Gestational Breast Cancer - a Review of Outcomes, Pathophysiology, and Model Systems.

The onset of pregnancy marks the start of offspring development, and represents the key physiological event that induces re-organization and specialization of breast tissue. Such drastic tissue remodeling has also been linked to epithelial cell transformation and the establishment of breast cancer (BC). While patient outcomes for BC overall continue to improve across subtypes, prognosis remains dismal for patients with gestational breast cancer (GBC) and post-partum breast cancer (PPBC), as pregnancy and lactation pose additional complications and barriers to several gold standard clinical approaches. Moreover, delayed diagnosis and treatment, coupled with the aggressive time-scale in which GBC metastasizes, inevitably contributes to the higher incidence of disease recurrence and patient mortality. Therefore, there is an urgent and evident need to better understand the factors contributing to the establishment and spreading of BC during pregnancy. In this review, we provide a literature-based overview of the diagnostics and treatments available to patients with BC more broadly, and highlight the treatment deficit patients face due to gestational status. Further, we review the current understanding of the molecular and cellular mechanisms driving GBC, and discuss recent advances in model systems that may support the identification of targetable approaches to block BC development and dissemination during pregnancy. Our goal is to provide an updated perspective on GBC, and to inform critical areas needing further exploration to improve disease outcome.

Protocol to culture and image pancreatic ductal adenocarcinoma tumor slices to study T cell migration.

Here, we describe a protocol for culture and live cell imaging of tumor slices. This approach studies carcinoma and immune cell dynamics in complex tumor microenvironments (TME) with nonlinear optical imaging platforms. Using a tumor-bearing mouse model of pancreatic ductal adenocarcinoma (PDA), we detail steps to isolate, activate, and label CD8+ T lymphocytes and later introduce them to live murine PDA tumor slice explants. The techniques described in this protocol can improve our understanding of cell migration in complex microenvironments ex vivo. For complete details on the use and execution of this protocol, please refer to Tabdanov et al. (2021).1.

Clinical applications of 3D normal and breast cancer organoids: A review of concepts and methods.

While mouse models and two-dimensional (2D) cell culture systems have dominated as research tools for cancer biology, three-dimensional (3D) cultures have gained traction as a new approach that retains features of in vivo biology within an in vitro system. Over time, 3D culture systems have evolved from spheroids and tumorspheres to organoids, and by doing so, they have become more complex and representative of original tissue. Such technological improvements have mostly benefited the study of heterogeneous solid tumors, like those found in breast cancer (BC), by providing an attractive avenue for scalable drug testing and biobank generation. Experimentally, organoids have been used in the BC field to dissect mechanisms related to cellular invasion and metastasis-and through co-culture methods-epithelial interactions with stromal and immune cells. In addition, organoid studies of wild-type mouse models and healthy donor samples have provided insight into the basic developmental cellular and molecular biology of the mammary gland, which may inform one's understanding of the initial stages of cancer development and progression.

Stromal architecture directs early dissemination in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is an extremely metastatic and lethal disease. Here, in both murine and human PDA, we demonstrate that extracellular matrix architecture regulates cell extrusion and subsequent invasion from intact ductal structures through tumor-associated collagen signatures (TACS). This results in early dissemination from histologically premalignant lesions and continual invasion from well-differentiated disease, and it suggests TACS as a biomarker to aid in the pathologic assessment of early disease. Furthermore, we show that pancreatitis results in invasion-conducive architectures, thus priming the stroma prior to malignant disease. Analysis in potentially novel microfluidic-derived microtissues and in vivo demonstrates decreased extrusion and invasion following focal adhesion kinase (FAK) inhibition, consistent with decreased metastasis. Thus, data suggest that targeting FAK or strategies to reengineer and normalize tumor microenvironments may have roles not only in very early disease, but also for limiting continued dissemination from unresectable disease. Likewise, it may be beneficial to employ stroma-targeting strategies to resolve precursor diseases such as pancreatitis in order to remove stromal architectures that increase risk for early dissemination.

Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments.

Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.

RNA-protein interactions govern antiviral specificity and encapsidation of broad spectrum anti-HIV reverse transcriptase aptamers.

RNA aptamers that bind HIV-1 reverse transcriptase (RT) inhibit HIV-1 replication, but little is known about potential aptamer-specific viral resistance. During replication, RT interacts with diverse nucleic acids. Thus, the genetic threshold for eliciting resistance may be high for aptamers that make numerous contacts with RT. To evaluate the impact of RT-aptamer binding specificity on replication, we engineered proviral plasmids encoding diverse RTs within the backbone of HIV-1 strain NL4-3. Viruses inhibited by pseudoknot aptamers were rendered insensitive by a naturally occurring R277K variant, providing the first demonstration of aptamer-specific resistance in cell culture. Naturally occurring, pseudoknot-insensitive viruses were rendered sensitive by the inverse K277R mutation, establishing RT as the genetic locus for aptamer-mediated HIV-1 inhibition. Non-pseudoknot RNA aptamers exhibited broad-spectrum inhibition. Inhibition was observed only when virus was produced in aptamer-expressing cells, indicating that encapsidation is required. HIV-1 suppression magnitude correlated with the number of encapsidated aptamer transcripts per virion, with saturation occurring around 1:1 stoichiometry with packaged RT. Encapsidation specificity suggests that aptamers may encounter dimerized GagPol in the cytosol during viral assembly. This study provides new insights into HIV-1's capacity to escape aptamer-mediated inhibition, the potential utility of broad-spectrum aptamers to overcome resistance, and molecular interactions that occur during viral assembly.

Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification.

The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon covalent modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated covalent modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational modifications of proteins. Ribozyme-catalyzed covalent modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits.

Nucleobase modification by an RNA enzyme.

Ribozymes can catalyze phosphoryl or nucleotidyl transfer onto ribose hydroxyls of RNA chains. We report a single ribozyme that performs both reactions, with a nucleobase serving as initial acceptor moiety. This unprecedented combined reaction was revealed while investigating potential contributions of ribose hydroxyls to catalysis by kinase ribozyme K28. For a 58nt, cis-acting form of K28, each nucleotide could be replaced with the corresponding 2΄F analog without loss of activity, indicating that no particular 2΄OH is specifically required. Reactivities of two-stranded K28 variants with oligodeoxynucleotide acceptor strands devoid of any 2΄OH moieties implicate modification on an internal guanosine N-2, rather than a ribose hydroxyl. Product mass suggests formation of a GDP(S) adduct along with a second thiophosphorylation, implying that the ribozyme catalyzes both phosphoryl and nucleotidyl transfers. This is further supported by transfer of radiolabels into product from both α and γ phosphates of donor molecules. Furthermore, periodate reactivity of the final product signifies acquisition of a ribose sugar with an intact 2΄-3΄ vicinal diol. Neither nucleobase modification nor nucleotidyl transfer has previously been reported for a kinase ribozyme, making this a first-in-class ribozyme. Base-modifying ribozymes may have played important roles in early RNA world evolution by enhancing nucleic acid functions.