A subset of cytotoxic effector memory T cells enhances CAR T cell efficacy in a model of pancreatic ductal adenocarcinoma.

In humans, the natural killer (NK) cell marker CD161 identifies several subsets of T cells, including a polyclonal CD8 αß T cell receptor-expressing subset with characteristic specificity for tissue-localized viruses. This subset also displays enhanced cytotoxic and memory phenotypes. Here, we characterized this unique T cell subset and determined its potential suitability for use in chimeric antigen receptor (CAR) T cell therapy. In mice, gene expression profiling among the CD161-equivalent CD8+ T cell populations (CD8+NK1.1+) revealed substantial up-regulation of granzymes, perforin, killer lectin-like receptors, and innate signaling molecules in comparison to CD8+NK1.1- T cells. Adoptive transfer of CD8+NK1.1+ cells from previously exposed animals offered substantially enhanced protection and improved survival against melanoma tumors and influenza infection compared to CD8+NK1.1- cells. Freshly isolated human CD8+CD61+ T cells exhibited heightened allogeneic killing activity in comparison to CD8+CD61- T cells or total peripheral blood mononuclear cells (PBMCs). To determine whether this subset might improve the antitumor efficacy of CAR T cell therapy against solid tumors, we compared bulk PBMCs, CD8+CD161-, and CD8+CD161+ T cells transduced with a human epidermal growth factor receptor-2 (HER2)-specific CAR construct. In vitro, CD8+CD161+ CAR-transduced T cells killed HER2+ targets faster and with greater efficiency. Similarly, these cells mediated enhanced in vivo antitumor efficacy in xenograft models of HER2+ pancreatic ductal adenocarcinoma, exhibiting elevated expression of granzymes and reduced expression of exhaustion markers. These data suggest that this T cell subset presents an opportunity to improve CAR T cell therapy for the treatment of solid tumors.

MHC class I and II peptide homology regulates the cellular immune response.

Mammalian immune responses are initiated by "danger" signals--immutable molecular structures known as PAMPs. When detected by fixed, germline encoded receptors, pathogen-associated molecular pattern (PAMPs) subsequently inform the polarization of downstream adaptive responses depending upon identity and localization of the PAMP. Here, we report the existence of a completely novel "PAMP" that is not a molecular structure but an antigenic pattern. This pattern--the incidence of peptide epitopes with stretches of 100% sequence identity bound to both dendritic cell (DC) major histocompatibility (MHC) class I and MHC class II--strongly induces TH 1 immune polarization and activation of the cellular immune response. Inherent in the existence of this PAMP is the concomitant existence of a molecular sensor complex with the ability to scan and compare amino acid sequence identities of bound class I and II peptides. We provide substantial evidence implicating the multienzyme aminoacyl-tRNA synthetase (mARS) complex and its AIMp1 structural component as the key constituents of this complex. The results demonstrate a wholly novel mechanism by which T-helper (TH ) polarization is governed and provide critical information for the design of vaccination strategies intended to provoke cell-mediated immunity.

CD8+CD161+ T-Cells: Cytotoxic Memory Cells With High Therapeutic Potential.

NK1.1 and its human homolog CD161 are expressed on NK cells, subsets of CD4+ and CD8+ T cells, and NKT cells. While the expression of NK1.1 is thought to be inhibitory to NK cell function, it is reported to play both costimulatory and coinhibitory roles in T-cells. CD161 has been extensively studied and characterized on subsets of T-cells that are MR1-restricted, IL-17 producing CD4+ (TH17 MAIT cells) and CD8+ T cells (Tc17 cells). Non-MAIT, MR1-independent CD161-expressing T-cells also exist and are characterized as generally effector memory cells with a stem cell like phenotype. Gene expression analysis of this enigmatic subset indicates a significant enhancement in the expression of cytotoxic granzyme molecules and innate like stress receptors in CD8+NK1.1+/CD8+CD161+ cells in comparison to CD8+ cells that do not express NK1.1 or CD161. First identified and studied in the context of viral infection, the role of CD8+CD161+ T-cells, especially in the context of tumor immunology, is still poorly understood. In this review, the functional characteristics of the CD161-expressing CD8+ T cell subset with respect to gene expression profile, cytotoxicity, and tissue homing properties are discussed, and application of this subset to immune responses against infectious disease and cancer is considered.

Beyond T-Cells: Functional Characterization of CTLA-4 Expression in Immune and Non-Immune Cell Types.

The immune response consists of a finely-tuned program, the activation of which must be coupled with inhibitory mechanisms whenever initiated. This ensures tight control of beneficial anti-pathogen and anti-tumor responses while preserving tissue integrity, promoting tissue repair, and safeguarding against autoimmunity. A cogent example of this binary response is in the mobilization of co-stimulatory and co-inhibitory signaling in regulating the strength and type of a T-cell response. Of particular importance is the costimulatory molecule CD28 which is countered by CTLA-4. While the role of CD28 in the immune response has been thoroughly elucidated, many aspects of CTLA-4 biology remain controversial. The expression of CD28 is largely constrained to constitutive expression in T-cells and as such, teasing out its function has been somewhat simplified by a limited and specific expression profile. The expression of CTLA-4, on the other hand, while reported predominantly in T-cells, has also been described on a diverse repertoire of cells within both lymphoid and myeloid lineages as well as on the surface of tumors. Nonetheless, the function of CTLA-4 has been mostly described within the context of T-cell biology. The focus on T-cell biology may be a direct result of the high degree of amino acid sequence homology and the co-expression pattern of CD28 and CTLA-4, which initially led to the discovery of CTLA-4 as a counter receptor to CD28 (for which a T-cell-activating role had already been described). Furthermore, observations of the outsized role of CTLA-4 in Treg-mediated immune suppression and the striking phenotype of T-cell hyperproliferation and resultant disease in CTLA-4-/- mice contribute to an appropriate T-cell-centric focus in the study of CTLA-4. Complete elucidation of CTLA-4 biology, however, may require a more nuanced understanding of its role in a context other than that of T-cells. This makes particular sense in light of the remarkable, yet limited utility of anti-CTLA-4 antibodies in the treatment of cancers and of CTLA-4-Ig in autoimmune disorders like rheumatoid arthritis. By fully deducing the biology of CTLA-4-regulated immune homeostasis, bottlenecks that hinder the widespread applicability of CTLA-4-based immunotherapies can be resolved.

Genetic Adjuvantation of a Cell-Based Therapeutic Vaccine for Amelioration of Chagasic Cardiomyopathy.

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a leading cause of heart disease ("chagasic cardiomyopathy") in Latin America, disproportionately affecting people in resource-poor areas. The efficacy of currently approved pharmaceutical treatments is limited mainly to acute infection, and there are no effective treatments for the chronic phase of the disease. Preclinical models of Chagas disease have demonstrated that antigen-specific CD8+ gamma interferon (IFN-γ)-positive T-cell responses are essential for reducing parasite burdens, increasing survival, and decreasing cardiac pathology in both the acute and chronic phases of Chagas disease. In the present study, we developed a genetically adjuvanted, dendritic cell-based immunotherapeutic for acute Chagas disease in an attempt to delay or prevent the cardiac complications that eventually result from chronic T. cruzi infection. Dendritic cells transduced with the adjuvant, an adenoviral vector encoding a dominant negative isoform of Src homology region 2 domain-containing tyrosine phosphatase 1 (SHP-1) along with the T. cruzi Tc24 antigen and trans-sialidase antigen 1 (TSA1), induced significant numbers of antigen-specific CD8+ IFN-γ-positive cells following injection into BALB/c mice. A vaccine platform transduced with the adenoviral vector and loaded in tandem with the recombinant protein reduced parasite burdens by 76% to >99% in comparison to a variety of different controls and significantly reduced cardiac pathology in a BALB/c mouse model of live Chagas disease. Although no statistical differences in overall survival rates among cohorts were observed, the data suggest that immunotherapeutic strategies for the treatment of acute Chagas disease are feasible and that this approach may warrant further study.

Phase I trial of antigen-targeted autologous dendritic cell-based vaccine with in vivo activation of inducible CD40 for advanced prostate cancer.

This phase I trial reports the safety and activity of BPX101, a second-generation antigen-targeted autologous antigen presenting cell (APC) vaccine in men with metastatic castration-resistant prostate cancer (mCRPC). To manufacture BPX101, APCs collected in a single leukapheresis were transduced with adenoviral vector Ad5f35 encoding inducible human (ih)-CD40, followed by incubation with protein PA001, which contains the extracellular domain of human prostate-specific membrane antigen. The ih-CD40 represents a modified chimeric version of the dendritic cell (DC) co-stimulatory molecule, CD40, which responds to a bioinert membrane-permeable activating dimerizer drug, rimiducid (AP1903), permitting temporally controlled, lymphoid-localized, DC-specific activation. Eighteen men with progressive mCRPC following ≤1 prior chemotherapy regimen were enrolled to evaluate three doses of BPX101 (4 × 106, 12.5 × 106 and 25 × 106 cells) administered intradermally every 2-4 weeks followed by rimiducid (0.4 mg/kg) intravenous (IV) infusion 24 h after each BPX101 dose. There were no dose-limiting toxicities. Immune upregulation as well as anti-tumor activity was observed with PSA declines, objective tumor regressions and robust efficacy of post-trial therapy. This novel antigen-targeted and in vivo activated immunotherapy platform may warrant further development as monotherapy and as a component of rational combinations.

Future of fundamental discovery in US biomedical research.

Young researchers are crucially important for basic science as they make unexpected, fundamental discoveries. Since 1982, we find a steady drop in the number of grant-eligible basic-science faculty [principal investigators (PIs)] younger than 46. This fall occurred over a 32-y period when inflation-corrected congressional funds for NIH almost tripled. During this time, the PI success ratio (fraction of basic-science PIs who are R01 grantees) dropped for younger PIs (below 46) and increased for older PIs (above 55). This age-related bias seems to have caused the steady drop in the number of young basic-science PIs and could reduce future US discoveries in fundamental biomedical science. The NIH recognized this bias in its 2008 early-stage investigator (ESI) policy to fund young PIs at higher rates. We show this policy is working and recommend that it be enhanced by using better data. Together with the National Institute of General Medical Sciences (NIGMS) Maximizing Investigators' Research Award (MIRA) program to reward senior PIs with research time in exchange for less funding, this may reverse a decades-long trend of more money going to older PIs. To prepare young scientists for increased demand, additional resources should be devoted to transitional postdoctoral fellowships already offered by NIH.

AIMp1 Potentiates TH1 Polarization and Is Critical for Effective Antitumor and Antiviral Immunity.

Dendritic cells (DCs) must integrate a broad array of environmental cues to exact control over downstream immune responses including TH polarization. The multienzyme aminoacyl-tRNA synthetase complex component AIMp1/p43 responds to cellular stress and exerts pro-inflammatory functions; however, a role for DC-expressed AIMp1 in TH polarization has not previously been shown. Here, we demonstrate that the absence of AIMp1 in bone marrow-derived DC (BMDC) significantly impairs cytokine and costimulatory molecule expression, p38 MAPK signaling, and TH1 polarization of cocultured T-cells while significantly dysregulating immune-related gene expression. These deficits resulted in significantly compromised BMDC vaccine-mediated protection against melanoma. AIMp1 within the host was also critical for innate and adaptive antiviral immunity against influenza virus infection in vivo. Cancer patients with AIMp1 expression levels in the highest tertiles exhibited a 70% survival advantage at 15-year postdiagnosis as determined by bioinformatics analysis of nearly 9,000 primary human tumor samples in The Cancer Genome Atlas database. These data establish the importance of AIMp1 for the effective governance of antitumor and antiviral immune responses.

Chemo-immunotherapy mediates durable cure of orthotopic KrasG12D/p53-/- pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States, exhibiting a five-year overall survival (OS) of only 7% despite aggressive standard of care. Recent advances in immunotherapy suggest potential application of immune-based treatment approaches to PDAC. To explore this concept further, we treated orthotopically established K-rasG12D/p53-/- PDAC tumors with gemcitabine and a cell-based vaccine previously shown to generate durable cell-mediated (TH1) immunity. Tumor progression was monitored by IVIS. The results indicated that the combination of chemotherapy and dendritic cell (DC) vaccination was effective in eliminating tumor, preventing metastasis and recurrence, and significantly enhancing OS. No animal that received the combination therapy relapsed, while mice that received gemcitabine-only or vaccine-only regimens relapsed and progressed. Analysis of circulating PBMC demonstrated that mice receiving the combination therapy exhibited significantly elevated levels of CD8+IFNγ+CCR7+NK1.1+ T-cells with significantly reduced levels of exhausted GITR+CD8+ T-cells after the cessation of treatment. Retro-orbital tumor re-challenge of surviving animals at six-months post-treatment demonstrated durable antitumor immunity only among mice that had received the combination therapy. CD8+ splenocytes derived from surviving mice that had received the combination therapy were sorted into NK1.1pos and NK1.1neg populations and adoptively transferred into naive recipients. Transfer of only 1,500 CD8+NK1.1pos T-cells was sufficient to mediate tumor rejection whereas transfer of 1,500 CD8+NK1.1neg T-cells imparted only minimal effects. The data suggest that addition of a TH1 DC vaccine regimen as an adjuvant to existing therapies can mediate eradication of tumors and offer durable protection against PDAC.

MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity.

Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways. When incorporated into a DNA vaccine, signaling by the MC adjuvant increased antigen-specific CD8+ T cells and promoted elimination of pre-established tumors. Interestingly, MC-enhanced vaccine efficacy did not require direct-expression of either antigen or adjuvant by local antigen-presenting cells, but rather our data supports a key role for MC function in "atypical" antigen-presenting cells of skin. In particular, MC adjuvant-modified keratinocytes increased inflammatory cytokine secretion, upregulated surface MHC class I, and were able to increase in vitro and in vivo priming of antigen-specific CD8+ T cells. Furthermore, in the absence of critical CD8α+/CD103+ cross-priming dendritic cells, MC was still able to promote immune priming in vivo, albeit at a reduced level. Altogether, our data support a mechanism by which MC signaling activates an inflammatory phenotype in atypical antigen-presenting cells within the cutaneous vaccination site, leading to an enhanced CD8+ T cell response against DNA vaccine-encoded antigens, through both CD8α+/CD103+ dendritic cell-dependent and independent pathways.

Dendritic Cell-Secreted Cytotoxic T-Lymphocyte-Associated Protein-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.

The remarkable functional plasticity of professional antigen-presenting cells (APCs) allows the adaptive immune system to respond specifically to an incredibly diverse array of potential pathogenic insults; nonetheless, the specific molecular effectors and mechanisms that underpin this plasticity remain poorly characterized. Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), the target of the blockbuster cancer immunotherapeutic ipilimumab, is one of the most well-known and well-studied members of the B7 superfamily and negatively regulates T cell responses by a variety of known mechanisms. Although CTLA-4 is thought to be expressed almost exclusively among lymphoid lineage hematopoietic cells, a few reports have indicated that nonlymphoid APCs can also express the CTLA-4 mRNA transcript and that transcript levels can be regulated by external stimuli. In this study, we substantially build upon these critical observations, definitively demonstrating that mature myeloid lineage dendritic cells (DC) express significant levels of intracellular CTLA-4 that they constitutively secrete in microvesicular structures. CTLA-4(+) microvesicles can competitively bind B7 costimulatory molecules on bystander DC, resulting in downregulation of B7 surface expression with significant functional consequences for downstream CD8(+) T-cell responses. Hence, the data indicate a previously unknown role for DC-derived CTLA-4 in immune cell functional plasticity and have significant implication for the design and implementation of immunomodulatory strategies intended to treat cancer and infectious disease.

Integrative Pathway Analysis of Metabolic Signature in Bladder Cancer: A Linkage to The Cancer Genome Atlas Project and Prediction of Survival.

PURPOSE: We used targeted mass spectrometry to study the metabolic fingerprint of urothelial cancer and determine whether the biochemical pathway analysis gene signature would have a predictive value in independent cohorts of patients with bladder cancer. MATERIALS AND METHODS: Pathologically evaluated, bladder derived tissues, including benign adjacent tissue from 14 patients and bladder cancer from 46, were analyzed by liquid chromatography based targeted mass spectrometry. Differential metabolites associated with tumor samples in comparison to benign tissue were identified by adjusting the p values for multiple testing at a false discovery rate threshold of 15%. Enrichment of pathways and processes associated with the metabolic signature were determined using the GO (Gene Ontology) Database and MSigDB (Molecular Signature Database). Integration of metabolite alterations with transcriptome data from TCGA (The Cancer Genome Atlas) was done to identify the molecular signature of 30 metabolic genes. Available outcome data from TCGA portal were used to determine the association with survival. RESULTS: We identified 145 metabolites, of which analysis revealed 31 differential metabolites when comparing benign and tumor tissue samples. Using the KEGG (Kyoto Encyclopedia of Genes and Genomes) Database we identified a total of 174 genes that correlated with the altered metabolic pathways involved. By integrating these genes with the transcriptomic data from the corresponding TCGA data set we identified a metabolic signature consisting of 30 genes. The signature was significant in its prediction of survival in 95 patients with a low signature score vs 282 with a high signature score (p = 0.0458). CONCLUSIONS: Targeted mass spectrometry of bladder cancer is highly sensitive for detecting metabolic alterations. Applying transcriptome data allows for integration into larger data sets and identification of relevant metabolic pathways in bladder cancer progression.

The angiopoietin-TIE2 pathway is a potential therapeutic target in urothelial carcinoma.

BACKGROUND: Angiopoietin/Tyrosine Kinase-2 (ANG/TIE2), Fibroblast Growth Factor-1 (FGFR1) and Vascular Endothelial Growth Factor Receptors (VEGFRs) promote growth of urothelial carcinoma (UC). We examined the pre-clinical activity of CEP-11981, a tyrosine kinase inhibitor of TIE2, FGFR1 and VEGFR-1-3, in UC. MATERIALS AND METHODS: The in vitro activity of CEP-11981 was evaluated in four human UC cell lines. The effect of daily oral CEP-11981 was examined in RT4 xenografts, which also underwent immunohistochemistry (IHC) for cleaved caspase-3, Cluster of Differentiation (CD)-31, VEGFR2 and TIE2. RESULTS: In vitro activity was not detected. Preliminary in vivo experiments suggested that CEP-11981 at both 5 mg/kg and 10 mg/kg induced similar regression of xenografts, greater than those at 2.5 mg/kg daily. Given the lower toxicity at 5 mg/kg, we performed a confirmatory experiment with 5 mg/kg, which significantly inhibited xenografts compared to controls (p<0.05). IHC of xenografts demonstrated no differences in CD31, cleaved caspase-3 or VEGFR2, but TIE2 was significantly down-regulated (p=0.008) by CEP-11981. CONCLUSION: CEP-11981 demonstrated a significant pre-clinical activity against human UC xenografts, which was attributable primarily to effects on TIE2 receptor.

The preclinical activity of lenalidomide in indolent urothelial carcinoma.

BACKGROUND: Lenalidomide is an IMiD® immunomodulatory drug, which may warrant evaluation in urothelial carcinoma (UC). MATERIALS AND METHODS: The in vitro and in vivo activity of lenalidomide was evaluated in human and murine UC cell lines. Tumors were evaluated by immunohistochemistry for (CD31), cleaved caspase-3 (CC3) and CD3+/CD20+ lymphocyte infiltration. Cereblon, a molecular target of lenalidomide was analyzed by immunohistochemistry. RESULTS: Significant pro-apoptotic activity, and reduction of cell viability was seen at low micromolar concentrations of lenalidomide against indolent human RT4 UC cells in vitro. Cereblon expression was quantitatively lower in sensitive RT4 cells compared to resistant 5637 cells. In RT4 xenografts, lenalidomide significantly reduced tumor size and CD31 expression, and increased expression of CC3 (p<0.05). Cereblon expression increased in lenalidomide-treated RT4 xenografts (p<0.05). CONCLUSION: Lenalidomide demonstrated preclinical activity against superficially-invasive low-grade UC cells attributable to direct tumor cell apoptosis and anti-angiogenic activity. Clinical trials are warranted in patients with indolent UC.

Activity of CEP-9722, a poly (ADP-ribose) polymerase inhibitor, in urothelial carcinoma correlates inversely with homologous recombination repair response to DNA damage.

As loss of DNA-repair proteins is common in urothelial carcinoma (UC), a rationale can be made to evaluate the activity of poly (ADP-ribose) polymerase (PARP) inhibitors to exploit synthetic lethality. We aimed to preclinically evaluate a PARP inhibitor, CEP-9722, and its active metabolite, CEP-8983, in UC. The activity of CEP-8983 was evaluated using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay against human UC cell lines. Flow cytometry, COMET assay, and western blot were performed to assess apoptosis, DNA damage, and DNA-repair proteins, respectively. RT4 xenografts received placebo or CEP-9722 (100 or 200 mg/kg/day) orally. Xenografts were subjected to immunohistochemistry for apoptosis [cleaved caspase (cc)-3] and angiogenesis (CD31). CEP-8983 (1 µmol/l) reduced the viability of RT4 and T24 cells by 20%, but did not reduce the viability of 5637 and TCC-SUP cells. Apoptosis and necrosis occurred in 9.7 and 9.1% of RT4 and 5637 cells, respectively. RT4 cells showed greater DNA damage compared with 5637 cells. Increased DNA damage occurred with combination versus CEP-8983 or cisplatin alone in RT4 and 5637 cells. T24 and RT4 showed the least RAD51 foci 8 h following radiation, whereas TCC-SUP and 5637 robustly induced RAD51 foci. CEP-9722 showed dose-dependent antitumor activity in RT4 xenografts; 200 mg/kg daily was better than control (P=0.04) and 100 mg/kg was not (P=0.26). Immunohistochemistry of xenografts showed a significant increase in cc-3 and decrease in CD31 with both doses (P<0.05). Biomarker-driven evaluation of PARP inhibitors in UC is justified as the activity of CEP-9722 correlated inversely with homologous recombination repair response to DNA damage.

FGFR1-WNT-TGF-ß signaling in prostate cancer mouse models recapitulates human reactive stroma.

The reactive stroma surrounding tumor lesions performs critical roles ranging from supporting tumor cell proliferation to inducing tumorigenesis and metastasis. Therefore, it is critical to understand the cellular components and signaling control mechanisms that underlie the etiology of reactive stroma. Previous studies have individually implicated fibroblast growth factor receptor 1 (FGFR1) and canonical WNT/ß-catenin signaling in prostate cancer progression and the initiation and maintenance of a reactive stroma; however, both pathways are frequently found to be coactivated in cancer tissue. Using autochthonous transgenic mouse models for inducible FGFR1 (JOCK1) and prostate-specific and ubiquitously expressed inducible ß-catenin (Pro-Cat and Ubi-Cat, respectively) and bigenic crosses between these lines (Pro-Cat × JOCK1 and Ubi-Cat × JOCK1), we describe WNT-induced synergistic acceleration of FGFR1-driven adenocarcinoma, associated with a pronounced fibroblastic reactive stroma activation surrounding prostatic intraepithelial neoplasia (mPIN) lesions found both in in situ and reconstitution assays. Both mouse and human reactive stroma exhibited increased transforming growth factor-ß (TGF-ß) signaling adjacent to pathologic lesions likely contributing to invasion. Furthermore, elevated stromal TGF-ß signaling was associated with higher Gleason scores in archived human biopsies, mirroring murine patterns. Our findings establish the importance of the FGFR1-WNT-TGF-ß signaling axes as driving forces behind reactive stroma in aggressive prostate adenocarcinomas, deepening their relevance as therapeutic targets.

Superresolution microscopy with quantum emitters.

The optical diffraction limit imposes a bound on imaging resolution in classical optics. Over the last twenty years, many theoretical schemes have been presented for overcoming the diffraction barrier in optical imaging using quantum properties of light. Here, we demonstrate a quantum superresolution imaging method taking advantage of nonclassical light naturally produced in fluorescence microscopy due to photon antibunching, a fundamentally quantum phenomenon inhibiting simultaneous emission of multiple photons. Using a photon counting digital camera, we detect antibunching-induced second and third order intensity correlations and perform subdiffraction limited quantum imaging in a standard wide-field fluorescence microscope.

A conventional preclinical schedule of cisplatin is more effective than a metronomic frequent bolus schedule for urothelial carcinoma.

OBJECTIVES: Given the frequent inability to administer a conventional 3-weekly schedule of cisplatin to human subjects with urothelial carcinoma, we evaluated a frequent bolus metronomic schedule in a preclinical system of transitional cell carcinoma (TCC) of the urothelium. We hypothesized that the anti-angiogenic and anti-cell-migratory activity of lower concentrations of cisplatin may confer similar anti-tumor activity and demonstrate less nephrotoxicity than conventional cytotoxic concentrations. MATERIALS AND METHODS: We evaluated the activity of cisplatin in vitro against human urothelial carcinoma (5637) and endothelial cells (human umbilical vein endothelial cells, HUVECs). The MTT assay was employed to assess viability, and flow cytometry with Annexin-FITC labeling was employed to assess apoptosis. Cell migration in monolayer culture was assessed by scratch assay. Murine xenografts (n = 12 per group) bearing measurable subcutaneous tumors of 5637 cells were administered either no therapy, cisplatin 2 mg/kg/3 days a week (metronomic) or 6 mg/kg/ once a week (standard) intraperitoneal (i.p.) for 4 weeks. Blood was collected from 5 mice per group at baseline and at the end of therapy to measure changes in serum creatinine. RESULTS: Significant antiproliferative activity, but poor pro-apoptotic activity, was observed against 5637 urothelial carcinoma cells in vitro by MTT assay with cisplatin at concentrations lower than those attainable in vivo. Anti-proliferative activity against HUVECs required higher concentrations than attainable in vivo. Anti-migratory activity was observed against 5637 and HUVECs at earlier time points and with concentrations lower than anti-proliferative concentrations. In murine 5637 xenografts, standard cisplatin was significantly better at inhibiting tumor growth than the no treatment control (P = 0.005), while metronomic therapy was not better than control (P = 0.22). Standard cisplatin was also significantly nephrotoxic (P = 0.016), while metronomic cisplatin (P = 0.374) or no therapy (P = 0.178) did not demonstrate significant nephrotoxicity compared with baseline. CONCLUSIONS: Cisplatin exhibited anti-cell-migratory activity against urothelial carcinoma and endothelial cells at lower than cytotoxic concentrations. However, a standard preclinical schedule was better than control in vivo for inhibiting tumor growth, while a metronomic schedule was not better. Despite the lower nephrotoxicity of the metronomic schedule, its lower anti-tumor activity suggests that a standard clinical schedule of cisplatin should not be routinely substituted by a split schedule without definitive clinical data.

Colloidal quantum dots as saturable fluorophores.

Although colloidal quantum dots (QDs) exhibit excellent photostability under mild excitation, intense illumination makes their emission increasingly intermittent, eventually leading to photobleaching. We study fluorescence of two commonly used types of QDs under pulsed excitation with varying power and repetition rate. The photostability of QDs is found to improve dramatically at low repetition rates, allowing for prolonged optical saturation of QDs without apparent photodamage. This observation suggests that QD blinking is facilitated by absorption of light in a transient state with a microsecond decay time. Enhanced photostability of generic quantum dots under intense illumination opens up new prospects for fluorescence microscopy and spectroscopy.