Publications

Noncanonical T cell responses are associated with protection from tuberculosis in mice and humans.

Abstract (Expand)

While control of Mycobacterium tuberculosis (Mtb) infection is generally understood to require Th1 cells and IFNgamma, infection produces a spectrum of immunological and pathological phenotypes in … diverse human populations. By characterizing Mtb infection in mouse strains that model the genetic heterogeneity of an outbred population, we identified strains that control Mtb comparably to a standard IFNgamma-dependent mouse model but with substantially lower lung IFNgamma levels. We report that these mice have a significantly altered CD4 T cell profile that specifically lacks the terminal effector Th1 subset and that this phenotype is detectable before infection. These mice still require T cells to control bacterial burden but are less dependent on IFNgamma signaling. Instead, noncanonical immune features such as Th17-like CD4 and gammadeltaT cells correlate with low bacterial burden. We find the same Th17 transcriptional programs are associated with resistance to Mtb infection in humans, implicating specific non-Th1 T cell responses as a common feature of Mtb control across species.

Authors: M. K. Proulx, C. D. Wiggins, C. J. Reames, C. Wu, M. C. Kiritsy, P. Xu, J. C. Gallant, P. S. Grace, B. A. Fenderson, C. M. Smith, C. S. Lindestam Arlehamn, G. Alter, D. A. Lauffenburger, C. M. Sassetti

Date Published: 7th Jul 2025

Publication Type: Journal

Antibody-Fab and -Fc features promote Mycobacterium tuberculosis restriction.

Abstract (Expand)

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), a leading cause of death by an infectious disease globally, has no efficacious vaccine. Antibodies are implicated in M. tuberculosis … control, but the mechanisms of action remain poorly understood. We assembled a library of monoclonal antibodies (mAb) and screened for M. tuberculosis-restrictive activity in mice, identifying protective antibodies targeting diverse antigens. To dissect the mechanism of mAb-mediated M. tuberculosis restriction, we optimized a protective lipoarabinomannan-specific mAb, generating Fc variants. In vivo analysis of these Fc variants revealed a role for Fc-effector function in M. tuberculosis restriction. Restrictive Fc variants altered distribution of M. tuberculosis across innate immune cells. Single-cell transcriptomics highlighted distinctly activated pathways within innate immune cell subpopulations, identifying early activation of neutrophils as a key signature of mAb-mediated M. tuberculosis restriction. Therefore, antibody-mediated restriction of M. tuberculosis is associated with reorganization of the tissue-level immune response to infection and depends on the collaboration of antibody Fab and Fc.

Authors: P. S. Grace, J. M. Peters, J. Sixsmith, R. Lu, E. B. Irvine, C. Luedeman, B. A. Fenderson, A. Vickers, M. D. Slein, T. McKitrick, M. H. Wei, R. D. Cummings, A. Wallace, L. A. Cavacini, A. Choudhary, M. K. Proulx, C. Sundling, G. Kallenius, R. Reljic, J. D. Ernst, A. Casadevall, C. Locht, A. Pinter, C. M. Sassetti, B. D. Bryson, S. M. Fortune, G. Alter

Date Published: 10th Jun 2025

Publication Type: Journal

Robust IgM responses following intravenous vaccination with Bacille Calmette–Guérin associate with prevention of Mycobacterium tuberculosis infection in macaques

Abstract

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Authors: Edward B. Irvine, Anthony O’Neil, Patricia A. Darrah, Sally Shin, Alok Choudhary, Wenjun Li, William Honnen, Smriti Mehra, Deepak Kaushal, Hannah Priyadarshini Gideon, JoAnne L. Flynn, Mario Roederer, Robert A. Seder, Abraham Pinter, Sarah Fortune, Galit Alter

Date Published: 22nd Nov 2021

Publication Type: Journal

Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control

Abstract

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Authors: Hannah P. Gideon, Travis K. Hughes, Constantine N. Tzouanas, Marc H. Wadsworth, Ang Andy Tu, Todd M. Gierahn, Joshua M. Peters, Forrest F. Hopkins, Jun-Rong Wei, Conner Kummerlowe, Nicole L. Grant, Kievershen Nargan, Jia Yao Phuah, H. Jacob Borish, Pauline Maiello, Alexander G. White, Caylin G. Winchell, Sarah K. Nyquist, Sharie Keanne C. Ganchua, Amy Myers, Kush V. Patel, Cassaundra L. Ameel, Catherine T. Cochran, Samira Ibrahim, Jaime A. Tomko, Lonnie James Frye, Jacob M. Rosenberg, Angela Shih, Michael Chao, Edwin Klein, Charles A. Scanga, Jose Ordovas-Montanes, Bonnie Berger, Joshua T. Mattila, Rajhmun Madansein, J. Christopher Love, Philana Ling Lin, Alasdair Leslie, Samuel M. Behar, Bryan Bryson, JoAnne L. Flynn, Sarah M. Fortune, Alex K. Shalek

Date Published: 1st May 2022

Publication Type: Journal

Immune cells in bronchoalveolar lavage fluid of Ugandan adults who resist versus those who develop latent Mycobacterium tuberculosis infection

Abstract

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Authors: Bonnie A. Thiel, William Worodria, Sophie Nalukwago, Mary Nsereko, Ingvar Sanyu, Lalitha Rejani, Josephine Zawedde, David H. Canaday, Catherine M. Stein, Keith A. Chervenak, LaShaunda L. Malone, Ronald Kiyemba, Richard F. Silver, John L. Johnson, Harriet Mayanja-Kizza, W. Henry Boom

Date Published: 1st Apr 2021

Publication Type: Journal

Specific CD4+ T cell phenotypes associate with bacterial control in people who ‘resist’ infection with Mycobacterium tuberculosis

Abstract

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Authors: Meng Sun, Jolie M. Phan, Nathan S. Kieswetter, Huang Huang, Krystle K. Q. Yu, Malisa T. Smith, Yiran E. Liu, Chuangqi Wang, Sanjana Gupta, Gerlinde Obermoser, Holden Terry Maecker, Akshaya Krishnan, Sundari Suresh, Neha Gupta, Mary Rieck, Peter Acs, Mustafa Ghanizada, Shin-Heng Chiou, Purvesh Khatri, W. Henry Boom, Thomas R. Hawn, Catherine M. Stein, Harriet Mayanja-Kizza, Mark M. Davis, Chetan Seshadri

Date Published: 1st Jul 2024

Publication Type: Journal

CD8+ lymphocytes are critical for early control of tuberculosis in macaques

Abstract

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Authors: Caylin G. Winchell, Sarah K. Nyquist, Michael C. Chao, Pauline Maiello, Amy J. Myers, Forrest Hopkins, Michael Chase, Hannah P. Gideon, Kush V. Patel, Joshua D. Bromley, Andrew W. Simonson, Roisin Floyd-O’Sullivan, Marc Wadsworth, Jacob M. Rosenberg, Rockib Uddin, Travis Hughes, Ryan J. Kelly, Josephine Griffo, Jaime Tomko, Edwin Klein, Bonnie Berger, Charles A. Scanga, Joshua Mattila, Sarah M. Fortune, Alex K. Shalek, Philana Ling Lin, JoAnne L. Flynn

Date Published: 16th Oct 2023

Publication Type: Journal

Age and sex influence antibody profiles associated with tuberculosis progression.

Abstract

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Authors: Leela R. L. Davies, Chuangqi Wang, Pia Steigler, Kathryn A. Bowman, Stephanie Fischinger, Mark Hatherill, Michelle Fisher, Stanley Kimbung Mbandi, Miguel Rodo, Tom H. M. Ottenhoff, Hazel M. Dockrell, Jayne S. Sutherland, Harriet Mayanja-Kizza, W. Henry Boom, Gerhard Walzl, Stefan H. E. Kaufmann, Elisa Nemes, Thomas J. Scriba, Douglas Lauffenburger, Galit Alter, Sarah M. Fortune

Date Published: 24th Apr 2024

Publication Type: Journal

Humoral correlates of protection against <i>Mycobacterium tuberculosis</i> following intravenous Bacille Calmette-Guérin vaccination in rhesus macaques.

Abstract

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Authors: Edward B. Irvine, Patricia A. Darrah, Shu Wang, Chuangqi Wang, Ryan P. McNamara, Mario Roederer, Robert A. Seder, Douglas A. Lauffenburger, JoAnne L. Flynn, Sarah M. Fortune, Galit Alter

Date Published: 2nd Aug 2023

Publication Type: Journal

Fc-engineered antibodies promote neutrophil-dependent control of Mycobacterium tuberculosis

Abstract

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Authors: Edward B. Irvine, Angel Nikolov, Mehak Z. Khan, Joshua M. Peters, Richard Lu, Jaimie Sixsmith, Aaron Wallace, Esther van Woudenbergh, Sally Shin, Wiktor Karpinski, Jeff C. Hsiao, Arturo Casadevall, Bryan D. Bryson, Lisa Cavacini, Patricia S. Grace, Galit Alter, Sarah M. Fortune

Date Published: 22nd Aug 2024

Publication Type: Journal

CD4+ T cells re-wire granuloma cellularity and regulatory networks to promote immunomodulation following Mtb reinfection

Abstract

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Authors: Joshua D. Bromley, Sharie Keanne C. Ganchua, Sarah K. Nyquist, Pauline Maiello, Michael Chao, H. Jacob Borish, Mark Rodgers, Jaime Tomko, Kara Kracinovsky, Douaa Mugahid, Son Nguyen, Qianchang Dennis Wang, Jacob M. Rosenberg, Edwin C. Klein, Hannah P. Gideon, Roisin Floyd-O’Sullivan, Bonnie Berger, Charles A. Scanga, Philana Ling Lin, Sarah M. Fortune, Alex K. Shalek, JoAnne L. Flynn

Date Published: No date defined

Publication Type: Journal

Markov field network model of multi-modal data predicts effects of immune system perturbations on intravenous BCG vaccination in macaques

Abstract

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Authors: Shu Wang, Amy J. Myers, Edward B. Irvine, Chuangqi Wang, Pauline Maiello, Mark A. Rodgers, Jaime Tomko, Kara Kracinovsky, H. Jacob Borish, Michael C. Chao, Douaa Mugahid, Patricia A. Darrah, Robert A. Seder, Mario Roederer, Charles A. Scanga, Philana Ling Lin, Galit Alter, Sarah M. Fortune, JoAnne L. Flynn, Douglas A. Lauffenburger

Date Published: 1st Dec 2024

Publication Type: Journal

High-dose intravenous BCG vaccination induces enhanced immune signaling in the airways

Abstract

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Authors: Joshua M. Peters, Edward B. Irvine, Mohau S. Makatsa, Jacob M. Rosenberg, Marc H. Wadsworth, Travis K. Hughes, Matthew S. Sutton, Sarah K. Nyquist, Joshua D. Bromley, Rajib Mondal, Mario Roederer, Robert A. Seder, Patricia A. Darrah, Galit Alter, Chetan Seshadri, JoAnne L. Flynn, Alex K. Shalek, Sarah M. Fortune, Bryan D. Bryson

Date Published: 2025

Publication Type: Journal

Intravenous BCG-mediated protection against tuberculosis requires CD4+ T cells and CD8α+ lymphocytes

Abstract

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Authors: Andrew W. Simonson, Joseph J. Zeppa, Allison N. Bucsan, Michael C. Chao, Supriya Pokkali, Forrest Hopkins, Michael R. Chase, Andrew J. Vickers, Matthew S. Sutton, Caylin G. Winchell, Amy J. Myers, Cassaundra L. Ameel, Ryan J. Kelly, Ben Krouse, Luke E. Hood, Jiaxiang Li, Chelsea C. Lehman, Megha Kamath, Jaime Tomko, Mark A. Rodgers, Rachel Donlan, Harris Chishti, H. Jacob Borish, Edwin Klein, Charles A. Scanga, Sarah M. Fortune, Philana Ling Lin, Pauline Maiello, Mario Roederer, Patricia A. Darrah, Robert A. Seder, JoAnne L. Flynn

Date Published: 1st Feb 2025

Publication Type: Journal

Cross-species transcriptomics translation reveals a role for the unfolded protein response in Mycobacterium tuberculosis infection.

Abstract

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Authors: Krista M. Pullen, Ryan Finethy, Seung-Hyun B. Ko, Charlotte J. Reames, Christopher M. Sassetti, Douglas A. Lauffenburger

Date Published: 15th Feb 2025

Publication Type: Journal

Systematic deconstruction of myeloid cell signaling in tuberculosis granulomas reveals IFN-γ, TGF-β, and time are associated with conserved myeloid diversity

Abstract

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Authors: Joshua M. Peters, Hannah P. Gideon, Travis K. Hughes, Cal Gunnarson, Pauline Maiello, Douaa Mugahid, Sarah K. Nyquist, Joshua D. Bromley, Paul C. Blainey, Beth F. Junecko, Molly L. Nelson, Douglas A. Lauffenburger, Philana Ling Lin, JoAnne L. Flynn, Alex K. Shalek, Sarah M. Fortune, Joshua T. Mattila, Bryan D. Bryson

Date Published: 1st May 2024

Publication Type: Journal

IFN-γ- GranzymeB + Natural killer cells are induced by IV BCG vaccination and associated with protection against tuberculosis in rhesus macaques.

Abstract

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Authors: Mohau S. Makatsa, Emma Bishop, Allison N. Bucsan, Matthew S. Sutton, Chelsea C. Lehman, Molly Robertson, Krystle K.Q. Yu, Joshua M. Peters, Bryan D. Bryson, Mario Roederer, Robert A. Seder, Patricia A. Darrah, Chetan Seshadri

Date Published: 1st Nov 2025

Publication Type: Journal

Protection against reinfection with <i>Mycobacterium tuberculosis</i> extends across heterologous Mtb lineages.

Abstract

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Authors: Andrew W Simonson, Michael C Chao, Luke E Hood, Rachel A Donlan, Forrest Hopkins, Michael R Chase, Andrew J Vickers, Alanna Callendrello, Edwin Klein, H Jacob Borish, Marshall Malin, Pauline Maiello, Charles A Scanga, Philana Ling Lin, Sarah M Fortune, JoAnne L Flynn

Date Published: 31st Jul 2025

Publication Type: Journal

BCG vaccination elicits protection against Mtb infection mediated by two phases of T cell immunity

Abstract

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Authors: Abiola F. Ogunsola, Rocky Lai, Kelly Cavallo, Gillian L. Beamer, Samuel M. Behar

Date Published: 30th Oct 2025

Publication Type: Journal

Impact of Fibrinogen, Fibrin Thrombi, and Thrombin on Cancer Cell Extravasation Using In Vitro Microvascular Networks

Abstract

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Authors: Emmanouil Angelidakis, Sophia Chen, Shun Zhang, Zhengpeng Wan, Roger D. Kamm, Sarah E. Shelton

Date Published: 1st May 2023

Publication Type: Journal

Patient-specific vascularized tumor model: Blocking monocyte recruitment with multispecific antibodies targeting CCR2 and CSF-1R

Abstract

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Authors: Huu Tuan Nguyen, Ellen L. Kan, Mouhita Humayun, Nadia Gurvich, Giovanni S. Offeddu, Zhengpeng Wan, Mark F. Coughlin, Diana C. Renteria, Andreas Loew, Susan Wilson, Christie Zhang, Vivian Vu, Sharon Wei Ling Lee, Seng-Lai Tan, David Barbie, Jonathan Hsu, Mark Robert Gillrie, Roger D. Kamm

Date Published: 2025

Publication Type: Journal

Utilizing convolutional neural networks for discriminating cancer and stromal cells in three-dimensional cell culture images with nuclei counterstain

Abstract

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Authors: Huu Tuan Nguyen, Nicholas Pietraszek, Sarah E. Shelton, Kwabena Arthur, Roger D. Kamm

Date Published: 1st Aug 2024

Publication Type: Journal

Personalized Vascularized Models of Breast Cancer Desmoplasia Reveal Biomechanical Determinants of Drug Delivery to the Tumor

Abstract

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Authors: Giovanni S. Offeddu, Elena Cambria, Sarah E. Shelton, Kristina Haase, Zhengpeng Wan, Luca Possenti, Huu Tuan Nguyen, Mark R. Gillrie, Dean Hickman, Charles G. Knutson, Roger D. Kamm

Date Published: 1st Jul 2024

Publication Type: Journal

Polyploidy of MDA-MB-231 cells drives increased extravasation with enhanced cell-matrix adhesion

Abstract

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Authors: Satomi Hirose, Tatsuya Osaki, Roger D. Kamm

Date Published: 1st Jul 2024

Publication Type: Journal

Chromatin conformation, gene transcription, and nucleosome remodeling as an emergent system

Abstract

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Authors: Luay M. Almassalha, Marcelo Carignano, Emily Pujadas Liwag, Wing Shun Li, Ruyi Gong, Nicolas Acosta, Cody L. Dunton, Paola Carrillo Gonzalez, Lucas M. Carter, Rivaan Kakkaramadam, Martin Kröger, Kyle L. MacQuarrie, Jane Frederick, I Chae Ye, Patrick Su, Tiffany Kuo, Karla I. Medina, Josh A Pritchard, Andrew Skol, Rikkert Nap, Masato Kanemaki, Vinayak Dravid, Igal Szleifer, Vadim Backman

Date Published: 2025

Publication Type: Journal

Confined cell migration along extracellular matrix space in vivo

Abstract

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Authors: Oleksandr Chepizhko, Josep-Maria Armengol-Collado, Stephanie Alexander, Esther Wagena, Bettina Weigelin, Luca Giomi, Peter Friedl, Stefano Zapperi, Caterina A. M. La Porta

Date Published: 1st Dec 2024

Publication Type: Journal

Chemo-mechanical diffusion waves explain collective dynamics of immune cell podosomes

Abstract

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Authors: Ze Gong, Koen van den Dries, Rodrigo A. Migueles-Ramírez, Paul W. Wiseman, Alessandra Cambi, Vivek B. Shenoy

Date Published: 1st May 2023

Publication Type: Journal

Remodeling of self-assembled microvascular networks under long term flow

Abstract

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Authors: Marie Floryan, Elena Cambria, Adriana Blazeski, Mark F. Coughlin, Zhengpeng Wan, Giovanni Offeddu, Vinayak Vinayak, Aayush Kant, Vivek Shenoy, Roger D. Kamm

Date Published: 1st Mar 2025

Publication Type: Journal

Myofibroblasts reduce angiogenesis and vasculogenesis in a vascularized microphysiological model of lung fibrosis

Abstract

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Authors: Elena Cambria, Adriana Blazeski, Eunkyung Clare Ko, Tran Thai, Shania Dantes, David A. Barbie, Sarah E. Shelton, Roger D. Kamm

Date Published: 2025

Publication Type: Journal

Benthic Fluxes of Fluorescent Dissolved Organic Material, Salt, and Heat Measured by Multiple-Sensor Aquatic Eddy Covariance.

Abstract (Expand)

Aquatic eddy covariance (AEC) is an in situ technique for measuring fluxes in marine and freshwater systems that is based on the covariance of velocity and concentration measurements. To date, AEC has … mainly been applied to the measurement of benthic oxygen fluxes. Here, development of a fast multiple-channel sensor enables the use of AEC for measurement of benthic fluxes of fluorescent material, salt, and heat at three distinct sites in Massachusetts, USA, including the Connecticut River, the Concord River, and Upper Mystic Lake. Benthic fluxes of salt, useful as a tracer for groundwater input (submarine groundwater discharge), were consistent with independent measurements made with seepage meters. Eddy fluxes of heat were consistent with the balance of incoming solar radiation and thermal conduction at the sediment surface. Benthic eddy fluxes of fluorescent dissolved organic material (FDOM) revealed a substantial net downward flux in the humic-rich Concord River, suggesting that microbial consumption of dissolved organic carbon in the sediment was significant. Simultaneous measurement of several fluxes expands the utility of AEC as a biogeochemical tool while enabling checks for mutual consistency among data channels.

Authors: I. H. Hu, H. F. Hemond

Date Published: 20th Nov 2022

Publication Type: Journal

Colorimetric Detection of Aqueous N-Nitrosodimethylamine via Photonitrosation of a Naphtholsulfonate Indicator.

Abstract (Expand)

N-Nitrosamines are contaminants found throughout the environment, including in drinking water, and many nitrosamines are likely potent carcinogens. Correspondingly, there is a need for rapid and … cost-effective in-field detection methods that can provide timely information about their contamination levels in water. This study details a colorimetric assay for detecting aqueous N-nitrosodimethylamine (NDMA) by photochemical nitrosation of a commercial naphtholsulfonate, to offer an attractive alternative to traditional laboratory-based analysis. The resulting naphthoquinone-oxime coordinates to aqueous iron(II) ions to form a green complex, allowing for direct visual detection. Characterization via Mossbauer and electron paramagnetic resonance (EPR) spectroscopy, alongside single-crystal structure determination, provides comprehensive structure information on the iron indicator complex. Optimization of detection conditions, including UV irradiation and response times, led to an improved colorimetric detection method with a limit of detection of 0.66 ppm for NDMA. The practical applicability and selectivity of this colorimetric detection scheme make it a promising candidate for the development of field-deployable sensors for NDMA in environmental water samples.

Authors: J. C. Beard, C. H. Wang, A. Sridharan, R. G. Croy, J. M. Essigmann, T. M. Swager

Date Published: 27th Sep 2024

Publication Type: Journal

CometChip enables parallel analysis of multiple DNA repair activities.

Abstract (Expand)

DNA damage can be cytotoxic and mutagenic, and it is directly linked to aging, cancer, and other diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA … repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these applications of the CometChip platform, we expand the utility of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.

Authors: J. Ge, L. P. Ngo, S. Kaushal, I. J. Tay, E. Thadhani, J. E. Kay, P. Mazzucato, D. N. Chow, J. L. Fessler, D. M. Weingeist, R. W. Sobol, L. D. Samson, S. R. Floyd, B. P. Engelward

Date Published: 9th Aug 2021

Publication Type: Journal

DNA adduct and mutational profiles reveal a threshold of cellular defenses against N-nitrosodimethylamine administered to mice in drinking water.

Abstract (Expand)

N-Nitrosodimethylamine (NDMA) is classified as an animal and probable human carcinogen. Murine liver DNA adducts, mutations, O (6)-methylguanine DNA methyltransferase (MGMT), and CYP2E1 were evaluated … following chronic administration of NDMA in drinking water. In a dose-escalation study, 7-methylguanine (m7G) increased linearly with NDMA dose. O (6)-Methylguanine (m6G) remained near background for NDMA doses up to approximately 1 ppm, beyond which its level, and corresponding mutations, rose steeply. An extended study was done with 5 ppm NDMA, in which adducts were measured at 3 and 10 weeks and mutations at 10 weeks. We found that both sexes experienced elevated levels of point mutations closely tracking with the levels of m6G, which emerged as the dominant mutagenic adduct under chronic dosing with NDMA. Homologous recombination-mediated chromosomal rearrangements, however, did not increase over background. Mutational analysis over 96 trinucleotide contexts revealed predominantly GC-->AT mutations in 5'-purine-G-3' contexts in a pattern matching human COSMIC cancer mutational signature SBS11, with secondary features resembling SBS119 (AT-->GC). Moreover, we identified the m6G level ( approximately 2000 adducts/diploid genome) above which its dedicated repair protein, MGMT, became saturated. The coordinated application of DNA adduct, mutational, and biochemical analyses provides a new approach for studying mechanisms of carcinogenesis, with relevance to early cancer detection and cancer management.

Authors: N. E. Gubina, L. B. Volk, A. F. Dormitzer, E. M. Michelsen, L. J. Pribyl, J. J. Corrigan, E. D. Dalvie, A. L. Armijo, M. Norales, N. A. Bugher, K. M. Schonvisky, D. L. Plata, B. P. Engelward, R. G. Croy, J. M. Essigmann, B. I. Fedeles

Date Published: 15th Jul 2026

Publication Type: Journal

Excision of mutagenic replication-blocking lesions suppresses cancer but promotes cytotoxicity and lethality in nitrosamine-exposed mice.

Abstract (Expand)

N-Nitrosodimethylamine (NDMA) is a DNA-methylating agent that has been discovered to contaminate water, food, and drugs. The alkyladenine DNA glycosylase (AAG) removes methylated bases to initiate the … base excision repair (BER) pathway. To understand how gene-environment interactions impact disease susceptibility, we study Aag-knockout (Aag(-/-)) and Aag-overexpressing mice that harbor increased levels of either replication-blocking lesions (3-methyladenine [3MeA]) or strand breaks (BER intermediates), respectively. Remarkably, the disease outcome switches from cancer to lethality simply by changing AAG levels. To understand the underlying basis for this observation, we integrate a suite of molecular, cellular, and physiological analyses. We find that unrepaired 3MeA is somewhat toxic, but highly mutagenic (promoting cancer), whereas excess strand breaks are poorly mutagenic and highly toxic (suppressing cancer and promoting lethality). We demonstrate that the levels of a single DNA repair protein tip the balance between blocks and breaks and thus dictate the disease consequences of DNA damage.

Authors: J. E. Kay, J. J. Corrigan, A. L. Armijo, I. S. Nazari, I. N. Kohale, D. K. Torous, S. L. Avlasevich, R. G. Croy, D. N. Wadduwage, S. E. Carrasco, S. D. Dertinger, F. M. White, J. M. Essigmann, L. D. Samson, B. P. Engelward

Date Published: 16th Mar 2021

Publication Type: Journal

Interaction of N-nitrosamines with binuclear copper complexes for luminescent detection.

Abstract (Expand)

Cu(I) from tetrakis(acetonitrile)copper(I) hexafluorophosphate ([Cu(MeCN)(4)]PF(6)) was complexed with five structurally related phosphines containing N-heterocycles. The interactions between the … resulting complexes and some N-nitrosamines were studied using X-ray crystallography as well as emission spectroscopy. Upon complexation, three phosphine ligands bridge two Cu(I) centers to give paddlewheel type structures that displayed a range of emission wavelengths spanning the visible region. N-Nitrosodimethylamine (NDMA) was shown to coordinate to one of the two copper centers in some of the paddlewheel complexes in the solid state and this interaction also quenches their emissions in solution. The influence of the weakly coordinating anion on crystal and spectroscopic properties of one of the paddlewheel complexes was also examined using tetrakis(acetonitrile)copper(I) perchlorate ([Cu(MeCN)(4)]ClO(4)) as an alternative Cu(I) source. Similarly, copper(II) perchlorate hexahydrate (Cu(ClO(4))(2).6H(2)O) was used for complexation to observe the impact of metal oxidation state on the two aforementioned properties. Lastly, the spectroscopic properties of the complex between Ph(2)P(1-Isoquinoline) and Cu(I) was shown to exhibit solvent dependence when the counterion is ClO(4)(-). These Cu(I) complexes are bench stable solids and may be useful materials for developing a fluorescence based detection method for N-nitrosamines.

Authors: H. Feng, S. L. Luo, R. G. Croy, J. M. Essigmann, T. M. Swager

Date Published: 7th Mar 2023

Publication Type: Journal

Molecular origins of mutational spectra produced by the environmental carcinogen N-nitrosodimethylamine and S(N)1 chemotherapeutic agents.

Abstract (Expand)

DNA-methylating environmental carcinogens such as N-nitrosodimethylamine (NDMA) and certain alkylators used in chemotherapy form O (6)-methylguanine (m6G) as a functionally critical intermediate. NDMA … is a multi-organ carcinogen found in contaminated water, polluted air, preserved foods, tobacco products, and many pharmaceuticals. Only ten weeks after exposure to NDMA, neonatally-treated mice experienced elevated mutation frequencies in liver, lung and kidney of approximately 35-fold, 4-fold and 2-fold, respectively. High-resolution mutational spectra (HRMS) of liver and lung revealed distinctive patterns dominated by GC-->AT mutations in 5'-Pu-G-3' contexts, very similar to human COSMIC mutational signature SBS11. Commonly associated with alkylation damage, SBS11 appears in cancers treated with the DNA alkylator temozolomide (TMZ). When cells derived from the mice were treated with TMZ, N-methyl-N-nitrosourea, and streptozotocin (two other therapeutic methylating agents), all displayed NDMA-like HRMS, indicating mechanistically convergent mutational processes. The role of m6G in shaping the mutational spectrum of NDMA was probed by removing MGMT, the main cellular defense against m6G. MGMT-deficient mice displayed a strikingly enhanced mutant frequency, but identical HRMS, indicating that the mutational properties of these alkylators is likely owed to sequence-specific DNA binding. In sum, the HRMS of m6G-forming agents constitute an early-onset biomarker of exposure to DNA methylating carcinogens and drugs.

Authors: A. L. Armijo, P. Thongararm, B. I. Fedeles, J. Yau, J. E. Kay, J. J. Corrigan, M. Chancharoen, S. Chawanthayatham, L. D. Samson, S. E. Carrasco, B. P. Engelward, J. G. Fox, R. G. Croy, J. M. Essigmann

Date Published: 31st Mar 2023

Publication Type: Journal

Novel In Vivo CometChip Reveals NDMA-Induced DNA Damage and Repair in Multiple Mouse Tissues.

Abstract (Expand)

The comet assay is a versatile assay for detecting DNA damage in eukaryotic cells. The assay can measure the levels of various types of damage, including DNA strand breaks, abasic sites and alkali-sensitive … sites. Furthermore, the assay can also be modified to include purified DNA glycosylases so that alkylated and oxidized bases can be detected. The CometChip is a higher throughput version of the traditional comet assay and has been used to study cultured cells. Here, we have tested its utility for studies of DNA damage present in vivo. We show that the CometChip is effective in detecting DNA damage in multiple tissues of mice exposed to the direct-acting methylating agent methylmethane sulfonate (MMS) and to the metabolically activated methylating agent N-nitrosodimethylamine (NDMA), which has been found to contaminate food, water, and drugs. Specifically, results from MMS-exposed mice demonstrate that DNA damage can be detected in cells from liver, lung, kidney, pancreas, brain and spleen. Results with NDMA show that DNA damage is detectable in metabolically competent tissues (liver, lung, and kidney), and that DNA repair in vivo can be monitored over time. Additionally, it was found that DNA damage persists for many days after exposure. Furthermore, glycosylases were successfully incorporated into the assay to reveal the presence of damaged bases. Overall, this work demonstrates the efficacy of the in vivo CometChip and reveals new insights into the formation and repair of DNA damage caused by MMS and NDMA.

Authors: N. A. Owiti, J. J. Corrigan, L. J. Pribyl, J. E. Kay, B. P. Engelward

Date Published: 4th Oct 2022

Publication Type: Journal

Optical Detection of Interleukin-6 Using Liquid Janus Emulsions Using Hyperthermophilic Affinity Proteins.

Abstract (Expand)

When equal volumes of two immiscible liquids are mixed (e.g., a hydrocarbon and a fluorocarbon), Janus droplets can form in an aqueous solution. In a gravity-aligned Janus droplet, the boundary between … the two phases is flat and thus optically transparent when viewed from above. When tipped due to interactions with an analyte (i.e., agglutination), the resulting change in refraction and reflection yields an optical signal that can be detected and quantified. This study reports the detection and quantitation of interleukin-6 (IL-6) using emulsions functionalized at the hydrocarbon:aqueous interface with engineered proteins that bind IL-6 at high affinity and specificity. Hyperthermophilic affinity proteins (rcSso7d) are derived from thermophiles, giving them excellent thermal stability. Two rcSso7d affinity protein variants were synthesized with a noncanonical azide-functionalized amino acid to enable click chemistry to novel polymeric anchors embedded in the hydrocarbon phase. The two binding proteins recognize different epitopes, enabling the detection of both monomeric and dimeric IL-6 via agglutination. It is noteworthy that the rsSso7d protein variants, in addition to having superior thermal stability and facile recombinant synthesis in E. coli, show superior performance when compared to commercial antibodies for IL-6.

Authors: M. Chen, E. I. Corless, B. P. Engelward, T. M. Swager

Date Published: 3rd Sep 2024

Publication Type: Journal

Persistent interferon signaling and clonal expansion mark early events in DNA methylation damage-induced liver cancer.

Abstract (Expand)

N-Nitrosodimethylamine (NDMA), a probable human carcinogen, induces toxic and mutagenic O (6)-methylguanine lesions that are repaired by the O (6)-methylguanine methyltransferase (MGMT). To elucidate … mechanisms of NDMA-induced liver cancer progression, we performed longitudinal analyses of phenomic, transcriptomic, and phosphoproteomic changes in wild-type and MGMT-deficient mice, observing amplified responses in the deficient genotype. Early molecular rewiring indicative of a DNA damage response was detected by phosphoproteomic and transcriptomic profiling within days post-exposure. Transcriptomic analyses identified a persistent and robust interferon response as the dominant activated pathway. This chronic interferon signaling, which remained unresolved, correlated with extensive clonal expansion, an early hallmark of oncogenesis. Spatial transcriptomics further revealed pathway alterations favoring tumorigenesis within clonally expanded cells. These findings delineate the cascade of molecular events triggered by acute early-life NDMA exposure, culminating in cancer development months later. Our study unveils potential predictive biomarkers and strategies for disease mitigation.

Authors: L. J. Pribyl, J. E. Kay, J. J. Corrigan, L. B. Volk, M. Norales, N. A. Owiti, E. A. Kowal, I. N. Kohale, I. S. Nazari, M. R. Swanson, A. C. Moise, D. Ma, S. S. Levine, E. Michelsen, R. G. Croy, T. Ragan, D. K. Torous, S. L. Avlasevich, S. D. Dertinger, S. E. Carrasco, L. D. Samson, J. M. Essigmann, F. M. White, B. P. Engelward

Date Published: 3rd Oct 2025

Publication Type: Journal

Reversible Electrochemical Sensor for NDMA: Leveraging Molecularly Imprinted Polymers for Enhanced Sensitivity and Selectivity.

Abstract (Expand)

Herein, we present the development and evaluation of a molecularly imprinted polymer (MIP) sensor for the sensitive and selective detection of N-nitrosodimethylamine (NDMA) in aqueous environments. MIP … coatings over electrochemically active electrodes enable NDMA detection with a notably low detection limit of 1.16 ppb. Our findings demonstrate that the dual-monomer system employed in the MIP fabrication enhances both the selectivity and sensitivity toward NDMA. Additionally, the reversibility of the sensor was confirmed via a chronoamperometry regeneration process. Furthermore, the sensor's robustness was demonstrated across various water samples, as well as on different electrode materials, highlighting its potential for practical and reliable water quality monitoring applications.

Authors: Z. Guo, H. Feng, T. M. Swager

Date Published: 28th Feb 2025

Publication Type: Journal

Chemo-mechanical diffusion waves explain collective dynamics of immune cell podosomes.

Abstract (Expand)

Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes … probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.

Authors: Z. Gong, K. van den Dries, R. A. Migueles-Ramirez, P. W. Wiseman, A. Cambi, V. B. Shenoy

Date Published: 22nd May 2023

Publication Type: Journal

Chromatin conformation, gene transcription, and nucleosome remodeling as an emergent system.

Abstract (Expand)

In single cells, variably sized nanoscale chromatin structures are observed, but it is unknown whether these form a cohesive framework that regulates RNA transcription. Here, we demonstrate that the … human genome is an emergent, self-assembling, reinforcement learning system. Conformationally defined heterogeneous, nanoscopic packing domains form by the interplay of transcription, nucleosome remodeling, and loop extrusion. We show that packing domains are not topologically associated domains. Instead, packing domains exist across a structure-function life cycle that couples heterochromatin and transcription in situ, explaining how heterochromatin enzyme inhibition can produce a paradoxical decrease in transcription by destabilizing domain cores. Applied to development and aging, we show the pairing of heterochromatin and transcription at myogenic genes that could be disrupted by nuclear swelling. In sum, packing domains represent a foundation to explore the interactions of chromatin and transcription at the single-cell level in human health.

Authors: L. M. Almassalha, M. Carignano, E. P. Liwag, W. S. Li, R. Gong, N. Acosta, C. L. Dunton, P. C. Gonzalez, L. M. Carter, R. Kakkaramadam, M. Kroger, K. L. MacQuarrie, J. Frederick, I. C. Ye, P. Su, T. Kuo, K. I. Medina, J. A. Pritchard, A. Skol, R. Nap, M. Kanemaki, V. Dravid, I. Szleifer, V. Backman

Date Published: 10th Jan 2025

Publication Type: Journal

Confined cell migration along extracellular matrix space in vivo.

Abstract (Expand)

Collective migration of cancer cells is often interpreted using concepts derived from the physics of active matter, but the experimental evidence is mostly restricted to observations made in vitro. … Here, we study collective invasion of metastatic cancer cells injected into the mouse deep dermis using intravital multiphoton microscopy combined with a skin window technique and three-dimensional quantitative image analysis. We observe a multicellular but low-cohesive migration mode characterized by rotational patterns which self-organize into antiparallel persistent tracks with orientational nematic order. We analyze the deformations induced by the cells in the extracellular matrix and find broadly distributed strain bands with a prevalence of compression. A model of active nematic hydrodynamics is able to describe several statistical features of the experimentally observed flow, suggesting that collective cancer cell invasion can be interpreted as a nematic active fluid in the turbulent regime. Our results help elucidate the migration patterns of cancer cells in vivo and provide quantitative guidance for the development of realistic in vitro and in silico models for collective cell migration.

Authors: O. Chepizhko, J. M. Armengol-Collado, S. Alexander, E. Wagena, B. Weigelin, L. Giomi, P. Friedl, S. Zapperi, C. A. M. La Porta

Date Published: 7th Jan 2025

Publication Type: Journal

Impact of Fibrinogen, Fibrin Thrombi, and Thrombin on Cancer Cell Extravasation Using In Vitro Microvascular Networks.

Abstract (Expand)

A bidirectional association exists between metastatic dissemination and the hypercoagulable state associated with many types of cancer. As such, clinical studies have provided evidence that markers … associated with elevated levels of coagulation and fibrinolysis correlate with decreased patient survival. However, elucidating the mechanisms underpinning the effects of different components of the coagulation system on metastasis formation is challenging both in animal models and 2D models lacking the complex cellular interactions necessary to model both thrombosis and metastasis. Here, an in vitro, 3D, microvascular model for observing the formation of fibrin thrombi is described, which is in turn used to study how different aspects of the hypercoagulable state associated with cancer affect the endothelium. Using this platform, cancer cells expressing ICAM-1 are shown to form a fibrinogen-dependent bridge and transmigrate through the endothelium more effectively. Cancer cells are also demonstrated to interact with fibrin thrombi, using them to adhere, spread, and enhance their extravasation efficiency. Finally, thrombin is also shown to enhance cancer cell extravasation. This system presents a physiologically relevant model of fibrin clot formation in the human microvasculature, enabling in-depth investigation of the cellular interactions between cancer cells and the coagulation system affecting cancer cell extravasation.

Authors: E. Angelidakis, S. Chen, S. Zhang, Z. Wan, R. D. Kamm, S. E. Shelton

Date Published: 28th Jul 2023

Publication Type: Journal

Myofibroblasts reduce angiogenesis and vasculogenesis in a vascularized microphysiological model of lung fibrosis.

Abstract (Expand)

Lung fibrosis, characterized by chronic and progressive scarring, has no cure. Hallmarks are the accumulation of myofibroblasts and extracellular matrix, as well as vascular remodeling. The crosstalk … between myofibroblasts and vasculature is poorly understood, with conflicting reports on whether angiogenesis and vessel density are increased or decreased in lung fibrosis. We developed a microphysiological system that recapitulates the pathophysiology of lung fibrosis and disentangles myofibroblast-vascular interactions. Lung myofibroblasts maintained their phenotype in 3D without exogenous TGF-beta and displayed anti-angiogenic and anti-vasculogenic activities when cultured with endothelial cells in a microfluidic device. These effects, including decreased endothelial sprouting, altered vascular morphology, and increased vascular permeability, were mediated by increased TGF-beta1 and reduced VEGF secretion. Pharmacological interventions targeting these cytokines restored vascular morphology and permeability, demonstrating the potential of this model to screen anti-fibrotic drugs. This system provides insights into myofibroblast-vascular crosstalk in lung fibrosis and offers a platform for therapeutic development.

Authors: E. Cambria, A. Blazeski, E. C. Ko, T. Thai, S. Dantes, D. A. Barbie, S. E. Shelton, R. D. Kamm

Date Published: 14th Jan 2025

Publication Type: Journal

Patient-specific vascularized tumor model: Blocking monocyte recruitment with multispecific antibodies targeting CCR2 and CSF-1R.

Abstract (Expand)

Tumor-associated inflammation drives cancer progression and therapy resistance, often linked to the infiltration of monocyte-derived tumor-associated macrophages (TAMs), which are associated with poor … prognosis in various cancers. To advance immunotherapies, testing on immunocompetent pre-clinical models of human tissue is crucial. We have developed an in vitro model of microvascular networks with tumor spheroids or patient tissues to assess monocyte trafficking into tumors and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via CCL7 and CCL2, mediated by CSF-1R. Additionally, a multispecific antibody targeting CSF-1R, CCR2, and neutralizing TGF-beta (CSF1R/CCR2/TGF-beta Ab) repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and blocks monocyte migration. This antibody also inhibits monocyte recruitment in patient-specific vascularized tumor models. In summary, this vascularized tumor model recapitulates the monocyte recruitment cascade, enabling functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment.

Authors: H. T. Nguyen, E. L. Kan, M. Humayun, N. Gurvich, G. S. Offeddu, Z. Wan, M. F. Coughlin, D. C. Renteria, A. Loew, S. Wilson, C. Zhang, V. Vu, S. W. L. Lee, S. L. Tan, D. Barbie, J. Hsu, M. R. Gillrie, R. D. Kamm

Date Published: 3rd Sep 2024

Publication Type: Journal

Personalized Vascularized Models of Breast Cancer Desmoplasia Reveal Biomechanical Determinants of Drug Delivery to the Tumor.

Abstract (Expand)

Desmoplasia in breast cancer leads to heterogeneity in physical properties of the tissue, resulting in disparities in drug delivery and treatment efficacy among patients, thus contributing to high … disease mortality. Personalized in vitro breast cancer models hold great promise for high-throughput testing of therapeutic strategies to normalize the aberrant microenvironment in a patient-specific manner. Here, tumoroids assembled from breast cancer cell lines (MCF7, SKBR3, and MDA-MB-468) and patient-derived breast tumor cells (TCs) cultured in microphysiological systems including perfusable microvasculature reproduce key aspects of stromal and vascular dysfunction causing impaired drug delivery. Models containing SKBR3 and MDA-MB-468 tumoroids show higher stromal hyaluronic acid (HA) deposition, vascular permeability, interstitial fluid pressure (IFP), and degradation of vascular HA relative to models containing MCF7 tumoroids or models without tumoroids. Interleukin 8 (IL8) secretion is found responsible for vascular dysfunction and loss of vascular HA. Interventions targeting IL8 or stromal HA normalize vascular permeability, perfusion, and IFP, and ultimately enhance drug delivery and TC death in response to perfusion with trastuzumab and cetuximab. Similar responses are observed in patient-derived models. These microphysiological systems can thus be personalized by using patient-derived cells and can be applied to discover new molecular therapies for the normalization of the tumor microenvironment.

Authors: G. S. Offeddu, E. Cambria, S. E. Shelton, K. Haase, Z. Wan, L. Possenti, H. T. Nguyen, M. R. Gillrie, D. Hickman, C. G. Knutson, R. D. Kamm

Date Published: 16th Oct 2024

Publication Type: Journal

Polyploidy of MDA-MB-231 cells drives increased extravasation with enhanced cell-matrix adhesion.

Abstract (Expand)

Metastasis, the leading cause of cancer-related deaths, involves a complex cascade of events, including extravasation. Despite extensive research into metastasis, the mechanisms underlying extravasation … remain unclear. Molecular targeted therapies have advanced cancer treatment, yet their efficacy is limited, prompting exploration into novel therapeutic targets. Here, we showed the association of polyploidy in MDA-MB-231 breast cancer cells and their extravasation, using microfluidic systems to reproduce the in vivo microvascular environment. We observed enhanced extravasation in polyploid cells alongside upregulated expression of genes involved in cell-substrate adhesion and cell mechanical dynamics. These findings offer insights into the relationship between polyploidy and extravasation, highlighting potential targets for cancer therapy.

Authors: S. Hirose, T. Osaki, R. D. Kamm

Date Published: 2nd Jul 2024

Publication Type: Journal

Remodeling of self-assembled microvascular networks under long term flow.

Abstract (Expand)

The incorporation of a functional perfusable microvascular network (MVN) is a common requirement for most organ on-chip-models. Long-term perfusion of MVNs is often required for the maturation of organ … phenotypes and disease pathologies and to model the transport of cells and drugs entering organs. In our microphysiological system, we observe that flow can recover perfusion in regressed MVNs and maintain perfusable MVNs for at least 51 days. Throughout the 51 days, however, the MVNs are continuously remodeling to align with the direction of bulk flow and only appear to attain morphological homeostasis with the use of maintenance medium without growth factors. We observed that the flow resistance of the MVNs decreases over time, and using a computational model, we show that stable vessels have higher flow rates and velocities compared to regressing vessels. Cytokine analysis suggests that static conditions generate an inflammatory state, and that continuous flow reduces inflammation over an extended period. Finally, through bulk RNA sequencing we identify that both the endothelial and fibroblast cells are actively engaged in vascular and matrix remodeling due to flow and that these effects persist for at least 2 weeks. This MPS can be applied to study hemodynamically driven processes, such as metastatic dissemination or drug distribution, or to model long-term diseases previously not captured by MPS, such as chronic inflammation or aging-associated diseases.

Authors: M. Floryan, E. Cambria, A. Blazeski, M. F. Coughlin, Z. Wan, G. Offeddu, V. Vinayak, A. Kant, V. Shenoy, R. D. Kamm

Date Published: 18th Mar 2025

Publication Type: Journal

Utilizing convolutional neural networks for discriminating cancer and stromal cells in three-dimensional cell culture images with nuclei counterstain.

Abstract (Expand)

SIGNIFICANCE: Accurate cell segmentation and classification in three-dimensional (3D) images are vital for studying live cell behavior and drug responses in 3D tissue culture. Evaluating diverse cell … populations in 3D cell culture over time necessitates non-toxic staining methods, as specific fluorescent tags may not be suitable, and immunofluorescence staining can be cytotoxic for prolonged live cell cultures. AIM: We aim to perform machine learning-based cell classification within a live heterogeneous cell culture population grown in a 3D tissue culture relying only on reflectance, transmittance, and nuclei counterstained images obtained by confocal microscopy. APPROACH: In this study, we employed a supervised convolutional neural network (CNN) to classify tumor cells and fibroblasts within 3D-grown spheroids. These cells are first segmented using the marker-controlled watershed image processing method. Training data included nuclei counterstaining, reflectance, and transmitted light images, with stained fibroblast and tumor cells as ground-truth labels. RESULTS: Our results demonstrate the successful marker-controlled watershed segmentation of 84% of spheroid cells into single cells. We achieved a median accuracy of 67% (95% confidence interval of the median is 65-71%) in identifying cell types. We also recapitulate the original 3D images using the CNN-classified cells to visualize the original 3D-stained image's cell distribution. CONCLUSION: This study introduces a non-invasive toxicity-free approach to 3D cell culture evaluation, combining machine learning with confocal microscopy, opening avenues for advanced cell studies.

Authors: H. T. Nguyen, N. Pietraszek, S. E. Shelton, K. Arthur, R. D. Kamm

Date Published: 26th Aug 2024

Publication Type: Journal

Organoid co-culture model of the human endometrium in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk.

Abstract (Expand)

BACKGROUND: The human endometrium undergoes recurring cycles of growth, differentiation, and breakdown in response to sex hormones. Dysregulation of epithelial-stromal communication during hormone-mediated … signaling may be linked to myriad gynecological disorders for which treatments remain inadequate. Here, we describe a completely defined, synthetic extracellular matrix that enables co-culture of human endometrial epithelial and stromal cells in a manner that captures healthy and disease states across a simulated menstrual cycle. METHODS: We parsed cycle-dependent endometrial integrin expression and matrix composition to define candidate cell-matrix interaction cues for inclusion in a polyethylene glycol (PEG)-based hydrogel crosslinked with matrix metalloproteinase-labile peptides. We semi-empirically screened a parameter space of biophysical and molecular features representative of the endometrium to define compositions suitable for hormone-driven expansion and differentiation of epithelial organoids, stromal cells, and co-cultures of the two cell types. FINDINGS: Each cell type exhibited characteristic morphological and molecular responses to hormone changes when co-encapsulated in hydrogels tuned to a stiffness regime similar to the native tissue and functionalized with a collagen-derived adhesion peptide (GFOGER) and a fibronectin-derived peptide (PHSRN-K-RGD). Analysis of cell-cell crosstalk during interleukin 1B (IL1B)-induced inflammation revealed dysregulation of epithelial proliferation mediated by stromal cells. CONCLUSIONS: Altogether, we demonstrate the development of a fully synthetic matrix to sustain the dynamic changes of the endometrial microenvironment and support its applications to understand menstrual health and endometriotic diseases. FUNDING: This work was supported by The John and Karine Begg Foundation, the Manton Foundation, and NIH U01 (EB029132).

Authors: J. S. Gnecco, A. Brown, K. Buttrey, C. Ives, B. A. Goods, L. Baugh, V. Hernandez-Gordillo, M. Loring, K. B. Isaacson, L. G. Griffith

Date Published: 11th Aug 2023

Publication Type: Journal

Structural and systems characterization of phosphorylation on metabolic enzymes identifies sex-specific metabolic reprogramming in obesity.

Abstract (Expand)

Coordination of adaptive metabolism through signaling networks is essential for cellular bioenergetics and homeostasis. Phosphorylation of metabolic enzymes provides a rapid, efficient, and dynamic … mechanism to regulate metabolic networks. Our structural analysis stratified phosphosites on metabolic enzymes based on proximity to functional and dimerization domains. Most phosphosites occur on oxidoreductases and are enriched near substrate, cofactor, active sites, or dimer interfaces. Despite low stoichiometry, phosphotyrosine (pY) is overrepresented in functional domains. Using high-fat diet (HFD)-induced obesity in C57BL/6J mice and multiomics, we measured HFD-induced sex-specific dysregulation of pY and metabolites, which was reversible with the antioxidant butylated hydroxyanisole (BHA). Computational modeling revealed predictive pY sites for HFD- or BHA-induced metabolite changes. We characterized functional roles for predictive pY sites on glutathione S-transferase pi 1 (GSTP1), isocitrate dehydrogenase 1 (IDH1), and uridine monophosphate synthase (UMPS) using CRISPR interference (CRISPRi) rescue and stable isotope tracing. Our findings reveal mechanisms whereby cellular signaling fine-tunes enzyme activity and metabolism.

Authors: T. Y. Tamir, S. Chaudhary, A. X. Li, S. E. Trojan, C. T. Flower, P. Vo, Y. Cui, J. C. Davis, R. Mukkamala, F. N. Venditti, A. L. Hillis, A. Toker, M. G. Vander Heiden, J. B. Spinelli, N. J. Kennedy, R. J. Davis, F. M. White

Date Published: 5th Jun 2025

Publication Type: Journal

Validation and quantification of peptide antigens presented on MHCs using SureQuant.

Abstract (Expand)

Vaccines and immunotherapies that target peptide-major histocompatibility complexes (peptide-MHCs) have the potential to address multiple unmet medical needs in cancer and infectious disease. Designing … vaccines and immunotherapies to target peptide-MHCs requires accurate identification of target peptides in infected or cancerous cells or tissue, and may require absolute or relative quantification to identify abundant targets and measure changes in presentation under different treatment conditions. Internal standard parallel reaction monitoring (also known as 'SureQuant') can be used to validate and/or quantify MHC peptides previously identified by using untargeted methods such as data-dependent acquisition. SureQuant MHC has three main use cases: (i) conclusive confirmation of the identities of putative MHC peptides via comparison with an internal synthetic stable isotope labeled (SIL) peptide standard; (ii) accurate relative quantification by using pre-formed heavy isotope-labeled peptide-MHC complexes (hipMHCs) containing SIL peptides as internal controls for technical variation; and (iii) absolute quantification of each target peptide by using different amounts of hipMHCs loaded with synthetic peptides containing one, two or three SIL amino acids to provide an internal standard curve. Absolute quantification can help determine whether the abundance of a peptide-MHC is sufficient for certain therapeutic modalities. SureQuant MHC therefore provides unique advantages for immunologists seeking to confidently validate antigenic targets and understand the dynamics of the MHC repertoire. After synthetic standards are ordered (3-4 weeks), this protocol can be carried out in 3-4 days and is suitable for individuals with mass spectrometry experience who are comfortable with customizing instrument methods.

Authors: O. Leddy, Y. Cui, R. Ahn, L. Stopfer, E. Choe, D. H. Kim, M. Roerden, S. Spranger, B. D. Bryson, F. M. White

Date Published: 13th May 2025

Publication Type: Journal

Failed cellular surveillance enables pathogenic matrix deposition in a COL2A1-related osteoarthritis.

Abstract (Expand)

Mutations in the COL2A1 gene, encoding procollagen-II, cause various chondrodysplasias, including precocious osteoarthritis with mild spondyloepiphyseal dysplasia engendered by the p.Arg719Cys substitution. … The molecular mechanisms underlying these disorders remain incompletely understood, largely owing to the absence of models faithfully recapitulating the human disease. Here, we developed an in vitro human cartilage model using isogenic induced pluripotent stem cell (iPSC) lines carrying either wild-type or Arg719Cys COL2A1. Directed differentiation into chondrocytes yielded cartilage tissues that were analyzed by immunohistochemistry, electron microscopy, SDS-PAGE, and RNA-sequencing. Tissues derived from Arg719Cys heterozygotes displayed a deficient matrix, closely reflecting the human disease phenotype. Arg719Cys procollagen-II was excessively post-translationally modified and partially retained within the endoplasmic reticulum (ER), leading to ER distention. Notably, despite introduction of an aberrant cysteine residue-expected to engage redox-sensitive folding and quality control pathways-Arg719Cys procollagen-II was not detectably recognized by the ER proteostasis network. The resulting inability to mount a quality control response, including absent activation of the unfolded protein response, indicates a failure in cellular surveillance. As a result, malformed procollagen-II both accumulates intracellularly and is secreted, contributing to the deposition of a structurally compromised extracellular matrix that drives disease pathology. The iPSC-derived cartilage model presented here provides a genetically defined, expandable, and human-based system for dissecting mechanisms of failed proteostasis in collagenopathies. These findings shed light on the types of substitutions in procollagen that cells do or do not recognize, and underscore the therapeutic potential of targeting cellular surveillance and collagen quality control pathways in COL2A1-related disorders and beyond.

Authors: K. M. Yammine, S. Mirda Abularach, M. Xiong, S. Y. Kim, A. A. Bikovtseva, V. L. Butty, R. P. Schiavoni, J. F. Bateman, S. R. Lamande, M. D. Shoulders

Date Published: 3rd Sep 2025

Publication Type: Journal

Tyrosine phosphoproteome profiling identifies cell-intrinsic signals limiting the efficacy of tyrosine kinase inhibitor therapies.

Abstract (Expand)

Tyrosine kinases (TKs) are frequently mutated or overexpressed in cancer, and TK inhibitors (TKIs) are an important therapeutic modality against TK-driven cancers, but many patients show an underwhelming … response to TKIs prescribed on the basis of tumor genotype. To find cell-intrinsic TK signaling patterns which might be predictive of poor response to TKI therapies, we used high-sensitivity multiplexed mass spectrometry to quantify endogenous levels of 1,222 phosphotyrosine (pY) sites across the proteomes of TK-driven human cancer cell lines with variable response to genotype-matched TKIs. In direct comparisons between TKI-tolerant and TKI-sensitive lines with a common driver TK, we found that TKI treatment was equally effective at blocking driver TK signaling, and higher basal activity of the driver TK did not always predict higher sensitivity to TKI. All tolerant lines showed a dampened proteome-wide pY response to TKI exposure compared to sensitive lines, suggesting that tumor cells with more robust TK signaling are less vulnerable to driver TK blockade. We found that each tolerant line depends on a unique set of compensatory TKs and signaling axes but are unified by hyperactivity of at least one of the SRC family kinases (SFKs) or the related ABL1/2 kinases, both at rest and under TKI treatment, despite the absence of SFK or ABL genetic mutations. In time- and dose-resolved drug combination experiments, SFK/ABL inhibitors were potently synergistic with all TKIs tested, demonstrating that elevated SFK/ABL signaling is a conserved bottleneck for maximal TKI efficacy which could be exploited therapeutically.

Authors: C. T. Flower, F. M. White

Date Published: 31st Mar 2026

Publication Type: Journal

Signaling and transcriptional dynamics underlying early adaptation to oncogenic BRAF inhibition.

Abstract (Expand)

A major contributor to poor sensitivity to anti-cancer kinase inhibitor therapy is drug-induced cellular adaptation, whereby remodeling of signaling and gene regulatory networks permits a drug-tolerant … phenotype. Here, we resolve the scale and kinetics of critical subcellular events following oncogenic kinase inhibition and preceding cell cycle re-entry, using mass spectrometry-based phosphoproteomics and RNA sequencing (RNA-seq) to monitor the dynamics of thousands of growth- and survival-related signals over the first minutes, hours, and days of oncogenic BRAF inhibition in human melanoma cells. We observed sustained inhibition of the BRAF-ERK axis, gradual downregulation of cell cycle signaling, and three distinct, reversible phase transitions toward quiescence. Statistical inference of kinetically defined regulatory modules revealed a dominant compensatory induction of SRC family kinase (SFK) signaling, promoted in part by excess reactive oxygen species, rendering cells sensitive to co-treatment with an SFK inhibitor in vitro and in vivo, underscoring the translational potential for assessing early drug-induced adaptive signaling. A record of this paper's transparent peer review process is included in the supplemental information.

Authors: C. T. Flower, C. Liu, H. Y. Chuang, X. Ye, H. Cheng, J. R. Heath, W. Wei, F. M. White

Date Published: 16th Apr 2025

Publication Type: Journal

Rewiring of cortical glucose metabolism fuels human brain cancer growth.

Abstract (Expand)

The brain avidly consumes glucose to fuel neurophysiology(1). Cancers of the brain, such as glioblastoma, relinquish physiological integrity and gain the ability to proliferate and invade healthy … tissue(2). How brain cancers rewire glucose use to drive aggressive growth remains unclear. Here we infused (13)C-labelled glucose into patients and mice with brain cancer, coupled with quantitative metabolic flux analysis, to map the fates of glucose-derived carbon in tumour versus cortex. Through direct and comprehensive measurements of carbon and nitrogen labelling in both cortex and glioma tissues, we identify profound metabolic transformations. In the human cortex, glucose carbons fuel essential physiological processes, including tricarboxylic acid cycle oxidation and neurotransmitter synthesis. Conversely, gliomas downregulate these processes and scavenge alternative carbon sources such as amino acids from the environment, repurposing glucose-derived carbons to generate molecules needed for proliferation and invasion. Targeting this metabolic rewiring in mice through dietary amino acid modulation selectively alters glioblastoma metabolism, slows tumour growth and augments the efficacy of standard-of-care treatments. These findings illuminate how aggressive brain tumours exploit glucose to suppress normal physiological activity in favour of malignant expansion and offer potential therapeutic strategies to enhance treatment outcomes.

Authors: A. J. Scott, A. Mittal, B. Meghdadi, A. O'Brien, J. Bailleul, P. Sravya, A. Achreja, W. Zhou, J. Xu, A. Lin, K. Wilder-Romans, N. Liang, A. U. Kothari, N. Korimerla, D. M. Edwards, Z. Wu, J. Feng, S. Su, L. Zhang, P. Sajjakulnukit, A. C. Andren, J. O. Park, J. Ten Hoeve, V. Tarnal, K. A. Redic, N. R. Qi, J. L. Fischer, E. Yang, M. S. Regan, S. A. Stopka, G. Baquer, K. Suresh, J. N. Sarkaria, T. S. Lawrence, S. Venneti, N. Y. R. Agar, E. Vlashi, C. A. Lyssiotis, W. N. Al-Holou, D. Nagrath, D. R. Wahl

Date Published: 9th Oct 2025

Publication Type: Journal

Quantitative Cell Type-Specific Immunopeptidome Analysis of Macrophage and Tumor Coevolution Reveals Therapeutic MHC-I Peptides in Glioblastoma.

Abstract (Expand)

Immune checkpoint inhibitors have shown impressive performance in treating several types of solid tumors. However, they have been ineffective in glioblastoma (GBM), in part due to the immunosuppressive … tumor microenvironment created by GBM-associated macrophages (GAM). To uncover MHC-I peptide antigens for targeted immunotherapy, we performed cell type-specific immunopeptidome analysis on primary macrophages and GBM tumor cells in a coculture system to profile MHC-I-associated antigen presentation at the tumor-macrophage interface. Coculturing tumor cells and macrophages induced increased presentation of peptides derived from proteins associated with cytokine signaling pathways on macrophages and from proteins associated with the Rho GTPase pathway on GBM tumor cells. In vivo expression was validated for a cohort of coculture-induced GAMs or GBM-associated peptides selected as potential immunotherapy targets, and an mRNA vaccine was developed encoding six peptides from GAMs and GBM tumor cells. Two doses of vaccination generated an antigen-specific immune response, significantly delayed GBM tumor growth, and in some cases eradicated tumors. These results demonstrate the translational potential of coculture-induced MHC peptide antigens as therapeutic targets for GBM/GAM-targeting vaccines. SIGNIFICANCE: Immunopeptidomic analysis identified altered expression of antigens during macrophage-tumor coevolution that could be targeted with an mRNA vaccine to significantly inhibit glioblastoma growth, revealing potential immunotherapeutic strategies for treating tumors.

Authors: Y. Cui, K. Phuong, N. S. Abdelfattah, H. M. Temple, L. Maiorino, B. J. Kim, J. Dye, K. K. H. Yu, S. Spranger, D. J. Irvine, F. M. White

Date Published: 15th Dec 2025

Publication Type: Journal

Immunopeptidomics can inform the design of mRNA vaccines for the delivery of Mycobacterium tuberculosis MHC class II antigens.

Abstract (Expand)

No currently licensed vaccine reliably prevents pulmonary tuberculosis (TB), a leading cause of infectious disease mortality. Developing effective new vaccines requires identifying which Mycobacterium … tuberculosis (Mtb) proteins are presented on major histocompatibility complex class II (MHC-II) by infected human phagocytes (target cells) and defining their capacity for recognition by CD4(+) T cells. Vaccine designs must elicit T cell responses recognizing the same peptide-MHC complexes presented by infected cells. Although many human CD4(+) T cell Mtb epitopes have been described, presentation on MHC-II by infected cells in most cases has not been directly evaluated. Using mass spectrometry (MS), we demonstrated that Mtb type VII secretion system (T7SS) substrates are enriched in the MHC-II repertoire of Mtb-infected human monocyte-derived phagocytes and that many of these antigens are immunogenic in people with prior evidence of Mtb infection. We next used MS to guide TB messenger RNA (mRNA) vaccine design, increasing the presentation of target MHC-II epitopes by orders of magnitude by incorporating design features that mirror aspects of antigen presentation dynamics in infected phagocytes. Our results provide a strategy for TB vaccine design that is guided by bottom-up unbiased discovery. Our approach combines targeted evaluation of antigen presentation in human cells paired with rapid iterative testing of mRNA vaccine designs to optimize antigen presentation before animal studies or human clinical trials.

Authors: O. Leddy, P. Ogongo, J. Huffaker, M. Gan, R. Milligan, S. Mahmud, H. M. Ni, Y. Yuki, K. Bobosha, L. Wassie, M. Carrington, Q. Liu, J. D. Ernst, F. M. White, B. D. Bryson

Date Published: 5th Nov 2025

Publication Type: Journal

Targeting infection-specific peptides in immunopeptidomics studies for vaccine target discovery.

Abstract (Expand)

Vaccine-elicited T cell responses can contribute to immune protection against emerging infectious disease risks such as antimicrobial-resistant (AMR) microbial pathogens and viruses with pandemic … potential, but rapidly identifying appropriate targets for T cell priming vaccines remains challenging. Mass spectrometry (MS) analysis of peptides presented on MHCs can identify potential targets for protective T cell responses in a proteome-wide manner. However, pathogen-derived peptides are outnumbered by self-peptides in the MHC repertoire and may be missed in untargeted MS analyses. Here, we present a novel approach, termed PathMHC, that uses computational analysis of untargeted MS data followed by targeted MS to discover novel pathogen-derived MHC peptides more efficiently than untargeted methods alone. We applied this workflow to identify MHC peptides derived from multiple microbes, including potential vaccine targets presented on MHC-I by human dendritic cells infected with Mycobacterium tuberculosis (Mtb), finding that all Mtb peptides detected in the MHC-I repertoire derived from proteins exported by type VII secretion systems. PathMHC will facilitate antigen discovery campaigns for vaccine development.

Authors: O. Leddy, Y. Yuki, M. Carrington, B. D. Bryson, F. M. White

Date Published: 6th Oct 2025

Publication Type: Journal

Interstitial flow promotes the formation of functional microvascular networks in vitro through upregulation of matrix metalloproteinase-2.

Abstract (Expand)

Self-organized microvascular networks (MVNs) have become key to the development of many microphysiological models. However, the self-organizing nature of this process combined with variations between … types or batches of endothelial cells (ECs) often lead to inconsistency or failure to form functional MVNs. Since interstitial flow (IF) has been reported to play a beneficial role in angiogenesis, vasculogenesis, and 3D capillary morphogenesis, we systematically investigated the role IF plays during neovessel formation in a customized single channel microfluidic chip for which IF has been fully characterized. Compared to static conditions, MVNs formed under IF have higher vessel density and diameters and greater network perfusability. Through a series of inhibitory experiments, we demonstrated that IF treatment improves vasculogenesis by ECs through upregulation of matrix metalloproteinase-2 (MMP-2). We then successfully implemented a novel strategy involving the interplay between IF and MMP-2 inhibitor to regulate morphological parameters of the self-organized MVNs, with vascular permeability and perfusability well maintained. The revealed mechanism and proposed methodology were further validated with a brain MVN model. Our findings and methods have the potential to be widely utilized to boost the development of various organotypic MVNs and could be incorporated into related bioengineering applications where perfusable vasculature is desired.

Authors: S. Zhang, Z. Wan, G. Pavlou, A. X. Zhong, L. Xu, R. D. Kamm

Date Published: 21st Oct 2022

Publication Type: Journal

Wnt signaling modulates mechanotransduction in the epidermis to drive hair follicle regeneration.

Abstract (Expand)

Most wounds form scars without hair follicles. However, in the wound-induced hair neogenesis (WIHN) model of skin regeneration, wounds regenerate hair follicles if tissue rigidity is optimal. Although … WIHN depends on Wnt signaling, whether Wnt performs a mechanoregulatory role that contributes to regeneration remains uncharacterized. Here, we demonstrate that Wnt signaling affects mechanosensitivity at both cellular and tissue levels to drive WIHN. Atomic force microscopy revealed an attenuated substrate rigidity response in epidermal but not dermal cells of healing wounds. Super-resolution microscopy and nanoneedle probing of intracellular compartments in live human keratinocytes revealed that Wnt-induced chromatin remodeling triggers a 10-fold drop in nuclear rigidity without jeopardizing the nucleocytoskeletal mechanical coupling. Mechanistically, Wnt signaling orchestrated a massive reorganization of actin architecture and recruited adherens junctions to generate a mechanical syncytium-a cohesive contractile unit with superior capacity for force coordination and collective durotaxis. Collectively, our findings unveil Wnt signaling's mechanoregulatory role that manipulates the machinery of mechanotransduction to drive regeneration.

Authors: A. S. W. Oak, A. Bagchi, M. J. Brukman, J. Toth, J. Ford, Y. Zheng, A. Nace, R. Yang, J. C. Hsieh, J. E. Hayden, G. Ruthel, A. Ray, E. Kim, V. B. Shenoy, G. Cotsarelis

Date Published: 21st Feb 2025

Publication Type: Journal

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