. In vivo, the combination of isiPI3K and IGF1R inhibitor induced stable disease in mice bearing either tumors generated by the HNCHPV+ UM-SCC47 cell line or HPV+ patient-derived xenografts. These findings indicate that IGF2 and the IGF2/IGF1R pathway may constitute new targets for combination therapies to enhance the efficacy of PI3K inhibitors for the treatment of HNCHPV+.

Evidence of intestinal inflammation in patients with cystic fibrosis.

OBJECTIVES: Treatment with pancreatic enzymes fails to completely correct malabsorption and gastrointestinal symptoms in patients with cystic fibrosis (CF). The aim of the present study was to examine the small intestine of patients with CF without overt evidence of gastrointestinal disease using capsule endoscopy (CE). METHODS: Patients with CF received the agile patency capsule and, depending on the result of that procedure, then underwent standard CE using the PillCam SB capsule (Given Imaging, Yokneam, Israel). A stool specimen was taken on the same day as the CE for determination of the calprotectin level. RESULTS: Forty-two patients with CF ages 10 to 36 years were included; 29 had pancreatic insufficiency. One patient failed to excrete the patency capsule after 36 hours and was withdrawn from the study. Pulmonary function was mild to moderate with FEV1 68.5% +/- 16% predicted. Review of the CE videos showed that most of the patients had varying degrees of diffuse areas of inflammatory findings in the small bowel including edema, erythema, mucosal breaks, and frank ulcerations. There were no adverse events. Fecal calprotectin levels were markedly high in patients with pancreatic insufficiency, 258 microg/g (normal <50). CONCLUSIONS: Small bowel mucosal pathology may be detected using CE in most of the patients with CF. The high fecal calprotectin levels found are suggestive of mucosal inflammation, which may correlate with the CE findings. Additional study is required to examine the possible relation of these mucosal lesions, which may be part of a newly identified enteropathy associated with CF, with persistent intestinal malabsorption in many of these patients.

Single-Molecule Arrays for Ultrasensitive Detection of Blood-Based Biomarkers for Immunotherapy.

Single-molecule array (Simoa) technology enables ultrasensitive protein detection that is suited to the development of peripheral blood-based assays for assessing immuno-oncology responses. We adapted a panel of Simoa assays to measure systemic cytokine levels from plasma and characterized physiologic variation in healthy individuals and preanalytic variation arising from processing and handling of patient samples. Insights from these preclinical studies led us to a well-defined set of Simoa assay conditions, a specimen processing protocol, and a data processing approach that we describe here. Simoa enables accurate quantitation of soluble immune signaling molecules in an unprecedented femtomolar range, opening up the potential for liquid biopsy-type approaches in immuno-oncology. We are using the method described here to distinguish PD-1 inhibitor nonresponders as early as after one dose after therapy and envision applications in characterizing PD-1 inhibitor resistance and detection of immune-related adverse effects.