The Cancer Biology and Molecular Medicine Team

Research Lab Overview

The Jovanovic-Talisman Lab within Beckman Research Institute of City of Hope employs novel, quantitative imaging techniques and nanobiology to investigate biological mechanisms, develop new diagnostic assays, and advance therapeutics.

Quantitative single molecule localization microscopy (qSMLM)

Talisman Lab Figure1 — Golfetto, Wakefield, et al., J Am Chem Soc. 2018,140,12785

To investigate biological processes that are critical to the progression of cancer and other diseases we use quantitative single molecule localization microscopy (qSMLM). qSMLM is a fluorescence-based imaging technique that evaluates single molecules with nanoscale precision. In typical qSMLM experiments, target molecules are detected with fluorescent reporters like optical highlighter proteins or antibodies labeled with photoswitchable dyes. Since these imaging agents have intricate photophysical properties, one major challenge in the field has been relating the number of localizations of reporters to the detected number of target proteins. To address this, we recently developed a new surface assay for molecular isolation (SAMI), that allows us to robustly count individual molecules. We use SAMI and other methodological advancements to study important biological mechanisms, aid in development of novel therapeutic agents, and advance diagnostics.

qSMLM for imaging of patient tissues

Talisman Lab Figure 2 — Tobin, Wakefield, et al., Sci Rep. 2018, 11, 15154

We advance qSMLM to assess patient specimens: cells from excised tissues and extracellular vesicles from biofluids.

Touch Prep — Tobin, Wakefield et al., Sci. Rep., 2018, 11, 15154
Wakefield, Tobin et al., Methods Mol. Biol., 2022, 2394, 231
Maddox et al., Cancers, 2022, 14, 2795

We apply advanced qSMLM methods to quantify both the density and nano-organization of membrane receptors in cancer. We are currently focusing on assessing G-protein coupled receptors and receptor tyrosine kinases. Recently, we used fluorescently labeled trastuzumab to quantify human epidermal growth factor receptor 2 (HER2) in breast cancer. We have combined qSMLM with tissue touch preparation to assess human tumor samples. We developed a straightforward analytical protocol and algorithms designed for rapid data analysis; these advancements allowed us to obtain quantitative results for patients within one day. Results from these studies demonstrated a significant correlation between classical HER2 screening (i.e., fluorescence in situ hybridization, FISH) and HER2 assessment with qSMLM. Moreover, we recently assessed how the molecular features of HER2 varied with the therapy response. According to our results, the therapy response was associated with high detected HER2 densities and clustering. In the future, qSMLM could provide key data to complement the current diagnostic standards.

Cancer enriched EVs — Lennon et al., J Extracell. Vesicles, 2019, 8, 1685634
Lennon et al. Clin. Transl. Med., 2022, 12, e979

Both healthy and diseased cells continuously shed extracellular vesicles (EVs). These membrane encapsulated nanoscale particles regulate intercellular communications through their cargo (e.g., nucleotides, metabolites, lipids, and proteins). EVs contain a wealth of biological information and can reflect disease pathology. Moreover, EVs can be collected frequently and non-invasively from almost any accessible biofluid, such as blood, saliva, and urine. EVs are thus an attractive target for the early detection and monitoring of various diseases.

Patient biofluids contain complex mixtures of EVs; they originate from a wide variety of cells and tissues. A major challenge has been the detection of disease-specific EVs or tissue-specific EVs within the background of ‘other’ EVs. To address this, we are advancing methodology to isolate and characterize EV populations enriched in receptors that have high expression in either specific tissue/cell type or disease. Our qSMLM approach uniquely allows us to assess both the size and the molecular content of individual EVs with high sensitivity. Since limited information on EVs is currently available at the molecular level, we expect these studies to help address EV heterogeneity and advance EVs as biomarker sources in a clinical setting.
.

qSMLM for probing molecular mechanisms/advancing drug development

We have applied qSMLM to probe nano-organization of opioid receptors in alcohol use disorders and to delineate the mechanisms of nucleocytoplasmic transport.
New drugs are urgently needed to treat alcohol use disorders (AUDs). Long-term alcohol use can lead to cancer and diabetes; it can significantly shorten lifespan. However, current therapies to treat this disease are at best marginally effective. We have used qSMLM to assess how physiologically relevant concentrations of alcohol perturb the dynamic organization of signaling domains that harbor opioid receptors. Additionally, we are studying how the organization of these domains is affected when we administer potential therapeutic reagents.

The nucleus and cytoplasm communicate through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope. NPCs are selectively permeable to certain macromolecules and thus regulate nucleocytoplasmic transport. To efficiently regulate selective transport, each NPC can shuttle thousands of molecules every second. Errors in this high throughput process can lead to large-scale cellular dysregulation observed in a number of diseases, including neurodegenerative disorders and solid tumors. Despite these high stakes, resolution of specific mechanism(s) of transport has been challenging. To advance our understanding of these mechanisms, we apply qSMLM to obtain single molecule data on NPC barrier mimics and on NPCs from intact nuclei. The resolution of nucleocytoplasmic transport mechanisms may ultimately provide novel targets and opportunities for drug development.

Principal Investigator: Tijana Jovanovic-Talisman, Ph.D.

Associate Professor, Department of Molecular Medicine

Research Focus