The poster session will take place on Thursday, June 5th, from 4:30 - 5:30 PM in the Ryan Family Atrium. The session will be split so that odd-numbered posters will present for the first 30 minutes, and even-numbered posters will present for the second 30 minutes, to allow presenters an opportunity to enjoy the poster session as well.
Poster board size is 72”H x 60”W.
All Trainee (Undergrad, Grad Student and Postdoc) Poster presenters will be considered for a poster prize. Please review the poster judging criteria here.
1) CD8+ T Cell Migration Mechanics revealed by high-resolution imaging
Aamir Ansari, University of Minnesota
CD8+ effector T cells have an incredible ability to migrate anywhere in the body. How T cells choose a specific migration mechanism and adapt to the changes in the environment remains elusive. Unraveling these modes requires understanding how they generate force —such as F-actin polymerization, myosin-driven contractility, and pressure-based forces— and how they transmit this force, either through specific or nonspecific interactions with their surroundings. F-actin and pressure-based forces produce characteristic protrusions, which can be identified from their membrane-cortex dynamics. The nature of the force transmission mechanism can be deduced by studying the extracellular environment dynamics during the protrusion formation and retraction phase. In this study, we used this technique to pinpoint the modes T cells use in mouse brain tissues and 3D collagen gels for reference. Using edge-velocity-map and F-actin level correlation, we found that T cells can form F-actin- and pressure-based protrusions and switch between the two. Next, we measured protrusion and retraction speeds and correlated them with the cell centroid displacement. Theoretically, rapid pressure-based protrusion can grant T cells very high speed; however, inadequate force transmission —due to the inability of the cortex and cortex-linked adhesion proteins to advance quickly enough—renders this advantage futile. Next, using super-resolution measurement of the gap between the membrane and the cortex, we found that the relative magnitude of osmotic-driven water influx at the cell's leading edge along with other factors (thermal fluctuations and membrane-cortex linkers breakage) determines this gap and the type of protrusions. We further aim to test this hypothesis using drug perturbation, engineered cell lines (e.g., Moesin-KO CD8+ T cells), and possibly using a mechanistic biophysical model. Overall, the findings suggest osmosis (a possible migration mode in itself) as a common influencer of two other major migration modes. This unique connection suggests "nanoblebbing", with faster membrane extension and a tunable gap optimized for enhanced F-actin polymerization while curbing rapid retraction, could effectively boost T-cell migration in the tumor microenvironment.
Other authors: Roberto Matilla, Marie Juzans, Janis Burkhardt, Paolo Provenzano, David Odde
2) High-Throughput 3D Imaging of Cleared Tissues Reveals Early Metastatic Pathways in Invasive Lobular Carcinoma
Hazel Borges, UT Southwestern Medical Center
Invasive lobular carcinoma (ILC) is a distinct subtype of breast cancer, due to loss of e-cadherin, that presents a unique pattern of late metastatic spread to distant organs such as bone. Understanding early metastatic dissemination of ILC remains a challenge due to the limitations of traditional 2D histology. In this study, we demonstrate how solvent-based tissue clearing (BABB) and high-resolution axially swept light sheet microscopy(ctASLM) enables volumetric imaging of intact organs, revealing novel metastatic pathways in a xenograft mouse model of ILC. We focus on GR^high and GR^low subpopulations within MM134 ILC cells,defined by differential expression of glucocorticoid receptor (GR). GR is a nuclear transcription factor activated by stress hormone, cortisol. GR-high cells exhibit low proliferation, enhanced survival, and metabolic flexibility, enabling them to adapt to a stressful microenvironment through the activation of oxidative phosphorylation (OxPhos) and related pathways. These properties may facilitate early metastatic seeding and outgrowth in distal tissues. Here, we were able to detect rare GR-high tumor cells within optically cleared mammary glands and femurs at early disease stage. Their spatial distribution suggests early escape from the primary site and preferential colonization of bone, showing a potential early escape route and preference for the bone microenvironment. Our goal is to understand the molecular mechanism that enables ILC cells to disseminate via blood and lymphatic vessels by linking GR-driven phenotypes to metastatic potential. To scale this analysis and enable high-throughput volumetric imaging we integrated ASLM with an automated robotic platform. This system will allow us to create a scalable platform for the identification and quantification of aggressive tumor cells, paving the way for new biomarkers and therapeutic strategies targeting early metastatic events in ILC.
Other authors: Baylee Porter, Suzanne Conzen, Kevin Dean
3) Self-Returning Excluded Volume (SR-EV) Model Captures Chromatin Organization Across Scales
Marcelo Carignano, Northwestern University
We present the Self-Returning Excluded Volume (SR-EV) model to capture chromatin 3D structure, based on stochastic rules and physical interactions. The model depends primarily on two parameters: one that controls the folding frequency and another that determines the overall chromatin volume fraction. Using SR-EV, we can rapidly generate millions of 3D configurations with nucleosome resolution at whole chromosomal scales. Model predictions were experimentally validated with Chromatin Scanning Transmission Electron microscopy (ChromSTEM), demonstrating both qualitative and quantitative agreement with the ground-truth nanoscale structure of the human genome. Using this high throughput model, we demonstrate the genome folds into a heterogeneous system, with densely packed domains interspersed with dilute regions. SR-EV configurations can be used for quantification of the physical features of the genome in 3D. This allows investigation of the properties of individual nucleosomes as well as features of genome connectivity and geometry. We demonstrate robust agreement with experimental data for both single configurations and ensemble averages. We conclude by showing how SR-EV framework can provide information on chromatin accessibility, connectivity, and 3D interactions with chromatin modifying enzymes for both single cell and ensemble features across modalities. It provides the capacity to interrogate chromatin structure-function both in a population and at the single cell level across modalities including single molecule localization microscopy (SMLM), chromatin conformation capture (HI-C), and chromatin immunoprecipitation sequencing (ChIP-Seq).
Other authors: M. Kroeger, L.M. Almassalha, V. Backman, I. Szleifer
4) Chromatinomics as a new multi-modal biomarkers that captures chromatin packing domain upregulation and transcriptional plasticity in advanced adenoma
Andrew Chang, Northwestern University
Recent studies highlight chromatin conformation as a key regulator of transcriptional plasticity. Chromatin chains (~kbp) fold into 50-1,000 kbp chromatin packing domains (PDs), which exhibit a mass-fractal structure where genomic length (N) scales with radius (R) as N=RD, with packing scaling D<3. PDs act as transcriptional memory elements, and their upregulation is associated with increased transcriptional plasticity. Utilizing the etiological field carcinogenesis, epi/genetic changes preceding focal neoplasia can be detected from an accessible source of biomarkers such as the rectal mucosa.
We define Chromatinomics as multi-modal biomarkers capturing PD upregulation and the resulting transcriptional plasticity. Using chromatin-sensitive partial wave spectroscopic (csPWS) microscopy, we quantified PD packing scaling (Dn), a physical descriptor of chromatin organization. We explored the capacity of csPWS in measuring chromatin fractal composition and density at high spatial confidence. Transcriptional plasticity was assessed through single-time-point biomarkers of transcriptional divergence and heterogeneity of gene expressions.
Rectal biopsies from 24 colonoscopy patients (15 controls, 9 AA cases) revealed significant differences in chromatinomics markers. Dn was higher in AA patients (AUC=0.84, sensitivity=88%, specificity=80%). RNA-sequencing-derived Slope of Differential Transcriptional Response (SDTR) for miRNA (AUC=0.84) and mRNA (AUC=0.76) and transcriptional heterogeneity (AUC=0.78) when combined with Dn achieved robust performance (AUC=0.96, sensitivity=89%, specificity=95%). Chromatinomics showed excellent sensitivity differentiating AA and healthy with low susceptibility to overfitting as demonstrated by bootstrapped AUCs (95% CI [0.86–1.0]) confirming its robustness in early CRC screening.
Other authors: Yuanzhe (Patrick) Su, Andrew Chang, Sravya Prabhala, Anik Ghosh
5) Visualizing Subcellular Features with Highly Multiplexed Expansion Microscopy
Seweryn Gałecki, UT Southwestern Medical Center
The spatial organization of proteins and organelles within eukaryotic cells orchestrates its fundamental vital processes. Simultaneous, super-resolution visualization of multiple biological structures is essential for better understanding both: physiological mechanisms, and those leading to disease. However, despite significant advancements in optics, spectral overlap typically restricts biological imaging to four fluorophores, and often lacks the resolution needed to visualize events at sub-diffraction scales. To overcome these challenges, we introduce Cyclically Multiplexed Expansion Microscopy (Cy-ExM) - a novel approach that integrates cryo-fixation of the specimen, expansion microscopy, and cyclic immunofluorescence labeling. Cy-ExM enabling highly multiplexed imaging of proteins, cytoskeletal architectures, and cellular organelles with preservation of their native spatial organization. We validate our approach in various mammalian cell lines, using different optical platforms, including spinning disk, oblique plane, and axially swept light-sheet microscopy modalities. Our method doesn't require specialized DNA-barcoded, or photoswitchable fluorophores, making it an accessible tool for multiplexed volumetric imaging, suitable for broad use in standard laboratory settings. Cy-ExM sheds new light on the molecular origins of oncogenesis, offering deeper insights into cellular ultrastructure, while holding potential for multiplexed diagnostics and unraveling the complexities of the tumor microenvironment.
Other authors: Seweryn Gałecki, Qionghua Shen, Daniel Stoddard, Bo-Jui Chang, Bingying Chen, Daniella Nicastro, Reto Fiolka, Kevin M. Dean.
6) Quantitative Mitochondrial Analysis of Brequinar’s Effect on Platinum Resistant Ovarian Cancer Cells
Xingyue Hao, Northwestern University
High grade serious ovarian cancer (HGSOC) is lethal due to the development of cisplatin-resistant cells, which enable resistance to platinum-based chemotherapy. Recent findings suggest that cisplatin-resistant cells are dependent on de novo pyrimidine synthesis and as such are vulnerable to inhibitors of this pathway, like Brequinar, which has been found to disrupt mitochondrial metabolic activity in cisplatin-resistant ovarian cancer cells and inhibit tumor growth. The effect of Brequinar on mitochondria structural are not well understood as few methods exist to quantify mitochondrial morphological alterations at nanoscale resolutions. Three-dimensional single-molecule localization microscopy (SMLM) can visualize such nanoscale alterations, allowing for quantitative measurements of mitochondrial structure. Using SMLM, we quantified mitochondrial morphologies in both OVCAR5 cells to examine how Brequinar affects mitochondrial structure.
OVCAR5 cisplatin-resistant and cisplatin-sensitive cells were both treated with DMSO and 1uM of Brequinar, respectively. Cells were fixed, stained with TOM-20, a mitochondrial membrane protein, and imaged using our SMLM system. We measured individual mitochondrial length, width, and volume. To normalize these values, we calculated the aspect ratio, which is defined as the ratio between mitochondrial length and width.
We found that though there was no significant change in aspect ratio between cell types, Brequinar treatment caused a significant increase in aspect ratio (p
Other authors: Yinu Wang, Horacio Cardenas, Daniela Matei, Hao F. Zhang
7) Modeling Protein Localization Dynamics in Heterogeneous Chromatin Distribution
Rivaan Kakkaramadam, Northwestern University
Chromatin is organized in heterogeneous domains with dense heterochromatic cores and decompacted euchromatic peripheries. Transcribing RNA polymerase prefers to localize within domain edges over freely open areas. This validates modeling-sourced predictions of a nonmonotonic relation between chromatin density and polymerase binding due to tradeoffs in entropic benefits and diffusion barriers. Heterochromatin proteins also trend smaller than euchromatin proteins, enabling access to compacted areas and thus reinforcing the organization of heterochromatin and euchromatin. Altogether, these findings suggest the transcriptome results from a dynamic interplay of chromatin, chromatin modifiers, and transcription. Cancer cells rearrange domain organization during chemotherapy to upregulate chemoresistance genes, so this network would be a promising tool in chemotherapeutic design. Recently, the Szleifer Lab built the Self-Returning Excluded Volume (SR-EV) algorithm, which can reproduce the statistical organization of chromatin domains observed in various cell lines via superresolution imaging. This presents an opportunity to model the means by which domain organization regulates protein localization. In performing molecular dynamics simulations of proteins in SREV-generated chromatin, we find that protein size enables the preferential localization of large euchromatin proteins like SETD2 in chromatin-decompacted regions and small heterochromatin proteins like SUV39H2 in chromatin-compacted regions. We also present preliminary work in modeling the “ideal zone” of chromatin that a given chromatin regulator (e.g., heterochromatin protein HDAC3, euchromatin protein SETD2, and RNA polymerase II) is expected to localize to based on the protein structure. From this, we gain clarifying insights on chromatin structure as a chemotherapeutic target to inform design of chromatin engineering-based treatment.
Other authors: Marcelo Carignano, Rikkert Nap, Igal Szleifer, Vadim Backman
8) Uncovering the role of heterochromatin protein 1 alpha in stabilizing chromatin packing domains
Tiffany Kuo, Northwestern University
The human genome is organized in a hierarchical manner to facilitate gene expression and overall genome function. Prior work has shown the existence of chromatin packing domains that demonstrate a mass scaling behavior. These packing domains range from 50 to 200 nm in size and follow a three-rule framework for assembly, stabilization, and function. Transcription and cohesin/CTCF-mediated loop formation create nascent domains. The size of nucleosome remodeling enzymes plays a major role in allowing heterochromatin enzymes to more easily penetrate areas of high density in the cores of domains to facilitate maturation. Furthermore, transcription has a nonmonotonic dependence of RNA polymerase on local crowding conditions and happens in optimal conditions. By using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy and Stochastic Optical Reconstruction Microscopy (STORM), this study aims to uncover how heterochromatin protein 1 alpha (HP1α), a critical chromatin crosslinking protein that binds to H3K9me3, is involved in stabilizing chromatin packing domains. By exploring how HP1α is involved in domain formation and maturation, we hope to manipulate transcriptional memory in various disease contexts, such as aging and cancer. This can be achieved by stabilizing packing domains to maintain long-term memory or degrading packing domains to reprogram cellular states.
Other authors: Tiffany Kuo, Nicolas Acosta, I Chae Ye, Samantha Niemi, Claudia V. Arriaga, Emily Pujadas Liwag, Cody L. Dunton, Jane Frederick, Paola Carillo Gonzalez, Luay M. Almassalha, Vadim Backman
9) Mapping the metastatic behavior of Ewing sarcoma in the zebrafish xenograft model
Ingrid Lekk, Children's Hospital Los Angeles
Ewing sarcoma (EwS) is a malignant solid tumor of bone and soft tissues, to which effective and targeted therapies are lacking but desperately needed. Metastatic forms of EwS confer a very poor prognosis in children and adolescents. However, insufficient understanding of the biology of metastasis has prevented the development of more effective therapies for disseminated EwS. Our aim is to live-image human EwS cells in interaction with various zebrafish microenvironments to study the main events in metastatic behavior: intra- and extravasation, colonization of new locations and the role of tumor extracellular vesicles (EV) in tumor spread. Using the FishAtlas pipeline developed at UT Southwestern for high throughput analysis of metastatic patterns, we show that human EwS cell line originating from a metastatic site (lymph node) shows more widespread distribution and extravasation compared to cell lines from primary tumors. Moreover, we can detect fluorescently labelled EwS EVs in the bloodstream of live zebrafish embryos and track their uptake by the cells in the tumor microenvironment. Combining the statistical power of FishAtlas and the high-magnification live-imaging advantages of zebrafish we have built a model to study the cellular and molecular mechanisms of EwS metastasis in a live organism.
Other authors: Hanieh Mazloom Farzibaf, Gaudenz Danuser, James Amatruda
10) Electron Microscopy Techniques at Intermediate-Scale for Chromatin Packing Domains
Wing Shun Li, Northwestern University
Electron microscopy is a well-established imaging technique in biological research, capable of resolving structures ranging from protein molecules with angstrom resolution to micron-scale cellular components. Imaging the ~100-200 nm chromatin nanodomain structure however presents a unique challenge. It requires neither the highest resolution nor the largest imaging volume, but no current methods can effectively address this intermediate scale. In this poster, we present our development of imaging techniques to bridge the gap between high-resolution imaging and large-volume imaging in electron microscopy.
Other authors: Ruyi Gong, Reiner Bleher, Paul JM Smeets, Geng Wang, Roberto dos Reis, Vinayak P. Dravid, Vadim Backman
11) Immune Engineering Strategies to Enhance T Lymphocyte Dynamics in Pancreatic Ductal Adenocarcinoma
Priyanila Magesh, University of Minnesota
Pancreatic Ductal Adenocarcinoma (PDA) stands as one of the leading contributors to cancer-associated mortality in the United States, in part, due to treatment resistance. Nevertheless, there is strong potential with Chimeric Antigen Receptor (CAR) T cell therapy, which harnesses T cells from the patient’s body modified with synthetic cell-surface receptors to target cancer cells efficiently. However, these CAR T cells face challenges in PDA: they display limited persistence, and poor tumor infiltration due to the dense desmoplastic stroma, and the immunosuppressive nature of the tumor microenvironment (TME). Hence, my research aims to identify and genetically engineer key focal adhesion-related genes in CAR T cells, enhancing their ability to sample within the tumor by overcoming the fibrotic stromal barriers.
Preliminary experiments reveal that T cell migration on ECM substrates follows a biphasic pattern, suggesting that tumor microenvironment rich in adhesion ligands may hinder T cell motility. We initially screened for potential targets using bulk RNA sequencing of meso-CAR T cells, selectively identifying upregulated adhesion associated genes such as integrins alpha-4, alpha-L, beta-1, and beta-2. To evaluate their effect on T cell migration, we performed two-photon time-series imaging of T cells moving in 3D ECM-like gels while blocking the target receptors with function-blocking antibodies. We observed that integrins alpha-4 and alpha-L blocking increases 3D speed, motility coefficient, and persistence. Targeting these integrins also results in unique morphology changes quantified by circularity and surface area in CAR T cells. Similarly, integrin-blockade migration experiments on mouse and human tumor slices, mimicking the TME, confirmed that integrin alpha-L blockade positively correlates with enhanced motility characteristics and sampling. Therefore, employing the CRISPR Cas9 knockout strategy to genetically modify the integrin alpha-L adhesion receptor could enhance the therapeutic potential of CAR T cells to navigate the desmoplastic stroma and infiltrate tumors in vivo, eventually offering an effective therapeutic for PDA patients.
Other authors: Paolo Provenzano
12) Investigating the structural organization of chromatin in cancer stem cells
Karla Medina, Northwestern University
The three-dimensional (3D) organization of chromatin and its dynamic mass distribution within the nucleus are critical in determining cellular phenotype and function. Tight regulation of this chromatin landscape is essential for normal development, and its disruption is closely linked to oncogenesis. Cancer stem cells (CSCs), which display altered chromatin structure, are believed to drive tumor recurrence through their stem-like and tumor-initiating capacities. ChromSTEM analysis has demonstrated that chromatin self-assembles into distinct yet interconnected packing domains with mass-fractal properties, with notable diGerences in chromatin compaction between healthy BJ fibroblasts and lung cancer cells. Mechanisms such as cohesin-CTCF-mediated looping, molecular diGusion constraints, and transcriptional forces likely contribute to domain formation, yet the complete molecular composition of chromatin domains remains undefined. This work focuses on: (1) characterizing specific diGerences in chromatin structure within domains across CSC and non-CSC models, (2) investigating the molecular organization underlying transitions from non-CSCs to CSCs, and (3) exploring reprogramming strategies to modify chromatin domain organization to enhance CSC targeting and therapeutic responsiveness.
Other authors: Karla I. Medina, Wing Shun Li, Ruyi Gong, Nicolas Acosta, Yinu Wang, Daniela Matei, Vadim Backman
13) The impact of charge regulation and ionic intranuclear environment on chromatin charge and structure.
Rikkert Nap, Northwestern University
Chromatin is composed of DNA, a negatively charged polyacid and histone proteins, which contain positively chargeable amino acid residues. These histone residues only partially neutralize the highly negative charge of DNA, and chromatin as a whole retains an overall net negative charge. The interplay between these electrostatic forces and chromatin’s structural organization is not fully understood. Here, we theoretically investigate how the intranuclear environment influences the charge of a nucleosome core particle (NCP)—the fundamental unit of chromatin consisting of DNA wrapped around a core of histone proteins. The molecular-based theory explicitly considers the size, shape, conformation, charge, and chemical state of all molecular species—thereby linking the structural state with the chemical/charged state of the system. We investigate how variations in monovalent and divalent salt concentrations, as well as pH, affect the charge distribution across different regions of an NCP and quantify the impact of charge regulation. Our findings highlight the critical role of divalent magnesium ions on the charge, electrostatic energy, and the counterion cloud that surrounds an NCP. Notably, as magnesium concentration increases, charge neutralization, and even charge inversion is predicted—in line with experimental observation of NCPs. This strong Mg-dependence of the nucleosome charge state arises from ion bridges between two neighboring DNA-phosphates and one Mg2+ ion. Moving forward, we aim to extend our investigation to small nucleosome arrays tethered to the nuclear lamina, examining how their change and structural organization respond to changes in monovalent and divalent magnesium concentrations.
Other authors: Rikkert J. Nap, Paola Carrillo Gonzalez, Aria Coraor, Ranya K. A. Virk, Marcelo A. Carignano, Juan J. de Pablo, Vadim Backman, and Igal Szleifer.
14) TME-CART: a computational discovery platform for cell-based immunotherapy in pancreatic ductal adenocarcinoma
Guhan Qian, University of Minnesota
Pancreatic ductal adenocarcinoma (PDA) is characterized by profound desmoplasia that promotes immune exclusion and impedes T cell infiltration. In this study, we present an integrated experimental and computational framework aimed at elucidating the infiltration dynamics of engineered CD8+ T cells within the PDA tumor microenvironment (TME). Utilizing KPCT genetically engineered mouse models (GEMMs) and ex vivo tumor slices, we employed multiplexed multiphoton microscopy to capture high-resolution, real-time interactions of T cells in relation to their immediate and broader environment. Our findings indicate a dual role for collagen within the extracellular matrix (ECM), where it serves as a directional contact guidance cue that facilitates rapid, ballistic T cell migration along pre-defined collagen fiber tracks while simultaneously imposing migration anisotropy that limits off-axis movement and creates localized immune exclusion zones. Colocalized with collagen fibers, CD11b+ myeloid cells are identified as key modulators of T cell behavior by engaging in MHC-I-mediated antigen presentation and activating the PD-1/PD-L1 immune checkpoint, ultimately leading to T cell sequestration. To quantitatively dissect immune infiltration barriers, we developed TME-CARTographer (TME-CART), behavior analysis, and interpretable deep learning, offering granular insights into distinct T cell-TME interplay. Leveraging local interpretable model-agnostic explanations (LIME) and partial dependence plots (PDPs), we reveal both univariate and multivariate T cell and TME factors that differentiate Msln CD8+ T cells +/- PD-1 blockade compared to naïve CD8+ T cells. Subsequently, we depleted CD11b+ myeloid cells in tumor, which reduced myeloid-mediated immunosuppression, enhanced T cell dispersal, and promoted tumor sampling and a less suppressed T cell phenotype.
Other authors: Hongrong Zhang, Ingunn Stromnes, Paolo Provenzano
15) Quantifying the Spatial Distribution of Post-Translational Histone Modifications Using 3D Spectroscopic Single-Molecule Localization Microscopy
George Rabadi, Northwestern University
Super-resolution microscopy has enabled studies that probe protein spatial distribution at the nanoscale. This, in turn, has made it possible to study the distribution of post-translational modifications in the nucleus and how these distributions change in response to diseases like cancer. Currently, there is a lack of studies that examine the interactions of multiple histone modifications in a single nucleus. We thus quantified the individual distribution and clustering behaviors of H3K27me3 and H3K27ac and classified the level of contact between these histone modifications using spectroscopic single-molecule localization microscopy (sSMLM). To validate this quantification, we developed a simulated ground truth chromatin model upon which we simulated probe binding, fluorophore blinking dynamics, noise, and image reconstruction. Together, this system was used to detect changes in clustering behavior with degrees of cancer malignancy and with drug-induced perturbations in methylation machinery.
Other authors: George Rabadi, Benjamin Brenner, Marcelo Carignano, Luay Almassalha, Daniela Matei, Vadim Backman, Igal Szleifer, Cheng Sun, Hao F. Zhang
16) Nanoscale Imaging of Cancer Onset, Progression, and Metastasis
Qionghua Nicole Shen, UT Southwestern Medical Center
Cancer progression and metastasis arise from a complex interplay of genetic mutations, subcellular morphology, and mechanical forces within the tumor microenvironment. While genomic approaches have revealed key oncogenic drivers, growing evidence suggests that subcellular alterations, such as nuclear deformation, cytoskeletal remodeling, and mitochondrial function, not only reflect these mutations but actively modulate cancer cells’ metastatic potential and therapeutic resistance. Although researchers acknowledge the significance of such phenomena, it remains challenging to accurately capture those events across multiple scales in intact tissue samples.
To address this, we developed a multi-scale imaging platform which combines optimized iterative expansion microscopy with high-resolution volumetric imaging. This workflow enables comprehensive visualization of intact tumor tissues, spanning from macroscopic tissue architecture down to the subcellular structures and molecular components. It also supports quantitative analysis of molecular and organellar features, including nuclear mechanics and cytoskeletal organization, within their native context. By integrating spatial structure with functional insights, our method offers a powerful platform to uncover how physical forces and subcellular morphology drive mutation-linked cancer progression and metastatic behavior across multiple biological scales.
Other authors: Felix Zhou, Doreen Idonije, Hazel Borges, Tai Ngo, Kevin Dean
17) Maximizing photon utilization in spectroscopic single-molecule localization microscopy using symmetrically dispersed dual-wedge prisms
Menglin Shi, Northwestern Univeristy
Single-molecule localization microscopy (SMLM) achieves super-resolution imaging by analyzing fluorescence emissions from individual molecules. Spectroscopic SMLM (sSMLM) further enables simultaneous single-molecule spectroscopy by splitting the emitted photons for independent spatial and spectral analyses but suffers from reduced spatial and spectral precision due to reduced photon budgets after splitting. To improve these precisions, we develop a symmetrically dispersed dual-wedge prism (SDDWP)-sSMLM that maximizes photon utilization for both spatial and spectral analyses. We equally divided fluorescence photons and symmetrically dispersed them using two identical dual-wedge prisms (DWPs). Then, we computationally extracted the fluorophores' spatial position and spectral characteristics using all photons in both channels. Theoretical analysis and experimental validation showed spatial and spectral precisions of 9.0 nm and 0.4 nm, respectively, representing 27% and 48% improvements over our previous sSMLM. Using a single wavelength laser excitation, we performed multiplexed super-resolution imaging of peroxisomes, microtubules, and mitochondria in Hela cells respectively labeled with DY-634, AF647, and CF660C, whose emission spectra are highly overlapping. By spectrally tagging individual nanoparticles, we further demonstrated massively parallel tracking of single nanoparticles at a concentration five-fold higher than the state-of-the-art. Furthermore, all optical components are assembled into a single compact unit that can be easily integrated with existing SMLM systems.
Other authors: Wei-Hong Yeo, Benjamin Brenner, Menglin Shi, Youngseop Lee, Junghun Kweon, Cheng Sun, Hao F. Zhang
18) Nanoscale multimodal imaging platform for chromatin study
Geng Wang, Northwestern University
The three-dimensional organization of the chromatin is critically involved in the regulation of gene expression and is highly complex. Large-scale chromatin alterations are linked to cancer, neurological and autoimmune disorders, and other complex diseases. However, chromatin structure spans length scales from nanometers to micrometers, no individual technique can fully elucidate the chromatin organization and its relation to molecular function at all spatial and temporal scales. To investigate chromatin conformation, we developed a multitechnique nanoscale chromatin imaging platform, which incorporates 3D chromatin scanning transmission electron microscopy (ChromSTEM), multi-label spectroscopic single-molecule localization microscopy (sSMLM), spectroscopic intrinsic-contrast photon-localization optical nanoscopy (SICLON), and partial wave spectroscopic microscopy (PWS). Our platform enables the examination of spatio-temporal chromatin packing changes from individual DNA strands to whole chromatin in hundreds of live cells. PWS provides real-time, label-free live cell imaging with high throughput, sensing nanometer-scale chromatin structures within their packing context. Multi-label sSMLM identifies the precise locations of individual molecules, uncovering chromatin's structural complexity, though targeting highly dense chromatin polymer with volume concentrations between 0.2 and 0.8 remains challenging. SICLON leverages the endogenous photoswitching of DNA and its spectroscopic capability to facilitate label-free DNA imaging, potentially mitigating label sparsity issues. ChromSTEM offers detailed measurement of DNA organization and density, providing a ground truth for the other methods. By consolidating these techniques, this platform can complement the genomic information provided by chromatin conformation capture and other sequencing-based techniques. Integrating this imaging platform with molecular and chromatin assays such as Hi-C and single-cell sequencing, we can relate chromatin organization to its function and the regulation of gene expression.
Other authors: Ruyi Gong, Nicolas Acosta, Yuanzhe Su, Wingshun Li, Luay Almassalha, Vadim Backman
19) Chromatin Organization Governs Transcriptional Response and Plasticity of Cancer Stem Cells
Yinu Wang, Northwestern University
Chromatin organization regulates transcription to influence cellular plasticity and cell fate. We explored whether chromatin nanoscale packing domains are involved in stemness and response to chemotherapy. Using an optical spectroscopic nanosensing technology, partial wave spectroscopy, we show that ovarian cancer-derived cancer stem cells (CSCs) display upregulation of nanoscale chromatin packing domains (P
Other authors: Yinu Wang, Jane Frederick, Karla Isabel Medina, Elizabeth Thomas Bartom, Luay Matthew Almassalha, Yaqi Zhang, Greta Wodarcyk, Hao Huang, I Chae Ye, Ruyi Gong, Cody Levi Dunton, Alex Duval, Paola Carrillo Gonzalez, Joshua Pritchard, John Carinato, Iuliia Topchu, Junzui Li, Zhe Ji, Mazhar Adli, Vadim Backman, and Daniela Matei
20) 3D spheroid modeling reveals matrix density-driven regulation of Ewing sarcoma cell invasion
Manon Watzky, Children's Hospital Los Angeles
Ewing sarcoma (EwS) is a pediatric bone and soft tissue cancer driven by chromosomal translocations that generate fusion oncogenes, primarily EWSR1::FLI1. Metastasis is the most significant prognostic factor and remains poorly understood due to the limited models that capture its dynamics. Moreover, EwS has a low mutational burden, suggesting that disease progression may involve adaptive responses rather than genetic selection.
To study how EwS cells adapt to microenvironmental cues, I developed a 3D spheroid model using cell lines derived from primary tumors and metastatic lesions. Spheroids are embedded in Collagen I matrices at varying densities, mimicking early metastasis, where cells detach from the primary mass and invade the surrounding extracellular matrix (ECM).
IncuCyte and two-photon time-lapse imaging show that all spheroids initiate ECM invasion within 72 hours post-embedding. Primary tumor cells exhibit homogeneous invasion, while metastatic cells display heterogeneous patterns, with both rounded and elongated cells detaching from spheroids and primarily invading the ECM as single cells. Notably, increasing matrix density suppresses invasion across all cell lines and leads to a reduction in initial spheroid size, indicating physical confinement. These results suggest greater plasticity or distinct invasive subpopulations in metastatic cells and highlight the strong influence of ECM properties on EwS cell behavior.
At the molecular level, preliminary data reveal that invasive cells at the spheroid edge show reduced EWSR1::FLI1 levels. This 3D model offers a platform to dissect the mechanical and molecular mechanisms of EwS invasion, and future work will explore how ECM shapes spheroid organization and EWSR1::FLI1 dynamics.
Other authors: James Amatruda
21) iCLAP: A novel high-plex immunostaining strategy for tumor microenviorment mapping
Fan Wu, Johns Hopkins University
Multiplex immunolabeling techniques, such as cyclic immunofluorescence (CyCIF), imaging mass cytometry (IMC), and co-detection by indexing (CODEX), enable high-plex spatial analysis of complex tissues. However, many key protein regulators are present in low abundance in tissues, limiting detection sensitivity, including senescence markers, secretory proteins, and transcription factors. While tyramide signal amplification (TSA) enhances fluorescence signals for improved sensitivity, its multiplexing capacity is constrained. Here, we introduce integrable Co-detection of Low Abundant Proteins (iCLAP), a novel workflow that achieves both high sensitivity and high multiplexed detection within the same section of formalin-fixed, paraffin-embedded (FFPE) tissues. iCLAP employs cyclic TSA-based staining for low-abundance proteins, followed by optimized fluorophore inactivation method to enable additional rounds of staining. This approach allows integration with other multiplex immunolabeling methods. Combining iCLAP with immunofluorescence (iCLAP-IF) enabled six-plex detection of low abundance senescence markers in archival FFPE tissues, revealing distinct expression patterns at the single-cell level. We further integrated iCLAP with high-plex techniques (CyCIF, CODEX, and IMC), achieving 40+ plex detections while preserving sensitivity. Our analysis uncovered spatially distinct senescence marker expression profiles in pancreatic islets and acinar cells. To demonstrate robustness and broad utility, we validated applicability of iCLAP across multiple tissue types. Overall, iCLAP provides a scalable solution for highly sensitive and highly multiplexed protein detection in FFPE tissues, advancing spatial biology
Other authors: Shuyuan Zheng, Yani Chen, Peijia Ye,Moo Joong Kim, Seojin Lee, Shriya Pillan, Ruihan Yuan, Kyu Sang Han, Qingfeng Zhu, Sarah M. Shin, Courtney D. Cannon, Gabriele Pierre, Birgit Schilling, Laura D. Wood, Won Jin Ho, Robert A. Anders, Denis Wirtz, Pei-Hsun Wu
22) Chromatin Modulation as a Strategy to Enhance Chemotherapy Response in Cancer Cells
I Chae Ye, Northwestern University
Cancer cells can rapidly adapt to cytotoxic stress through non-genetic mechanisms. In particular, chromatin organization and the epigenetic landscape modulate gene accessibility, influencing transcriptional shifts associated with chemoresistance.
Here, we identify a novel class of therapeutic, Transcriptional Plasticity Regulators (TPRs), that suppress chromatin states promoting plasticity in cancer cells. Using live-cell chromatin imaging, we screened for fast-acting TPRs and validated their effects on chromatin organization via super-resolution microscopy. Pretreatment with TPRs enhanced chemotherapy-induced cell death across multiple cancer cell lines and chemotherapeutic agents in vitro. Among these, celecoxib emerged as the most effective and significantly improved treatment outcomes in a patient-derived xenograft (PDX) model.
To distinguish celecoxib’s chromatin-modulating effects from its known anti-inflammatory role as a COX-2 inhibitor, we employed COX-2-deficient HCT116 cells and confirmed the synergistic effect with chemotherapy. We further compared celecoxib to other NSAIDs lacking chromatin-modulating properties and observed a loss of synergy, supporting a chromatin-dependent mechanism. Additionally, analysis of the PDX tumor microenvironment revealed no significant differences, suggesting that celecoxib’s effect is intrinsic to cancer cell transcriptional plasticity.
In summary, our findings highlight the pivotal role of chromatin architecture and transcriptional regulation in cancer cell adaptation to therapy. Targeting these non-genetic determinants with TPRs offers a promising strategy to overcome chemoresistance and improve clinical outcomes in cancer treatment.
Other authors: I Chae Ye, Jane Frederick, Ranya Virk, Luay Almassalha, David VanDerway, Ruyi Gong, Saira John, Tiffany Kuo, Karla Medina, Vasundhara Agrawal, Nicholas Anthony, Guillermo Ameer, Igal Szleifer, Vadim Backman
23) Random Labeling Single-Molecule Localization Microscopy
Wei Hong Yeo, Northwestern University
Single-molecule localization microscopy (SMLM) has revolutionized optical imaging by achieving resolutions down to ~20 nm. However, further improvements are needed to bridge the gap with electron microscopy (EM) for sub-nanometer resolution. We introduce a novel approach combining photon-accumulation enhanced reconstruction (PACER) with random labeling, which maximizes spectral heterogeneity by labeling proteins with a diverse set of fluorophores. This method enhances classification accuracy and spatial precision, achieving resolutions approaching 1 nm. We validate our approach through simulations and experimental imaging of nuclear pore complexes (NPCs), demonstrating significant improvements in localization precision and clustering performance. Our results highlight the potential of PACER with random labeling for high-resolution imaging of complex biological structures.
Other authors: Benjamin Brenner, Junghun Kweon, Cheng Sun, Hao F. Zhang
24) γδ CAR-T cells show enhanced migration into pancreatic cancer
Chris Zahm, University of Minnesota
Current immunotherapies have revolutionized the treatment of blood cancers, but solid tumors remain a challenge. Pancreatic ductal adenocarcinoma (PDAC) is particularly difficult due to multiple levels of immunosuppression and the failure of immune cells to effectively infiltrate their dense fibrotic stroma. As such, chimeric antigen receptor (CAR) T cell therapies targeting PDAC must be engineered with enhanced migratory abilities to penetrate the complicated TME. In this study we assessed the migratory capacity and metabolism of γδ CAR-T cells and explore their use as a cellular therapy for PDAC. Using multiphoton microscopy we generated fluorescent, second harmonic generation (SHG), and fluoresce lifetime (FLIM) images of γδ and αβ CAR-T cells in 3D matrices and compared their migration kinetics and metabolic profiles. We found that γδ CAR-Ts moved faster and further through collagen and showed increased motility coefficients in both collagen and spheroids of Panc-1 or Mia-Paca cells when compared to αβ-CAR-Ts. Furthermore, NAD(P)H FLIM revealed the metabolic profile of γδ CAR-T’s as more oxidative, resembling a stem memory phenotype while the αβ CAR were more glycolytic, resembling later stage cytotoxic T cells. Taken together these data indicate that γδ CAR-T’s may have an enhanced ability to migrate into and persist for longer periods in the PDAC TME when compared to their αβ counterparts. While in vivo studies are needed this data signifies the use of γδ CAR-T cells to treat PDAC and suggests they may play an essential role in the future of cellular therapies.
Other authors: Christopher D. Zahm, Shambojit Roy, Joseph G. Skeate, Ethan Niemeyer, Beau R. Webber, Branden S. Moriarity, Paolo P. Provenzano
25) Reprogramming T Cell Motility and Function Through the RhoA Pathway
Hongrong Zhang, University of Minnesota
Despite being the 14th most common cancer, pancreatic cancer—particularly pancreatic ductal adenocarcinoma (PDA)—is projected to become the second leading cause of cancer-related deaths due to late diagnosis, aggressive progression, and limited treatment options. T cell-based immunotherapy offers a promising alternative. Indeed, higher densities of peritumoral CD8⁺ T cells are associated with improved survival in PDA and other solid tumors. However, T cell presence alone is insufficient; effective anti-tumor immunity requires robust infiltration, sustained tumor engagement, and cytotoxic activity. Enhancing T cell trafficking, intratumoral migration, and interaction with tumor cells is therefore critical for improving immunotherapy efficacy. In this study, we engineered CD8⁺ T cells to express a constitutively active RhoA mutant using CRISPR/Cas9 genome editing and evaluated their migratory behavior, metabolic profile, differentiation status, and tumor interaction. Engineered T cells exhibited significantly increased total RhoA and RhoA-GTP levels without affecting proliferation or viability, indicating that sustained RhoA activation does not impose a metabolic burden. Functionally, these cells exhibited enhanced motility in 3D collagen matrices and live PDA tumor slices, adopting an amoeboid migration phenotype with increased metabolic activity and cortical tension. To assess therapeutic relevance, we incorporated the RhoA mutation into mesothelin-targeting CAR T cells (mesoCAR).
While cytotoxicity remained comparable in 2D assays, RhoA mutant mesoCAR T cells showed significantly higher tumor interaction frequency in 3D cultures, suggesting improved tumor search efficiency. Overall, these suggest RhoA as a potent driver of T cell motility, offering a novel approach to overcome physical barriers in PDA and enhance immunotherapy outcomes.
Other authors: Zhongming Chen, Guhan Qian, Chris Zahm, Branden Moriarity, Paolo Provenzano