Morphologic examination of tissue biopsies is essential for histopathological diagnosis. However, accurate and scalable cellular quantification in human samples remains challenging. Here, we present a deep learning-based approach for antigen-specific cellular morphometrics in human kidney biopsies, which combines indirect immunofluorescence imaging with U-Net-based architectures for image-to-image translation and dual segmentation tasks, achieving human-level accuracy. In the kidney, podocyte loss represents a hallmark of glomerular injury and can be estimated in diagnostic biopsies. Thus, we profiled over 27,000 podocytes from 110 human samples, including patients with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN), an immune-mediated disease with aggressive glomerular damage and irreversible loss of kidney function. We identified previously unknown morphometric signatures of podocyte depletion in patients with ANCA-GN, which allowed patient classification and, in combination with routine clinical tools, showed potential for risk stratification. Our approach enables robust and scalable molecular morphometric analysis of human tissues, yielding deeper biological insights into the human kidney pathophysiology.
Marina Zimmermann, Martin Klaus, Milagros N. Wong, Ann-Katrin Thebille, Lukas Gernhold, Christoph Kuppe, Maurice Halder, Jennifer Kranz, Nicola Wanner, Fabian Braun, Sonia Wulf, Thorsten Wiech, Ulf Panzer, Christian F. Krebs, Elion Hoxha, Rafael Kramann, Tobias B. Huber, Stefan Bonn, Victor G. Puelles
With the advent of cancer immunology, mass cytometry has been increasingly employed to characterize the responses to cancer therapies and the tumor microenvironment (TME). One of its most notable applications is efficient multiplexing of samples into batches by dedicating a number of metal isotope channels to barcodes, enabling robust data acquisition and analysis. Barcoding is most effective when markers are present in all cells of interest. While CD45 has been shown to be a reliable marker for barcoding all immune cells in a given sample, a strategy to reliably barcode mouse cancer cells has not been demonstrated. To this end, we identified CD29 and CD98 as markers widely expressed by commonly used mouse cancer cell lines. We conjugated anti-CD29 and anti-CD98 antibodies to cadmium or indium metals and validated their utility in 10-plex barcoding of live cells. Finally, we established a novel barcoding system incorporating the combination of CD29, CD98, and CD45 to multiplex ten tumors from subcutaneous MC38 and KPC tumor models, while successfully recapitulating the known contrast in the PD1-PDL1 axis between the two models. The ability to barcode tumor cells along with immune cells empowers the interrogation of the tumor-immune interactions in mouse TME studies.
Soren Charmsaz, Nicole Gross, Elizabeth Jaffee, Won Jin Ho
Complexity of lung microenvironment and changes in cellular composition during disease make it exceptionally hard to understand molecular mechanisms driving development of chronic lung diseases. Although recent advances in cell-type resolved approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows storage of lung tissue with high viability, permits thorough sample quality check before cell isolation, and befits sequencing-based profiling. We demonstrate that cryopreservation enables isolation of multiple cell types from both healthy and diseased lungs. Basal cells from cryopreserved airways retain their differentiation ability, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for -omics profiling of lung cells. Moreover, we obtained high-quality single-cell RNA sequencing data of cells from cryopreserved human lung, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our workflow is well-suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to viable human tissue.
Maria Llamazares Prada, Elisa Espinet, Vedrana Mijošek, Uwe Schwartz, Pavlo Lutsik, Raluca Tamas, Mandy Richter, Annika Behrendt, Stephanie T. Pohl, Naja P. Benz, Thomas Muley, Arne Warth, Claus Peter Heußel, Hauke Winter, Jonathan J. M. Landry, Felix J.F. Herth, Tinne C.J. Mertens, Harry Karmouty-Quintana, Ina Koch, Vladimir Benes, Jan O. Korbel, Sebastian M. Waszak, Andreas Trumpp, David M. Wyatt, Heiko F. Stahl, Christoph Plass, Renata Z. Jurkowska
To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole genome sequencing (WGS) from both double- and single-strand DNA library preparations (DSP and SSP), as well as using Q-PCR. The size profile in healthy individuals was remarkably homogenous when using either DSP sequencing (DSP-S) or SSP sequencing (SSP-S). Our findings also confirmed that cfDNA size profile shows a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes and chromatin of higher molecular size (>1,000bp) can be estimated as 67.5-80%, 9.4-11.5% and 8.5-21.0%, respectively. Thus, our data on WGS (N=7) and Q-PCR (N=116 taken together suggests that only a minor proportion of cfDNA is bigger than that existing in mono-nucleosome or transcription factor complexes circulating in blood. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97-98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between healthy metastatic colorectal cancer (mCRC) patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content. Size profile analysis, or ‘fragmentomics’, has shown significant potential to improve diagnostics and cancer screening.
Cynthia Sanchez, Benoit Roch, Thilbault Mazard, Philippe Blache, Zahra Al Amir Dache, Brice Pastor, Ekaterina Pisareva, Rita Tanos, Alain R. Thierry
Progress in our understanding of MR1-restricted Mucosa-associated Invariant T (MAIT) cells has raised an interest in harnessing these cells for immunotherapy. The innate-like response characteristics, abundance in the blood, donor-unrestricted nature, and tropism for tissues make MAIT cells suitable candidates for adoptive cell transfer therapies. However, reliable methods and tools to utilize MAIT cells in such approaches are lacking. Here, we established methodology for efficient expansion of human MAIT cells in culture with high purity and yield, preserved functional response toward their natural ligand, and with increased cytotoxic potential. The cultured MAIT cells retained their effector memory characteristics without signs of terminal differentiation, and expressed a more diverse set of chemokine receptors potentially widening their already broad tissue tropism. To investigate the potential of MAIT cells in a context outside their main role in controlling bacterial infection, we engineered cultured MAIT cells with a new TCR specificity to mediate effective antiviral HLA class I-restricted effector function. In summary, we developed robust and effective methodology for the expansion of human MAIT cells with enhanced cytolytic capacity, and for their engineering with a new specificity. These findings form a basis for the development of MAIT cells as a platform for adoptive immunotherapy.
Tiphaine Parrot, Katie Healy, Caroline Boulouis, Michał J. Sobkowiak, Edwin Leeansyah, Soo Aleman, Antonio Bertoletti, Margaret Sällberg Chen, Johan K. Sandberg
MC4R mutations represent the largest monogenic cause of obesity, resulting mainly from receptor misfolding and intracellular retention by the cellular quality control system. The present study aimed at determining whether pharmacological chaperones (PC) that restore folding and plasma membrane trafficking by stabilizing near native protein conformation, may represent valid therapeutic avenues for the treatment of melanocortin type 4 receptor (MC4R) linked obesity.To test the therapeutic PC potential, we engineered humanized MC4R mouse models expressing either the wild type (WT) human MC4R or a prevalent obesity-causing mutant (R165W). Administration of a PC able to rescue cell surface expression and functional activity of R165W-hMC4R in cells, restored the anorexigenic response of the R165W-hMC4R obese mice to melanocortin agonist, providing a proof-of-principle for the therapeutic potential of MC4R-targetting PC in vivo. Interestingly, the expression of the WT-hMC4R in mice revealed lower sensitivity of the human receptor to alpha-melanocyte-stimulating hormone (α-MSH) but not β-MSH or MTII, resulting in a lower penetrance obese phenotype in the WT-hMC4R versus R165W-hMC4R mice. In conclusion, we created two new obesity models, one hypomorph highlighting species differences, and one amorphic that provides a pre-clinical model to test the therapeutic potential of PC to treat MC4R-linked obesity.
Patricia René, Damien Lanfray, Denis Richard, Michel Bouvier
Recent in vivo tracer studies demonstrated that targeted mass spectrometry (MS) on the Q Exactive Orbitrap could determine the metabolism of HDL proteins 100s-fold less abundant than APOA1. In this study, we demonstrate that the Orbitrap Lumos can measure tracer in proteins whose abundances are 1000s-fold less than APOA1, specifically the lipid transfer proteins PLTP, CETP, and LCAT. Relative to the Q Exactive, the Lumos improved tracer detection by reducing tracer enrichment compression, thereby providing consistent enrichment data across multiple HDL sizes from six participants. We determined by compartmental modeling that PLTP is secreted in medium and large HDL (alpha2, 1, and 0), and is transferred from medium to larger sizes during circulation from where it is catabolized. CETP is secreted mainly in alpha1 and alpha2, and remains in these sizes during circulation. LCAT is secreted mainly in medium and small HDL (alpha2, 3, prebeta). Unlike PLTP and CETP, LCAT appearance on HDL is markedly delayed compared to APOA1 and the other transfer proteins, indicating that LCAT may reside for a time outside of systemic circulation before attaching to HDL in plasma. The determination of these lipid transfer proteins’ unique metabolic structures was possible due to advances in MS technologies.
Sasha A. Singh, Allison B. Andraski, Hideyuki Higashi, Lang Ho Lee, Ashisha Ramsaroop, Frank M. Sacks, Masanori Aikawa
Human lung adenocarcinoma (LUAD) in current or former smokers exhibits a high tumor mutational burden (TMB) and distinct mutational signatures. Syngeneic mouse models of clinically relevant smoking-related LUAD are lacking. We established and characterized a tobacco-associated transplantable murine LUAD cell line, designated FVBW-17, from a LUAD induced by the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in the FVB/N mouse strain. Whole exome sequencing of FVBW-17 cells identified tobacco-associated KrasG12D and Trp53 mutations and a similar mutation profile to that of classic alkylating agents with a TMB >500. FVBW-17 cells transplanted subcutaneously, via tail vein and orthotopically generated tumors in FVB/N mice that were histologically similar to human LUAD. FVBW-17 tumors expressed PD-L1, were infiltrated with CD8+ T cells, and responsive to anti-PD-L1 therapy. FVBW-17 cells were also engineered to express green fluorescent protein and luciferase to facilitate the detection and quantification of tumor growth. Distant metastases to lung, spleen, liver, and kidney were observed from subcutaneously transplanted tumors. This novel cell line is a robust representation of human smoking-related LUAD biology and provides a much needed pre-clinical model in which to test promising new agents and combinations including immune-based therapies.
Laura P. Stabile, Vinod Kumar, Autumn Gaither-Davis, Eric H.B. Huang, Frank P. Vendetti, Princey Devadassan, Sanja Dacic, Riyue Bao, Richard A. Steinman, Timothy F. Burns, Christopher J. Bakkenist
Adoptive cell therapy involves the infusion of tumor-reactive T cells into patients with cancer to provide antitumor immunity. The ex vivo expansion and differentiation of such T cells are key parameters that affect their therapeutic potential. Human T cells are presently expanded in culture through the use of anti-CD3 and anti-CD28 mAbs immobilized on beads, expressed on cells, or assembled in the context of soluble antibody complexes. Here we report the design of a small, bispecific single-chain variable fragment construct agonizing both CD3 and CD28 pathways. This soluble T cell expansion protein, termed T-CEP, activates, expands, and differentiates human T cells ex vivo at concentrations in the femtomolar range. Importantly, T-CEP promotes the preferential growth of human CD8+ T cells over the course of 12 days in comparison with methods involving immobilized anti-CD3 mAb/soluble anti-CD28 mAb or soluble anti-CD3/CD28 mAb complexes. The differentiation profile of the resulting human T cell population is also singularly affected by T-CEP, favoring the expansion of a preferred CD8+CD27+ T cell phenotype. The activity profile of T-CEP on human T cells ex vivo suggests its use in generating human T cell populations that are more suited for adoptive cell therapy.
Esther I. Matus, Amanda Sparkes, Jean Gariépy
The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass-spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV positive, 11 MCV negative) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV Large T antigen, CDKN2AIP, SERPINB5 and TRIM29) that discriminates MCV positive and negative MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be up-regulated in virus-infected samples. Remarkably, four (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify novel viral sequences in infectious tumors when nucleic acid-based methods are not feasible.
Tuna Toptan, Pamela S. Cantrell, Xuemei Zeng, Yang Liu, Mai Sun, Nathan A. Yates, Yuan Chang, Patrick S. Moore
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