Liangliang Hao

Immunomodulatory nucleic acids have extraordinary promise for treating disease, yet clinical progress has been limited by a lack of tools to safely increase activity in patients. Immunomodulatory nucleic acids act by agonizing or antagonizing endosomal toll-like receptors (TLR3, TLR7/8, and TLR9), proteins involved in innate immune signaling. Immunomodulatory spherical nucleic acids (SNAs) that stimulate (immunostimulatory, IS-SNA) or regulate (immunoregulatory, IR-SNA) immunity by engaging TLRs have been designed, synthesized, and characterized. Compared with free oligonucleotides, IS-SNAs exhibit up to 80-fold increases in potency, 700-fold higher antibody titers, 400-fold higher cellular responses to a model antigen, and improved treatment of mice with lymphomas. IR-SNAs exhibit up to eightfold increases in potency and 30% greater reduction in fibrosis score in mice with nonalcoholic steatohepatitis (NASH). Given the clinical potential of SNAs due to their potency, defined chemical nature, and good tolerability, SNAs are attractive new modalities for developing immunotherapies.

Abstract Acidosis is a fundamental feature of the tumor microenvironment, which directly regulates tumor cell invasion by affecting immune cell function, clonal cell evolution, and drug resistance. Despite the important association of tumor microenvironment acidosis with tumor cell invasion, relatively little is known regarding which areas within a tumor are acidic and how acidosis influences gene expression to promote invasion. Here, we injected a labeled pH-responsive peptide to mark acidic regions within tumors. Surprisingly, acidic regions were not restricted to hypoxic areas and overlapped with highly proliferative, invasive regions at the tumor–stroma interface, which were marked by increased expression of matrix metalloproteinases and degradation of the basement membrane. RNA-seq analysis of cells exposed to low pH conditions revealed a general rewiring of the transcriptome that involved RNA splicing and enriched for targets of RNA binding proteins with specificity for AU-rich motifs. Alternative splicing of Mena and CD44, which play important isoform-specific roles in metastasis and drug resistance, respectively, was sensitive to histone acetylation status. Strikingly, this program of alternative splicing was reversed in vitro and in vivo through neutralization experiments that mitigated acidic conditions. These findings highlight a previously underappreciated role for localized acidification of tumor microenvironment in the expression of an alternative splicing-dependent tumor invasion program. Significance: This study expands our understanding of acidosis within the tumor microenvironment and indicates that acidosis induces potentially therapeutically actionable changes to alternative splicing.

Abstract The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell–derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell–derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell–derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets. Significance: This study provides insights into the biological roles of cancer cell–derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.

Synthetic biomarkers, bioengineered sensors that generate molecular reporters in diseased microenvironments, represent an emerging paradigm in precision diagnostics. Despite the utility of DNA barcodes as a multiplexing tool, their susceptibility to nucleases in vivo has limited their utility. Here we exploit chemically stabilized nucleic acids to multiplex synthetic biomarkers and produce diagnostic signals in biofluids that can be 'read out' via CRISPR nucleases. The strategy relies on microenvironmental endopeptidase to trigger the release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed urine. Our data suggest that DNA-encoded nanosensors can non-invasively detect and differentiate disease states in transplanted and autochthonous murine cancer models. We also demonstrate that CRISPR-Cas amplification can be harnessed to convert the readout to a point-of-care paper diagnostic tool. Finally, we employ a microfluidic platform for densely multiplexed, CRISPR-mediated DNA barcode readout that can potentially evaluate complex human diseases rapidly and guide therapeutic decisions.

Layer-by-layer nanoparticles (NPs) are modular drug delivery vehicles that incorporate multiple functional materials through sequential deposition of polyelectrolytes onto charged nanoparticle cores. Herein, we combined the multicomponent features and tumor targeting capabilities of layer-by-layer assembly with functional biosensing peptides to create a new class of nanotheranostics. These NPs encapsulate a high weight percentage of siRNA while also carrying a synthetic biosensing peptide on the surface that is cleaved into a urinary reporter upon exposure to specific proteases overexpressed in the tumor microenvironment. Importantly, this biosensor reports back on a molecular signature characteristic to metastatic tumors and associated with poor prognosis, MMP9 protease overexpression. This nanotheranostic mediates noninvasive urinary-based diagnostics in mouse models of three different cancers with simultaneous gene silencing in flank and metastatic mouse models of ovarian cancer.