Big Ideas, Small Features Incubator Awardees
A 3-D Bioprinted Model of Calcific Aortic Valve Disease for Drug Screening and Mechanistic Studies
Principal Investigator: Elena Aikawa, MD. Brigham and Women's Hospital
Calcific aortic valve disease (CAVD) claims 17,000 lives in the US alone, annually. No drug-based therapy is available. The only effective treatment is invasive and costly aortic valve replacement for late-stage disease patients. This study will use a novel 3D-bioprinted model of CAVD and an innovative drug-delivery platform to elucidate the underlying mechanisms of CAVD and identify potential therapeutic targets.
Advanced imaging and nanoscale technologies at the HCBI and CNS will greatly enhance our insight into mechanisms of CAVD. To illuminate the mechanisms of CAVD, a 3D-bioprinted CAVD model will be used to track differentially expressed microRNAs recently identified in our screening of heart valve tissues from CAVD patients. This 3D-bioprinted CAVD system will allow us to evaluate the temporal association of novel microRNAs with pathological cellular changes observed in CAVD using fluorescent in situ hybridization and fluorescent phenotypic markers. We will then test whether these microRNAs can be targeted to therapeutically improve CAVD cell phenotypes. Confocal microscopy will track microRNA delivery from a polymer-nanoparticle(PNP)-hydrogel drug delivery system into cells incorporated into CAVD model. The functional response of calcified cells to microRNA- therapeutics (mimics and antagomirs) will be assessed by visualizing microcalcifications in the CAVD model using a near-infrared fluorescent calcium tracer.
This research will validate a patentable PNP-hydrogel delivery platform in a unique 3D-bioprinted model of CAVD and identify functional combinations of miRNA therapeutics with a main goal to provide treatment for patients suffering from CAVD, a disease with no available drug strategies.
Optimization of Drug Delivery by Tumor-Targeting Layer-by Layer Nanoparticles Using Advanced Microscopic Technologies: Phase II
Principal Investigator: David Frank, MD, PhD, Dana-Farber Cancer Institute
The goal of this project is to target tumor cells specifically, based on unique properties of their plasma membranes. We have found that the activation of common oncogenic signaling molecules, such as mutated K-Ras or STAT3, leads to stereotyped changes in cell membrane lipid composition. As these oncogenic proteins have eluded direct targeting (often being referred to as “undruggable”), this finding provides an opportunity to deliver targeted or cytotoxic agents preferentially to tumor cells by exploiting this property. To exploit this change in lipids, which generally leads to increased expression of positively-charged lipids on the cell surface, we, and our collaborator Dr. Paula Hammond from MIT, have generated a family of unique layer-by-layer nanoparticles coated with polyanions in the external shell, to generate a negative charge. In the first year of funding of this grant, we quantitated the uptake of these nanoparticles into non-transformed MCF-10A mammary epithelial cells or these cells transformed with activated STAT3 (as occurs in approximately 70% of breast cancers). We have also performed parallel experiments with non-transformed pancreatic epithelial cells and the same cells into which mutated K-Ras has been introduced (as occurs in greater than 90% of pancreatic cancers). From these experiments, we have identified variants of poly-glutamic acid coated nanoparticles that have shown significantly enhanced uptake into the transformed cell populations relative to the non-transformed cells. In a second year of funding, we would extend these observations to image nanoparticle delivery in tumor systems grown in three-dimensions, explore the potential for combined diagnostic and therapeutic use of tumor-targeting nanoparticles, and assess novel therapeutic strategies exploiting this delivery system in combination with radiation therapy.
A Microscopy-Based Platform For Rapid, At-will Antimicrobial Resistance Testing
Principal Investigator: James Kirby, MD, Beth Israel Deaconess Medical Center
Antibiotic resistance is compromising our ability to treat bacterial infections. Clinical microbiology laboratories guide appropriate treatment through antimicrobial susceptibility testing (AST) of patient isolates. However, increasingly, pathogens are developing resistance to a broad range of antimicrobials, requiring AST of less commonly used or recently introduced agents for which no commercially available or FDA-cleared testing methods exist. Agar or broth dilution are gold standard methods for AST that can be used to test any antimicrobial; however, labor and technical complexity precludes their use in hospital-based clinical laboratories. Therefore, bacterial isolates often must be sent to a reference laboratory with a 4-6 day delay in results. Further, even standard methods require overnight incubation prior to readout. Therefore, there exists a significant AST testing gap in which current methodologies cannot adequately address the need for rapid results in the face of unpredictable susceptibility profiles. Our laboratory has recently verified inkjet, digital dispensing technology as a novel platform to facilely perform reference AST for any antimicrobial at will. In this proposal, we aim to harness technical assets and expertise at HCBI/IDAC to leapfrog current technology through: (1) development of a method for microscopic imaging of bacterial replication in solid-phase 384-well microplate AST format, thereby determining susceptibility for any drug in <4 hours and (2) verification of the clinical performance of the new assay using well-characterized clinical isolates. We anticipate establishing a prototype method that will address the AST testing gap and thereby help our health system more effectively address the antimicrobial resistance threat.
Metabolic Imaging of Mouse Embryos to Determine Safety of 1-Photon FLIM for Clinical Applications in In Vitro Fertilization
Principal Investigator: Daniel Needleman, PhD, Harvard University Faculty of Arts and Sciences
Mitochondrial dysfunction has long been associated with reduced reproductive potential. More than 200 publications link mitochondrial function with in vitro fertilization (IVF) success. 67% of all IVF cycles fail, making the process economically and emotionally costly to patients and the health system. Developing an effective and accurate embryo selection tool has long been a primary goal in clinical reproductive research, as it would have a dramatic impact on IVF success rates. Non-invasive assessment of mitochondrial health could provide the means to such a tool.
We have established that we can non-invasively assess mitochondrial function of oocytes by measuring NADH and FAD fluorescence using Fluorescence Lifetime Imaging Microscopy (FLIM). Experiments thus far have been performed on a 2-photon system in the Needleman Lab, and we have demonstrated the safety of 2-photon FLIM for use in oocytes and embryos.
The aim of this proposed research is to assess the safety and feasibility of a 1-photon FLIM system for generating FLIM measurements of NADH and FAD. To accelerate translation of our research to the clinical realm, we must better understand phototoxicity of 1-photon microscopy systems to determine whether they are clinically viable. We will achieve this aim by varying photodosage for mouse oocytes and mouse embryos, and by measuring Reactive Oxygen Species levels, potential DNA damage, and live birth outcomes.
Advanced Microscopy Incubator Awardees
3D-Microscopy for Classification and Risk Prediction in Early Breast Neoplasia
This work builds directly off the accomplishments from our Harvard Catalyst Advanced Microscopy pilot project, in which we successfully developed a protocol that takes as input an archival pathology specimen, performs tissue processing and clarification, fluorescent staining, Lightsheet Microscopy imaging, 3D feature extraction, and statistical analyses to identify 3D morphologic phenotypes associated with progressive stages of breast neoplasia and to construct accurate, automated classification models. Now, we will build on this preliminary data to integrate our approach with a recently developed method (Expansion Microscopy) and to apply this advanced microscopy and image analysis platform to a large retrospective cohort of benign breast diseases and ductal carcinoma in situ from the Nurses’ Health Study. This unique cohort will enable us to assess the ability of LSM-derived morphological features to outperform traditional pathological assessment for predicting risk of future breast cancer from pathological analysis of breast tissue specimens.
A. John Iafrate
Highly Multiplexed FISH for In Situ Genomics
We propose to continue development of a clinical grade high-throughput DNA-FISH platform that will enable the automated copy number analysis of a large panel of genes in formalin-fixed paraffin embedded (FFPE) tumor biopsy samples and in isolated circulating tumor cells (CTCs).
Advanced Imaging Incubator Awardees
Development and validation of novel tissue biomarkers for guiding neurosurgical resection of brain tumors
Principal Investigator: Alexandra Golby, MD, Brigham and Women's Hospital
An extension of Dr. Golby's innovator award. In this study Stimulated Raman scattering (SRS) microscopy is a cutting edge imaging technology that has the potential to enable surgeons to reliably detect cancerous tissues during surgery. SRS microscopy enables high-resolution, cellular-level imaging of biological tissues based on the intrinsic spectroscopic properties of their macromolecular components such as lipids, proteins and DNA.
Diagnosis of keratoconus using biomechanical metrics via Brillouin microscopy
Principal Investigator: Andy Yun, PhD, Massachusetts General Hospital
During a pilot clinical study funded by Harvard Catalyst, we have developed a clinical instrument based on Brillouin microscopy (Scarcelli & Yun, Nature Photonics 2008), which can measure corneal elasticity at high 3D resolution without contacting or perturbing the eye. Leveraging on this novel technology, the overall goal of this research program is introduce elasticity-based metrics for keratoconus diagnosis and CXL therapy monitoring. Our central hypothesis is that Brillouin stiffness is a suitable indicator of corneal biomechanical stability and thus can serve for early diagnosis of keratoconus progression and for evaluation of CXL treatment outcome. This hypothesis is based on human data we collected during the pilot keratoconus study and animal data demonstrating the ability of our technology to quantitatively assess the mechanical outcome of CXL procedure (Scarcelli, Pineda & Yun, IOVS 2013).