A Randomized Trial of Two Remote Health Care Delivery Models on the Uptake of Genetic Testing and Impact on Patient-Reported Psychological Outcomes in Families With Pancreatic Cancer: The Genetic Education, Risk Assessment, and Testing (GENERATE) Study.

BACKGROUND & AIMS: Genetic testing uptake for cancer susceptibility in family members of patients with cancer is suboptimal. Among relatives of patients with pancreatic ductal adenocarcinoma (PDAC), The GENetic Education, Risk Assessment, and TEsting (GENERATE) study evaluated 2 online genetic education/testing delivery models and their impact on patient-reported psychological outcomes. METHODS: Eligible participants had ≥1 first-degree relative with PDAC, or ≥1 first-/second-degree relative with PDAC with a known pathogenic germline variant in 1 of 13 PDAC predisposition genes. Participants were randomized by family, between May 8, 2019, and June 1, 2021. Arm 1 participants underwent a remote interactive telemedicine session and online genetic education. Arm 2 participants were offered online genetic education only. All participants were offered germline testing. The primary outcome was genetic testing uptake, compared by permutation tests and mixed-effects logistic regression models. We hypothesized that Arm 1 participants would have a higher genetic testing uptake than Arm 2. Validated surveys were administered to assess patient-reported anxiety, depression, and cancer worry at baseline and 3 months postintervention. RESULTS: A total of 424 families were randomized, including 601 participants (n = 296 Arm 1; n = 305 Arm 2), 90% of whom completed genetic testing (Arm 1 [87%]; Arm 2 [93%], P = .014). Arm 1 participants were significantly less likely to complete genetic testing compared with Arm 2 participants (adjusted ratio [Arm1/Arm2] 0.90, 95% confidence interval 0.78-0.98). Among participants who completed patient-reported psychological outcomes questionnaires (Arm 1 [n = 194]; Arm 2 [n = 206]), the intervention did not affect mean anxiety, depression, or cancer worry scores. CONCLUSIONS: Remote genetic education and testing can be a successful and complementary option for delivering genetics care. (Clinicaltrials.gov, number NCT03762590).

A 3D Perfusable Platform for In Vitro Culture of Patient Derived Xenografts.

Many advanced cancer models, such as patient-derived xenografts (PDXs), offer significant benefits in their preservation of the native tumor's heterogeneity and susceptibility to treatments, but face significant barriers to use in their reliance on a rodent host for propagation and screening. PDXs remain difficult to implement in vitro, particularly in configurations that enable both detailed cellular analysis and high-throughput screening (HTS). Complex multilineage co-cultures with stromal fibroblasts, endothelium, and other cellular and structural components of the tumor microenvironment (TME) further complicate ex vivo implementation. Herein, the culture of multiple prostate cancer (PCa)-derived PDX models as 3D clusters within engineered biomimetic hydrogel matrices, in a HTS-compatible multiwell microfluidic format, alongside bone marrow-derived stromal cells and a perfused endothelial channel. Polymeric hydrogel matrices are customized for each cell type, enabling cell survival in vitro and facile imaging across all conditions. PCa PDXs demonstrate unique morphologies and reliance on TME partners, retention of known phenotype, and expected sensitivity or resistance to standard PCa therapeutics. This novel integration of technologies provides a fully human model, and expands the information to be gathered from each specimen, while avoiding the time and labor involved with animal-based testing.

Novel Models of Genetic Education and Testing for Pancreatic Cancer Interception: Preliminary Results from the GENERATE Study.

Up to 10% of patients with pancreatic ductal adenocarcinoma (PDAC) carry underlying germline pathogenic variants in cancer susceptibility genes. The GENetic Education Risk Assessment and TEsting (GENERATE) study aimed to evaluate novel methods of genetic education and testing in relatives of patients with PDAC. Eligible individuals had a family history of PDAC and a relative with a germline pathogenic variant in APC, ATM, BRCA1, BRCA2, CDKN2A, EPCAM, MLH1, MSH2, MSH6, PALB2, PMS2, STK11, or TP53 genes. Participants were recruited at six academic cancer centers and through social media campaigns and patient advocacy efforts. Enrollment occurred via the study website (https://GENERATEstudy.org) and all participation, including collecting a saliva sample for genetic testing, could be done from home. Participants were randomized to one of two remote methods that delivered genetic education about the risks of inherited PDAC and strategies for surveillance. The primary outcome of the study was uptake of genetic testing. From 5/8/2019 to 5/6/2020, 49 participants were randomized to each of the intervention arms. Overall, 90 of 98 (92%) of randomized participants completed genetic testing. The most frequently detected pathogenic variants included those in BRCA2 (N = 15, 17%), ATM (N = 11, 12%), and CDKN2A (N = 4, 4%). Participation in the study remained steady throughout the onset of the Coronavirus disease (COVID-19) pandemic. Preliminary data from the GENERATE study indicate success of remote alternatives to traditional cascade testing, with genetic testing rates over 90% and a high rate of identification of germline pathogenic variant carriers who would be ideal candidates for PDAC interception approaches. PREVENTION RELEVANCE: Preliminary data from the GENERATE study indicate success of remote alternatives for pancreatic cancer genetic testing and education, with genetic testing uptake rates over 90% and a high rate of identification of germline pathogenic variant carriers who would be ideal candidates for pancreatic cancer interception.

Hyperpolarized MRI with silicon micro and nanoparticles: Principles and applications.

Silicon-based micro and nanoparticles are ideally suited for use as biomedical imaging agents because of their biocompatibility, biodegradability, and simple surface chemistry that facilitates drug loading and targeting. A method to hyperpolarize silicon particles using dynamic nuclear polarization (DNP), which increases magnetic resonance (MR) imaging signals by several orders-of-magnitude through enhanced nuclear spin alignment, was developed to allow silicon particles to function as contrast agents for in vivo magnetic resonance imaging. In this review, we describe the application of the DNP technique to silicon particles and nanoparticles for background-free real-time molecular MR imaging. This review provides a summary of the state-of-the-science in silicon particle hyperpolarization with a detailed protocol for hyperpolarizing silicon particles. This information will foster awareness and spur interest in this emerging area of nanoimaging and provide a path to new developments and discoveries to further advance the field. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

Tumor necrosis factor-α and interferon-γ stimulate MUC16 (CA125) expression in breast, endometrial and ovarian cancers through NFκB.

Transmembrane mucins (TMs) are restricted to the apical surface of normal epithelia. In cancer, TMs not only are over-expressed, but also lose polarized distribution. MUC16/CA125 is a high molecular weight TM carrying the CA125 epitope, a well-known molecular marker for human cancers. MUC16 mRNA and protein expression was mildly stimulated by low concentrations of TNFα (2.5 ng/ml) or IFNγ (20 IU/ml) when used alone; however, combined treatment with both cytokines resulted in a moderate (3-fold or less) to large (> 10-fold) stimulation of MUC16 mRNA and protein expression in a variety of cancer cell types indicating that this may be a general response. Human cancer tissue microarray analysis indicated that MUC16 expression directly correlates with TNFα and IFNγ staining intensities in certain cancers. We show that NFκB is an important mediator of cytokine stimulation of MUC16 since siRNA-mediated knockdown of NFκB/p65 greatly reduced cytokine responsiveness. Finally, we demonstrate that the 250 bp proximal promoter region of MUC16 contains an NFκB binding site that accounts for a large portion of the TNFα response. Developing methods to manipulate MUC16 expression could provide new approaches to treating cancers whose growth or metastasis is characterized by elevated levels of TMs, including MUC16.

Transmembrane Mucin Expression and Function in Embryo Implantation and Placentation.

Transmembrane mucins (TMs) are extremely large, complex glycoproteins that line the apical surfaces of simple epithelia including those of the female reproductive tract. TMs provide a physical barrier consistent with their role as part of the innate immune system. This barrier function must be overcome in the context of embryo implantation to permit blastocyst attachment. Three major TMs have been identified in uterine epithelia of multiple species: MUC1, MUC4, and MUC16. MUC1 has been found in all species studied to date, whereas expression of MUC4 and MUC16 have been less well studied and may be species specific. The strategies for removing mucins to permit embryo attachment also vary in a species-specific way and include both hormonal suppression of TM gene expression and membrane clearance via cell surface proteases. Studies emerging from the cancer literature indicate that TMs can modulate a surprisingly wide variety of signal transduction processes. Furthermore, various cell surface proteins have been identified that bind either the oligosaccharide or protein motifs of TMs suggesting that these molecules may support cell attachment in some contexts, including trophoblast interactions with cells of the immune system. The intimate association of TMs at sites of embryo-maternal interaction and the varied functions these complex molecules can play make them key players in embryo implantation and placentation processes.

Multilayered, Hyaluronic Acid-Based Hydrogel Formulations Suitable for Automated 3D High Throughput Drug Screening of Cancer-Stromal Cell Cocultures.

Validation of a high-throughput compatible 3D hyaluronic acid hydrogel coculture of cancer cells with stromal cells. The multilayered hyaluronic acid hydrogels improve drug screening predictability as evaluated with a panel of clinically relevant chemotherapeutics in both prostate and endometrial cancer cell lines compared to 2D culture.

Construction and analyses of elastically coupled multiple-motor systems.

Precision analyses of the collective motor behaviors have become important to dissecting mechanisms underlying the trafficking of subcellular commodities in eukaryotic cells. Here, we describe a synthetic approach to create structurally defined multiple protein complexes containing two elastically coupled motor molecules. Motors are connected using a simple DNA-scaffolding molecule and DNA-conjugated, artificial protein polymers that function as tunable elastic linkers. The procedure to self-assemble these components produces complexes in high synthetic yield and allows individual multiple-motor systems to be interrogated at the single-complex level. Methods to evaluate cooperative motor responses in a static optical trap are also discussed. While enabling the average transport properties of single/noninteracting and coupled motors to be compared, these procedures can provide insight into the extent to which motors cooperate productively via load sharing as well as the roles loading-rate-dependent phenomena play in collective motor functions.

Hemozoin-generated vapor nanobubbles for transdermal reagent- and needle-free detection of malaria.

Successful diagnosis, screening, and elimination of malaria critically depend on rapid and sensitive detection of this dangerous infection, preferably transdermally and without sophisticated reagents or blood drawing. Such diagnostic methods are not currently available. Here we show that the high optical absorbance and nanosize of endogenous heme nanoparticles called "hemozoin," a unique component of all blood-stage malaria parasites, generates a transient vapor nanobubble around hemozoin in response to a short and safe near-infrared picosecond laser pulse. The acoustic signals of these malaria-specific nanobubbles provided transdermal noninvasive and rapid detection of a malaria infection as low as 0.00034% in animals without using any reagents or drawing blood. These on-demand transient events have no analogs among current malaria markers and probes, can detect and screen malaria in seconds, and can be realized as a compact, easy-to-use, inexpensive, and safe field technology.

The MUC1 Ectodomain: A Novel and Efficient Target for Gold Nanoparticle Clustering and Vapor Nanobubble Generation.

MUC1 is a large, heavily glycosylated transmembrane glycoprotein that is proposed to create a protective microenvironment in many adenocarcinomas. Here we compare MUC1 and the well studied cell surface receptor target, EGFR, as gold nanoparticle (AuNP) targets and their subsequent vapor nanobubble generation efficacy in the human epithelial cell line, HES. Although EGFR and MUC1 were both highly expressed in these cells, TEM and confocal images revealed MUC1 as a superior target for nanoparticle intracellular accumulation and clustering. The MUC1-targeted AuNP intracellular clusters also generated significantly larger vapor nanobubbles. Our results demonstrate the promising opportunities MUC1 offers to improve the efficacy of targeted nanoparticle based approaches.

Improved cellular specificity of plasmonic nanobubbles versus nanoparticles in heterogeneous cell systems.

The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level.

The absence of tertiary interactions in a self-assembled DNA crystal structure.

DNA is a highly effective molecule for controlling nanometer-scale structure. The convenience of using DNA lies in the programmability of Watson-Crick base-paired secondary interactions, useful both to design branched molecular motifs and to connect them through sticky-ended cohesion. Recently, the tensegrity triangle motif has been used to self-assemble three-dimensional crystals whose structures have been determined; sticky ends were reported to be the only intermolecular cohesive elements in those crystals. A recent communication in this journal suggested that tertiary interactions between phosphates and cytosine N(4) groups are responsible for intermolecular cohesion in these crystals, in addition to the secondary and covalent interactions programmed into the motif. To resolve this issue, we report experiments challenging this contention. Gel electrophoresis demonstrates that the tensegrity triangle exists in conditions where cytosine-PO(4) tertiary interactions seem ineffective. Furthermore, we have crystallized a tensegrity triangle using a junction lacking the cytosine suggested for involvement in tertiary interactions. The unit cell is isomorphous with that of a tensegrity triangle crystal reported earlier. This structure has been solved by molecular replacement and refined. The data presented here leave no doubt that the tensegrity triangle crystal structures reported earlier depend only on base pairing and covalent interactions for their formation.

Transmembrane mucins as novel therapeutic targets.

Membrane-tethered mucin glycoproteins are abundantly expressed at the apical surfaces of simple epithelia, where they play important roles in lubricating and protecting tissues from pathogens and enzymatic attack. Notable examples of these mucins are MUC1, MUC4 and MUC16 (also known as cancer antigen 125). In adenocarcinomas, apical mucin restriction is lost and overall expression is often highly increased. High-level mucin expression protects tumors from killing by the host immune system, as well as by chemotherapeutic agents, and affords protection from apoptosis. Mucin expression can increase as the result of gene duplication and/or in response to hormones, cytokines and growth factors prevalent in the tumor milieu. Rises in the normally low levels of mucin fragments in serum have been used as markers of disease, such as tumor burden, for many years. Currently, several approaches are being examined that target mucins for immunization or nanomedicine using mucin-specific antibodies.

Two kinesins transport cargo primarily via the action of one motor: implications for intracellular transport.

The number of microtubule motors attached to vesicles, organelles, and other subcellular commodities is widely believed to influence their motile properties. There is also evidence that cells regulate intracellular transport by tuning the number and/or ratio of motor types on cargos. Yet, the number of motors responsible for cargo motion is not easily characterized, and the extent to which motor copy number affects intracellular transport remains controversial. Here, we examined the load-dependent properties of structurally defined motor assemblies composed of two kinesin-1 molecules. We found that a group of kinesins can produce forces and move with velocities beyond the abilities of single kinesin molecules. However, such capabilities are not typically harnessed by the system. Instead, two-kinesin assemblies adopt a range of microtubule-bound configurations while transporting cargos against an applied load. The binding arrangement of motors on their filament dictates how loads are distributed within the two-motor system, which in turn influences motor-microtubule affinities. Most configurations promote microtubule detachment and prevent both kinesins from contributing to force production. These results imply that cargos will tend to be carried by only a fraction of the total number of kinesins that are available for transport at any given time, and provide an alternative explanation for observations that intracellular transport depends weakly on kinesin number in vivo.

The mechanochemistry of integrated motor protein complexes.

The assembly of molecular motor proteins into multi-unit protein complexes plays an important role in determining the intracellular transport and trafficking properties of many subcellular commodities. Yet, it is not known how proteins within these complexes interact and function collectively. Considering the established ties between motor transport and diseases, it has become increasingly important to investigate the functional properties of these essential transport 'motifs'. Doing so requires that the composite motile and force-generating properties of multi-unit motor assemblies are characterized. However, such analyses are typically confounded by a lack of understanding of the links between the structural and mechanical properties of many motor complexes. New experimental challenges also emerge when one examines motor cooperation. Distributions in the mechanical microstates available to motor ensembles must be examined in order to fully understand the transport behavior of multi-motor complexes. Furthermore, mechanisms by which motors communicate must be explored to determine whether motor groups can move cargo together in a truly cooperative fashion. Resolving these issues requires the development of experimental methods that allow the dynamics of complex systems of transport proteins to be monitored with the same precision available to single-molecule biophysical assays. Herein, we discuss key fundamental principles governing the function of motor complexes and their relation to mechanisms that regulate intracellular cargo transport. We also outline new experimental strategies to resolve these essential features of intracellular transport.

From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal.

We live in a macroscopic three-dimensional (3D) world, but our best description of the structure of matter is at the atomic and molecular scale. Understanding the relationship between the two scales requires a bridge from the molecular world to the macroscopic world. Connecting these two domains with atomic precision is a central goal of the natural sciences, but it requires high spatial control of the 3D structure of matter. The simplest practical route to producing precisely designed 3D macroscopic objects is to form a crystalline arrangement by self-assembly, because such a periodic array has only conceptually simple requirements: a motif that has a robust 3D structure, dominant affinity interactions between parts of the motif when it self-associates, and predictable structures for these affinity interactions. Fulfilling these three criteria to produce a 3D periodic system is not easy, but should readily be achieved with well-structured branched DNA motifs tailed by sticky ends. Complementary sticky ends associate with each other preferentially and assume the well-known B-DNA structure when they do so; the helically repeating nature of DNA facilitates the construction of a periodic array. It is essential that the directions of propagation associated with the sticky ends do not share the same plane, but extend to form a 3D arrangement of matter. Here we report the crystal structure at 4 A resolution of a designed, self-assembled, 3D crystal based on the DNA tensegrity triangle. The data demonstrate clearly that it is possible to design and self-assemble a well-ordered macromolecular 3D crystalline lattice with precise control.

Negative interference dominates collective transport of kinesin motors in the absence of load.

The collective function of motor proteins is known to be important for the directed transport of many intracellular cargos. However, understanding how multiple motors function as a group remains challenging and requires new methods that enable determination of both the exact number of motors participating in motility and their organization on subcellular cargos. Here we present a biosynthetic method that enables exactly two kinesin-1 molecules to be organized on linear scaffolds that separate the motors by a distance of 50 nm. Tracking the motions of these complexes revealed that while two motors produce longer average run lengths than single kinesins, the system effectively behaves as though a single-motor attachment state dominates motility. It is proposed that negative motor interference derived from asynchronous motor stepping and the communication of forces between motors leads to this behavior by promoting the rapid exchange between different microtubule-bound configurations of the assemblies.

Design of DNA-conjugated polypeptide-based capture probes for the anchoring of proteins to DNA matrices.

A new method for protein surface functionalization was developed that utilizes DNA-conjugated artificial polypeptides to capture recombinant target proteins from the solution phase and direct their deposition onto DNA-functionalized matrices. Protein capture is accomplished through the coiled-coil association of an engineered pair of heterodimeric leucine zippers. Incorporating half of the zipper complex directly into the polypeptides and labeling these polymers with ssDNA enables the polypeptide conjugates to form intermediate linkages that connect the target proteins securely to DNA-functionalized supports. This synthetic route provides an important alternative to conventional DNA-conjugation techniques by allowing proteins to be outfitted site-specifically with ssDNA while minimizing the need for postexpression processing. We demonstrate these attributes by (i) using the capture probes to prepare protein microarrays, (ii) demonstrating control over enzyme activity via deposition of DNA, and, (iii) synthesizing finite-sized, multiprotein complexes that are templated on designed DNA scaffolds in near quantitative yield.