bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Single-Cell Gene Expression Analyses Reveal Distinct Self-Renewing and Proliferating Subsets in the Leukemia Stem Cell Compartment in Acute Myeloid Leukemia
Authors: Sachs Karen , Sarver Aaron L. , Noble-Orcutt Klara E. , LaRue Rebecca S. , Antony Marie Lue , Chang Daniel , Lee Yoonkyu , Navis Connor M. , Hillesheim Alexandria L. , Nykaza Ian R. , Ha Ngoc A. , Hansen Conner J. , Karadag Fatma K. , Bergerson Rachel J. , Verneris Michael R. , Meredith Matthew M. , Schomaker Matthew L. , Linden Michael A. , Myers Chad L. , Largaespada David A. , Sachs Zohar
Published: March 2023

Abstract
Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRASG12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes “LSC-specific” genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML.
Significance:
These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML.
Data from Fibroblasts from Distinct Pancreatic Pathologies Exhibit Disease-Specific Properties
Authors: Barrera Lawrence N. , Evans Anthony , Lane Brian , Brumskill Sarah , Oldfield Frances E. , Campbell Fiona , Andrews Timothy , Lu Zipeng , Perez-Mancera Pedro A. , Liloglou Triantafillos , Ashworth Milton , Jalali Mehdi , Dawson Rebecca , Nunes Quentin , Phillips Phoebe A. , Timms John F. , Halloran Christopher , Greenhalf William , Neoptolemos John P. , Costello Eithne
Published: March 2023

Abstract
Although fibrotic stroma forms an integral component of pancreatic diseases, whether fibroblasts programmed by different types of pancreatic diseases are phenotypically distinct remains unknown. Here, we show that fibroblasts isolated from patients with pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP), periampullary tumors, and adjacent normal (NA) tissue (N = 34) have distinct mRNA and miRNA profiles. Compared with NA fibroblasts, PDAC-associated fibroblasts were generally less sensitive to an antifibrotic stimulus (NPPB) and more responsive to positive regulators of activation such as TGFβ1 and WNT. Of the disease-associated fibroblasts examined, PDAC- and CP-derived fibroblasts shared greatest similarity, yet PDAC-associated fibroblasts expressed higher levels of tenascin C (TNC), a finding attributable to miR-137, a novel regulator of TNC. TNC protein and transcript levels were higher in PDAC tissue versus CP tissue and were associated with greater levels of stromal activation, and conditioned media from TNC-depleted PDAC-associated fibroblasts modestly increased both PDAC cell proliferation and PDAC cell migration, indicating that stromal TNC may have inhibitory effects on PDAC cells. Finally, circulating TNC levels were higher in patients with PDAC compared with CP. Our characterization of pancreatic fibroblast programming as disease-specific has consequences for therapeutic targeting and for the manner in which fibroblasts are used in research.
Significance:
Primary fibroblasts derived from various types of pancreatic diseases possess and retain distinct molecular and functional characteristics in culture, providing a series of cellular models for treatment development and disease-specific research.
Data from In Situ DESI-MSI Lipidomic Profiles of Breast Cancer Molecular Subtypes and Precursor Lesions
Authors: Santoro Adriana Leandra , Drummond Rodrigo D. , Silva Israel Tojal , Ferreira Severino S. , Juliano Luiz , Vendramini Pedro H. , Lemos Monique Batista da Costa , Eberlin Marcos N. , Andrade Victor Piana
Published: March 2023

Abstract
Clinically meaningful molecular subtypes for classification of breast cancers have been established, however, initiation and progression of these subtypes remain poorly understood. The recent development of desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) facilitates the convergence of analytical chemistry and traditional pathology, allowing chemical profiling with minimal tissue pretreatment in frozen samples. Here, we characterized the chemical composition of molecular subtypes of breast cancer with DESI-MSI. Regions of interest were identified, including invasive breast cancer (IBC), ductal carcinoma in situ (DCIS), and adjacent benign tissue (ABT), and metabolomic profiles at 200 μm elaborated using Biomap software and the Lasso method. Top ions identified in IBC regions included polyunsaturated fatty acids, deprotonated glycerophospholipids, and sphingolipids. Highly saturated lipids, as well as antioxidant molecules [taurine (m/z 124.0068), uric acid (m/z 167.0210), ascorbic acid (m/z 175.0241), and glutathione (m/z 306.0765)], were able to distinguish IBC from ABT. Moreover, luminal B and triple-negative subtypes showed more complex lipid profiles compared with luminal A and HER2 subtypes. DCIS and IBC were distinguished on the basis of cell signaling and apoptosis-related ions [fatty acids (341.2100 and 382.3736 m/z) and glycerophospholipids (PE (P-16:0/22:6, m/z 746.5099, and PS (38:3), m/z 812.5440)]. In summary, DESI-MSI identified distinct lipid composition between DCIS and IBC and across molecular subtypes of breast cancer, with potential implications for breast cancer pathogenesis.
Significance:
These findings present the first in situ metabolomic findings of the four molecular subtypes of breast cancer, DCIS, and normal tissue, and add to the understanding of their pathogenesis.
Data from Metastatic Conditioning of Myeloid Cells at a Subcutaneous Synthetic Niche Reflects Disease Progression and Predicts Therapeutic Outcomes
Authors: Oakes Robert S. , Bushnell Grace G. , Orbach Sophia M. , Kandagatla Pridvi , Zhang Yining , Morris Aaron H. , Hall Matthew S. , LaFaire Petrina , Decker Joseph T. , Hartfield Rachel M. , Brooks Michael D. , Wicha Max S. , Jeruss Jacqueline S. , Shea Lonnie D.
Published: March 2023

Abstract
Monitoring metastatic events in distal tissues is challenged by their sporadic occurrence in obscure and inaccessible locations within these vital organs. A synthetic biomaterial scaffold can function as a synthetic metastatic niche to reveal the nature of these distal sites. These implanted scaffolds promote tissue ingrowth, which upon cancer initiation is transformed into a metastatic niche that captures aggressive circulating tumor cells. We hypothesized that immune cell phenotypes at synthetic niches reflect the immunosuppressive conditioning within a host that contributes to metastatic cell recruitment and can identify disease progression and response to therapy. We analyzed the expression of 632 immune-centric genes in tissue biopsied from implants at weekly intervals following inoculation. Specific immune populations within implants were then analyzed by single-cell RNA-seq. Dynamic gene expression profiles in innate cells, such as myeloid-derived suppressor cells, macrophages, and dendritic cells, suggest the development of an immunosuppressive microenvironment. These dynamics in immune phenotypes at implants was analogous to that in the diseased lung and had distinct dynamics compared with blood leukocytes. Following a therapeutic excision of the primary tumor, longitudinal tracking of immune phenotypes at the implant in individual mice showed an initial response to therapy, which over time differentiated recurrence versus survival. Collectively, the microenvironment at the synthetic niche acts as a sentinel by reflecting both progression and regression of disease.
Significance:
Immune dynamics at biomaterial implants, functioning as a synthetic metastatic niche, provides unique information that correlates with disease progression.
Data from Extracellular ATP and Purinergic P2Y2 Receptor Signaling Promote Liver Tumorigenesis in Mice by Exacerbating DNA Damage
Authors: Schulien Isabel , Hockenjos Birgit , van Marck Veerle , Ayata C. Korcan , Follo Marie , Thimme Robert , Hasselblatt Peter
Published: March 2023

Abstract
Release of ATP to the extracellular compartment and subsequent activation of purinergic receptors is a conserved mechanism mediating inflammatory responses and cell fate decisions in various organs including the liver. Previous findings suggest that extracellular ATP may promote liver tumorigenesis, however, the underlying mechanisms are poorly understood. Therefore, our aim was to dissect the functions of extracellular ATP and P2Y2 receptors (P2Y2R) during hepatocarcinogenesis. Liver tumors were induced in wild-type and P2y2r−/− knockout mice by intraperitoneal diethylnitrosamine (DEN) injection. Tumorigenesis was analyzed after 8 to 10 months and molecular analyses were performed at different stages of tumorigenesis in vivo, as well as in primary mouse hepatocytes in vitro. Liver tumor incidence and tumor numbers were strongly reduced in P2y2r−/− mice, whereas tumor size and morphology were comparable to wild-type controls, suggesting that P2Y2R contributes to tumor initiation. Mechanistically, hepatocyte proliferation in DEN-treated P2y2r−/− mice was reduced, which correlated with reduced c-JUN and CCND1 but increased p21 expression. Moreover, DNA damage as determined by hepatocellular expression of γH2A.X and of genes related to genotoxic stress, as well as STAT3 phosphorylation, was reduced in the absence of P2y2r. Administration of genotoxic agents to primary hepatocytes in vitro confirmed that DNA damage was indeed exacerbated by extracellular ATP, subsequent P2Y2R activation, and downstream intracellular calcium-dependent signal transduction. In conclusion, our data reveal that extracellular ATP and subsequent P2Y2R function stimulate DNA damage responses and hepatocyte proliferation, thereby promoting hepatocarcinogenesis. Targeting this pathway may be an attractive approach for chemoprevention of hepatocellular carcinoma.
Significance:
Extracellular ATP and subsequent P2Y2 receptor function stimulate DNA damage responses and hepatocyte proliferation, thereby promoting hepatocarcinogenesis in mice. Targeting this pathway may be an attractive approach for chemoprevention of hepatocellular carcinoma.
Data from Long Noncoding RNA DRAIC Inhibits Prostate Cancer Progression by Interacting with IKK to Inhibit NF-κB Activation
Authors: Saha Shekhar , Kiran Manjari , Kuscu Canan , Chatrath Ajay , Wotton David , Mayo Marty W. , Dutta Anindya
Published: March 2023

Abstract
DRAIC is a 1.7 kb spliced long noncoding RNA downregulated in castration-resistant advanced prostate cancer. Decreased DRAIC expression predicts poor patient outcome in prostate and seven other cancers, while increased DRAIC represses growth of xenografted tumors. Here, we show that cancers with decreased DRAIC expression have increased NF-κB target gene expression. DRAIC downregulation increased cell invasion and soft agar colony formation; this was dependent on NF-κB activation. DRAIC interacted with subunits of the IκB kinase (IKK) complex to inhibit their interaction with each other, the phosphorylation of IκBα, and the activation of NF-κB. These functions of DRAIC mapped to the same fragment containing bases 701–905. Thus, DRAIC lncRNA inhibits prostate cancer progression through suppression of NF-κB activation by interfering with IKK activity.
Significance:
A cytoplasmic tumor-suppressive lncRNA interacts with and inhibits a major kinase that activates an oncogenic transcription factor in prostate cancer.
Data from An Integrative Gene Expression and Mathematical Flux Balance Analysis Identifies Targetable Redox Vulnerabilities in Melanoma Cells
Authors: Paudel B. Bishal , Lewis Joshua E. , Hardeman Keisha N. , Hayford Corey E. , Robbins Charles J. , Stauffer Philip E. , Codreanu Simona G. , Sherrod Stacy D. , McLean John A. , Kemp Melissa L. , Quaranta Vito
Published: March 2023

Abstract
Melanomas harboring BRAF mutations can be treated with BRAF inhibitors (BRAFi), but responses are varied and tumor recurrence is inevitable. Here we used an integrative approach of experimentation and mathematical flux balance analyses in BRAF-mutated melanoma cells to discover that elevated antioxidant capacity is linked to BRAFi sensitivity in melanoma cells. High levels of antioxidant metabolites in cells with reduced BRAFi sensitivity confirmed this conclusion. By extending our analyses to other melanoma subtypes in The Cancer Genome Atlas, we predict that elevated redox capacity is a general feature of melanomas, not previously observed. We propose that redox vulnerabilities could be exploited for therapeutic benefits and identify unsuspected combination targets to enhance the effects of BRAFi in any melanoma, regardless of mutational status.
Significance:
An integrative bioinformatics, flux balance analysis, and experimental approach identify targetable redox vulnerabilities and show the potential for modulation of cancer antioxidant defense to augment the benefits of existing therapies in melanoma.
Data from NOX4 Inhibition Potentiates Immunotherapy by Overcoming Cancer-Associated Fibroblast-Mediated CD8 T-cell Exclusion from Tumors
Authors: Ford Kirsty , Hanley Christopher J. , Mellone Massimiliano , Szyndralewiez Cedric , Heitz Freddy , Wiesel Philippe , Wood Oliver , Machado Maria , Lopez Maria-Antoinette , Ganesan Anusha-Preethi , Wang Chuan , Chakravarthy Ankur , Fenton Tim R. , King Emma V. , Vijayanand Pandurangan , Ottensmeier Christian H. , Al-Shamkhani Aymen , Savelyeva Natalia , Thomas Gareth J.
Published: March 2023

Abstract
Determining mechanisms of resistance to αPD-1/PD-L1 immune-checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities [anticancer vaccination, TC1 (HPV E7 DNA vaccine), αPD-1, and MC38] and found that CAFs broadly suppressed response by specifically excluding CD8+ T cells from tumors (not CD4+ T cells or macrophages); CD8+ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8+ T cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a nondepleting antibody overcame the CD8+ T-cell exclusion effect without affecting Tregs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and compared this with TGFβ1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition [GKT137831 (Setanaxib)] of NOX4 “normalized” CAF to a quiescent phenotype and promoted intratumoral CD8+ T-cell infiltration, overcoming the exclusion effect; TGFβ1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition and could improve outcome in a broad range of cancers.
Significance:
NOX4 is critical for maintaining the immune-suppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAF-mediated CD8+ T-cell exclusion.
Data from HPV16 E5 Mediates Resistance to PD-L1 Blockade and Can Be Targeted with Rimantadine in Head and Neck Cancer
Authors: Miyauchi Sayuri , Sanders P. Dominick , Guram Kripa , Kim Sangwoo S. , Paolini Francesca , Venuti Aldo , Cohen Ezra E.W. , Gutkind J. Silvio , Califano Joseph A. , Sharabi Andrew B.
Published: March 2023

Abstract
There is a critical need to understand mechanisms of resistance and to develop combinatorial strategies to improve responses to checkpoint blockade immunotherapy (CBI). Here, we uncover a novel mechanism by which the human papillomavirus (HPV) inhibits the activity of CBI in head and neck squamous cell carcinoma (HNSCC). Using orthotopic HNSCC models, we show that radiation combined with anti–PD-L1 immunotherapy significantly enhanced local control, CD8+ memory T cells, and induced preferential T-cell homing via modulation of vascular endothelial cells. However, the HPV E5 oncoprotein suppressed immune responses by downregulating expression of major histocompatibility complex and interfering with antigen presentation in murine models and patient tumors. Furthermore, tumors expressing HPV E5 were rendered entirely resistant to anti–PD-L1 immunotherapy, and patients with high expression of HPV16 E5 had worse survival. The antiviral E5 inhibitor rimantadine demonstrated remarkable single-agent antitumor activity. This is the first report that describes HPV E5 as a mediator of resistance to anti–PD-1/PD-L1 immunotherapy and demonstrates the antitumor activity of rimantadine. These results have broad clinical relevance beyond HNSCC to other HPV-associated malignancies and reveal a powerful mechanism of HPV-mediated immunosuppression, which can be exploited to improve response rates to checkpoint blockade.
Significance:
This study identifies a novel mechanism of resistance to anti–PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients.
Data from Circulating Immune Cell Composition and Cancer Risk: A Prospective Study Using Epigenetic Cell Count Measures
Authors: Le Cornet Charlotte , Schildknecht Konstantin , Rossello Chornet Araceli , Fortner Renée T. , González Maldonado Sandra , Katzke Verena A. , Kühn Tilman , Johnson Theron , Olek Sven , Kaaks Rudolf
Published: March 2023

Abstract
Although ample evidence indicates that immune cell homeostasis is an important prognostic outcome determinant in patients with cancer, few studies have examined whether it also determines cancer risk among initially healthy individuals. We performed a case–cohort study including incident cases of breast (n = 207), colorectal (n = 111), lung (n = 70), and prostate (n = 201) cancer as well as a subcohort (n = 465) within the European Prospective Investigation into Cancer and Nutrition-Heidelberg cohort. Relative counts of neutrophils, monocytes, and lymphocyte sublineages were measured by qRT-PCR. HRs and 95% confidence intervals were used to measure the associations between relative counts of immune cell and cancer risks. When relative counts of immune cell types were taken individually, a significant positive association was observed between relative counts of FOXP3+ regulatory T cells (Tregs) and lung cancer risk, and significant inverse associations were observed between relative CD8+ counts and risks of lung and breast cancer (overall and ER+ subtype). Multivariable models with mutual adjustments across immune markers showed further significant positive associations between higher relative FOXP3+ T-cell counts and increased risks of colorectal and breast cancer (overall and ER− subtype). No associations were found between immune cell composition and prostate cancer risk. These results affirm the relevance of elevated FOXP3+ Tregs and lower levels of cytotoxic (CD8+) T cells as risk factors for tumor development.
Significance:
This epidemiologic study supports a role for both regulatory and cytotoxic T cells in determining cancer risk among healthy individuals.

See related commentary by Song and Tworoger, p. 1801
Data from Glucose-6-Phosphate Dehydrogenase Is Not Essential for K-Ras–Driven Tumor Growth or Metastasis
Authors: Ghergurovich Jonathan M. , Esposito Mark , Chen Zihong , Wang Joshua Z. , Bhatt Vrushank , Lan Taijin , White Eileen , Kang Yibin , Guo Jessie Yanxiang , Rabinowitz Joshua D.
Published: March 2023

Abstract
The enzyme glucose-6-phosphate dehydrogenase (G6PD) is a major contributor to NADPH production and redox homeostasis and its expression is upregulated and correlated with negative patient outcomes in multiple human cancer types. Despite these associations, whether G6PD is essential for tumor initiation, growth, or metastasis remains unclear. Here, we employ modern genetic tools to evaluate the role of G6PD in lung, breast, and colon cancer driven by oncogenic K-Ras. Human HCT116 colorectal cancer cells lacking G6PD exhibited metabolic indicators of oxidative stress, but developed into subcutaneous xenografts with growth comparable with that of wild-type controls. In a genetically engineered mouse model of non–small cell lung cancer driven by K-Ras G12D and p53 deficiency, G6PD knockout did not block formation or proliferation of primary lung tumors. In MDA-MB-231–derived human triple-negative breast cancer cells implanted as orthotopic xenografts, loss of G6PD modestly decreased primary site growth without ablating spontaneous metastasis to the lung and moderately impaired the ability of breast cancer cells to colonize the lung when delivered via tail vein injection. Thus, in the studied K-Ras tumor models, G6PD was not strictly essential for tumorigenesis and at most modestly promoted disease progression.
Significance:
K-Ras–driven tumors can grow and metastasize even in the absence of the oxidative pentose pathway, a main NADPH production route.
Data from Lymphoma Angiogenesis Is Orchestrated by Noncanonical Signaling Pathways
Authors: Gloger Marleen , Menzel Lutz , Grau Michael , Vion Anne-Clemence , Anagnostopoulos Ioannis , Zapukhlyak Myroslav , Gerlach Kerstin , Kammertöns Thomas , Hehlgans Thomas , Zschummel Maria , Lenz Georg , Gerhardt Holger , Höpken Uta E. , Rehm Armin
Published: March 2023

Abstract
Tumor-induced remodeling of the microenvironment relies on the formation of blood vessels, which go beyond the regulation of metabolism, shaping a maladapted survival niche for tumor cells. In high-grade B-cell lymphoma, angiogenesis correlates with poor prognosis, but attempts to target established proangiogenic pathways within the vascular niche have been inefficient. Here, we analyzed Myc-driven B-cell lymphoma–induced angiogenesis in mice. A few lymphoma cells were sufficient to activate the angiogenic switch in lymph nodes. A unique morphology of dense microvessels emerged without obvious tip cell guidance and reliance on blood endothelial cell (BEC) proliferation. The transcriptional response of BECs was inflammation independent. Conventional HIF1α or Notch signaling routes prevalent in solid tumors were not activated. Instead, a nonconventional hypersprouting morphology was orchestrated by lymphoma-provided VEGFC and lymphotoxin (LT). Interference with VEGF receptor-3 and LTβ receptor signaling pathways abrogated lymphoma angiogenesis, thus revealing targets to block lymphomagenesis.
Significance:
In lymphoma, transcriptomes and morphogenic patterns of the vasculature are distinct from processes in inflammation and solid tumors. Instead, LTβR and VEGFR3 signaling gain leading roles and are targets for lymphomagenesis blockade.
Data from Clonal ZEB1-Driven Mesenchymal Transition Promotes Targetable Oncologic Antiangiogenic Therapy Resistance
Authors: Chandra Ankush , Jahangiri Arman , Chen William , Nguyen Alan T. , Yagnik Garima , Pereira Matheus P. , Jain Saket , Garcia Joseph H. , Shah Sumedh S. , Wadhwa Harsh , Joshi Rushikesh S. , Weiss Jacob , Wolf Kayla J. , Lin Jung-Ming G. , Müller Sören , Rick Jonathan W. , Diaz Aaron A. , Gilbert Luke A. , Kumar Sanjay , Aghi Manish K.
Published: March 2023

Abstract
Glioblastoma (GBM) responses to bevacizumab are invariably transient with acquired resistance. We profiled paired patient specimens and bevacizumab-resistant xenograft models pre- and post-resistance toward the primary goal of identifying regulators whose targeting could prolong the therapeutic window, and the secondary goal of identifying biomarkers of therapeutic window closure. Bevacizumab-resistant patient specimens and xenografts exhibited decreased vessel density and increased hypoxia versus pre-resistance, suggesting that resistance occurs despite effective therapeutic devascularization. Microarray analysis revealed upregulated mesenchymal genes in resistant tumors correlating with bevacizumab treatment duration and causing three changes enabling resistant tumor growth in hypoxia. First, perivascular invasiveness along remaining blood vessels, which co-opts vessels in a VEGF-independent and neoangiogenesis-independent manner, was upregulated in novel biomimetic 3D bioengineered platforms modeling the bevacizumab-resistant microenvironment. Second, tumor-initiating stem cells housed in the perivascular niche close to remaining blood vessels were enriched. Third, metabolic reprogramming assessed through real-time bioenergetic measurement and metabolomics upregulated glycolysis and suppressed oxidative phosphorylation. Single-cell sequencing of bevacizumab-resistant patient GBMs confirmed upregulated mesenchymal genes, particularly glycoprotein YKL-40 and transcription factor ZEB1, in later clones, implicating these changes as treatment-induced. Serum YKL-40 was elevated in bevacizumab-resistant versus bevacizumab-naïve patients. CRISPR and pharmacologic targeting of ZEB1 with honokiol reversed the mesenchymal gene expression and associated stem cell, invasion, and metabolic changes defining resistance. Honokiol caused greater cell death in bevacizumab-resistant than bevacizumab-responsive tumor cells, with surviving cells losing mesenchymal morphology. Employing YKL-40 as a resistance biomarker and ZEB1 as a target to prevent resistance could fulfill the promise of antiangiogenic therapy.
Significance:
Bevacizumab resistance in GBM is associated with mesenchymal/glycolytic shifts involving YKL-40 and ZEB1. Targeting ZEB1 reduces bevacizumab-resistant GBM phenotypes.
Data from Extensive Clonal Branching Shapes the Evolutionary History of High-Risk Pediatric Cancers
Authors: Andersson Natalie , Bakker Bjorn , Karlsson Jenny , Valind Anders , Holmquist Mengelbier Linda , Spierings Diana C.J. , Foijer Floris , Gisselsson David
Published: March 2023

Abstract
Darwinian evolution of tumor cells remains underexplored in childhood cancer. We here reconstruct the evolutionary histories of 56 pediatric primary tumors, including 24 neuroblastomas, 24 Wilms tumors, and 8 rhabdomyosarcomas. Whole-genome copy-number and whole-exome mutational profiling of multiple regions per tumor were performed, followed by clonal deconvolution to reconstruct a phylogenetic tree for each tumor. Overall, 88% of the tumors exhibited genetic variation among primary tumor regions. This variability typically emerged through collateral phylogenetic branching, leading to spatial variability in the distribution of more than 50% (96/173) of detected diagnostically informative genetic aberrations. Single-cell sequencing of 547 individual cancer cells from eight solid pediatric tumors confirmed branching evolution to be a fundamental underlying principle of genetic variation in all cases. Strikingly, cell-to-cell genetic diversity was almost twice as high in aggressive compared with clinically favorable tumors (median Simpson index of diversity 0.45 vs. 0.88; P = 0.029). Similarly, a comparison of multiregional sampling data from a total of 274 tumor regions showed that new phylogenetic branches emerge at a higher frequency per sample and carry a higher mutational load in high-risk than in low-risk tumors. Timelines based on spatial genetic variation showed that the mutations most influencing relapse risk occur at initiation of clonal expansion in neuroblastoma and rhabdomyosarcoma, whereas in Wilms tumor, they are late events. Thus, from an evolutionary standpoint, some high-risk childhood cancers are born bad, whereas others grow worse over time.
Significance:
Different pediatric cancers with a high risk of relapse share a common generic pattern of extensively branching evolution of somatic mutations.
Data from Cdkn2a Loss in a Model of Neurofibroma Demonstrates Stepwise Tumor Progression to Atypical Neurofibroma and MPNST
Authors: Chaney Katherine E. , Perrino Melissa R. , Kershner Leah J. , Patel Ami V. , Wu Jianqiang , Choi Kwangmin , Rizvi Tilat A. , Dombi Eva , Szabo Sara , Largaespada David A. , Ratner Nancy
Published: March 2023

Abstract
Plexiform neurofibromas are benign nerve sheath Schwann cell tumors characterized by biallelic mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene. Atypical neurofibromas show additional frequent loss of CDKN2A/Ink4a/Arf and may be precursor lesions of aggressive malignant peripheral nerve sheath tumors (MPNST). Here we combined loss of Nf1 in developing Schwann cells with global Ink4a/Arf loss and identified paraspinal plexiform neurofibromas and atypical neurofibromas. Upon transplantation, atypical neurofibromas generated genetically engineered mice (GEM)-PNST similar to human MPNST, and tumors showed reduced p16INK4a protein and reduced senescence markers, confirming susceptibility to transformation. Superficial GEM-PNST contained regions of nerve-associated plexiform neurofibromas or atypical neurofibromas and grew rapidly on transplantation. Transcriptome analyses showed similarities to corresponding human tumors. Thus, we recapitulated nerve tumor progression in NF1 and provided preclinical platforms for testing therapies at each tumor grade. These results support a tumor progression model in which loss of NF1 in Schwann cells drives plexiform neurofibromas formation, additional loss of Ink4a/Arf contributes to atypical neurofibromas formation, and further changes underlie transformation to MPNST.
Significance:
New mouse models recapitulate the stepwise progression of NF1 tumors and will be useful to define effective treatments that halt tumor growth and tumor progression in NF1.
Data from Twist1-Induced Epithelial Dissemination Requires Prkd1 Signaling
Authors: Georgess Dan , Padmanaban Veena , Sirka Orit Katarina , Coutinho Kester , Choi Alex , Frid Gabriela , Neumann Neil M. , Inoue Takanari , Ewald Andrew J.
Published: March 2023

Abstract
Dissemination is an essential early step in metastasis but its molecular basis remains incompletely understood. To define the essential targetable effectors of this process, we developed a 3D mammary epithelial culture model, in which dissemination is induced by overexpression of the transcription factor Twist1. Transcriptomic analysis and ChIP-PCR together demonstrated that protein kinase D1 (Prkd1) is a direct transcriptional target of Twist1 and is not expressed in the normal mammary epithelium. Pharmacologic and genetic inhibition of Prkd1 in the Twist1-induced dissemination model demonstrated that Prkd1 was required for cells to initiate extracellular matrix (ECM)–directed protrusions, release from the epithelium, and migrate through the ECM. Antibody-based protein profiling revealed that Prkd1 induced broad phosphorylation changes, including an inactivating phosphorylation of β-catenin and two microtubule depolymerizing phosphorylations of Tau, potentially explaining the release of cell–cell contacts and persistent activation of Prkd1. In patients with breast cancer, TWIST1 and PRKD1 expression correlated with metastatic recurrence, particularly in basal breast cancer. Prkd1 knockdown was sufficient to block dissemination of both murine and human mammary tumor organoids. Finally, Prkd1 knockdown in vivo blocked primary tumor invasion and distant metastasis in a mouse model of basal breast cancer. Collectively, these data identify Prkd1 as a novel and targetable signaling node downstream of Twist1 that is required for epithelial invasion and dissemination.
Significance:
Twist1 is a known regulator of metastatic cell behaviors but not directly targetable. This study provides a molecular explanation for how Twist1-induced dissemination works and demonstrates that it can be targeted.
Data from A Premalignant Cell-Based Model for Functionalization and Classification of PTEN Variants
Authors: Chao Jesse T. , Hollman Rocio , Meyers Warren M. , Meili Fabian , Matreyek Kenneth A. , Dean Pamela , Fowler Douglas M. , Haas Kurt , Roskelley Calvin D. , Loewen Christopher J.R.
Published: March 2023

Abstract
As sequencing becomes more economical, we are identifying sequence variations in the population faster than ever. For disease-associated genes, it is imperative that we differentiate a sequence variant as either benign or pathogenic, such that the appropriate therapeutic interventions or surveillance can be implemented. PTEN is a frequently mutated tumor suppressor that has been linked to the PTEN hamartoma tumor syndrome. Although the domain structure of PTEN and the functional impact of a number of its most common tumor-linked mutations have been characterized, there is a lack of information about many recently identified clinical variants. To address this challenge, we developed a cell-based assay that utilizes a premalignant phenotype of normal mammary epithelial cells lacking PTEN. We measured the ability of PTEN variants to rescue the spheroid formation phenotype of PTEN−/− MCF10A cells maintained in suspension. As proof of concept, we functionalized 47 missense variants using this assay, only 19 of which have clear classifications in ClinVar. We utilized a machine learning model trained with annotated genotypic data to classify variants as benign or pathogenic based on our functional scores. Our model predicted with high accuracy that loss of PTEN function was indicative of pathogenicity. We also determined that the pathogenicity of certain variants may have arisen from reduced stability of the protein product. Overall, this assay outperformed computational predictions, was scalable, and had a short run time, serving as an ideal alternative for annotating the clinical significance of cancer-associated PTEN variants.
Significance:
Combined three-dimensional tumor spheroid modeling and machine learning classifies PTEN missense variants, over 70% of which are currently listed as variants of uncertain significance.
Data from CD44 Promotes PD-L1 Expression and Its Tumor-Intrinsic Function in Breast and Lung Cancers
Authors: Kong Tim , Ahn Ryuhjin , Yang Kangning , Zhu Xianbing , Fu Zheng , Morin Geneviève , Bramley Rachel , Cliffe Nikki C. , Xue Yibo , Kuasne Hellen , Li Qinghao , Jung Sungmi , Gonzalez Anne V. , Camilleri-Broet Sophie , Guiot Marie-Christine , Park Morag , Ursini-Siegel Josie , Huang Sidong
Published: March 2023

Abstract
The PD-L1 (CD274) immune-checkpoint ligand is often upregulated in cancers to inhibit T cells and elicit immunosuppression. Independent of this activity, PD-L1 has recently been shown to also exert a cancer cell–intrinsic function promoting tumorigenesis. Here, we establish this tumor-intrinsic role of PD-L1 in triple-negative breast cancer (TNBC) and non–small cell lung cancer (NSCLC). Using FACS-assisted shRNA screens, we identified the cell-surface adhesion receptor CD44 as a key positive regulator of PD-L1 expression in these cancers. Mechanistically, CD44 activated PD-L1 transcription in part through its cleaved intracytoplasmic domain (ICD), which bound to a regulatory region of the PD-L1 locus containing a consensus CD44-ICD binding site. Supporting this genetic interaction, CD44 positively correlated with PD-L1 expression at the mRNA and protein levels in primary tumor samples of TNBC and NSCLC patients. These data provide a novel basis for CD44 as a critical therapeutic target to suppress PD-L1 tumor–intrinsic function.
Significance:
CD44 is a potential target to suppress PD-L1 function in TNBC. This finding has the potential to open a new area of therapy for TNBC.
Data from Organoids Reveal That Inherent Radiosensitivity of Small and Large Intestinal Stem Cells Determines Organ Sensitivity
Authors: Martin Maria Laura , Adileh Mohammad , Hsu Kuo-Shun , Hua Guoqiang , Lee Sang Gyu , Li Christy , Fuller John D. , Rotolo Jimmy A. , Bodo Sahra , Klingler Stefan , Haimovitz-Friedman Adriana , Deasy Joseph O. , Fuks Zvi , Paty Philip B. , Kolesnick Richard N.
Published: March 2023

Abstract
Tissue survival responses to ionizing radiation are nonlinear with dose, rather yielding tissue-specific descending curves that impede straightforward analysis of biologic effects. Apoptotic cell death often occurs at low doses, while at clinically relevant intermediate doses, double-strand break misrepair yields mitotic death that determines outcome. As researchers frequently use a single low dose for experimentation, such strategies may inaccurately depict inherent tissue responses. Cutting edge radiobiology has adopted full dose survival profiling and devised mathematical algorithms to fit curves to observed data to generate highly reproducible numerical data that accurately define clinically relevant inherent radiosensitivities. Here, we established a protocol for irradiating organoids that delivers radiation profiles simulating the organ of origin. This technique yielded highly similar dose–survival curves of small and large intestinal crypts in vivo and their cognate organoids analyzed by the single-hit multi-target (SHMT) algorithm, outcomes reflecting the inherent radiation profile of their respective Lgr5+ stem cell populations. As this technological advance is quantitative, it will be useful for accurate evaluation of intestinal (patho)physiology and drug screening.
Significance:
These findings establish standards for irradiating organoids that deliver radiation profiles that phenocopy the organ of origin.

See related commentary by Muschel et al., p. 927
Data from Proteomic Profiling of the ECM of Xenograft Breast Cancer Metastases in Different Organs Reveals Distinct Metastatic Niches
Authors: Hebert Jess D. , Myers Samuel A. , Naba Alexandra , Abbruzzese Genevieve , Lamar John M. , Carr Steven A. , Hynes Richard O.
Published: March 2023

Abstract
Metastasis causes most cancer-related deaths, and one poorly understood aspect of metastatic cancer is the adaptability of cells from a primary tumor to create new niches and survive in multiple, different secondary sites. We used quantitative mass spectrometry to analyze the extracellular matrix (ECM), a critical component of metastatic niches, in metastases to the brain, lungs, liver, and bone marrow, all derived from parental MDA-MB-231 triple-negative breast cancer cells. Tumor and stromal cells cooperated in forming niches; stromal cells produced predominantly core, structural ECM proteins and tumor cells produced a diverse array of ECM-associated proteins, including secreted factors and modulators of the matrix. In addition, tumor and stromal cells together created distinct niches in each tissue. Downregulation of SERPINB1, a protein elevated in brain metastases, led to a reduction in brain metastasis, suggesting that some niche-specific ECM proteins may be involved in metastatic tropism.
Significance:
Tumor and stromal cells together create distinct ECM niches in breast cancer metastases to various tissues, providing new insight into how tumor cells adapt to survive in different tissue environments.
Data from Postdiagnostic Fruit and Vegetable Consumption and Breast Cancer Survival: Prospective Analyses in the Nurses' Health Studies
Authors: Farvid Maryam S. , Holmes Michelle D. , Chen Wendy Y. , Rosner Bernard A. , Tamimi Rulla M. , Willett Walter C. , Eliassen A. Heather
Published: March 2023

Abstract
Fruits and vegetables contain many bioactive components that may contribute to improved survival after diagnosis of breast cancer, however, evidence to date is insufficient. We prospectively assessed the associations of postdiagnostic fruit and vegetable consumption with breast cancer–specific and all-cause mortality among 8,927 women with stage I–III breast cancer identified during follow-up of the Nurses' Health Study (NHS; 1980–2010) and NHSII (1991–2011), using a validated food frequency questionnaire completed every 4 years after diagnosis. We prospectively documented 2,521 deaths, including 1,070 from breast cancer through follow-up until 2014 in the NHS and 2015 in the NHSII. Total fruit and vegetable and total vegetable consumption was related to lower all-cause [HRQ5vsQ1, 0.82; 95% confidence interval (CI), 0.71–0.94; Ptrend = 0.004, and HRQ5vsQ1, 0.84; 95% CI, 0.72–0.97; Ptrend = 0.001, respectively], but not breast cancer–specific mortality. Total fruit consumption was not related to breast cancer–specific or all-cause mortality. Greater intake of green leafy and cruciferous vegetables was associated with lower all-cause mortality. Each 2 servings/week of blueberries was associated with a 25% (HR, 0.75; 95% CI, 0.60–0.94) lower breast cancer–specific and a 17% (HR, 0.83; 95% CI, 0.72–0.96) lower all-cause mortality. In contrast, higher fruit juice consumption was associated with higher breast cancer–specific (HRQ5vsQ1, 1.33; 95% CI, 1.09–1.63; Ptrend = 0.002) and all-cause mortality (HRQ5vsQ1, 1.19; 95% CI, 1.04–1.36; Ptrend = 0.003). Apple juice largely accounted for these higher risks and orange juice was not associated with risk. Higher postdiagnostic fruit and vegetable consumption among breast cancer survivors was not associated with breast cancer–specific mortality. However, our findings suggest that higher vegetable consumption, particularly green leafy and cruciferous vegetables, was associated with better overall survival among patients with breast cancer. Higher fruit juice consumption, but not orange juice, was associated with poorer breast cancer–specific and all-cause survival.
Significance:
A large-scale study shows that high fruit and vegetable consumption may be associated with better overall survival among breast cancer patients, while high fruit juice consumption may be associated with poorer porgnosis.
Data from The PET-Tracer 89Zr-Df-IAB22M2C Enables Monitoring of Intratumoral CD8 T-cell Infiltrates in Tumor-Bearing Humanized Mice after T-cell Bispecific Antibody Treatment
Authors: Griessinger Christoph M. , Olafsen Tove , Mascioni Alessandro , Jiang Ziyue Karen , Zamilpa Charles , Jia Fang , Torgov Michael , Romero Jason M. , Marchioni Filippo , Satpayev Daulet , Lee Chenyu , Zhang Green , Nayak Tapan K. , Pincha Mudita , Amann Maria , Mohan Preethi L.B. , Richard Marine , Nicolini Valeria G. , Sam Johannes , Claus Christina , Ferrara Claudia , Brünker Peter , Bacac Marina , Umana Pablo , Rüttinger Dominik , Wilson Ian A. , Gudas Jean , Klein Christian , Tessier Jean J.L.
Published: March 2023

Abstract
CD8-expressing T cells are the main effector cells in cancer immunotherapy. Treatment-induced changes in intratumoral CD8+ T cells may represent a biomarker to identify patients responding to cancer immunotherapy. Here, we have used a 89Zr-radiolabeled human CD8-specific minibody (89Zr-Df-IAB22M2C) to monitor CD8+ T-cell tumor infiltrates by PET. The ability of this tracer to quantify CD8+ T-cell tumor infiltrates was evaluated in preclinical studies following single-agent treatment with FOLR1-T-cell bispecific (TCB) antibody and combination therapy of CEA-TCB (RG7802) and CEA-targeted 4-1BB agonist CEA-4-1BBL. In vitro cytotoxicity assays with peripheral blood mononuclear cells and CEA-expressing MKN-45 gastric or FOLR1-expressing HeLa cervical cancer cells confirmed noninterference of the anti-CD8-PET-tracer with the mode of action of CEA-TCB/CEA-4-1BBL and FOLR1-TCB at relevant doses. In vivo, the extent of tumor regression induced by combination treatment with CEA-TCB/CEA-4-1BBL in MKN-45 tumor–bearing humanized mice correlated with intratumoral CD8+ T-cell infiltration. This was detectable by 89Zr-IAB22M2C-PET and γ-counting. Similarly, single-agent treatment with FOLR1-TCB induced strong CD8+ T-cell infiltration in HeLa tumors, where 89Zr-Df-IAB22M2C again was able to detect CD8 tumor infiltrates. CD8-IHC confirmed the PET imaging results. Taken together, the anti-CD8-minibody 89Zr-Df-IAB22M2C revealed a high sensitivity for the detection of intratumoral CD8+ T-cell infiltrates upon either single or combination treatment with TCB antibody–based fusion proteins. These results provide further evidence that the anti-CD8 tracer, which is currently in clinical phase II, is a promising monitoring tool for intratumoral CD8+ T cells in patients treated with cancer immunotherapy.
Significance:
Monitoring the pharmacodynamic activity of cancer immunotherapy with novel molecular imaging tools such as 89Zr-Df-IAB22M2C for PET imaging is of prime importance to identify patients responding early to cancer immunotherapy.
Data from Mitochondrial Haplotype of the Host Stromal Microenvironment Alters Metastasis in a Non-cell Autonomous Manner
Authors: Brinker Amanda E. , Vivian Carolyn J. , Beadnell Thomas C. , Koestler Devin C. , Teoh Shao Thing , Lunt Sophia Y. , Welch Danny R.
Published: March 2023

Abstract
Mitochondria contribute to tumor growth through multiple metabolic pathways, regulation of extracellular pH, calcium signaling, and apoptosis. Using the Mitochondrial Nuclear Exchange (MNX) mouse models, which pair nuclear genomes with different mitochondrial genomes, we previously showed that mitochondrial SNPs regulate mammary carcinoma tumorigenicity and metastatic potential in genetic crosses. Here, we tested the hypothesis that polymorphisms in stroma significantly affect tumorigenicity and experimental lung metastasis. Using syngeneic cancer cells (EO771 mammary carcinoma and B16-F10 melanoma cells) injected into wild-type and MNX mice (i.e., same nuclear DNA but different mitochondrial DNA), we showed mt-SNP–dependent increases (C3H/HeN) or decreases (C57BL/6J) in experimental metastasis. Superoxide scavenging reduced experimental metastasis. In addition, expression of lung nuclear-encoded genes changed specifically with mt-SNP. Thus, mitochondrial–nuclear cross-talk alters nuclear-encoded signaling pathways that mediate metastasis via both intrinsic and extrinsic mechanisms.
Significance:
Stromal mitochondrial polymorphisms affect metastatic colonization through reactive oxygen species and mitochondrial–nuclear cross-talk.