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1. Ultra-rapid somatic variant detection via real-time targeted amplicon sequencing

   https://doi.org/10.1038/s42003-022-03657-6  

   Wadden, Jack; Newell, Brandon S.; Bugbee, Joshua; John, Vishal; Bruzek, Amy K.; Dickson, Robert P.; Koschmann, Carl; Blaauw, David; Narayanasamy, Satish; Das, Reetuparna (July 2022, Communications Biology)

   Abstract Molecular markers are essential for cancer diagnosis, clinical trial enrollment, and some surgical decision making, motivating ultra-rapid, intraoperative variant detection. Sequencing-based detection is considered the gold standard approach, but typically takes hours to perform due to time-consuming DNA extraction, targeted amplification, and library preparation times. In this work, we present a proof-of-principle approach for sub-1 hour targeted variant detection using real-time DNA sequencers. By modifying existing protocols, optimizing for diagnostic time-to-result, we demonstrate confirmation of a hot-spot mutation from tumor tissue in ~52 minutes. To further reduce time, we explore rapid, targeted Loop-mediated Isothermal Amplification (LAMP) and design a bioinformatics tool—LAMPrey—to process sequenced LAMP product. LAMPrey’s concatemer aware alignment algorithm is designed to maximize recovery of diagnostically relevant information leading to a more rapid detection versus standard read alignment approaches. Using LAMPrey, we demonstrate confirmation of a hot-spot mutation (250x support) from tumor tissue in less than 30 minutes. 

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2. In Situ Imaging of O-Linked β-N-Acetylglucosamine Using On-Tissue Hydrolysis and MALDI Mass Spectrometry

   https://doi.org/10.3390/cancers15041224  

   Escobar, Edwin E.; Seeley, Erin H.; Serrano-Negrón, Jesús E.; Vocadlo, David J.; Brodbelt, Jennifer S. (February 2023, Cancers)

   Post-translational O-glycosylation of proteins via the addition of N-acetylglucosamine (O-GlcNAc) is a regulator of many aspects of cellular physiology. Processes driven by perturbed dynamics of O-GlcNAcylation modification have been implicated in cancer development. Variability in O-GlcNAcylation is emerging as a metabolic biomarker of many cancers. Here, we evaluate the use of MALDI-mass spectrometry imaging (MSI) to visualize the location of O-GlcNAcylated proteins in tissue sections by mapping GlcNAc that has been released by the enzymatic hydrolysis of glycoproteins using an O-GlcNAc hydrolase. We use this strategy to monitor O-GlcNAc within hepatic VX2 tumor tissue. We show that increased O-GlcNAc is found within both viable tumor and tumor margin regions, implicating GlcNAc in tumor progression. 

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3. Micromechanical remodeling of the extracellular matrix by invading tumors: anisotropy and heterogeneity

   https://doi.org/10.1039/D2SM01100J  

   Naylor, Austin; Zheng, Yu; Jiao, Yang; Sun, Bo (December 2022, Soft Matter)

   Altered tissue mechanics is an important signature of invasive solid tumors. While the phenomena have been extensively studied by measuring the bulk rheology of the extracellular matrix (ECM) surrounding tumors, micromechanical remodeling at the cellular scale remains poorly understood. By combining holographic optical tweezers and confocal microscopy on in vitro tumor models, we show that the micromechanics of collagen ECM surrounding an invading tumor demonstrate directional anisotropy, spatial heterogeneity and significant variations in time as tumors invade. To test the cellular mechanisms of ECM micromechanical remodeling, we construct a simple computational model and verify its predictions with experiments. We find that collective force generation of a tumor stiffens the ECM and leads to anisotropic local mechanics such that the extension direction is more rigid than the compression direction. ECM degradation by cell-secreted matrix metalloproteinase softens the ECM, and active traction forces from individual disseminated cells re-stiffen the matrix. Together, these results identify plausible biophysical mechanisms responsible for the remodeled ECM micromechanics surrounding an invading tumor. 

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4. Engineered anti-prostate cancer CAR-neutrophils from human pluripotent stem cells

   https://doi.org/10.1016/j.regen.2023.100074  

   Harris, Jackson D.; Chang, Yun; Syahirah, Ramizah; Lian, Xiaojun Lance; Deng, Qing; Bao, Xiaoping (May 2023, Journal of Immunology and Regenerative Medicine)

   Immunotherapy is a powerful technique where immune cells are modified to improve cytotoxicity against cancerous cells to treat cancers that do not respond to surgery, chemotherapy, or radiotherapy. Expressing chimeric antigen receptor (CAR) in immune cells, typically T lymphocytes, is a practical modification that drives an immune response against cancerous tissue. CAR-T efficacy is suboptimal in solid tumors due to the tumor microenvironment (TME) that limits T lymphocyte cytotoxicity. In this study, we demonstrate that neutrophils differentiated from human pluripotent stem cells modified with AAVS1-inserted CAR constructs showed a robust cytotoxic effect against prostate-specific membrane antigen (PSMA) expressing LNCaP cells as a model for prostate cancer in vitro. Our results suggest that engineered CAR can significantly enhance the neutrophil anti-tumor effect, providing a new avenue in treating prostate cancers. 

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5. A microphysiological system reveals neutrophil contact-dependent attenuation of pancreatic tumor progression by CXCR2 inhibition-based immunotherapy

   https://doi.org/10.1038/s41598-024-64780-4  

   Shao, Shuai; Delk, Nikki_A; Jones, Caroline_N (June 2024, Scientific Reports)

   Abstract Cancer cells recruit neutrophils from the bloodstream into the tumor tissue, where these immune cells promote the progression of numerous solid tumors. Studies in mice suggest that blocking neutrophil recruitment to tumors by inhibition of neutrophil chemokine receptor CXCR2 could be a potential immunotherapy for pancreatic cancer. Yet, the mechanisms by which neutrophils promote tumor progression in humans, as well as how CXCR2 inhibition could potentially serve as a cancer therapy, remain elusive. In this study, we developed a human cell-based microphysiological system to quantify neutrophil-tumor spheroid interactions in both “separated” and “contact” scenarios. We found that neutrophils promote the invasion of tumor spheroids through the secretion of soluble factors and direct contact with cancer cells. However, they promote the proliferation of tumor spheroids solely through direct contact. Interestingly, treatment with AZD-5069, a CXCR2 inhibitor, attenuates invasion and proliferation of tumor spheroids by blocking direct contact with neutrophils. Our findings also show that CXCR2 inhibition reduces neutrophil migration toward tumor spheroids. These results shed new light on the tumor-promoting mechanisms of human neutrophils and the tumor-suppressive mechanisms of CXCR2 inhibition in pancreatic cancer and may aid in the design and optimization of novel immunotherapeutic strategies based on neutrophils. 

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