Sangeeta N. Bhatia

Bile flow is an essential feature of the liver, and disruption of this process contributes to a range of liver pathologies. Efficient bile transport requires coordinated organization between hepatocytes and cholangiocytes at the hepatobiliary junction, a structure that remains poorly captured in existing in vitro models of liver disease. Here, we present a 3D multicellular spheroid-based model of the human hepatobiliary junction. Building on advances in organoid and spheroid engineering, we co-aggregate human hepatocytes and intrahepatic cholangiocytes, supported by murine fibroblasts, into adult hepatobiliary organoids (aHBOs). aHBOs directionally transport bile from hepatocyte canaliculi to cholangiocyte-lined ductule-like structures, visualized through a high-throughput imaging assay. Hepatobiliary junction formation and bile flow dynamics are quantified over time using a fluorescent bile acid analog and AI-assisted image analysis. When subjected to hypoxia-reoxygenation, aHBOs exhibit disrupted bile transport and distinct cell-type-specific responses, enabling interrogation of hepatocyte and cholangiocyte vulnerability to transplant-associated biliary hypoxia. Our findings suggest a reversible reduction in hepatocyte canalicular function under hypoxia, followed by selective cholangiocyte death upon reoxygenation, potentially contributing to biliary dysfunction after ischemic injury. This human-derived, scalable platform provides a phenotypically relevant model for dissecting mechanisms of biliary dysfunction and discovering therapeutics for hypoxic liver injury and cholestatic diseases.

Fibrosis contributes to ∼40% of mortality in the industrialized world. Fibrosis in the liver can spontaneously resolve when injury terminates. In this study, we establish a robust mouse model of fibrosis regression in MASH (metabolic dysfunction-associated steatohepatitis), a highly prevalent chronic liver disease worldwide, and perform single-cell and in situ molecular profiling to define the molecular drivers of fibrosis regression. Prediction of cell-cell communication identifies a Wnt9b-Sfrp2 crosstalk that emerges as fibrosis resolves, the perturbation of which attenuates spontaneous fibrosis regression. We further identify a subset of liver endothelial cells termed "Endo4" as the source of Wnt9b. Immunostaining for the Endo4 marker VWF using tissue clearing and 3D imaging reveals VWF+ vasculature juxtaposing activated hepatic stellate cells that penetrate deep into the fibrotic septa and exhibit in situ protease activity, establishing them as de facto scar-associated endothelial cells and a regulatory node in murine MASH fibrosis regression.

Abstract Proteases, enzymes that play critical roles in health and disease, exert their function through the cleavage of peptide bonds. Identifying substrates that are efficiently and selectively cleaved by target proteases is essential for studying protease activity and for harnessing it in protease-activated diagnostics and therapeutics. However, the vast design space of possible substrates (c.a. 20 10 amino acid combinations for a 10-mer peptide) and the limited accessibility of high-throughput activity profiling tools hinder the speed and success of substrate design. We present CleaveNet, an end-to-end AI pipeline for the design of protease substrates. Applied to matrix metalloproteinases, CleaveNet enhances the scale, tunability, and efficiency of substrate design. CleaveNet generates peptide substrates that exhibit sound biophysical properties and capture not only well-established but also previously-uncharacterized cleavage motifs. To control substrate design, CleaveNet incorporates a conditioning tag that steers peptide generation towards desired cleavage profiles, enabling targeted design of efficient and selective substrates. CleaveNet-generated substrates were validated experimentally through a large-scale in vitro screen, even in the challenging case of designing highly selective substrates for MMP13. We envision that CleaveNet will accelerate our ability to study and capitalize on protease activity, paving the way for in silico design tools across enzyme classes.

Despite the promise of engineered tissue implants for the treatment of organ failure, scaling of these constructs to sizes of therapeutic relevance remains a barrier to clinical translation. Here, we propose a strategy to circumvent this limitation: to instead implant a small-scale construct and then induce it to grow in situ after its engraftment into a host. Using engineered liver tissue as a proof-of-concept application, we integrated synthetic biology and tissue engineering tools to build liver tissues that can be expanded on-demand after implantation in vivo. To achieve this goal, we first identified the combination of Yes-associated protein (YAP) and growth factor (GF) signaling as sufficient to drive human hepatocyte proliferation in dense, three-dimensional engineered tissues. We then engineered control of these signaling axes using synthetic biology tools to drive human liver tissue expansion both in vitro and in vivo. As such, this work establishes a genetic strategy for generating large organ implants through bioengineered on-demand outgrowth via synthetic biology triggering (BOOST).

ABSTRACT Antibiotic-resistant (AMR) bacterial infections are a major global health threat. Despite the critical need for new antimicrobials, progress is constrained by protracted development timelines, as well as the requirement for chemical novelty to avoid cross-resistance. Although advances in high-throughput screening, genome mining, and machine learning have greatly accelerated antimicrobial discovery, insufficient separation between antibacterial efficacy and host toxicity remains a bottleneck, precluding the clinical development of many promising compounds. Here, we establish a generalizable, two-component strategy to engineer antimicrobial safety and mobilize otherwise inaccessible chemical space for antimicrobial therapy, using calicheamicin, a potent cytotoxin with unacceptable host toxicity, as a proof of concept. In the first arm, we engineer a conditionally-active drug conjugate that limits calicheamicin activity to infected tissue, thereby reducing systemic toxicity. In the second arm, we co-administer a re-engineered self-resistance enzyme from Micromonospora echinospora , the natural producer of calicheamicin, as an “antidote” to neutralize calicheamicin present outside of infected tissue, further mitigating off-target toxicity. The conditionally-active conjugate exhibits activity against Gram-negative and Gram-positive pathogens in response to a protease present within the infected microenvironment. When delivered in combination with the antidote, antibacterial efficacy is maintained while off-target toxicity is reduced in mouse models of Gram positive and negative bacterial pneumonia. We anticipate that our dual strategy, which engineers, rather than selects for enhanced drug safety, by combining conditional drug activity with antidote-driven neutralization of off-target effects, provides a generalizable framework for mobilizing other promising but toxic compounds as antimicrobials.

Abstract The human malaria parasite, Plasmodium falciparum , relies exclusively on Anopheles mosquitoes for transmission. Once ingested during blood feeding, most parasites die in the mosquito midgut lumen or during epithelium traversal 1 . How surviving ookinetes interact with midgut cells and form oocysts remains poorly understood, yet these steps are essential to initiate a remarkable growth process culminating in the production of thousands of infectious sporozoites 2 . Here, using single-cell RNA sequencing of both parasites and mosquito cells across different developmental stages and metabolic conditions, we unveil key transitions and mosquito–parasite interactions that occur in the midgut. Functional analyses uncover processes that regulate oocyst growth and identify the Plasmodium transcription factor PfSIP2 as essential for sporozoite infection of human hepatocytes. Combining shared mosquito–parasite barcode analysis with confocal microscopy, we reveal that parasites preferentially interact with midgut progenitor cells during epithelial crossing, potentially using their basal location as an exit landmark. Additionally, we show tight connections between extracellular late oocysts and surrounding muscle cells that may ensure parasite adherence to the midgut. We confirm our major findings in several mosquito–parasite combinations, including field-derived parasites. Our study provides fundamental insight into the molecular events that characterize previously inaccessible biological transitions and mosquito–parasite interactions, and identifies candidates for transmission-blocking strategies.

ABSTRACT Effective, rapid, and functionally perfusable vascularization remains a major challenge in tissue engineering. Current approaches to generating vasculature in vitro require multipart fabrication methods or complex and costly media supplements, limiting their scalability. Here, we demonstrate that exogenous electrical stimulation (ESTIM) offers a promising alternative by enhancing 3D vasculogenesis in engineered human tissues. Exposing 3D endothelial‐fibroblast cocultures to pulsed ESTIM promoted the formation of dense and branched vascular networks. In a microfluidic device model, we show that ESTIM induces the formation of a perfusable, interconnected vascular network, whereas unstimulated networks remain less mature. Importantly, we demonstrate that upon implantation, ESTIM‐pretreated vascular grafts exhibit elevated anastomosis with the host and perfusion with blood relative to the untreated grafts. In addition, we use ESTIM to promote engraftment of a vascularized 3D liver construct. Mechanistically, we find that ESTIM induces membrane hyperpolarization in endothelial cells via voltage‐gated potassium channels (K V ). Inhibiting K V s abrogated ESTIM's pro‐vasculogenic effects in endothelial cells. Conversely, pharmacologically activating hyperpolarization induced endothelial responses even without ESTIM, directly linking K V ‐mediated hyperpolarization as a key mechanism by which ESTIM drives vascular assembly and function. Ultimately, our work establishes ESTIM as a new orthogonal approach to promote the formation of perfusable vasculature in engineered tissues.

Abstract Cholestasis, or disruption in bile flow, is a common yet poorly understood feature of many liver diseases and injuries. Despite this, many engineered human tissue models of liver disease fail to recapitulate physiological bile flow. Here, we present a 3D multicellular spheroid-based model of the human hepatobiliary junction, the interface between hepatocytes and cholangiocytes often disrupted in liver disease that is required for directing bile excreted by hepatocytes into the biliary ductal system. Building on advances in organoid and spheroid engineering, we co-aggregate human hepatocytes and intrahepatic cholangiocytes into adult hepatobiliary organoids (aHBOs) that structurally connect and functionally transport bile. aHBOs directionally transport bile from hepatocyte bile canaliculi to cholangiocyte-lined ductules, which we visualize through a high-throughput imaging assay. Hepatobiliary junction formation and bile flow dynamics are quantified over time using fluorescent bile acid analogs and AI-assisted image analysis. When subjected to hypoxia-reoxygenation, aHBOs recapitulate features of biliary dysfunction that mimics the cholestasis and ischemia-reperfusion injury that complicates liver transplant. Our findings suggest that 1) a reversible reduction in hepatocyte canalicular function under hypoxia, followed by 2) selective cholangiocyte death upon reoxygenation, are processes that potentially contribute to biliary dysfunction upon ischemic injury. This human-derived, scalable platform provides a phenotypically-relevant in vitro model for dissecting biliary pathophysiology and lays the groundwork for a therapeutic discovery platform for post-transplant ischemic cholangiopathy and other cholestatic liver diseases.

Abstract Fibrosis, or scarring, can affect many organs including liver, lung, heart, kidney, intestines etc. and is responsible for ∼40% of mortality in the industrialized world. Compared to other organs, fibrosis in the liver typically resolves when the source of injury is extinguished. Elucidating the molecular mechanisms that underlie spontaneous fibrosis resolution in the liver may lead to novel antifibrotic strategies for all organs. In this study we established a robust mouse model of fibrosis regression in MASH (Metabolic dysfunction-Associated Steatohepatitis), a highly prevalent chronic liver diseases worldwide, and performed single cell and in situ molecular profiling of the liver to define novel drivers of fibrosis regression. As fibrosis regressed, we detected a reduction of inflammatory cells and an expansion of endothelial cells. Prediction of cell-cell communication using the Calligraphy pipeline identified a Wnt9b-Sfrp2 crosstalk that emerges as fibrosis resolved in our model. To establish the Wnt9b-Sfrp2 crosstalk as a driver of fibrosis resolution we treated mice with recombinant Sfrp2, which slowed spontaneous fibrosis regression compared to vehicle treated mice. From our single cell datasets we identified a subset of endothelial cells, termed “Endo4”, as the source of Wnt9b. Immunostaining of the Endo4 marker VWF using tissue clearing and 3D imaging revealed VWF+ vasculature enveloped by activated hepatic stellate cells (HSCs) that penetrated deep into the fibrotic septa, establishing Endo4 as de facto scar-associated endothelial cells and providing a structural basis of their cellular crosstalk with HSCs. Finally, using a recently developed in situ protease activity screen, prominent serine protease activity co-localized with both scar-associated Endo4 cells and HSCs. In summary, we uncovered an WNT-dependent endo-stellate crosstalk within the fibrotic niche as a novel regulatory node underlying murine MASH fibrosis regression, and a promising therapeutic target.

Abstract The systemic inhibition of IL-1b, a key mediator of pulmonary inflammation, has been shown to reduce the incidence of lung cancer in patients in years following treatment, but knowledge gaps surrounding its activation and role in the tumor microenvironment are hindering approaches for cancer interception. We developed a suite of activity-based technologies to probe inflammation in early lung cancer and identified a translational target candidate. We designed probes sensitive to various IL-1b-activating proteases and applied them to a murine model of inflammatory lung cancer, Kras/Trp53 -mutant with SIINFEKL expression (KPS). Our nanosensors revealed reduced cleavage of a caspase-1 reporter in the lungs of KPS mice treated with IL-1b antibody, as well as elevated caspase-1 expression and activity in naïve tumor tissue sections, highlighting the importance of caspase-1 processing of IL-1b during cancer development. We conducted a pre-clinical trial of a novel combination intervention by administering both IL-1b blockade and caspase-1 inhibition shortly after tumor induction. Following treatment, we observed significant reduction in lung cancer formation, including complete ablation of tumor incidence in nearly 20% of KPS mice. Our approach to understand the interplay of protease activity and cytokine activation supports development of new strategies to mitigate inflammation and intercept lung cancer progression.

Liquid biopsies offer the potential for early cancer detection as well as a non-invasive, real-time window into the evolving tumor as the cancer progresses (Turabi+, 2024 PMID: 39001494). In this context, circulating tumor DNA (ctDNA) - the fraction of cell-free DNA (cfDNA) shed by tumor cells into the bloodstream, has emerged as a promising candidate. It carries the genetic and epigenetic alterations specific to the originating tumor, making it an essential source of information for cancer diagnosis and monitoring. While in patients with advanced cancer and a higher tumor burden the proportion of ctDNA is >10%, and can even exceed 40% of the total cfDNA, this fraction is less than 1% in early-stage tumors making early detections challenging. We hypothesized that manipulating ctDNA biology could improve its recovery and developed a SPE-liposomal “priming agent" which transiently inhibit macrophage mediated clearance (Martin-Alonso/Tabrizi/Xiong+, 2024 PMID: 38236959). The targeting of SPE liposomes to liver macrophages was predominantly size-dependent, utilizing particles with the average hydrodynamic diameter between 230 and 260 nm. This range was designed to match the size of murine liver capillary fenestrae, such that the nanoparticles would preferentially target liver-resident macrophages over hepatocytes. However, effective macrophage priming still required a significant lipid load to increase cfDNA levels in circulation. To improve on our priming agents, we developed three distinct SPE-modified nanoparticles to target macrophage subpopulations in the liver and tumor. These next-generation priming agents enabled a 60-fold reduction in lipid dose while achieving a more than 1500-fold higher recovery of cfDNA compared to untreated mice. Additionally, we show that these nanoparticles provided a marked increase in the detection of mutant duplexes in a transplantation model of colon cancer lung metastases, Luc-MC26 tumor-bearing mice. In summary, we optimized macrophage-targeted nanoparticle-based strategies that significantly improved the recovery of cfDNA, and that enhanced early tumor detection rate. These technologies have the potential to improve the accuracy and sensitivity of liquid biopsies, as well as to extend our fundamental understanding of the in vivo dynamics of circulating nucleic acid structures. Citation Format: Kasturi Chakraborty, Carmen Martin-Alonso, Daniel Kim, Savan Patel, Sahil Patel, Zhenyi An, Shervin Tabrizi, Claire Sullivan, Lily Gao, J. Christopher Love, Viktor A. Adalsteinsson, Sangeeta N. Bhatia. Enhancing circulating tumor DNA recovery and liquid biopsy sensitivity with precision liposomal priming agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4564.

Pediatric pneumonia requires a rapid diagnostic technique that does not rely on sputum production. Recently engineered breath biomarkers that release infection-specific volatile organic compounds (VOCs) upon inhalation present an opportunity for breath-based diagnostics, but mass spectroscopy-based detection limits use at the point-of-need. We have developed a breath-based detection scheme with surface-enhanced Raman spectroscopy and gas-capturing tubes. Conducting area scans across SERS substrates using a 50x confocal microscope, we report detection in micromolar range concentrations of representative VOCs in breath samples. With advancements in portable Raman, this design proves promising for rapid, automated diagnosis at the point-of-care.

Conditional therapeutics that rely on disease microenvironment-specific triggers for activation are a promising strategy to improve therapeutic cargos. Among the investigated triggers, protease activity is used most often because of its dysregulation in several diseases. How to optimally fine-tune protease activation for different therapeutic cargos remains a challenge. Here, we designed nanobody-targeted conditional antimicrobial therapeutics to deliver a model therapeutic peptide and protein to the site of bacterial infection. We explored several parameters that influence proteolytic activation. We report the use of targeting nanobodies to enhance the activation of therapeutics that are otherwise activated inefficiently despite extensive optimization of the cleavable linker. Specifically, the pairing of Ly6G/C or ADAM10-targeting nanobodies with ADAM10-cleavable linkers improved activation via proximity-enabled reactivity. This study demonstrates a distinct role of active targeting in conditional therapeutic activation. More broadly, this optimization framework provides a guideline for the development of conditional therapeutics to treat various diseases in which protease activity is dysregulated.

Abstract Proteases, a class of enzymes that play critical roles in health and disease, exert their function through the cleavage of peptide bonds. Identifying substrates that are efficiently and selectively cleaved by target proteases is essential for studying protease activity and for harnessing their activity in protease-activated diagnostics and therapeutics. However, the vast design space of possible substrates (c.a. 20 10 unique amino acid combinations for a 10-mer peptide) and the limited accessibility of high-throughput activity profiling tools hinder the speed and success of substrate design. We present CleaveNet, an end-to-end AI pipeline for the design of protease substrates. Applied to matrix metalloproteinases, CleaveNet enhances the scale, tunability, and efficiency of substrate design. CleaveNet generates peptide substrates that exhibit sound biophysical properties and capture not only well-established but also novel cleavage motifs. To enable precise control over substrate design, CleaveNet incorporates a conditioning tag that enables generation of peptides guided by a target cleavage profile, enabling targeted design of efficient and selective substrates. CleaveNet-generated substrates were validated experimentally through a large-scale in vitro screen, even in the challenging case of designing highly selective substrates for MMP13. We envision that CleaveNet will accelerate our ability to study and capitalize on protease activity, paving the way for new in silico design tools across enzyme classes.

Abstract Liver disease affects millions annually in the United States, with orthotopic transplantation as the only curative option for many patients. However, the scarcity of donor organs highlights a need for alternative cell-based therapies. Hepatocyte-based approaches are promising due to the cells’ inherent synthetic, metabolic, and detoxifying functions, but they face critical barriers, including the lack of a scalable source of functional hepatocytes and poor engraftment. In this study, we developed a scalable process for expanding primary human hepatocytes (PHHs) while preserving their identity and function. By leveraging heterocellular aggregation with stromal cells, we generated cryopreserved “seed” constructs that maintained viability and function post-thaw. Seeds demonstrated enhanced metabolic and detoxification functions and robust engraftment across multiple anatomic sites outside of the liver. Our approach addresses key limitations of hepatocyte-based therapies, offering a stable, scalable, and clinically viable platform for liver cell therapy applications.

Abstract Macromolecular therapeutics designed for intracellular targets must overcome systemic delivery barriers, target cell membrane impermeability, and inefficient endosomal escape. Here, we engineer a class of conditional fusogenic liposomes (C-FLIPs) that harness the catalytic activity of extracellular proteases present in the pathological microenvironment. This context-specific activation enables on-target membrane fusion with cells in diseased tissue, resulting in cytosolic delivery of therapeutic payloads. We describe the cytoplasmic delivery of three prototypic macromolecular therapeutic classes: peptide degraders, cytotoxic proteins, and ribonucleoprotein particles (RNPs). We further develop C-FLIP to deliver granzyme B (GzmB) to the cytoplasm of cancer cells in vivo and induce pyroptosis in immunologically-inert tumors. Treatment with C-FLIP/GzmB reprograms the immunosuppressive tumor microenvironment and synergizes with checkpoint blockade to result in the regression of established tumors and induce immunological memory. This modular, non-viral, cytosolic delivery platform represents a promising approach to leverage pathological protease activity for targeted delivery of biologics.

Technical advances over the past two decades have enabled robust detection of cell-free DNA (cfDNA) in biological samples. Yet, higher clinical sensitivity is required to realize the full potential of liquid biopsies. This Opinion article argues that to overcome current limitations, the abundance of informative cfDNA molecules - such as circulating tumor DNA (ctDNA) - collected in a sample needs to increase. To accomplish this, new methods to modulate the biological processes that govern cfDNA production, trafficking and clearance in the body are needed, informed by a deeper understanding of cfDNA biology. Successful development of such methods could enable a major leap in the performance of liquid biopsies and vastly expand their utility across the spectrum of clinical care.

Abstract Inflammation is linked to prostate cancer progression. Inflammatory cell infiltrates are commonly observed in prostate biopsies, and inflammation-induced lesions (proliferative inflammatory atrophy, PIA) are precursors of prostate cancer. However, the mechanism by which inflammation impacts prostate cancer progression is poorly understood. Here, we investigated the significance of inflammation on prostate cancer progression using a cMyc-driven prostate adenocarcinoma mouse model (Hi-Myc). We observed a robust tumor-associated macrophage (TAM) infiltrate early during progression of Hi-Myc tumors. Depleting TAMs led to a decrease in both tumor weight and invasive area, demonstrating the functional importance of TAMs in tumor maintenance. To elucidate the molecular basis of how TAMs influence tumor progression, we collected Hi-Myc tumors throughout cancer development from the precursor stage of prostatic intraepithelial neoplasia to prostate adenocarcinoma and performed single-cell RNA sequencing (scRNA-seq). Our study revealed that a gene signature of strong IL-1β signaling activation was observed in TAMs from Hi-Myc tumors, but not in macrophages from wild-type prostates. Importantly, IL-1β neutralization led to delayed tumor progression with reduced tumor weight and invasive area. Furthermore, blocking IL-1β signaling decreased TAM infiltration, suggesting a positive feedback loop created by TAMs. To understand the effect of IL-1β on cancer cell invasion, we used a protease-dependent fluorescent probe to investigate the activity of major extracellular matrix degraders and found that IL-1β neutralization impairs MMP activity, likely through loss of expression by tumor cells and macrophages. To further investigate the targets of IL-1β signaling, we analyzed Il1r1 expression levels across all cell types and found the highest levels in cancer-associated fibroblasts (CAFs). Moreover, CAFs expressed elevated levels of the myeloid chemokines Ccl2, Csf1, Cxcl1, Cxcl2 as well as Il6 compared to fibroblasts from wild-type prostates. In vitro studies of wild-type prostate fibroblasts treated with recombinant IL-1β confirmed that IL-1β directly upregulates expression of these inflammatory cytokines and chemokines. In addition, IL-1β directly promotes proliferation of tumor-derived prostate cancer organoids in vitro. Overall, our study suggests that TAMs and CAFs cooperatively drive pro-tumorigenic IL-1β signaling in prostate cancer, demonstrating a direct mechanistic link between inflammation and prostate cancer progression. Citation Format: Young Sun Lee, Jimmy L. Zhao, Max Land, Joseph Chan, Perianne Smith, Roshan Sharma, Sanjay Kottapalli, Linda Fong, Zhenghao Chen, Cathy Wang, Jesse Kirkpatrick, Ava Soleimany, Samir Zaidi, Kayla Lawrence, Amanda Kulick, Teng Han, Zhen Sun, Philip Watson, Anuradha Gopalan, Ojasvi Chaudhary, Tianhao Xu, Ignas Masilionis, Ronan Chaligne, Dana Rathkopf, Michael Morris, Sangeeta Bhatia, Michael Haffner, Dana Pe'er, Charles Sawyers. Tumor-associated macrophages drive prostate cancer progression via IL-1β signaling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C006.

Abstract The human malaria parasite, Plasmodium falciparum , relies exclusively on Anopheles mosquitoes for transmission. Once ingested during blood feeding, most parasites die in the mosquito midgut lumen or during epithelium traversal 1 . How surviving ookinetes interact with midgut cells and form oocysts is poorly known, yet these steps are essential to initiate a remarkable growth process culminating in the production of thousands of infectious sporozoites 2 . Here, using single-cell RNA sequencing of both parasites and mosquito cells across different developmental stages and metabolic conditions, we unveil key transitions and mosquito-parasite interactions occurring in the midgut. Functional analyses uncover processes regulating oocyst growth and identify the transcription factor Pf SIP2 as essential for sporozoite infection of human hepatocytes. Combining shared mosquito-parasite barcode analysis with confocal microscopy, we reveal that parasites preferentially interact with midgut progenitor cells during epithelial crossing, potentially using their basal location as an exit landmark. Additionally, we show tight connections between extracellular late oocysts and surrounding muscle cells that may ensure parasites adherence to the midgut. We confirm our major findings in several mosquito-parasite combinations, including field-derived parasites. Our study provides fundamental insight into the molecular events characterizing previously inaccessible biological transitions and mosquito-parasite interactions, and identifies candidates for transmission-blocking strategies.

Critical aspects of physiology and cell function exhibit self-sustained ~24-hour variations termed circadian rhythms. In the liver, circadian rhythms play fundamental roles in maintaining organ homeostasis. Here, we established and characterized an in vitro liver experimental system in which primary human hepatocytes display self-sustained oscillations. By generating gene expression profiles of these hepatocytes over time, we demonstrated that their transcriptional state is dynamic across 24 hours and identified a set of cycling genes with functions related to inflammation, drug metabolism, and energy homeostasis. We designed and tested a treatment protocol to minimize atorvastatin- and acetaminophen-induced hepatotoxicity. Last, we documented circadian-dependent induction of pro-inflammatory cytokines when triggered by LPS, IFN-β, or Plasmodium infection in human hepatocytes. Collectively, our findings emphasize that the phase of the circadian cycle has a robust impact on the efficacy and toxicity of drugs, and we provide a test bed to study the timing and magnitude of inflammatory responses over the course of infection in human liver.

Abstract Conditional therapeutics that rely on disease microenvironment-specific triggers for activation are a promising strategy to improve therapeutic cargos. Among the investigated triggers, protease activity is used most often, because of its dysregulation in several diseases. How to optimally fine-tune protease activation for different therapeutic cargos remains a challenge. Here, we designed nanobody-targeted conditional antimicrobial therapeutics to deliver a model therapeutic peptide and protein to the site of bacterial infection. We explored several parameters that influence proteolytic activation. We report the use of targeting nanobodies to enhance the activation of therapeutics that are otherwise activated inefficiently, despite extensive optimization of the cleavable linker. Specifically, pairing of Ly6G/C or ADAM10-targeting nanobodies with ADAM10-cleavable linkers improved activation via proximity-enabled reactivity. More broadly, this optimization framework provides a guideline for the development of conditional therapeutics to treat various diseases where protease activity is dysregulated.

Liquid biopsies enable early detection and monitoring of diseases such as cancer, but their sensitivity remains limited by the scarcity of analytes such as cell-free DNA (cfDNA) in blood. Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. We sought to transiently augment the level of circulating tumor DNA (ctDNA) in a blood draw by attenuating its clearance in vivo. We report two intravenous priming agents given 1 to 2 hours before a blood draw to recover more ctDNA. Our priming agents consist of nanoparticles that act on the cells responsible for cfDNA clearance and DNA-binding antibodies that protect cfDNA. In tumor-bearing mice, they greatly increase the recovery of ctDNA and improve the sensitivity for detecting small tumors.

Although low-dose computed tomography screening improves lung cancer survival in at-risk groups, inequality remains in lung cancer diagnosis due to limited access to and high costs of medical imaging infrastructure. We designed a needleless and imaging-free platform, termed PATROL (point-of-care aerosolizable nanosensors with tumor-responsive oligonucleotide barcodes), to reduce resource disparities for early detection of lung cancer. PATROL formulates a set of DNA-barcoded, activity-based nanosensors (ABNs) into an inhalable format. Lung cancer-associated proteases selectively cleave the ABNs, releasing synthetic DNA reporters that are eventually excreted via the urine. The urinary signatures of barcoded nanosensors are quantified within 20 min at room temperature using a multiplexable paper-based lateral flow assay. PATROL detects early-stage tumors in an autochthonous lung adenocarcinoma mouse model with high sensitivity and specificity. Tailoring the library of ABNs may enable not only the modular PATROL platform to lower the resource threshold for lung cancer early detection tools but also the rapid detection of chronic pulmonary disorders and infections.

The pandemic outbreak of coronavirus (SARS CoV-2) has been going on over the last 3 years. The people are vaccinated with different vaccines targeting the S protein. Aim. Therefore, it is essential to have an assay that can detect different parts of the virus as a serological assay and can be performed as a point of care test. Hence, in this work, we decided to develop such an assay with the help of magnetic beads. Methods. The magnetic beads ELISA (MB ELISA) was developed in a microtube. The viral ligand-specific magnetic beads were used to detect the nucleoprotein (NP)-specific IgG antibodies in human plasma samples. The results were read with the naked eye as well as with professional ELISA readers. Results. 7 μL magnetic beads were suitable to detect the presence of NP-specific antibodies. The assay needs only a magnetic rack and a pipettor to be performed. The results were available within 30 min. The positive results were observed as yellow color visually but also read in ELISA reader as OD values. The sensitivity of this assay was 1:108 dilutions. The cross-reaction panel was negative with different pathogens and negative human plasma. Conclusions. This work may be the first report in literature about the development of a magnetic beads ELISA as a point of the care assay, which is reproducible, highly sensitive, robust, and easy to perform. It was used to detect the presence of NP-specific IgG antibodies in the plasma samples successfully. This assay can be used as a professional assay, where the results can be measured with an ELISA reader. This assay may be suitable in small clinics also under field conditions. It can be used to detect the SARS CoV-2 infection in vaccinated persons (S protein-based vaccines) along with non-vaccinated population in latent and active phase.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Abstract The late-stage detection of lung cancer leads to a high global mortality rate. Although low-dose computed tomography screening improves lung cancer survival in at-risk groups, this test still suffers from high rates of false positive results. In addition, inequality remains in the diagnosis of lung cancer as access to medical imaging infrastructure is limited. Here, we designed a needleless and imaging-free platform, termed PATROL ( p oint-of-care a erosolizable nanosensors with t umor- r esponsive ol igonucleotide barcodes), to increase detection accuracy, to reduce resource disparities for early detection of lung cancer, and to enable timely interception. PATROL formulates a set of DNA-barcoded, activity-based nanosensors (ABNs) into inhalable formats that can be delivered using clinical nebulizers or inhalers. Lung cancer-associated proteases in the tumor microenvironment selectively cleave the ABNs, releasing synthetic DNA reporters that are eventually excreted via the urine. The barcoded nanosensor signatures present in urine samples are quantified within 20 minutes using a multiplexable paper-based lateral flow assay at room temperature. PATROL detects early-stage tumors in an autochthonous lung adenocarcinoma mouse model with high sensitivity and specificity. Tailoring the library of ABNs may enable the modular PATROL platform to not only lower the resource thresholds required for early detection of lung cancer, but also enable rapid detection of chronic pulmonary disorders and infections.

Patients with pancreatic cancer commonly develop weight loss and muscle wasting. Whether adipose tissue and skeletal muscle losses begin before diagnosis and the potential utility of such losses for earlier cancer detection are not well understood. We quantify skeletal muscle and adipose tissue areas from computed tomography (CT) imaging obtained 2 months to 5 years before cancer diagnosis in 714 pancreatic cancer cases and 1748 matched controls. Adipose tissue loss is identified up to 6 months, and skeletal muscle wasting is identified up to 18 months before the clinical diagnosis of pancreatic cancer and is not present in the matched control population. Tissue losses are of similar magnitude in cases diagnosed with localized compared with metastatic disease and are not correlated with at-diagnosis circulating levels of CA19-9. Skeletal muscle wasting occurs in the 1-2 years before pancreatic cancer diagnosis and may signal an upcoming diagnosis of pancreatic cancer.

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.

Abstract Liquid biopsies using cell-free DNA (cfDNA) enable non-invasive detection and characterization of disease. Advances in sequencing methods have significantly improved the performance of liquid biopsies. Yet, despite these advances, sensitivity remains a fundamental challenge. In oncology, circulating tumor DNA (ctDNA) screening tests only detect 20-40% of stage I tumors and tests for minimal residual disease have only 25-50% sensitivity after surgery. The major barrier to better sensitivity is the intrinsic low level of ctDNA in plasma. Physical absence of tumor DNA molecules in a blood draw from a patient with low disease burden will result in a negative test, no matter the sensitivity of the ex vivo detection platform. To overcome this barrier, here we report a first-in-class intravenous DNA-binding priming agent that is given 2 hours prior to a blood draw to recover more ctDNA, boosting the detection of tumor mutations in plasma by 19-fold and increasing sensitivity from 6% to 84%. Given the rapid clearance of cfDNA from circulation, we reasoned that a priming agent that could bind and protect cfDNA from clearance could increase the tumor DNA recovered from plasma. We selected monoclonal antibodies (mAbs) as the class of molecules to use as cfDNA protectors given their persistence in circulation and ease of engineering. We identify a mAb that binds double-stranded DNA (dsDNA) and find on electrophoretic mobility shift assays that it binds both free and histone-bound dsDNA, the constituent components of cfDNA. We then demonstrate that this mAb can delay the clearance of dsDNA from plasma in vivo through co-injection of the mAb with free- and histone-bound dsDNA in mice. We further identify interactions with Fc-gamma-receptors as a key mediator of early clearance of dsDNA bound to the priming mAb. To address this early clearance and limit potential immune interactions, we engineer the mAb to abrogate its Fc effector function. The engineered variant decreases clearance of injected dsDNA by over 150-fold at one hour post-injection compared to dsDNA alone. We next evaluate the effect of our priming mAb on cancer detection. We use a targeted panel against 1,822 mutations in the MC26 murine colon carcinoma cell line to detect tumor mutations in the plasma of tumor bearing mice. The priming mAb results in 19-fold higher recovery of tumor DNA molecules compared to a control mAb. This improved recovery leads to detection of 77% of targeted sites in plasma compared to only 15% in the control group. In sensitivity analyses, higher recovery of mutant molecules improves sensitivity for cancer detection from 6% to 84% at 0.001% tumor fraction. In summary, we demonstrate an approach to overcome a key barrier in liquid biopsies. We envision that similar to contrast agents in clinical imaging, priming agents could significantly boost the diagnostic sensitivity of liquid biopsies and enable further applications across biomedicine. Citation Format: Shervin Tabrizi, Carmen Martin-Alonso, Kan Xiong, Timothy Blewett, Sainetra Sridhar, Zhenyi An, Sahil Patel, Sergio Rodriguez-Aponte, Christopher Naranjo, Douglas Shea, Todd Golub, Sangeeta N. Bhatia, Viktor A. Adalsteinsson, J. Christopher Love. A DNA-binding priming agent protects cell-free DNA and improves the sensitivity of liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3371.

<p>Intravital imaging of a subcutaneous pancreatic cancer xenograft (MIA PaCa-2) showing distribution of PEGylated iRGD TPNs (green) in tumor cell compartments surrounding abnormal tumor blood vessels. Collagen visualized through second-harmonic generation is displayed in magenta. Images in this video were captured at a depth of 60 μm from the edge of the exposed tumor. Each frame of 0.25 sec represents 5 min of elapsed time, starting 3 minutes after injection of the particles. Please refer to Figure S5 for reference scale bar.</p>

<p>Figure S1. In vitro optimization of iRGD TPNs in PDAC cell lines; Figure S2. Dual knockdown using TPNs; Figure S3. In vitro and in vivo properties and function of PEGylated iRGD TPNs; Figure S4. Overview of organoid image analysis; Figure S5. iRGD TPN-mediated delivery of siRNA in models of pancreatic cancer; Figure S6. iRGD-mediated TPN uptake and NRP-1 expression in the tumor vasculature; Figure S7. Stromal elements in a KPC-derived orthotopic tumor section following iRGD TPN administration.</p>

<p>Supplementary Fig. 1. Galectin-3 is present within tumors. Supplementary Fig. 2. Analysis of peripheral blood from tumor-bearing mice by flow cytometry. Supplementary Fig. 3. Galectin-3 expression on CD11b+ cells is not dependent on the presence of tumors. Supplementary Fig. 4. Galectin-3 presentation on CD11b+ leukocytes is not carbohydrate dependent. Supplementary Fig. 5. Tumor conditioned medium induces galectin-3-independent mobilization of CD11b+galectin-3+ leukocytes. Supplementary Fig. 6. Galectin-3 is not necessary for mobilization of galectin-3+CD11b+ leukocytes or metastatic seeding. Supplementary Fig. 7. IL-6 induces mobilization of galectin-3+CD11b+ leukocytes. Supplementary Fig. 8. IL-6 induces the mobilization of galectin-3+CD11b+ granulocytes. Supplementary Fig. 9. Galectin-3 expression by leukocytes or stromal cells is not necessary for IL-6-dependent mobilization of myeloid cells. Supplementary Fig. 10. The Thomsen-Friedenreich Antigen is elevated in metastatic cells and human lung cancer tissue. Supplementary Fig. 11. Gene expression microarray analysis of glycosyltransferases shows no evidence of differential regulation of transferases that generate galectin-3 ligands. Supplementary Fig. 12. Differential regulation of select glycosyltransferase activity prevents glycan elongation and preserves T-Antigen presentation. Supplementary Fig. 13. O-linked core 1 disaccharide synthesis is unaltered during metastatic progression, but branching and elongation affect T-Antigen surface presentation. Supplementary Fig. 14. Transfection of Gcnt3 and knockdown of St6galnac4. (A) qRT-PCR for Gcnt3 in 393M1 cells following transfection with Gcnt3. Supplementary Fig. 15. Transfection of Gcnt3 reduces T-Antigen expression and adhesion to galectin-3. Supplementary Fig. 16. Knockdown of St6GalNAcIV reduces the formation of metastases in vivo. Supplementary Fig. 17. Knockdown of St6galnac4 does not affect proliferation. Supplementary Fig. 18. Galectin-3 is elevated in mice bearing tumors.</p>

<div>Abstract<p>Pancreatic cancer is one of the leading causes of cancer-related death, with 5-year survival of 8.5%. The lack of significant progress in improving therapy reflects our inability to overcome the desmoplastic stromal barrier in pancreatic ductal adenocarcinoma (PDAC) as well as a paucity of new approaches targeting its genetic underpinnings. RNA interference holds promise in targeting key mutations driving PDAC; however, a nucleic acid delivery vehicle that homes to PDAC and breaches the stroma does not yet exist. Noting that the cyclic peptide iRGD mediates tumor targeting and penetration through interactions with α<sub>v</sub>β<sub>3/5</sub> integrins and neuropilin-1, we hypothesized that “tandem” peptides combining a cell-penetrating peptide and iRGD can encapsulate siRNA to form tumor-penetrating nanocomplexes (TPN) capable of delivering siRNA to PDAC. The use of directly conjugated iRGD is justified by receptor expression patterns in human PDAC biopsies. In this work, we optimize iRGD TPNs with polyethylene glycol (PEG)-peptide conjugates for systemic delivery to sites of disease. We show that TPNs effectively knockdown siRNA targets in PDAC cell lines and in an immunocompetent genetically engineered mouse model of PDAC. Furthermore, we validate their tumor-penetrating ability in three-dimensional organoids and autochthonous tumors. In murine therapeutic trials, TPNs delivering anti-<i>Kras</i> siRNA significantly delay tumor growth. Thus, iRGD TPNs hold promise in treating PDAC by not only overcoming physical barriers to therapy, but by leveraging the stroma to achieve knockdown of the gold-standard genetic target. Moreover, the modular construction of this delivery platform allows for facile adaptation to future genetic target candidates in pancreatic cancer. <i>Mol Cancer Ther; 17(11); 2377–88. ©2018 AACR</i>.</p></div>

<div>Abstract<p>Pancreatic cancer is one of the leading causes of cancer-related death, with 5-year survival of 8.5%. The lack of significant progress in improving therapy reflects our inability to overcome the desmoplastic stromal barrier in pancreatic ductal adenocarcinoma (PDAC) as well as a paucity of new approaches targeting its genetic underpinnings. RNA interference holds promise in targeting key mutations driving PDAC; however, a nucleic acid delivery vehicle that homes to PDAC and breaches the stroma does not yet exist. Noting that the cyclic peptide iRGD mediates tumor targeting and penetration through interactions with α<sub>v</sub>β<sub>3/5</sub> integrins and neuropilin-1, we hypothesized that “tandem” peptides combining a cell-penetrating peptide and iRGD can encapsulate siRNA to form tumor-penetrating nanocomplexes (TPN) capable of delivering siRNA to PDAC. The use of directly conjugated iRGD is justified by receptor expression patterns in human PDAC biopsies. In this work, we optimize iRGD TPNs with polyethylene glycol (PEG)-peptide conjugates for systemic delivery to sites of disease. We show that TPNs effectively knockdown siRNA targets in PDAC cell lines and in an immunocompetent genetically engineered mouse model of PDAC. Furthermore, we validate their tumor-penetrating ability in three-dimensional organoids and autochthonous tumors. In murine therapeutic trials, TPNs delivering anti-<i>Kras</i> siRNA significantly delay tumor growth. Thus, iRGD TPNs hold promise in treating PDAC by not only overcoming physical barriers to therapy, but by leveraging the stroma to achieve knockdown of the gold-standard genetic target. Moreover, the modular construction of this delivery platform allows for facile adaptation to future genetic target candidates in pancreatic cancer. <i>Mol Cancer Ther; 17(11); 2377–88. ©2018 AACR</i>.</p></div>

<p>Figure S1. In vitro optimization of iRGD TPNs in PDAC cell lines; Figure S2. Dual knockdown using TPNs; Figure S3. In vitro and in vivo properties and function of PEGylated iRGD TPNs; Figure S4. Overview of organoid image analysis; Figure S5. iRGD TPN-mediated delivery of siRNA in models of pancreatic cancer; Figure S6. iRGD-mediated TPN uptake and NRP-1 expression in the tumor vasculature; Figure S7. Stromal elements in a KPC-derived orthotopic tumor section following iRGD TPN administration.</p>

<p>Intravital imaging of a subcutaneous pancreatic cancer xenograft (MIA PaCa-2) showing distribution of PEGylated iRGD TPNs (green) in tumor cell compartments surrounding abnormal tumor blood vessels. Collagen visualized through second-harmonic generation is displayed in magenta. Images in this video were captured at a depth of 60 μm from the edge of the exposed tumor. Each frame of 0.25 sec represents 5 min of elapsed time, starting 3 minutes after injection of the particles. Please refer to Figure S5 for reference scale bar.</p>