3D imaging analysis on an organoid-based platform guides personalized treatment in pancreatic ductal adenocarcinoma

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Abstract

BACKGROUND. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with unpredictable responses to chemotherapy. Approaches to assay patient tumors before treatment and identify effective treatment regimens based on tumor sensitivities are lacking. We developed an organoid-based platform (OBP) to visually quantify patient derived organoid (PDO) responses to drug treatments and associated tumor-stromal modulation for personalized PDAC therapy. METHODS. We retrospectively quantified apoptotic responses and tumor-stromal cell proportions in patient-derived organoids (PDOs) via 3D immunofluorescence imaging through annexin A5, α-smooth muscle actin (α-SMA), and cytokeratin 19 (CK-19) levels. Simultaneously, an ex vivo organoid drug sensitivity assay (ODSA) was used to measure responses to standard of care (SOC) regimens. Differences between ODSA results and patient tumor responses were assessed by exact McNemar test. RESULTS. Immunofluorescent signals, organoid growth curves, and Ki-67 levels were measured and authenticated through the OBP for up to 14 days. ODSA drug responses were not different from patient tumor responses as reflected by CA19-9 reductions following neoadjuvant chemotherapy (P = 0.99). PDOs demonstrated unique apoptotic and tumor-stromal modulation profiles (P < 0.0001). α-SMA/CK-19 ratio levels > 1.0 were associated with improved outcomes (P = 0.0179), and longer parental patient survival by Kaplan-Meier analysis (P = 0.0046). CONCLUSION. Heterogenous apoptotic drug responses and tumor-stromal modulation are present in PDOs after SOC chemotherapy. Ratios of α-SMA and CK-19 levels in PDOs are associated with patient survival and the OBP could aid in the selection of personalized therapies to improve the efficacy of systemic therapy in PDAC patients.

Authors

Ya'an Kang, Jenying Deng, Jianhua Ling, Xinqun Li, Yi-Ju Chiang, Eugene J. Koay, Huamin Wang, Jared K. Burks, Paul J. Chiao, Mark W. Hurd, Manoop S. Bhutani, Jeffrey H. Lee, Brian R. Weston, Anirban Maitra, Naruhiko Ikoma, Ching-Wei D. Tzeng, Jeffrey E. Lee, Ronald A. DePinho, Robert A. Wolff, Shubham Pant, Florencia McAllister, Matthew H.G. Katz, Jason B. Fleming, Michael P. Kim

The Notch1/CD22 signaling axis disrupts Treg cell function in SARS-CoV2-associated multisystem inflammatory syndrome in children

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Abstract

Multisystem inflammatory syndrome in children (MIS-C) evolves in some pediatric patients following acute infection with SARS-CoV-2 by hitherto unknown mechanisms. Whereas acute-COVID-19 severity and outcome were previously correlated with Notch4 expression on regulatory T (Treg) cells, here we show that the Treg cells in MIS-C are destabilized through a Notch1-dependent mechanism. Genetic analysis revealed that MIS-C patients were enriched in rare deleterious variants impacting inflammation and autoimmunity pathways, including dominant-negative mutations in the Notch1 regulators NUMB and NUMBL leading to Notch1 upregulation. Notch1 signaling in Treg cells induced CD22, leading to their destabilization in a mTORC1-dependent manner and to the promotion of systemic inflammation. These results establish a Notch1-CD22 signaling axis that disrupts Treg cell function in MIS-C and point to distinct immune checkpoints controlled by individual Treg cell Notch receptors that shape the inflammatory outcome in SARS-CoV-2 infection.

Authors

Mehdi Benamar, Qian Chen, Janet Chou, Amélie M. Julé, Rafik Boudra, Paola Contini, Elena Crestani, Peggy S. Lai, Muyun Wang, Jason Fong, Shira Rockwitz, Pui Y. Lee, Tsz Man Fion Chan, Ekin Zeynep Altun, Eda Kepenekli, Elif Karakoc-Aydiner, Ahmet Ozen, Perran Boran, Fatih Aygun, Pınar Önal, Ayse Ayzit Kilinc Sakalli, Haluk Cokugras, Metin Yusuf Gelmez, Fatma B. Oktelik, Esin Cetin Aktas, Yuelin Zhong, Maria L. Taylor, Katherine Irby, Natasha B. Halasa, Elizabeth H. Mack, Sara Signa, Ignazia Prigione, Marco Gattorno, Nicola Cotugno, Donato Amodio, Raif S. Geha, Mary Beth Son, Jane W. Newburger, Pankaj B. Agrawal, Stefano Volpi, Paolo Palma, Ayca Kiykim, Adrienne Randolph, Gunnur Deniz, Safa Baris, Raffaele De Palma, Klaus Schmitz-Abe, Louis-Marie Charbonnier, Lauren A. Henderson, Talal A. Chatila

Trained immunity is induced in humans after immunization with an adenoviral vector COVID-19 vaccine

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Abstract

BACKGROUND. Heterologous effects of vaccines are mediated by ‘trained immunity’ whereby myeloid cells are metabolically and epigenetically reprogrammed resulting in heightened responses to subsequent insults. Adenovirus vaccine vector has been reported to induce trained immunity in mice. Therefore, we sought to determine if the ChAdOx1 nCoV-19 vaccine (AZD1222), which uses an adenoviral vector, could induce trained immunity in vivo in humans. METHODS. Ten healthy volunteers donated blood on the day before receiving the ChAdOx1 nCoV-19 vaccine and on day 14, 56 and 90 post vaccination. Monocytes were purified from PBMC; cell phenotype was determined by flow cytometry, expression of metabolic enzymes were quantified by RT-qPCR and production of cytokines and chemokine in response to stimulation ex vivo were analyzed by multiplex ELISA. RESULTS. Monocyte frequency and count were increased in peripheral blood up to 3 months post vaccination compared with their own pre-vaccine control. Expression of HLA-DR, CD40 and CD80 was enhanced on monocytes for up to 3 months following vaccination. Moreover, monocytes had increased expression of glycolysis-associated enzymes 2 months post vaccination. Upon stimulation ex vivo with unrelated antigens, monocytes produced increased IL-1β, IL-6, IL-10, CXCL1, and MIP-1α, and decreased TNF, compared with pre-vaccine controls. Resting monocytes produced more IFN-γ, IL-18, and MCP-1 up to 3 months post vaccination compared with pre-vaccine controls. CONCLUSION. These data provide evidence for the induction of trained immunity following a single dose of the ChAdOx1 nCoV-19 vaccine. FUNDING. This work was funded by The Health Research Board (EIA-2019-010) and Science Foundation Ireland Strategic Partnership Programme (Proposal ID 20/SPP/3685).

Authors

Dearbhla M. Murphy, Donal J. Cox, Sarah A. Connolly, Eamon P Breen, Aenea A.I. Brugman, James J. Phelan, Joseph Keane, Sharee A. Basdeo

Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2

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Abstract

SARS-CoV-2 infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increases susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. By identifying one rapalog (ridaforolimus) that is less potent in this regard, we demonstrate that rapalogs promote Spike-mediated entry into cells by triggering the degradation of antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increase virus entry inhibit the mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitates its nuclear translocation and triggers microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity.

Authors

Guoli Shi, Abhilash I. Chiramel, Tiansheng Li, Kin Kui Lai, Adam D. Kenney, Ashley Zani, Adrian C Eddy, Saliha Majdoul, Lizhi Zhang, Tirhas Dempsey, Paul A. Beare, Swagata Kar, Jonathan W. Yewdell, Sonja M Best, Jacob S. Yount, Alex A. Compton

Neutralizing antibody responses in patients hospitalized with SARS-CoV-2 Delta or Omicron infection

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Abstract

Authors

Susanne L. Linderman, Lilin Lai, Estefany L. Bocangel Gamarra, Max S.Y. Lau, Srilatha Edupuganti, Diya Surie, Mark W. Tenforde, James D. Chappell, Nicholas M. Mohr, Kevin W. Gibbs, Jay S. Steingrub, Matthew C. Exline, Nathan I. Shapiro, Anne E. Frosch, Nida Qadir, Meredith E. Davis-Gardner, M. Juliana McElrath, Adam S. Lauring, Mehul S. Suthar, Manish M. Patel, Wesley H. Self, Rafi Ahmed

Glutamine synthetase limits b-catenin-mutated liver cancer growth by maintaining nitrogen homeostasis and suppressing mTORC1

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Abstract

Glutamine synthetase (GS) catalyzes de novo synthesis of glutamine that facilitates cancer cell growth. In the liver, GS functions next to the urea cycle to remove ammonia waste. As dysregulated urea cycle is implicated in cancer development, the impact of GS’ ammonia clearance function has not been explored in cancer. Here we show that, oncogenic activation of beta-catenin led to decreased urea cycle and elevated ammonia waste burden. While beta-catenin induced the expression of GS, which is thought to be cancer-promoting, surprisingly, genetic ablation of hepatic GS accelerated the onset of liver tumors in several mouse models that involved β-catenin activation. Mechanistically, GS ablation exacerbated hyperammonemia and facilitated the production of glutamate-derived non-essential amino acids (NEAAs), which subsequently stimulated mTORC1. Pharmacological and genetic inhibition of mTORC1 and glutamic transaminases suppressed tumorigenesis facilitated by GS ablation. While HCC patients, especially those with CTNNB1 mutations, have an overall defective urea cycle and increased expression of GS, there exists a subset of patients with low GS expression that is associated with mTORC1 hyperactivation. Therefore, GS-mediated ammonia clearance serves as a tumor-suppressing mechanism in livers that harbor β-catenin activation mutations and a compromised urea cycle.

Authors

Weiwei Dai, Jianliang Shen, Junrong Yan, Alex J. Bott, Sara Maimouni, Heineken Q. Daguplo, Yujue Wang, Khoosheh Khayati, Jessie Yanxiang Guo, Lanjing Zhang, Yongbo Wang, Alexander Valvezan, Wen-Xing Ding, Xin Chen, Xiaoyang Su, Shenglan Gao, Wei-Xing Zong

The interferon-inducible protein viperin controls cancer metabolic reprogramming to enhance cancer progression

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Abstract

Metabolic reprogramming is an important cancer hallmark. However, the mechanisms driving metabolic phenotypes of cancer cells are unclear. Here, we showed that the interferon (IFN)-inducible protein, viperin, drives metabolic alteration in cancer cells. Viperin was observed in various types of cancer and inversely correlated with the survival rate of patients with gastric, lung, breast, renal, pancreatic, or brain cancer. By generating viperin knockdown or stably expressing cancer cells, we showed that viperin, but not a mutant lacking its iron-sulfur cluster-binding motif, increased lipogenesis and glycolysis via inhibition of fatty acid β-oxidation in cancer cells. In the tumor microenvironment, deficiency of fatty acids and oxygen as well as production of IFNs upregulated viperin expression via the PI3K/AKT/mTOR/HIF-1α and JAK/STAT pathways. Moreover, viperin was primarily expressed in cancer stem-like cells (CSCs) and functioned to promote metabolic reprogramming and enhance CSC properties, thereby facilitating tumor growth in xenograft mouse models. Collectively, our data indicate that viperin-mediated metabolic alteration drives the metabolic phenotype and progression of cancer.

Authors

Kyung Mi Choi, Jeong Jin Kim, Jihye Yoo, Ku Sul Kim, Youngeun Gu, John Eom, Haengdueng Jeong, Kyungeun Kim, Ki Taek Nam, Young Soo Park, Joon-Yong Chung, Jun-Young Seo

Meningeal dendritic cells drive neuropathic pain through elevation of the kynurenine metabolic pathway in mice

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Abstract

Neuropathic pain is one of the most important clinical consequences of injury to the somatosensory system. Nevertheless, the critical pathophysiological mechanisms involved in neuropathic pain development are poorly understood. In this study, we found that neuropathic pain is abrogated when the kynurenine metabolic pathway initiated by the enzyme indoleamine 2,3-dioxygenase (IDO1) is ablated pharmacologically or genetically. Mechanistically, it was found that IDO1-expressing dendritic cells (DCs) accumulated in the dorsal root leptomeninges and led to an increase in kynurenine levels in the spinal cord. In the spinal cord, kynurenine was metabolized by kynurenine-3-monooxygenase-expressing astrocytes into a pro-nociceptive metabolite 3-hydroxykynurenine. Ultimately, 3-hydroxyanthranilate 3,4-dioxygenase-derived quinolinic acid formed in the final step of the canonical KYNPATH was also involved in neuropathic pain development through the activation of the glutamatergic N-methyl-D-aspartate (NMDA) receptor. In conclusion, these data revealed a novel role for DCs driving neuropathic pain development through elevation of the kynurenine metabolic pathway. This novel paradigm offers potential new targets for drug development against this type of chronic pain.

Authors

Alexandre G.M. Maganin, Guilherme R. Souza, Miriam D. Fonseca, Alexandre H. Lopes, Rafaela M. Mano Guimarães, André Dagostin, Nerry T. Cecilio, Atlante S. Mendes, William A. Gonçalves, Conceição E.A. Silva, Francisco I. Fernandes Gomes, Lucas M. Mauriz Marques, Rangel L. Silva, Letícia M. Arruda, Denis A. Santana, Henrique Lemos, Lei Huang, Marcela Davoli-Ferreira, Danielle S. Santana-Coelho, Morena B. Sant'Anna, Ricardo Kusuda, Jhimmy Talbot, Gabriela Pacholczyk, Gabriela A. Buqui, Norberto Lopes, Jose C. Alves-Filho, Ricardo M. Leão, Jason C. O'Connor, Fernando Q. Cunha, Andrew Mellor, Thiago Cunha

Improved control of SARS-CoV-2 by treatment with nucleocapsid-specific monoclonal antibody

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Abstract

The SARS-CoV-2 spike protein is the main antigen in all approved COVID-19 vaccines and is also the only target for monoclonal antibody therapies. Immune responses to other viral antigens are generated after SARS-CoV-2 infection, but their contribution to the antiviral response remains unclear. Here, we interrogate whether nucleocapsid-specific antibodies can improve protection against SARSCoV-2. We first immunized mice with a nucleocapsid-based vaccine, and then transferred sera from these mice into naïve mice, followed by challenge with SARS-CoV-2. We show that mice that received nucleocapsid-specific sera or a nucleocapsid-specific monoclonal antibody (mAb) exhibited enhanced control of SARS-CoV-2. Nucleocapsid-specific antibodies elicited NK-mediated antibodydependent cellular cytotoxicity (ADCC) against infected cells. These findings provide the first demonstration in the coronavirus literature that antibody responses specific to the nucleocapsid protein can improve viral clearance, providing a rationale for the clinical evaluation of nucleocapsid-based monoclonal antibody therapies to treat COVID-19.

Authors

Tanushree Dangi, Sarah Sanchez, Jacob Class, Michelle C. Richner, Lavanya Visvabharathy, Young Rock Chung, Kirsten Bentley, Richard J. Stanton, Igor J. Koralnik, Justin M. Richner, Pablo Penaloza-MacMaster

RAD21 amplification epigenetically suppresses interferon signaling to promote immune evasion in ovarian cancer

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Abstract

Prevalent copy number alteration (CNA) is the most prominent genetic characteristic associated with ovarian cancer (OV) development, but its role in immune evasion has not been fully elucidated. In this study, we identified RAD21, a key component of the cohesin complex, as a frequently amplified oncogene that could modulate immnue response in OV. Through interrogating RAD21-regulated transcriptional program we found that RAD21 directly interacts with YAP/TEAD4 transcriptional co-repressors and recruits NuRD complex to suppress interferon (IFN) signaling. In multiple clinical cohorts, RAD21 overexpression is inversely correlated with IFN signature gene expression in OV. We further demonstrated in murine syngeneic tumor models that RAD21 ablation potentiated anti-PD-1 efficacy with increased intratumoral CD8+ T-cell effector activity. Our study identified a previously unrecognized RAD21-YAP/TEAD4-NuRD co-repressor complex in immune modulation, and thus provided a potential target and biomarker for precision immunotherapy in OV.

Authors

Peng Deng, Zining Wang, Jinghong Chen, Shini Liu, Xiaosai Yao, Shaoyan Liu, Lizhen Liu, Zhaoliang Yu, Yulin Huang, Zhongtang Xiong, Rong Xiao, Jiuping Gao, Weiting Liang, Jieping Chen, Hui Liu, Jing Han Hong, Jason Yongsheng Chan, Peiyong Guan, Jianfeng Chen, Yali Wang, Jiaxin Yin, Jundong Li, Min Zheng, Chao Zhang, Penghui Zhou, Tiebang Kang, Bin Tean Teh, Qiang Yu, Zhixiang Zuo, Qingping Jiang, Jihong Liu, Ying Xiong, Xiaojun Xia, Jing Tan