bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from SOX4 Is Essential for Prostate Tumorigenesis Initiated by PTEN Ablation
Authors: Bilir Birdal , Osunkoya Adeboye O. , Wiles W. Guy , Sannigrahi Soma , Lefebvre Veronique , Metzger Daniel , Spyropoulos Demetri D. , Martin W. David , Moreno Carlos S.
Published: April 2023

Abstract
Understanding remains incomplete of the mechanisms underlying initiation and progression of prostate cancer, the most commonly diagnosed cancer in American men. The transcription factor SOX4 is overexpressed in many human cancers, including prostate cancer, suggesting it may participate in prostate tumorigenesis. In this study, we investigated this possibility by genetically deleting Sox4 in a mouse model of prostate cancer initiated by loss of the tumor suppressor Pten. We found that specific homozygous deletion of Sox4 in the adult prostate epithelium strongly inhibited tumor progression initiated by homozygous loss of Pten. Mechanistically, Sox4 ablation reduced activation of AKT and β-catenin, leading to an attenuated invasive phenotype. Furthermore, SOX4 expression was induced by Pten loss as a result of the activation of PI3K–AKT–mTOR signaling, suggesting a positive feedback loop between SOX4 and PI3K–AKT–mTOR activity. Collectively, our findings establish that SOX4 is a critical component of the PTEN/PI3K/AKT pathway in prostate cancer, with potential implications for combination-targeted therapies against both primary and advanced prostate cancers. Cancer Res; 76(5); 1112–21. ©2015 AACR.
Data from STAT3 Establishes an Immunosuppressive Microenvironment during the Early Stages of Breast Carcinogenesis to Promote Tumor Growth and Metastasis
Authors: Jones Laura M. , Broz Miranda L. , Ranger Jill J. , Ozcelik John , Ahn Ryuhjin , Zuo Dongmei , Ursini-Siegel Josie , Hallett Michael T. , Krummel Matthew , Muller William J.
Published: April 2023

Abstract
Immunosurveillance constitutes the first step of cancer immunoediting in which developing malignant lesions are eliminated by antitumorigenic immune cells. However, the mechanisms by which neoplastic cells induce an immunosuppressive state to evade the immune response are still unclear. The transcription factor STAT3 has been implicated in breast carcinogenesis and tumor immunosuppression in advanced disease, but its involvement in early disease development has not been established. Here, we genetically ablated Stat3 in the tumor epithelia of the inducible PyVmT mammary tumor model and found that Stat3-deficient mice recapitulated the three phases of immunoediting: elimination, equilibrium, and escape. Pathologic analyses revealed that Stat3-deficient mice initially formed hyperplastic and early adenoma-like lesions that later completely regressed, thereby preventing the emergence of mammary tumors in the majority of animals. Furthermore, tumor regression was correlated with massive immune infiltration into the Stat3-deficient lesions, leading to their elimination. In a minority of animals, focal, nonmetastatic Stat3-deficient mammary tumors escaped immune surveillance after a long latency or equilibrium period. Taken together, our findings suggest that tumor epithelial expression of Stat3 plays a critical role in promoting an immunosuppressive tumor microenvironment during breast tumor initiation and progression, and prompt further investigation of Stat3-inhibitory strategies that may reactivate the immunosurveillance program. Cancer Res; 76(6); 1416–28. ©2015 AACR.
Data from Mass Spectrometry–Based Metabolomics Identifies Longitudinal Urinary Metabolite Profiles Predictive of Radiation-Induced Cancer
Authors: Cook John A. , Chandramouli Gadisetti V.R. , Anver Miriam R. , Sowers Anastasia L. , Thetford Angela , Krausz Kristopher W. , Gonzalez Frank J. , Mitchell James B. , Patterson Andrew D.
Published: April 2023

Abstract
Nonlethal exposure to ionizing radiation (IR) is a public concern due to its known carcinogenic effects. Although latency periods for IR-induced neoplasms are relatively long, the ability to detect cancer as early as possible is highly advantageous for effective therapeutic intervention. Therefore, we hypothesized that metabolites in the urine from mice exposed to total body radiation (TBI) would predict for the presence of cancer before a palpable mass was detected. In this study, we exposed mice to 0 or 5.4 Gy TBI, collected urine samples periodically over 1 year, and assayed urine metabolites by using mass spectrometry. Longitudinal data analysis within the first year post-TBI revealed that cancers, including hematopoietic, solid, and benign neoplasms, could be distinguished by unique urinary signatures as early as 3 months post-TBI. Furthermore, a distinction among different types of malignancies could be clearly delineated as early as 3 months post-TBI for hematopoietic neoplasms, 6 months for solid neoplasms, and by 1 year for benign neoplasms. Moreover, the feature profile for radiation-exposed mice 6 months post-TBI was found to be similar to nonirradiated control mice at 18 months, suggesting that TBI accelerates aging. These results demonstrate that urine feature profiles following TBI can identify cancers in mice prior to macroscopic detection, with important implications for the early diagnosis and treatment. Cancer Res; 76(6); 1569–77. ©2016 AACR.
Data from Integrative Genome-Scale Analysis Identifies Epigenetic Mechanisms of Transcriptional Deregulation in Unfavorable Neuroblastomas
Authors: Henrich Kai-Oliver , Bender Sebastian , Saadati Maral , Dreidax Daniel , Gartlgruber Moritz , Shao Chunxuan , Herrmann Carl , Wiesenfarth Manuel , Parzonka Martha , Wehrmann Lea , Fischer Matthias , Duffy David J. , Bell Emma , Torkov Alica , Schmezer Peter , Plass Christoph , Höfer Thomas , Benner Axel , Pfister Stefan M. , Westermann Frank
Published: April 2023

Abstract
The broad clinical spectrum of neuroblastoma ranges from spontaneous regression to rapid progression despite intensive multimodal therapy. This diversity is not fully explained by known genetic aberrations, suggesting the possibility of epigenetic involvement in pathogenesis. In pursuit of this hypothesis, we took an integrative approach to analyze the methylomes, transcriptomes, and copy number variations in 105 cases of neuroblastoma, complemented by primary tumor- and cell line–derived global histone modification analyses and epigenetic drug treatment in vitro. We found that DNA methylation patterns identify divergent patient subgroups with respect to survival and clinicobiologic variables, including amplified MYCN. Transcriptome integration and histone modification–based definition of enhancer elements revealed intragenic enhancer methylation as a mechanism for high-risk–associated transcriptional deregulation. Furthermore, in high-risk neuroblastomas, we obtained evidence for cooperation between PRC2 activity and DNA methylation in blocking tumor-suppressive differentiation programs. Notably, these programs could be re-activated by combination treatments, which targeted both PRC2 and DNA methylation. Overall, our results illuminate how epigenetic deregulation contributes to neuroblastoma pathogenesis, with novel implications for its diagnosis and therapy. Cancer Res; 76(18); 5523–37. ©2016 AACR.
Data from Redirecting Transport of Nanoparticle Albumin-Bound Paclitaxel to Macrophages Enhances Therapeutic Efficacy against Liver Metastases
Authors: Tanei Tomonori , Leonard Fransisca , Liu Xuewu , Alexander Jenolyn F. , Saito Yuki , Ferrari Mauro , Godin Biana , Yokoi Kenji
Published: April 2023

Abstract
Current treatments for liver metastases arising from primary breast and lung cancers are minimally effective. One reason for this unfavorable outcome is that liver metastases are poorly vascularized, limiting the ability to deliver therapeutics from the systemic circulation to lesions. Seeking to enhance transport of agents into the tumor microenvironment, we designed a system in which nanoparticle albumin-bound paclitaxel (nAb-PTX) is loaded into a nanoporous solid multistage nanovector (MSV) to enable the passage of the drug through the tumor vessel wall and enhance its interaction with liver macrophages. MSV enablement increased nAb-PTX efficacy and survival in mouse models of breast and lung liver metastasis. MSV-nAb-PTX also augmented the accumulation of paclitaxel and MSV in the liver, specifically in macrophages, whereas paclitaxel levels in the blood were unchanged after administering MSV-nAb-PTX or nAb-PTX. In vitro studies demonstrated that macrophages treated with MSV-nAb-PTX remained viable and were able to internalize, retain, and release significantly higher quantities of paclitaxel compared with treatment with nAb-PTX. The cytotoxic potency of the released paclitaxel was also confirmed in tumor cells cultured with the supernatants of macrophage treated with MSV-nAB-PTX. Collectively, our findings showed how redirecting nAb-PTX to liver macrophages within the tumor microenvironment can elicit a greater therapeutic response in patients with metastatic liver cancer, without increasing systemic side effects. Cancer Res; 76(2); 429–39. ©2016 AACR.
Data from Treatment of Invasive Brain Tumors Using a Chain-like Nanoparticle
Authors: Peiris Pubudu M. , Abramowski Aaron , Mcginnity James , Doolittle Elizabeth , Toy Randall , Gopalakrishnan Ramamurthy , Shah Shruti , Bauer Lisa , Ghaghada Ketan B. , Hoimes Christopher , Brady-Kalnay Susann M. , Basilion James P. , Griswold Mark A. , Karathanasis Efstathios
Published: April 2023

Abstract
Glioblastoma multiforme is generally recalcitrant to current surgical and local radiotherapeutic approaches. Moreover, systemic chemotherapeutic approaches are impeded by the blood–tumor barrier. To circumvent limitations in the latter area, we developed a multicomponent, chain-like nanoparticle that can penetrate brain tumors, composed of three iron oxide nanospheres and one drug-loaded liposome linked chemically into a linear chain-like assembly. Unlike traditional small-molecule drugs or spherical nanotherapeutics, this oblong-shaped, flexible nanochain particle possessed a unique ability to gain access to and accumulate at glioma sites. Vascular targeting of nanochains to the αvβ3 integrin receptor resulted in a 18.6-fold greater drug dose administered to brain tumors than standard chemotherapy. By 2 hours after injection, when nanochains had exited the blood stream and docked at vascular beds in the brain, the application of an external low-power radiofrequency field was sufficient to remotely trigger rapid drug release. This effect was produced by mechanically induced defects in the liposomal membrane caused by the oscillation of the iron oxide portion of the nanochain. In vivo efficacy studies conducted in two different mouse orthotopic models of glioblastoma illustrated how enhanced targeting by the nanochain facilitates widespread site-specific drug delivery. Our findings offer preclinical proof-of-concept for a broadly improved method for glioblastoma treatment. Cancer Res; 75(7); 1356–65. ©2015 AACR.
Data from Heme Oxygenase-1 Controls an HDAC4-miR-206 Pathway of Oxidative Stress in Rhabdomyosarcoma
Authors: Ciesla Maciej , Marona Paulina , Kozakowska Magdalena , Jez Mateusz , Seczynska Marta , Loboda Agnieszka , Bukowska-Strakova Karolina , Szade Agata , Walawender Magdalena , Kusior Magdalena , Stepniewski Jacek , Szade Krzysztof , Krist Bart , Yagensky Oleksandr , Urbanik Aleksandra , Kazanowska Bernarda , Dulak Jozef , Jozkowicz Alicja
Published: April 2023

Abstract
Rhabdomyosarcoma (RMS) is an aggressive soft tissue cancer characterized by disturbed myogenic differentiation. Here we report a role for the oxidative stress response factor HO-1 in progression of RMS. We found that HO-1 was elevated and its effector target miR-206 decreased in RMS cell lines and clinical primary tumors of the more aggressive alveolar phenotype (aRMS). In embryonal RMS (eRMS), HO-1 expression was induced by Pax3/7-FoxO1, an aRMS hallmark oncogene, followed by a drop in miR-206 levels. Inhibition of HO-1 by tin protoporphyrin (SnPP) or siRNA downregulated Pax3/7-FoxO1 target genes and induced a myogenic program in RMS. These effects were not mediated by altered myoD expression; instead, cells with elevated HO-1 produced less reactive oxygen species, resulting in nuclear localization of HDAC4 and miR-206 repression. HO-1 inhibition by SnPP reduced growth and vascularization of RMS tumors in vivo accompanied by induction of miR-206. Effects of SnPP on miR-206 expression and RMS tumor growth were mimicked by pharmacologic inhibition of HDAC. Thus, HO-1 inhibition activates an miR-206–dependent myogenic program in RMS, offering a novel therapeutic strategy for treatment of this malignancy. Cancer Res; 76(19); 5707–18. ©2016 AACR.
Data from Mitochondria-Targeted Analogues of Metformin Exhibit Enhanced Antiproliferative and Radiosensitizing Effects in Pancreatic Cancer Cells
Authors: Cheng Gang , Zielonka Jacek , Ouari Olivier , Lopez Marcos , McAllister Donna , Boyle Kathleen , Barrios Christy S. , Weber James J. , Johnson Bryon D. , Hardy Micael , Dwinell Michael B. , Kalyanaraman Balaraman
Published: April 2023

Abstract
Metformin (Met) is an approved antidiabetic drug currently being explored for repurposing in cancer treatment based on recent evidence of its apparent chemopreventive properties. Met is weakly cationic and targets the mitochondria to induce cytotoxic effects in tumor cells, albeit not very effectively. We hypothesized that increasing its mitochondria-targeting potential by attaching a positively charged lipophilic substituent would enhance the antitumor activity of Met. In pursuit of this question, we synthesized a set of mitochondria-targeted Met analogues (Mito-Mets) with varying alkyl chain lengths containing a triphenylphosphonium cation (TPP+). In particular, the analogue Mito-Met10, synthesized by attaching TPP+ to Met via a 10-carbon aliphatic side chain, was nearly 1,000 times more efficacious than Met at inhibiting cell proliferation in pancreatic ductal adenocarcinoma (PDAC). Notably, in PDAC cells, Mito-Met10 potently inhibited mitochondrial complex I, stimulating superoxide and AMPK activation, but had no effect in nontransformed control cells. Moreover, Mito-Met10 potently triggered G1 cell-cycle phase arrest in PDAC cells, enhanced their radiosensitivity, and more potently abrogated PDAC growth in preclinical mouse models, compared with Met. Collectively, our findings show how improving the mitochondrial targeting of Met enhances its anticancer activities, including aggressive cancers like PDAC in great need of more effective therapeutic options. Cancer Res; 76(13); 3904–15. ©2016 AACR.
Data from Cancer Differentiating Agent Hexamethylene Bisacetamide Inhibits BET Bromodomain Proteins
Authors: Nilsson Lisa M. , Green Lydia C. , Muralidharan Somsundar Veppil , Demir Dağsu , Welin Martin , Bhadury Joydeep , Logan Derek T. , Walse Björn , Nilsson Jonas A.
Published: April 2023

Abstract
Agents that trigger cell differentiation are highly efficacious in treating certain cancers, but such approaches are not generally effective in most malignancies. Compounds such as DMSO and hexamethylene bisacetamide (HMBA) have been used to induce differentiation in experimental systems, but their mechanisms of action and potential range of uses on that basis have not been developed. Here, we show that HMBA, a compound first tested in the oncology clinic over 25 years ago, acts as a selective bromodomain inhibitor. Biochemical and structural studies revealed an affinity of HMBA for the second bromodomain of BET proteins. Accordingly, both HMBA and the prototype BET inhibitor JQ1 induced differentiation of mouse erythroleukemia cells. As expected of a BET inhibitor, HMBA displaced BET proteins from chromatin, caused massive transcriptional changes, and triggered cell-cycle arrest and apoptosis in Myc-induced B-cell lymphoma cells. Furthermore, HMBA exerted anticancer effects in vivo in mouse models of Myc-driven B-cell lymphoma. This study illuminates the function of an early anticancer agent and suggests an intersection with ongoing clinical trials of BET inhibitor, with several implications for predicting patient selection and response rates to this therapy and starting points for generating BD2-selective BET inhibitors. Cancer Res; 76(8); 2376–83. ©2016 AACR.
Data from Gender-Specific Molecular and Clinical Features Underlie Malignant Pleural Mesothelioma
Authors: De Rienzo Assunta , Archer Michael A. , Yeap Beow Y. , Dao Nhien , Sciaranghella Daniele , Sideris Antonios C. , Zheng Yifan , Holman Alexander G. , Wang Yaoyu E. , Dal Cin Paola S. , Fletcher Jonathan A. , Rubio Renee , Croft Larry , Quackenbush John , Sugarbaker Peter E. , Munir Kiara J. , Battilana Jesse R. , Gustafson Corinne E. , Chirieac Lucian R. , Ching Soo Meng , Wong James , Tay Liang Chung , Rudd Stephen , Hercus Robert , Sugarbaker David J. , Richards William G. , Bueno Raphael
Published: April 2023

Abstract
Malignant pleural mesothelioma (MPM) is an aggressive cancer that occurs more frequently in men, but is associated with longer survival in women. Insight into the survival advantage of female patients may advance the molecular understanding of MPM and identify therapeutic interventions that will improve the prognosis for all MPM patients. In this study, we performed whole-genome sequencing of tumor specimens from 10 MPM patients and matched control samples to identify potential driver mutations underlying MPM. We identified molecular differences associated with gender and histology. Specifically, single-nucleotide variants of BAP1 were observed in 21% of cases, with lower mutation rates observed in sarcomatoid MPM (P < 0.001). Chromosome 22q loss was more frequently associated with the epithelioid than that nonepitheliod histology (P = 0.037), whereas CDKN2A deletions occurred more frequently in nonepithelioid subtypes among men (P = 0.021) and were correlated with shorter overall survival for the entire cohort (P = 0.002) and for men (P = 0.012). Furthermore, women were more likely to harbor TP53 mutations (P = 0.004). Novel mutations were found in genes associated with the integrin-linked kinase pathway, including MYH9 and RHOA. Moreover, expression levels of BAP1, MYH9, and RHOA were significantly higher in nonepithelioid tumors, and were associated with significant reduction in survival of the entire cohort and across gender subgroups. Collectively, our findings indicate that diverse mechanisms highly related to gender and histology appear to drive MPM. Cancer Res; 76(2); 319–28. ©2015 AACR.
Data from Lack of p53 Augments Antitumor Functions in Cytolytic T Cells
Authors: Banerjee Anirban , Thyagarajan Krishnamurthy , Chatterjee Shilpak , Chakraborty Paramita , Kesarwani Pravin , Soloshchenko Myroslawa , Al-Hommrani Mazen , Andrijauskaite Kristina , Moxley Kelly , Janakiraman Harinarayanan , Scheffel Matthew J. , Helke Kristi , Armenson Kent , Palanisamy Viswanathan , Rubinstein Mark P. , Mayer Elizabeth-Garrett , Cole David J. , Paulos Chrystal M. , Voelkel-Johnson Christina , Nishimura Michael I. , Mehrotra Shikhar
Published: April 2023

Abstract
Repetitive stimulation of T-cell receptor (TCR) with cognate antigen results in robust proliferation and expansion of the T cells, and also imprints them with replicative senescence signatures. Our previous studies have shown that life-span and antitumor function of T cells can be enhanced by inhibiting reactive oxygen species (ROS) or intervening with ROS-dependent JNK activation that leads to its activation-induced cell death. Because tumor suppressor protein p53 is also a redox active transcription factor that regulates cellular ROS generation that triggers downstream factor–mediating apoptosis, we determined if p53 levels could influence persistence and function of tumor-reactive T cells. Using h3T TCR transgenic mice, with human tyrosinase epitope–reactive T cells developed on p53 knockout (KO) background, we determined its role in regulating antitumor T-cell function. Our data show that as compared with h3T cells, h3T-p53 KO T cells exhibited enhanced glycolytic commitment that correlated with increased proliferation, IFNγ secretion, cytolytic capacity, expression of stemness gene signature, and decreased TGF-β signaling. This increased effector function correlated to the improved control of subcutaneously established murine melanoma after adoptive transfer of p53-KO T cells. Pharmacological inhibition of human TCR-transduced T cells using a combination of p53 inhibitors also potentiated the T-cell effector function and improved persistence. Thus, our data highlight the key role of p53 in regulating the tumor-reactive T-cell response and that targeting this pathway could have potential translational significance in adoptive T-cell therapy. Cancer Res; 76(18); 5229–40. ©2016 AACR.
Data from SIRT2-Mediated Deacetylation and Tetramerization of Pyruvate Kinase Directs Glycolysis and Tumor Growth
Authors: Park Seong-Hoon , Ozden Ozkan , Liu Guoxiang , Song Ha Yong , Zhu Yueming , Yan Yufan , Zou Xianghui , Kang Hong-Jun , Jiang Haiyan , Principe Daniel R. , Cha Yong-Il , Roh Meejeon , Vassilopoulos Athanassios , Gius David
Published: April 2023

Abstract
Sirtuins participate in sensing nutrient availability and directing metabolic activity to match energy needs with energy production and consumption. However, the pivotal targets for sirtuins in cancer are mainly unknown. In this study, we identify the M2 isoform of pyruvate kinase (PKM2) as a critical target of the sirtuin SIRT2 implicated in cancer. PKM2 directs the synthesis of pyruvate and acetyl-CoA, the latter of which is transported to mitochondria for use in the Krebs cycle to generate ATP. Enabled by a shotgun mass spectrometry analysis founded on tissue culture models, we identified a candidate SIRT2 deacetylation target at PKM2 lysine 305 (K305). Biochemical experiments including site-directed mutants that mimicked constitutive acetylation suggested that acetylation reduced PKM2 activity by preventing tetramerization to the active enzymatic form. Notably, ectopic overexpression of a deacetylated PKM2 mutant in Sirt2-deficient mammary tumor cells altered glucose metabolism and inhibited malignant growth. Taken together, our results argued that loss of SIRT2 function in cancer cells reprograms their glycolytic metabolism via PKM2 regulation, partially explaining the tumor-permissive phenotype of mice lacking Sirt2. Cancer Res; 76(13); 3802–12. ©2016 AACR.
Data from Pharmacological Inhibition of the Histone Lysine Demethylase KDM1A Suppresses the Growth of Multiple Acute Myeloid Leukemia Subtypes
Authors: McGrath John P. , Williamson Kaylyn E. , Balasubramanian Srividya , Odate Shobu , Arora Shilpi , Hatton Charlie , Edwards Thomas M. , O'Brien Thomas , Magnuson Steven , Stokoe David , Daniels Danette L. , Bryant Barbara M. , Trojer Patrick
Published: April 2023

Abstract
Lysine-specific demethylase 1 (KDM1A) is a transcriptional coregulator that can function in both the activation and repression of gene expression, depending upon context. KDM1A plays an important role in hematopoiesis and was identified as a dependency factor in leukemia stem cell populations. Therefore, we investigated the consequences of inhibiting KDM1A in a panel of cell lines representing all acute myelogenous leukemia (AML) subtypes using selective, reversible and irreversible KDM1A small-molecule inhibitors. Cell models of AML, CML, and T-ALL were potently affected by KDM1A inhibition, and cells bearing RUNX1-RUNX1T1 (AML1-ETO) translocations were especially among the most sensitive. RNAi-mediated silencing of KDM1A also effectively suppressed growth of RUNX1-RUNX1T1–containing cell lines. Furthermore, pharmacologic inhibition of KDM1A resulted in complete abrogation of tumor growth in an AML xenograft model harboring RUNX1–RUNX1T1 translocations. We unexpectedly found that KDM1A-targeting compounds not only inhibited the catalytic activity of the enzyme, but evicted KDM1A from target genes. Accordingly, compound-mediated KDM1A eviction was associated with elevated levels of local histone H3 lysine 4 dimethylation, and increased target gene expression, which was further accompanied by cellular differentiation and induction of cell death. Finally, our finding that KDM1A inhibitors effectively synergize with multiple conventional as well as candidate anti-AML agents affords a framework for potential future clinical application. Cancer Res; 76(7); 1975–88. ©2016 AACR.
Data from Androgen Receptor Upregulation Mediates Radioresistance after Ionizing Radiation
Authors: Spratt Daniel E. , Evans Michael J. , Davis Brian J. , Doran Michael G. , Lee Man Xia , Shah Neel , Wongvipat John , Carnazza Kathryn E. , Klee George G. , Polkinghorn William , Tindall Donald J. , Lewis Jason S. , Sawyers Charles L.
Published: April 2023

Abstract
Clinical trials have established the benefit of androgen deprivation therapy (ADT) combined with radiotherapy in prostate cancer. ADT sensitizes prostate cancer to radiotherapy-induced death at least in part through inhibition of DNA repair machinery, but for unknown reasons, adjuvant ADT provides further survival benefits. Here, we show that androgen receptor (AR) expression and activity are durably upregulated following radiotherapy in multiple human prostate cancer models in vitro and in vivo. Moreover, the degree of AR upregulation correlates with survival in vitro and time to tumor progression in animal models. We also provide evidence of AR pathway upregulation, measured by a rise in serum levels of AR-regulated hK2 protein, in nearly 20% of patients after radiotherapy. Furthermore, these men were three-fold more likely to experience subsequent biochemical failure. Collectively, these data demonstrate that radiotherapy can upregulate AR signaling after therapy to an extent that negatively affects disease progression and/or survival. Cancer Res; 75(22); 4688–96. ©2015 AACR.
Data from Cyclin A1 and P450 Aromatase Promote Metastatic Homing and Growth of Stem-like Prostate Cancer Cells in the Bone Marrow
Authors: Miftakhova Regina , Hedblom Andreas , Semenas Julius , Robinson Brian , Simoulis Athanasios , Malm Johan , Rizvanov Albert , Heery David M. , Mongan Nigel P. , Maitland Norman J. , Allegrucci Cinzia , Persson Jenny L.
Published: April 2023

Abstract
Bone metastasis is a leading cause of morbidity and mortality in prostate cancer. While cancer stem-like cells have been implicated as a cell of origin for prostate cancer metastasis, the pathways that enable metastatic development at distal sites remain largely unknown. In this study, we illuminate pathways relevant to bone metastasis in this disease. We observed that cyclin A1 (CCNA1) protein expression was relatively higher in prostate cancer metastatic lesions in lymph node, lung, and bone/bone marrow. In both primary and metastatic tissues, cyclin A1 expression was also correlated with aromatase (CYP19A1), a key enzyme that directly regulates the local balance of androgens to estrogens. Cyclin A1 overexpression in the stem-like ALDHhigh subpopulation of PC3M cells, one model of prostate cancer, enabled bone marrow integration and metastatic growth. Further, cells obtained from bone marrow metastatic lesions displayed self-renewal capability in colony-forming assays. In the bone marrow, cyclin A1 and aromatase enhanced local bone marrow-releasing factors, including androgen receptor, estrogen and matrix metalloproteinase MMP9 and promoted the metastatic growth of prostate cancer cells. Moreover, ALDHhigh tumor cells expressing elevated levels of aromatase stimulated tumor/host estrogen production and acquired a growth advantage in the presence of host bone marrow cells. Overall, these findings suggest that local production of steroids and MMPs in the bone marrow may provide a suitable microenvironment for ALDHhigh prostate cancer cells to establish metastatic growths, offering new approaches to therapeutically target bone metastases. Cancer Res; 76(8); 2453–64. ©2016 AACR.
Data from Tumor Suppressor HIPK2 Regulates Malignant Growth via Phosphorylation of Notch1
Authors: Ann Eun-Jung , Kim Mi-Yeon , Yoon Ji-Hye , Ahn Ji-Seon , Jo Eun-Hye , Lee Hye-Jin , Lee Hyun-Woo , Kang Hyeok-Gu , Choi Dong Wook , Chun Kyung-Hee , Lee Ji Shin , Choi Cheol Yong , Ferrando Adolfo A. , Lee Keesook , Park Hee-Sae
Published: April 2023

Abstract
The receptor Notch1 plays an important role in malignant progression of many cancers, but its regulation is not fully understood. In this study, we report that the kinase HIPK2 is responsible for facilitating the Fbw7-dependent proteasomal degradation of Notch1 by phosphorylating its intracellular domain (Notch1-IC) within the Cdc4 phosphodegron motif. Notch1-IC expression was higher in cancer cells than normal cells. Under genotoxic stress, Notch1-IC was phosphorylated constitutively by HIPK2 and was maintained at a low level through proteasomal degradation. HIPK2 phosphorylated the residue T2512 in Notch1-IC. Somatic mutations near this residue rendered Notch1-IC resistant to degradation, as induced either by HIPK2 overexpression or adriamycin treatment. In revealing an important mechanism of Notch1 stability, the results of this study could offer a therapeutic strategy to block Notch1-dependent progression in many types of cancer. Cancer Res; 76(16); 4728–40. ©2016 AACR.
Data from Tyrosine Phosphatase PTPRJ/DEP-1 Is an Essential Promoter of Vascular Permeability, Angiogenesis, and Tumor Progression
Authors: Fournier Patrick , Dussault Sylvie , Fusco Alfredo , Rivard Alain , Royal Isabelle
Published: April 2023

Abstract
The protein tyrosine phosphatase PTPRJ/DEP-1 has been implicated in negative growth regulation in endothelial cells, where its expression varies at transitions between proliferation and contact inhibition. However, in the same cells, DEP-1 has also been implicated in VEGF-dependent Src activation, permeability, and capillary formation, suggesting a positive role in regulating these functions. To resolve this dichotomy in vivo, we investigated postnatal angiogenesis and vascular permeability in a DEP-1–deficient mouse. In this study, we report that DEP-1 is required for Src activation and phosphorylation of its endothelial cell–specific substrate, VE-cadherin, after systemic injection of VEGF. Accordingly, VEGF-induced vascular leakage was abrogated in the DEP-1–deficient mice. Furthermore, capillary formation was impaired in murine aortic tissue rings or Matrigel plugs infused with VEGF. In the absence of DEP-1, angiogenesis triggered by ischemia or during tumor formation was defective, which in the latter case was associated with reduced tumor cell proliferation and increased apoptosis. Macrophage infiltration was also impaired, reflecting reduced vascular permeability in the tumors or a possible cell autonomous effect of DEP-1. Consequently, the formation of spontaneous and experimental lung metastases was strongly decreased in DEP-1–deficient mice. In clinical specimens of cancer, less vascularized tumors exhibited lower microvascular expression of DEP-1. Altogether, our results established DEP-1 as an essential driver of VEGF-dependent permeability, angiogenesis, and metastasis, suggesting a novel therapeutic route to cancer treatment. Cancer Res; 76(17); 5080–91. ©2016 AACR.
Supplementary Table 1 from Modeling of the Growth Kinetics of Occult Breast Tumors: Role in Interpretation of Studies of Prevention and Menopausal Hormone Therapy
Authors: Santen Richard J. , Yue Wei , Heitjan Daniel F.
Published: April 2023

PDF file, 48KB, Table with review of pevalence of contralateral occult tumors.
Related Article from Cabozantinib and Prostate Cancer: Inhibiting Seed and Disrupting Soil?
Authors: Lee Richard J. , Smith Matthew R.
Published: April 2023

Related Article from Cabozantinib and Prostate Cancer: Inhibiting Seed and Disrupting Soil?
Supplementary Figure S3 from Targeting Small Cell Lung Cancer Harboring PIK3CA Mutation with a Selective Oral PI3K Inhibitor PF-4989216
Authors: Walls Marlena , Baxi Sangita M. , Mehta Pramod P. , Liu Kevin K.-C. , Zhu JinJiang , Estrella Heather , Li Chunze , Zientek Michael , Zong Qing , Smeal Tod , Yin Min-Jean
Published: April 2023

PDF file 196K, Supplementary Figure S3: PF-4989216 induced cell cycle progression block and increased the sub-G1 population in SCLCs with PIK3CA mutation
Supplementary Table 4 from Unique DNA Methylation Loci Distinguish Anatomic Site and HPV Status in Head and Neck Squamous Cell Carcinoma
Authors: Lleras Roberto A. , Smith Richard V. , Adrien Leslie R. , Schlecht Nicolas F. , Burk Robert D. , Harris Thomas M. , Childs Geoffrey , Prystowsky Michael B. , Belbin Thomas J.
Published: April 2023

PDF file, 1946K, Validation of hypermethylated CpG loci identified in HNSCC using TCGA data.
Supplementary Figure Legend from Stearoyl-CoA Desaturase 1 Is a Novel Molecular Therapeutic Target for Clear Cell Renal Cell Carcinoma
Authors: von Roemeling Christina A. , Marlow Laura A. , Wei Johnny J. , Cooper Simon J. , Caulfield Thomas R. , Wu Kevin , Tan Winston W. , Tun Han W. , Copland John A.
Published: April 2023

Supplementary Figure Legend - PDF file 92K, Figure legends for Supplementary Figures
Supplementary Figure 7 from Stearoyl-CoA Desaturase 1 Is a Novel Molecular Therapeutic Target for Clear Cell Renal Cell Carcinoma
Authors: von Roemeling Christina A. , Marlow Laura A. , Wei Johnny J. , Cooper Simon J. , Caulfield Thomas R. , Wu Kevin , Tan Winston W. , Tun Han W. , Copland John A.
Published: April 2023

Supplementary Figure 7: temsirolimus and SCD1 inhibitor synergy soft agar - PDF file 3573K, Treatment of ccRCC cells with A939572 in combination with the mTOR inhibitor Temsirolimus synergistically inhibits tumor cell growth in soft agar
Supplementary Data from Stearoyl-CoA Desaturase 1 Is a Novel Molecular Therapeutic Target for Clear Cell Renal Cell Carcinoma
Authors: von Roemeling Christina A. , Marlow Laura A. , Wei Johnny J. , Cooper Simon J. , Caulfield Thomas R. , Wu Kevin , Tan Winston W. , Tun Han W. , Copland John A.
Published: April 2023

Supplementary Data from Stearoyl-CoA Desaturase 1 Is a Novel Molecular Therapeutic Target for Clear Cell Renal Cell Carcinoma
Supplementary Table 2 from MGMT-STP27 Methylation Status as Predictive Marker for Response to PCV in Anaplastic Oligodendrogliomas and Oligoastrocytomas. A Report from EORTC Study 26951
Authors: van den Bent Martin J. , Erdem-Eraslan Lale , Idbaih Ahmed , de Rooi Johan , Eilers Paul H.C. , Spliet Wim G.M. , den Dunnen Wilfred F.A. , Tijssen Cees , Wesseling Pieter , Sillevis Smitt Peter A.E. , Kros Johan M. , Gorlia Thierry , French Pim J.
Published: April 2023

Supplementary Table 2 - PDF file 58K, Comparison of baseline characteristics between patients of the EORTC26951 clinical trial that were included v. not included for genome wide methylation analysis
Supplementary Table S4 from Molecular Classification of Malignant Pleural Mesothelioma: Identification of a Poor Prognosis Subgroup Linked to the Epithelial-to-Mesenchymal Transition
Authors: de Reyniès Aurélien , Jaurand Marie-Claude , Renier Annie , Couchy Gabrielle , Hysi Ilir , Elarouci Nabila , Galateau-Sallé Françoise , Copin Marie-Christine , Hofman Paul , Cazes Aurélie , Andujar Pascal , Imbeaud Sandrine , Petel Fabien , Pairon Jean-Claude , Le Pimpec-Barthes Francoise , Zucman-Rossi Jessica , Jean Didier
Published: April 2023

XLS file 669K, This file contains supplementary table S4: Convergence between microRNA and mRNA deregulation
Supplementary Figure 3 from MGMT-STP27 Methylation Status as Predictive Marker for Response to PCV in Anaplastic Oligodendrogliomas and Oligoastrocytomas. A Report from EORTC Study 26951
Authors: van den Bent Martin J. , Erdem-Eraslan Lale , Idbaih Ahmed , de Rooi Johan , Eilers Paul H.C. , Spliet Wim G.M. , den Dunnen Wilfred F.A. , Tijssen Cees , Wesseling Pieter , Sillevis Smitt Peter A.E. , Kros Johan M. , Gorlia Thierry , French Pim J.
Published: April 2023

Supplementary Figure 3 - PDF file 186K, SAM analysis of survival within CIMP+ or CIMP- tumors
Supplementary Figure 2 from Prevailing Role of Contact Guidance in Intrastromal T-cell Trapping in Human Pancreatic Cancer
Authors: Hartmann Natalie , Giese Nathalia A. , Giese Thomas , Poschke Isabel , Offringa Rienk , Werner Jens , Ryschich Eduard
Published: April 2023

PDF file - 44KB, T cell migration in response to SDF-1 and IP-10 in collagen matrix of different densities with and without beta1-integrin blockade. Migration of activated PBTs in a collagen type I matrix was not changed through the uniform chemokine concentration and was independent of beta1-integrin blockade.
Supplementary Figure Legend from JMJD2B Promotes Epithelial–Mesenchymal Transition by Cooperating with β-Catenin and Enhances Gastric Cancer Metastasis
Authors: Zhao Li , Li Wenjuan , Zang Wen , Liu Zhifang , Xu Xia , Yu Han , Yang Qing , Jia Jihui
Published: April 2023

PDF file - 115K
Supplementary Figure 1 from JMJD2B Promotes Epithelial–Mesenchymal Transition by Cooperating with β-Catenin and Enhances Gastric Cancer Metastasis
Authors: Zhao Li , Li Wenjuan , Zang Wen , Liu Zhifang , Xu Xia , Yu Han , Yang Qing , Jia Jihui
Published: April 2023

PDF file - 111K, Supplementary Figure S1. The effect of JMJD2B knockdown on gastric cancer cell migration, invasion and metastasis.
Supplementary Table 3 from A Phase I Trial of Dasatinib, an Src-Family Kinase Inhibitor, in Combination with Paclitaxel and Carboplatin in Patients with Advanced or Recurrent Ovarian Cancer
Authors: Secord Angeles Alvarez , Teoh Deanna K. , Barry William T. , Yu Miao , Broadwater Gloria , Havrilesky Laura J. , Lee Paula S. , Berchuck Andrew , Lancaster Johnathan , Wenham Robert M.
Published: April 2023

PDF file, 84K, Differentially regulated genes.
Supplementary Figure 6 from Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma
Authors: Rader JulieAnn , Russell Mike R. , Hart Lori S. , Nakazawa Michael S. , Belcastro Lili T. , Martinez Daniel , Li Yimei , Carpenter Erica L. , Attiyeh Edward F. , Diskin Sharon J. , Kim Sunkyu , Parasuraman Sudha , Caponigro Giordano , Schnepp Robert W. , Wood Andrew C. , Pawel Bruce , Cole Kristina A. , Maris John M.
Published: April 2023

Supplementary Figure 6 - PDF file 75K, Statistical analysis of in vivo growth suppression. (A) Growth rates of BE2C, 1643, and EBC1 xenografts treated with 200 mg/kg LEE011 or vehicle, as determined by linear mixed effects analysis. (B) Summary table of statistics
Supplementary Information from Identification and Selective Degradation of Neopeptide-Containing Truncated Mutant Proteins in the Tumors with High Microsatellite Instability
Authors: Kim Won Kyu , Park Misun , Park Minhee , Kim Yun Ji , Shin Nara , Kim Hyun Ki , You Kwon Tae , Kim Hoguen
Published: April 2023

Supplementary Information - PDF file 587K, Figure S1. Semiquantitative RT-PCR analysis of the selected genes in 10 MSI-H and 2 MSS cell lines. Figure S2. Endogenous levels of wild type TTK, TCF7L2, and MARCKS proteins decrease according to the mutation status. Figure S3. Generation of an antibody specific for neopeptides of mutant MARCKS. Figure S4. Endogenous mutant TTK, TCF7L2, and MARCKS are detected after proteasome inhibition. Figure S5. Inhibition of proteasome degradation by MG132 increased the expression of the mutants derived from the cDNA expression constructs. Figure S6. Generation of neopeptides from frameshift mutations in 29 genes that displayed frequent insertion/deletion mutations. Table S1: Primers used for RT-PCR and construction of expression vectors. Table S2. List of genes containing coding mononucleotide repeats longer than 9 nt in the last exon
Supplementary Methods from A Novel Therapeutic Regimen to Eradicate Established Solid Tumors with an Effective Induction of Tumor-Specific Immunity
Authors: Tysome James R. , Li Xiaozhu , Wang Shengdian , Wang Pengju , Gao Dongling , Du Pan , Chen Dong , Gangeswaran Rathi , Chard Louisa S. , Yuan Ming , Alusi Ghassan , Lemoine Nicholas R. , Wang Yaohe
Published: April 2023

PDF file - 76K, Development of the antibody against Syrian Hamster CD3
Supplementary Figure 2 from A Novel Therapeutic Regimen to Eradicate Established Solid Tumors with an Effective Induction of Tumor-Specific Immunity
Authors: Tysome James R. , Li Xiaozhu , Wang Shengdian , Wang Pengju , Gao Dongling , Du Pan , Chen Dong , Gangeswaran Rathi , Chard Louisa S. , Yuan Ming , Alusi Ghassan , Lemoine Nicholas R. , Wang Yaohe
Published: April 2023

PDF file - 174K, Flow cytometry analysis of mAb 4F11 and 4F4 applied to 293T cells over-expressing EGFP-hamster CD3e fusion protein
Supplementary Figure 4 from The Use of Quantitative Real-Time Reverse Transcriptase PCR for 5′ and 3′ Portions of ALK Transcripts to Detect ALK Rearrangements in Lung Cancers
Authors: Wang Rui , Pan Yunjian , Li Chenguang , Hu Haichuan , Zhang Yang , Li Hang , Luo Xiaoyang , Zhang Jie , Fang Zhaoyuan , Li Yuan , Shen Lei , Ji Hongbin , Garfield David , Sun Yihua , Chen Haiquan
Published: April 2023

PDF file, 10567K, Identification of novel variants of EML4-ALK.
Supplementary Table 1 from The Nedd8-Activating Enzyme Inhibitor MLN4924 Thwarts Microenvironment-Driven NF-κB Activation and Induces Apoptosis in Chronic Lymphocytic Leukemia B Cells
Authors: Godbersen J. Claire , Humphries Leigh Ann , Danilova Olga V. , Kebbekus Peter E. , Brown Jennifer R. , Eastman Alan , Danilov Alexey V.
Published: April 2023

PDF file - 80KB, Gene expression profiling reveals a decrease in NFκB-driven gene signature pattern upon treatment with MLN4924. CLL cells were co-cultured with CD40L-expressing stroma for 18 hours and treated or not with 1 microM MLN4924 for additional 24 hours. RNA was isolated from the purified CLL B-cells and microarray analysis was performed. The table shows fold change in selected NFkappaB target genes.
Supplementary Table 1 from The Nedd8-Activating Enzyme Inhibitor MLN4924 Thwarts Microenvironment-Driven NF-κB Activation and Induces Apoptosis in Chronic Lymphocytic Leukemia B Cells
Authors: Godbersen J. Claire , Humphries Leigh Ann , Danilova Olga V. , Kebbekus Peter E. , Brown Jennifer R. , Eastman Alan , Danilov Alexey V.
Published: April 2023

Gene expression profiling reveals a decrease in NFÃŽÂºB-driven gene signature pattern upon treatment with MLN4924. CLL cells were co-cultured with CD40L-expressing stroma for 18 hours and treated or not with 1 ÃŽÂ¼M MLN4924 for additional 24 hours. RNA was isolated from the purified CLL B-cells and microarray analysis was performed. The table shows fold change in selected NFÃŽÂºB target genes.
Supplementary Figure 7 from The Nedd8-Activating Enzyme Inhibitor MLN4924 Thwarts Microenvironment-Driven NF-κB Activation and Induces Apoptosis in Chronic Lymphocytic Leukemia B Cells
Authors: Godbersen J. Claire , Humphries Leigh Ann , Danilova Olga V. , Kebbekus Peter E. , Brown Jennifer R. , Eastman Alan , Danilov Alexey V.
Published: April 2023

PDF file - 123KB, Effect of MLN4924 on Bim expression in CLL. CLL cells were co-cultured with CD40L-expressing stroma for 24 hours, followed by incubation with 1 microM MLN4924 or vehicle control. Cell were lysed at 24 hours and subjected to immunoblotting. Raji cells (American Type Culture Collection) were cultured in RPMI 1640 with 10% FBS without (first lane) or with (second lane) 50 microg/L phorbol 12-myristate 13-acetate for 30 min to serve as control for expression of phospho-Bim.
Supporting video 1 from A NOTCH3 Transcriptional Module Induces Cell Motility in Neuroblastoma
Authors: van Nes Johan , Chan Alvin , van Groningen Tim , van Sluis Peter , Koster Jan , Versteeg Rogier
Published: April 2023

Supporting video 1 - MOV file 1654K, IMR32-NOTCH3IC cells without doxycycline. Note the continuous presence of a single cell
Supplementary Tables from A NOTCH3 Transcriptional Module Induces Cell Motility in Neuroblastoma
Authors: van Nes Johan , Chan Alvin , van Groningen Tim , van Sluis Peter , Koster Jan , Versteeg Rogier
Published: April 2023

Supplementary Tables - XLS file 163K. This file contains 6 supplementary tables
Supplementary Figures from A NOTCH3 Transcriptional Module Induces Cell Motility in Neuroblastoma
Authors: van Nes Johan , Chan Alvin , van Groningen Tim , van Sluis Peter , Koster Jan , Versteeg Rogier
Published: April 2023

Supplementary Figures - PDF file 2758K, This file contains supplementary figure 1-7
Supplementary Figure 2 from Targeting miR-21 Inhibits In Vitro and In Vivo Multiple Myeloma Cell Growth
Authors: Leone Emanuela , Morelli Eugenio , Di Martino Maria T. , Amodio Nicola , Foresta Umberto , Gullà Annamaria , Rossi Marco , Neri Antonino , Giordano Antonio , Munshi Nikhil C. , Anderson Kenneth C. , Tagliaferri Pierosandro , Tassone Pierfrancesco
Published: April 2023

PDF file - 263K, Figure S2. miR-21 inhibitors do not affect growth of MM cell lines with low miR-21.
Supplementary Figures 1 - 13 from Synergistic Activity of Bortezomib and HDACi in Preclinical Models of B-cell Precursor Acute Lymphoblastic Leukemia via Modulation of p53, PI3K/AKT, and NF-κB
Authors: Bastian Lorenz , Hof Jana , Pfau Madlen , Fichtner Iduna , Eckert Cornelia , Henze Günter , Prada Javier , von Stackelberg Arend , Seeger Karl , Shalapour Shabnam
Published: April 2023

PDF file - 2592K, S1: Detailed information on the cell death analysis after VPA/BTZ treatment at multiple dose levels of each drug; S2: Synergistic effects of concomitant VPA/BTZ treatment in Nalm6 and primary BCP-ALL cells; S3: Cytotoxic effects of VPA/BTZ treatment on human PBMC; S4: Cytotoxic effects of VPA/BTZ treatment on murine BM-MNC; S5: Cytotoxic effects on murine splenocytes; S6: Additional information on apoptosis, cell cycle, VLA-4 expression, p53, and protein ubiquitination pathway; S7: PI3K/AKT inhibitor treatments in combination with VPA/BTZ; S8: Combination effect of VPA/BTZ in LC cocultures with MSC; S9: p65 and MDM2 in cocultures after VPA/BTZ treatment; S10: Information on the i.v.-xenograft experiment; S11: Treatment schemes and analyses of body weight and hematotoxicity in s.c.-xenograft experiments; S12: Tumor growth, method of cl. Casp. 3 quantification and expression of ERTR-7 in tumors of the s.c.-xenograft mouse model; S13: Detailed analysis of cell death after combinations of VPA/BTZ with chemotherapeutics.
Supplementary Figure 6 from Systemic Administration of a Novel Immune-Stimulatory Pseudovirion Suppresses Lung Metastatic Melanoma by Regionally Enhancing IFN-γ Production
Authors: Saga Kotaro , Tamai Katsuto , Yamazaki Takehiko , Kaneda Yasufumi
Published: April 2023

PDF file - 67K, Serum IFN-γ levels induced upon the systemic administration of scIL-12-HVJ-E and the scIL-12 and Fc-HVJ-E mixture.
Supplementary Figure 5 from Systemic Administration of a Novel Immune-Stimulatory Pseudovirion Suppresses Lung Metastatic Melanoma by Regionally Enhancing IFN-γ Production
Authors: Saga Kotaro , Tamai Katsuto , Yamazaki Takehiko , Kaneda Yasufumi
Published: April 2023

PDF file - 113K, Tissue localization of ΔHN-HVJ-E following intravenous injection, and generation of the murine F10 metastatic lung melanoma model.
Supplementary Figure 1 from Castration Therapy Results in Decreased Ku70 Levels in Prostate Cancer
Authors: Al-Ubaidi Firas L. T. , Schultz Niklas , Loseva Olga , Egevad Lars , Granfors Torvald , Helleday Thomas
Published: April 2023

PDF file - 100K, Figure S1 ADT induced Ku70 decrease correlates with a decrease of PSA in prostate tumours
Supplementary Figure 4 from Systemic Administration of a Novel Immune-Stimulatory Pseudovirion Suppresses Lung Metastatic Melanoma by Regionally Enhancing IFN-γ Production
Authors: Saga Kotaro , Tamai Katsuto , Yamazaki Takehiko , Kaneda Yasufumi
Published: April 2023

PDF file - 103K, Measurement of the ZZ-scIL-12 on Fc-HVJ-E.
Supplementary Figure 1 from Systemic Administration of a Novel Immune-Stimulatory Pseudovirion Suppresses Lung Metastatic Melanoma by Regionally Enhancing IFN-γ Production
Authors: Saga Kotaro , Tamai Katsuto , Yamazaki Takehiko , Kaneda Yasufumi
Published: April 2023

PDF file - 137K, FACS analysis of IFN-γ-positive splenocytes.