ABSTRACT
Head and neck squamous cell carcinomas (HNSCC) preferentially spread to regional cervical tissues and lymph nodes. Here, we hypothesized that lymphotoxin-ß (LTß), receptor LTßR, and NF-κB-inducing kinase (NIK), promote the aberrant activation of alternative NF-κB2/RELB pathway and genes, that enhance migration and invasion of HNSCC. Genomic and expression alterations of the alternative NF-kB pathway were examined in 279 HNSCC tumors from The Cancer Genome Atlas (TCGA) and a panel of HNSCC lines. LTßR is amplified or overexpressed in HNSCC of the larynx or oral cavity, while LTß, NIK, and RELB are overexpressed in cancers arising within lymphoid oropharyngeal and tonsillar sites. Similarly, subsets of HNSCC lines displayed overexpression of LTßR, NIK, and RELB proteins. Recombinant LTß, and siRNA depletion of endogenous LTßR and NIK, modulated expression of LTßR, NIK, and nuclear translocation of NF-κB2(p52)/RELB as well as functional NF-κB promoter reporter activity. Treatment with a NIK inhibitor (1,3[2H,4H]-Iso-Quinoline Dione) reduced the protein expression of NIK and NF-κB2(p52)/RELB, and blocked LTß induced nuclear translocation of RELB. NIK and RELB siRNA knockdown or NIK inhibitor slowed HNSCC migration or invation in vitro. LTß-induces expression of migration and metastasis related genes, including hepatocyte growth/scatter factor receptor MET. Knockdown of NIK or MET similarly inhibited the migration of HNSCC cell lines. This may help explain why HNSCC preferentially migrate to local lymph nodes, where LTß is expressed. Our findings show that LTß/LTßR promotes activation of the alternative NIK-NF-κB2/RELB pathway to enhance MET-mediated cell migration in HNSCC, which could be potential therapeutic targets in HNSCC.
Subject(s)
Carcinoma, Squamous Cell/secondary , Head and Neck Neoplasms/pathology , Lymphotoxin beta Receptor/metabolism , Lymphotoxin-alpha/metabolism , Lymphotoxin-beta/metabolism , Protein Serine-Threonine Kinases/metabolism , Transcription Factor RelB/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Humans , Lymphotoxin beta Receptor/genetics , Lymphotoxin-alpha/genetics , Lymphotoxin-beta/genetics , Prognosis , Protein Serine-Threonine Kinases/genetics , Signal Transduction , Transcription Factor RelB/genetics , Tumor Cells, Cultured , NF-kappaB-Inducing KinaseABSTRACT
BACKGROUND: Breast cancer is the most common invasive cancer among women. Currently, there are only a few models used for therapy selection, and they are often poor predictors of therapeutic response or take months to set up and assay. In this report, we introduce a microfluidic OrganoPlate® platform for extracellular matrix (ECM) embedded tumor culture under perfusion as an initial study designed to investigate the feasibility of adapting this technology for therapy selection. METHODS: The triple negative breast cancer cell lines MDA-MB-453, MDA-MB-231 and HCC1937 were selected based on their different BRCA1 and P53 status, and were seeded in the platform. We evaluate seeding densities, ECM composition (Matrigel®, BME2rgf, collagen I) and biomechanical (perfusion vs static) conditions. We then exposed the cells to a series of anti-cancer drugs (paclitaxel, olaparib, cisplatin) and compared their responses to those in 2D cultures. Finally, we generated cisplatin dose responses in 3D cultures of breast cancer cells derived from 2 PDX models. RESULTS: The microfluidic platform allows the simultaneous culture of 96 perfused micro tissues, using limited amounts of material, enabling drug screening of patient-derived material. 3D cell culture viability is improved by constant perfusion of the medium. Furthermore, the drug response of these triple negative breast cancer cells was attenuated by culture in 3D and differed from that observed in 2D substrates. CONCLUSIONS: We have investigated the use of a high-throughput organ-on-a-chip platform to select therapies. Our results have raised the possibility to use this technology in personalized medicine to support selection of appropriate drugs and to predict response to therapy in a real time fashion.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Culture Techniques/methods , Extracellular Matrix/metabolism , Microfluidics/methods , BRCA1 Protein/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cisplatin/pharmacology , Collagen , Drug Combinations , Female , Humans , Laminin , Mutation , Outcome Assessment, Health Care/methods , Paclitaxel/pharmacology , Phthalazines/pharmacology , Piperazines/pharmacology , Prognosis , Proteoglycans , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Bronchopulmonary fistula (BPF) is an abnormal connection between the pleural space and bronchial tree, potentially leading to fatal outcomes if untreated. While BPF commonly arises following lung surgery, it can also be linked to infections. This report details the case of a 47-year-old male with recent untreated bacterial pneumonia, who developed bilateral pneumothoraces with persistent air leaks, Pseudomonas and Aspergillus empyema, culminating in a right-sided BPF necessitating video-assisted thoracic surgery (VATS) decortication. The agenda of this presentation is to enhance early recognition of BPF, which can be presented subtly, to avert severe complications.
ABSTRACT
Coronavirus-19 emerged about 3 years ago and has proven to be a devastating disease, crippling communities worldwide and accounting for more than 6.31 million deaths. The true disease burden of COVID-19 will come to light in the upcoming years as we care for COVID-19 survivors with post-COVID-19 syndrome (PCS) with residual long-term symptoms affecting every organ system. Pulmonary fibrosis is the most severe long-term pulmonary manifestation of PCS, and due to the high incidence of COVID-19 infection rates, PCS-pulmonary fibrosis has the potential of becoming the next large-scale respiratory health crisis. To confront the potentially devastating effects of emerging post-COVID-19 pulmonary fibrosis, dedicated research efforts are needed to focus on surveillance, understanding pathophysiologic mechanisms, and most importantly, an algorithmic approach to managing these patients. We have performed a thorough literature review on post-COVID-19 pulmonary symptoms/imaging/physiology and present an algorithmic approach to these patients based on the best available data and extensive clinical experience.
ABSTRACT
Many advanced cancer models, such as patient-derived xenografts (PDXs), offer significant benefits in their preservation of the native tumor's heterogeneity and susceptibility to treatments, but face significant barriers to use in their reliance on a rodent host for propagation and screening. PDXs remain difficult to implement in vitro, particularly in configurations that enable both detailed cellular analysis and high-throughput screening (HTS). Complex multilineage co-cultures with stromal fibroblasts, endothelium, and other cellular and structural components of the tumor microenvironment (TME) further complicate ex vivo implementation. Herein, the culture of multiple prostate cancer (PCa)-derived PDX models as 3D clusters within engineered biomimetic hydrogel matrices, in a HTS-compatible multiwell microfluidic format, alongside bone marrow-derived stromal cells and a perfused endothelial channel. Polymeric hydrogel matrices are customized for each cell type, enabling cell survival in vitro and facile imaging across all conditions. PCa PDXs demonstrate unique morphologies and reliance on TME partners, retention of known phenotype, and expected sensitivity or resistance to standard PCa therapeutics. This novel integration of technologies provides a fully human model, and expands the information to be gathered from each specimen, while avoiding the time and labor involved with animal-based testing.
Subject(s)
Prostatic Neoplasms , Male , Animals , Humans , Heterografts , Prostatic Neoplasms/metabolism , Coculture Techniques , Prostate/pathology , Disease Models, Animal , Hydrogels , Tumor MicroenvironmentABSTRACT
TNFα is a key mediator of immune and radiotherapy-induced cytotoxicity, but many cancers, including head and neck squamous cell carcinomas (HNSCC), display TNF resistance due to activation of the canonical IKK-NF-κB/RELA pro-survival pathway. However, toxicities associated with direct targeting of the canonical pathway point to the need to identify mechanism(s) contributing to TNFα resistance and synthetic lethal targets to overcome such resistance in cancer cells. Here, RNAi screening for modulators of TNFα-NF-κB reporter activity and cell survival unexpectedly implicated the WEE1 and CDC2 G2-M checkpoint kinases. The IKKα/ß-RELA and WEE1-CDC2 signaling pathways are activated by TNFα and form a complex in cell lines derived from both human papillomavirus (-) and (+) subtypes of HNSCC. WEE1 inhibitor AZD1775 reduced IKK/RELA phosphorylation and the expression of NF-κB-dependent pro-survival proteins Cyclin D1 and BCL2. Combination of TNFα and AZD1775 enhanced caspase-mediated apoptosis in vitro, and combination treatment with radiotherapy and AZD1775 potentiated inhibition of HNSCC tumor xenograft growth in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insight into the interplay between NF-κB signaling and WEE1-mediated regulation of the G2-M cell-cycle checkpoint in HNSCC. IMPLICATIONS: Inhibiting WEE1 and IKK-RELA crosstalk could potentially enhance the effects of therapies mediated by TNFα with less systemic immune suppression and toxicity than observed with direct interruption of IKK-NF-κB/RELA signaling.
Subject(s)
Cell Cycle Proteins , Head and Neck Neoplasms , I-kappa B Kinase , Protein-Tyrosine Kinases , Transcription Factor RelA , Apoptosis , Cell Cycle Proteins/genetics , Cell Line, Tumor , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , NF-kappa B/metabolism , Protein-Tyrosine Kinases/genetics , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/genetics , Transcription Factor RelA/genetics , Tumor Necrosis Factor-alphaABSTRACT
We report the development, automation and validation of a 3D, microfluidic liver-on-a-chip for high throughput hepatotoxicity screening, the OrganoPlate LiverTox™. The model is comprised of aggregates of induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep) seeded in an extracellular matrix in the organ channel and co-cultured with endothelial cells and THP-1 monoblasts differentiated to macrophages seeded in the vascular channel of the 96 well Mimetas OrganoPlate 2-lane. A key component of high throughput screening is automation and we report a protocol to seed, dose, collect and replenish media and add assay reagents in the OrganoPlate 2-lane using a standard laboratory liquid handling robot. A combination of secretome measurements and image-based analysis was used to demonstrate stable 15 day cell viability, albumin and urea secretion. Over the same time-period, CYP3A4 activity increased and alpha-fetoprotein secretion decreased suggesting further maturation of the iHeps. Troglitazone, a clinical hepatotoxin, was chosen as a control compound for validation studies. Albumin, urea, hepatocyte nuclear size and viability staining provided Robust Z'factors > 0.2 in plates treated 72 h with 180 µM troglitazone compared with a vehicle control. The viability assay provided the most robust statistic for a Robust Z' factor = 0.6. A small library of 159 compounds with known liver effects was added to the OrganoPlate LiverTox model for 72 h at 50 µM and the Toxicological Prioritization scores were calculated. A follow up dose-response evaluation of select hits revealed the albumin assay to be the most sensitive in calculating TC50 values. This platform provides a robust, novel model which can be used for high throughput hepatotoxicity screening.
Subject(s)
Cell Culture Techniques/methods , High-Throughput Screening Assays/methods , Liver/drug effects , Microfluidics/methods , Toxicity Tests/methods , Cell Survival/drug effects , Cell Survival/physiology , Cytochrome P-450 CYP3A/metabolism , Dose-Response Relationship, Drug , Hepatocytes/drug effects , Hepatocytes/physiology , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/physiology , Liver/cytology , Liver/physiology , Troglitazone/toxicityABSTRACT
Coupling ultrafast light irradiation to surface nanoreliefs leads to periodic patterns, achieving record processing scales down to tens of nanometers. Driven by near-field interactions, the promising potential of the spontaneous pattern formation relies on the scaling up of one-step manufacturing processes. Here, we report the self-assembly of unconventional arrays of nanocavities of 20 nm diameter with a periodicity down to 60 nm upon ultrafast laser irradiation of a nickel surface. In stark contrast to laser-induced surface ripples, which are stochastic and suffer from a lack of regularity, the 2D patterns present an unprecedented uniformity on extreme scales. The onset of nanocavity arrays ordered in a honeycomb lattice is achieved by overcoming the anisotropic polarization response of the surface by a delayed action of cross-polarized laser pulses. The origin of this self-arrangement is identified as a manifestation of Marangoni convection instability in a nanoscale melt layer, destabilized by the laser-induced rarefaction wave.
ABSTRACT
BACKGROUND: Metastatic cancer is incurable and understanding the molecular underpinnings is crucial to improving survival for our patients. The IGF-1/Akt signaling pathway is often impaired in cancer leading to its progression and metastases. Diet modification is known to alter the IGF-1/Akt pathway and affect the expression of microRNA involved in tumor initiation, growth and metastases. Liver metastases are one of the most common type of metastases in breast and colon cancer. In the present study, we looked at the effect of diet modification on the expression of microRNA in normal liver and liver with breast cancer metastases using in vivo model. METHODOLOGY: 6-month-old C57BL/6 J mice were put on either an ad libitum (AL) diet, or 40% calorie restricted (CR) diet or were fasted for 24 h (FA) before sacrifice. MicroRNA array analysis, western blot and qRT-PCR were performed using liver tissue to compare the treatment groups. A breast cancer model was also used to study the changes in microRNA expression in liver of a group of BALB/c mice orthotopically injected with 4 T1 cells in the mammary fat pad, put on either an AL or 30% CR diet. Liver and primary tumor tissues were used to perform qRT-PCR to compare the treatment groups. RESULTS: MicroRNA array analysis showed significant changes in miRNA expression in both CR and FA conditions in normal liver. Expression of miR-29 and miR-30 family members was increased in both CR and FA. Western blot analysis of the normal liver tissue showed that CR and FA downregulated the IGF-1/Akt pathway and qRT-PCR showed that the expression of miR-29b, miR-29c, miR-30a and miR-30b were increased with CR and FA. Liver tissue collected from mice in the breast cancer model showed an increase in expression of miR-29b, miR-29c and miR-30b while tumor tissue showed increased expression of miR-29c, miR-30a and miR-30b. DISCUSSION: Members of the miR-29 family are known to target and suppress IGF-1, while members of the miR-30 family are known to target and suppress both IGF-1 and IGF-1R. In the present study, we observe that calorie restriction increased the expression of miR-29 and miR-30 in both the normal liver as well as the liver with breast cancer metastases. These findings suggest that dietary alterations may play a role in the treatment of liver metastasis, which should be evaluated further.
ABSTRACT
Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.
Subject(s)
Cell Culture Techniques , Heterografts , Hydrogels/chemistry , Microfluidics , Animals , Cell Survival , Centrifugation, Density Gradient , Disease Models, Animal , Humans , Image Processing, Computer-Assisted , Mice , Staining and LabelingABSTRACT
Head and neck squamous cell carcinomas (HNSCC) promote inflammation in the tumor microenvironment through aberrant NF-κB activation, but the genomic alterations and pathway networks that modulate NF-κB signaling have not been fully dissected. Here, we analyzed genome and transcriptome alterations of 279 HNSCC specimens from The Cancer Genome Atlas (TCGA) cohort and identified 61 genes involved in NF-κB and inflammatory pathways. The top 30 altered genes were distributed across 96% of HNSCC samples, and their expression was often correlated with genomic copy-number alterations (CNA). Ten of the amplified genes were associated with human papilloma virus (HPV) status. We sequenced 15 HPV- and 11 HPV+ human HNSCC cell lines, and three oral mucosa keratinocyte lines, and supervised clustering revealed that 28 of 61 genes exhibit altered expression patterns concordant with HNSCC tissues and distinct signatures related to their HPV status. RNAi screening using an NF-κB reporter line identified 16 genes that are induced by TNFα or Lymphotoxin-ß (LTß) and implicated in the classic and/or alternative NF-κB pathways. Knockdown of TNFR, LTBR, or selected downstream signaling components established cross-talk between the classic and alternative NF-κB pathways. TNFα and LTß induced differential gene expression involving the NF-κB, IFNγ, and STAT pathways, inflammatory cytokines, and metastasis-related genes. Improved survival was observed in HNSCC patients with elevated gene expression in T-cell activation, immune checkpoints, and IFNγ and STAT pathways. These gene signatures of NF-κB activation, which modulate inflammation and responses to the immune therapy, could serve as potential biomarkers in future clinical trials.
Subject(s)
Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/genetics , NF-kappa B/metabolism , Signal Transduction/genetics , Cell Line , Cell Proliferation , DNA Copy Number Variations , Databases, Genetic , Female , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/virology , Humans , Inflammation , Interferon-gamma/metabolism , Lymphotoxin-beta/metabolism , Male , NF-kappa B/genetics , Papillomavirus Infections/genetics , Papillomavirus Infections/immunology , Papillomavirus Infections/metabolism , Papillomavirus Infections/virology , STAT Transcription Factors/metabolism , Transcriptome , Tumor Necrosis Factor-alpha/metabolismABSTRACT
PURPOSE: To identify deregulated and inhibitory miRNAs and generate novel mimics for replacement nanomedicine for head and neck squamous cell carcinomas (HNSCC). EXPERIMENTAL DESIGN: We integrated miRNA and mRNA expression, copy number variation, and DNA methylation results from The Cancer Genome Atlas (TCGA), with a functional genome-wide screen. RESULTS: We reveal that the miR-30 family is commonly repressed, and all 5 members sharing these seed sequence similarly inhibit HNSCC proliferation in vitro. We uncover a previously unrecognized inverse relationship with overexpression of a network of important predicted target mRNAs deregulated in HNSCC, that includes key molecules involved in proliferation (EGFR, MET, IGF1R, IRS1, E2F7), differentiation (WNT7B, FZD2), adhesion, and invasion (ITGA6, SERPINE1). Reexpression of the most differentially repressed family member, miR-30a-5p, suppressed this mRNA program, selected signaling proteins and pathways, and inhibited cell proliferation, migration, and invasion in vitro. Furthermore, a novel miR-30a-5p mimic formulated into a targeted nanomedicine significantly inhibited HNSCC xenograft tumor growth and target growth receptors EGFR and MET in vivo. Significantly decreased miR-30a/e family expression was related to DNA promoter hypermethylation and/or copy loss in TCGA data, and clinically with decreased disease-specific survival in a validation dataset. Strikingly, decreased miR-30e-5p distinguished oropharyngeal HNSCC with poor prognosis in TCGA (P = 0.002) and validation (P = 0.007) datasets, identifying a novel candidate biomarker and target for this HNSCC subset. CONCLUSIONS: We identify the miR-30 family as an important regulator of signal networks and tumor suppressor in a subset of HNSCC patients, which may benefit from miRNA replacement nanomedicine therapy.
Subject(s)
Biomarkers, Tumor/metabolism , Genes, Tumor Suppressor , Head and Neck Neoplasms/pathology , MicroRNAs/administration & dosage , MicroRNAs/genetics , Nanoparticles/administration & dosage , Squamous Cell Carcinoma of Head and Neck/secondary , Animals , Apoptosis , Biomarkers, Tumor/genetics , Case-Control Studies , Cell Movement , Cell Proliferation , DNA Copy Number Variations , Female , Follow-Up Studies , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genomics , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Nanomedicine , Nanoparticles/chemistry , Prognosis , Prospective Studies , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/genetics , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
IV heroin use is associated with several well-described complications, including noncardiogenic pulmonary edema, aspiration pneumonitis, ARDS, pneumonia, lung abscess, septic pulmonary emboli, and atelectasis. Foreign-body granulomatosis may develop when drug users inject solutions containing crushed oral tablets in which talc is used as filler and can be complicated by pulmonary fibrosis. The effects are distinct from pulmonary edema, which may occur acutely with heroin injection. We describe the case of a young female patient who was an IV heroin user who also smoked cigarettes, and presented with progressive dyspnea, hypoxia, and bilateral lung infiltrates. The final pathologic diagnosis in this case was one that had not been previously reported in IV heroin users.
Subject(s)
Cryptogenic Organizing Pneumonia/etiology , Heroin Dependence/complications , Substance Abuse, Intravenous/complications , Adult , Cryptogenic Organizing Pneumonia/diagnostic imaging , Cryptogenic Organizing Pneumonia/pathology , Female , Granuloma, Foreign-Body/etiology , Granuloma, Foreign-Body/pathology , Humans , Tomography, X-Ray ComputedABSTRACT
Loss or mutation of TP53 has been linked to alterations in anti-tumor immunity as well as dysregulation of cell cycle and apoptosis. We explored immunologic effects and mechanisms following restoration of wild-type human TP53 cDNA in murine oral cancer cells using the therapeutic nanocomplex scL-53. We demonstrated scL-53 induces dose-dependent expression of TP53 and induction of apoptosis and immunogenic cell death. We further demonstrated both TP53-dependent and independent induction of tumor cell immunogenicity through the use of blocking mAbs, nanocomplex loaded with DNA plasmid with or without TP53 cDNA, empty nanocomplex and siRNA knockdown techniques. TP53-independent immune modulation was observed following treatment with nanocomplex loaded with DNA plasmid lacking TP53 cDNA and abrogated in STING-deficient tumor cells, supporting the presence of a cytoplasmic DNA sensing, STING-dependent type-I IFN response. Cooperatively, TP53- and STING-dependent alterations sensitized tumor cells to CTL-mediated lysis, which was further enhanced following reversal of adaptive immune resistance with PD-1 mAb. In vivo, combination scL-53 and PD-1 mAb resulted in growth control or rejection of established tumors that was abrogated in mice depleted of CD8+ cells or in STING deficient mice. Cumulatively, this work demonstrates 1) a direct anti-tumor effects of functional TP53; 2) non-redundant TP53- and STING-dependent induction of tumor cell immunogenicity following scL-53 treatment; and 3) that adaptive immune resistance following scL-53 treatment can be reversed with PD-based immune checkpoint blockade, resulting in the rejection or control of syngeneic murine tumors. These data strongly support the clinical combination of scL-53 and immune checkpoint blockade.
ABSTRACT
Intrinsic resistance to cytotoxic T-lymphocyte (CTL) killing limits responses to immune activating anti-cancer therapies. Here, we established that activation of the G2/M cell cycle checkpoint results in tumor cell cycle pause and protection from granzyme B-induced cell death. This was reversed with WEE1 kinase inhibition, leading to enhanced CTL killing of antigen-positive tumor cells. Similarly, but at a later time point, cell cycle pause following TNFα exposure was reversed with WEE1 kinase inhibition, leading to CTL transmembrane TNFα-dependent induction of apoptosis and necroptosis in bystander antigen-negative tumor cells. Results were reproducible in models of oral cavity carcinoma, melanoma and colon adenocarcinoma harboring variable Tp53 genomic alterations. WEE1 kinase inhibition sensitized tumors to PD-1 mAb immune checkpoint blockade in vivo, resulting in CD8+-dependent rejection of established tumors harboring antigen-positive or mixed antigen-positive and negative tumor cells. Together, these data describe activation of the G2/M cell cycle checkpoint in response to early and late CTL products as a mechanism of resistance to CTL killing, and provide pre-clinical rationale for the clinical combination of agents that inhibit cell cycle checkpoints and activate anti-tumor immunity.
ABSTRACT
Cell lines are important tools for biological and preclinical investigation, and establishing their relationship to genomic alterations in tumors could accelerate functional and therapeutic discoveries. We conducted integrated analyses of genomic and transcriptomic profiles of 15 human papillomavirus (HPV)-negative and 11 HPV-positive head and neck squamous cell carcinoma (HNSCC) lines to compare with 279 tumors from The Cancer Genome Atlas (TCGA). We identified recurrent amplifications on chromosomes 3q22-29, 5p15, 11q13/22, and 8p11 that drive increased expression of more than 100 genes in cell lines and tumors. These alterations, together with loss or mutations of tumor suppressor genes, converge on important signaling pathways, recapitulating the genomic landscape of aggressive HNSCCs. Among these, concurrent 3q26.3 amplification and TP53 mutation in most HPV(-) cell lines reflect tumors with worse survival. Our findings elucidate and validate genomic alterations underpinning numerous discoveries made with HNSCC lines and provide valuable models for future studies.
Subject(s)
Genome , Head and Neck Neoplasms/genetics , Transcriptome/genetics , Cell Line, Tumor , Chromosomes, Human/genetics , DNA Copy Number Variations/genetics , Gene Dosage , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/virology , Humans , Mutation/genetics , Neoplasm Invasiveness , Papillomaviridae/pathogenicity , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The HIV-1 Rev response element (RRE), a highly structured RNA sequence consisting of five stemloops, is found in all spliced and partially spliced human immunodeficiency virus (HIV) mRNA transcripts. The RRE interacts with HIV-encoded Rev protein, which facilitates exit of the transcripts from the nucleus to the cytoplasm. Because the Rev/RRE interaction is critical to virus function, it is considered a potential target for therapeutic drugs. We have investigated the interactions of antisense oligonucleotides with stem-loop II, a region that contains the high-affinity binding site for Rev. Oligo-2'-O-methylribonucleotides terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were targeted to the 5'- or 3'-side of stem-loop IIB, which is adjacent to the Rev binding site. Thermal denaturation experiments showed that oligonucleotides of this type form highly stable duplexes with complementary single-stranded RNA. Gel electrophoretic mobility shift assays (EMSA) showed that the oligonucleotides bound with high affinity and specificity at 37 degrees C to RRE stem-loop II RNA with apparent dissociation constants, K(D), in the low nM range. A 16-mer, 2-1mp, whose K(D) is 46 nM, competitively inhibited binding of Rev peptide to RRE stem-loop II RNA as shown by EMSA experiments. When transfected into HEK 293T cells, 2-1mp inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) by 60% at a concentration of 300 nM oligonucleotide. These results are consistent with a mechanism by which 2-1mp blocks access of Rev to the RRE/CAT transcript thus preventing nuclear export and subsequent translation.
Subject(s)
Gene Products, rev/metabolism , Genes, env , HIV-1/genetics , Oligoribonucleotides, Antisense/metabolism , RNA, Viral/metabolism , Cell Line , Electrophoretic Mobility Shift Assay , Gene Expression , HIV-1/metabolism , Humans , Nucleic Acid Conformation , RNA, Viral/geneticsABSTRACT
We report a case of a 65-year-old male patient who presented with gradually worsening dyspnea over 2 years. History was significant for smoking and the absence of any hazardous occupational exposure. The clinical findings at presentation included absent breath sounds and stony dullness on the left side, with tracheal deviation contralaterally and clubbing. A chest roentgenogram showed a left-sided opacity occupying almost the entire left hemithorax. A subsequent CT scan of the chest revealed an intrathoracic, extrapulmonary lesion producing a mediastinal shift. Surgical resection of the mass was performed, and pathology, along with immunohistochemical studies positive for CD34 and negative for epithelial markers, confirmed the diagnosis of solitary fibrous tumor of the pleura (SFTP). SFTP is a rare neoplasm, and diagnosis is often difficult. Suspicion of SFTP should arise in the setting of a patient presenting with a paucity of symptoms (except in the case of an accompanying paraneoplastic syndrome), the absence of exposure to asbestos, and a large mass with sharp margins and encapsulation seen on a chest radiograph.
Subject(s)
Dyspnea/etiology , Mesothelioma/diagnostic imaging , Pleural Neoplasms/diagnostic imaging , Diagnosis, Differential , Humans , Male , Mesothelioma/pathology , Mesothelioma/surgery , Pleural Neoplasms/pathology , Pleural Neoplasms/surgery , Tomography, X-Ray ComputedABSTRACT
Chest radiographs are sometimes taken before surgeries and interventional procedures on hospital admissions and outpatients. This manuscript summarizes the American College of Radiology review of the literature and recommendations on routinely performed chest radiographies in these settings. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every 3 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment.