Search

Single-cell level investigations such as DNA and RNA sequencing, protein analyses, and phenotypic studies require an input sample with a high rate of viability (>90%). If too many dead cells are present in the input, degraded proteins and ambient DNA or RNA can increase background noise that may lead to missing identification of crucial targets. Since the cost for single-cell sequencing experiments still remains high, it is critical to ensure the input sample is optimized for high viability to ensure cost-effective and reproducible data. Current bead assays or other sample viability enrichment techniques used in preparation for single-cell analyses typically result in a significant sample loss, sometimes sacrificing 80% of the starting sample, and these preparation steps require multiple passes (> 45 minutes time) and costly kits. These beads or kits can be biased and change the cell population, subject cells to unwanted stresses, and diminish sample integrity with the time needed for preparations. Here, we investigate sample cell viability enrichment on the Cyto R1 Platform, a label-free, cell enrichment, sorting, and recovery platform. At the core, the Cyto R1 uses Cyto Chips, microfluidic technologies with electrical fields, to phenotypically enrich and sort various cells based upon cells’ unique physical structures and subcellular features. Thus, the Cyto R1 ensures native cell recovery without any unwanted cell tagging to maximize sample integrity. In this work, a variety of single cell solutions derived from T cell lymphoma tumors, ascites fluid, and organ tissues were enriched for viable cells to obtain a final sample viability over 90%. Additionally, we aimed to achieve a minimum of 50,000 viable cells, a common target for single-cell sequencing, in as rapid a timeframe as possible. In one sample origin tested, murine T cell lymphomas, the average starting viability was 65 ± 6.5%, and the final viability after enrichment on the Cyto R1 Platform was 95.8 ± 1.8%. Similarly, murine ascites cell mixtures were enriched from average viabilities below 55% up to 91.6 ± 4.2%. For all samples tested, >50,000 viable cells were obtained in under 30 minutes. This work demonstrates that the Cyto R1 can effectively and efficiently enrich a variety of starting samples for high viability (>90%) as required for downstream assays and single-cell sequencing. Citation Format: Alexandra R. Hyler, Alicia M. Cole, Ridi Barua, Amiran K. Dzutsev, Eva M. Schmelz. Cyto R1 Platform enriches samples’ viability for single-cell sequencing and downstream assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1687.

Background: Monoclonal antibodies (mAb) targeting the programmed cell death protein 1 (PD-1) receptor provide durable long-term benefit in a subset of patients (pts) with advanced melanoma with response rates of 35-42% and 4-year progression-free survival (PFS) rate of 27%. Separately, the composition of the gut microbiota has been shown to correlate with anti-PD-1 efficacy in human cancer pts with melanoma, renal cell cancer and non-small cell lung cancer (NSCLC) although the precise organisms differ considerably across various studies. In preclinical models, responder-derived fecal microbiome and microbiome consortia produce anti-tumor responses. The effect of microbiome modulation in pts with anti-PD-1 refractory melanoma has not been evaluated. Methods: To evaluate whether primary resistance to anti-PD-1 immunotherapy could be overcome by intestinal microbiome modulation, we designed and conducted a phase II study (NCT03341143). We enrolled pts with primary refractory metastatic melanoma with best response of short-term stable disease (≤6 months) or progressive disease (PD) to prior anti-PD-1 based immunotherapy. Pts received single-administration of responder-derived fecal microbiota transplantation (R-FMT) together with pembrolizumab. Candidate donors were pts with advanced melanoma treated with anti-PD-1 immunotherapy with durable partial or complete response (PR, CR). Pembrolizumab was continued till intolerable toxicity or disease progression. Safety and clinical activity (based on RECIST v1.1) were main objectives; while progression-free survival (PFS) was a key secondary endpoint. Results: As of December 1, 2020, 16 pts with primary refractory melanoma were enrolled, of whom 15 were evaluable. LDH was elevated in 14/15 pts; and the median number of prior therapies was 2. Recipient pts were seromatched to receive a single R-FMT from one of eight candidate donors (5 CR; 3 PR; median PFS 58 months, range 43-70). R-FMT was administered via colonoscopy after bowel preparation with no use of antibiotics. Pembrolizumab was administered IV per label. R-FMT/pembrolizumab was well-tolerated, with no unusual toxicity signals. R-FMT induced rapid and durable microbiota perturbation in most pts; while 6 of 15 evaluable pts had evidence of clinical benefit. Response to R-FMT/pembrolizumab was associated with an increased abundance of taxa previously shown to be associated with response to anti-PD-1, increased CD8+ T cell activation, and decreased frequency of IL-8 expressing myeloid cells. Responders had distinct proteomic and metabolomic signatures, and transkingdom network analyses confirmed that the gut microbiome regulated these changes. Conclusions: In pts with anti-PD-1 primary refractory melanoma, R-FMT/pembrolizumab changed the gut microbiome and reprogrammed the tumor microenvironment to overcome resistance to anti-PD-1 immunotherapy. Response was associated with CD8 T cell induction and reduction of IL-8 expressing myeloid cells. Citation Format: Diwakar Davar, Amiran Dzutsev, John A. McCulloch, Richard R. Rodrigues, Joe-Marc Chauvin, Robert M. Morrison, Richelle N. Deblasio, Carmine Menna, Quanquan Ding, Ornella Pagliano, Bochra Zidi, Shuowen Zhang, Jonathan H. Badger, Marie Vetizou, Alicia M. Cole, Miriam R. Fernandes, Stephanie Prescott, Raquel G. Costa, Ascharya K. Balaji, Andrey Morgun, Ivan Vujkovic-Cvijin, Hong Wang, Amir A. Borhani, Marc B. Schwartz, Howard M. Dubner, Scarlett J. Ernst, Amy Rose, Yana G. Najjar, Yasmine Belkaid, John M. Kirkwood, Giorgio Trinchieri, Hassane M. Zarour. Efficacy of Responder-derived Fecal Microbiota Transplant (R-FMT) and Pembrolizumab in Anti-PD-1 Refractory Patients with Advanced Melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB062.

Cachexia is a multifactorial syndrome characterized by weight loss and tissue wasting, which is associated with reduced quality of life, responsiveness to chemotherapy, and decreased survival. We aim to determine whether changes in plasma fatty acid content in pancreatic ductal adenocarcinoma (PDAC): 1) are predictive of developing cachexia, and 2) whether these changes are influenced by other clinical parameters, like diabetes, that could contribute to malnutrition. Treatment-naïve metastatic PDAC subjects (n=51) were enrolled in NCT01280058 in which plasma samples and clinical data were collected prior to treatment. Subjects were assigned to either weight stable ( Citation Format: Kristyn Gumpper, Phil A. Hart, Martha Belury, Olivia Crowe, Rachel M. Cole, Niharika Badi, Alice Hinton, Mitchell L. Ramsey, Anne Noonan, Darwin L. Conwell, Zobeida Cruz-Monserrate. Plasma fatty acid levels in treatment-naïve metastatic pancreatic cancer patients are associated with clinical indicators of cachexia [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-022.

E-cigarette (E-cig) vaping has gained popularity among middle and high-school students within the last decade, being advertised as a safer alternative to cigarette smoke. While acute lung illness has been reported, it is too early to have epidemiologic data suggesting a role of E-cig on oral squamous cell carcinoma. However, recent studies point to its potentially adverse effects on the human epithelium and the oral human microbial flora. Staphylococcus aureus is a resident of the oral microbiota and its carriage has been shown to be significantly higher in the periodontal pocket of patients with actively progressing periodontitis, which is a chronic inflammatory disease and known precursor for oral squamous cell carcinoma (OSCC). Additionally, S. aureus has also been isolated from OSCC tissues. In this study, we aim to identify the response of S. aureus and the oral epithelium to E-cig vape exposure and, further, to determine the effect of E-cig vape on the interaction of S. aureus with the oral epithelium. To measure differences in the ability to form biofilm upon E-cig vape exposure with (3mg/mL) and without nicotine, we analyzed two clinical S. aureus strains isolated from healthy individuals following a 30s puff delivery and a 5-minute incubation. We observed that E-cig vape induced significantly higher biofilm formation than control air exposure in a specifically designed vape chamber regardless of nicotine content (p Citation Format: Alma R. Catala-Valentin, Matthew Caldwell, Alexander M. Cole, Sean Moore, Claudia Andl. E-cigarette vape and cigarette smoke potentially increase S. aureus oral colonization and inflammatory epithelial signaling [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B02.

Background: Tumor-specific T cells possess unique potential for cancer therapy but are limited by T cell exhaustion and anergy induced in the tumor microenvironment. Ex vivo manipulation of these T cells to maintain their full function is critical to their success clinically. Yet, limitations of existing ex vivo delivery approaches dramatically restrict their function and thus limit their therapeutic use. Methods: Genome-wide profiling was used to identify the impact of optimized electroporation treatment and the SQZ cell therapy platform on gene expression in human T cells. The profiling was paired with a 42 key T cell cytokine-multiplex analysis comprised of to assess perturbation of cytokine secretion. We then compared the in vivo functionality of immune checkpoint deleted antigen-specific T cells, modified by either electroporation or SQZ delivery of CRISPR/Cas9, and adoptively transferred into tumor bearing mice. Finally, genomic editing of tumor infiltrating leukocyte (TIL) derived T cells was compared using either electroporation or SQZ and subsequent effector response upon re-exposure to tumor cells. Results: Impactful disruptions in transcript expression after treatment with electroporation (17% of genes mis-regulated, FDR q Conclusions: This work demonstrates that functional modifications to tumor-specific T cells ex vivo can restore and improve their function upon re-exposure to tumor cells but that the delivery mechanism used is critical to the desired phenotype. The significant differences in outcomes from the two techniques tested here underscores the importance of understanding the impact of intracellular delivery methods on cell function for research and clinical applications. For both research and therapeutic applications with primary T cells, the functional consequences of the selected intracellular delivery technique and its impact on cell phenotype should be carefully evaluated. Citation Format: Luke Cassereau, Julie M. Cole, Roslyn Yi, Jacquelyn L. Hanson, Josh Bugge, Tia DiTommaso, Howard Bernstein, Armon Sharei. Tumor-specific T cell engineering for enhanced effector function via microfluidic delivery of bioactive molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1445.

Introduction Clinical research into human intraocular disease UM FFPE tissue sections is described. UM remains the most common intraocular malignancy in adults, with poor prognosis for UM within the choroid region and distant sites UM metastasis. Two imaging MS techniques Matrix Assisted Laser Desorption/Ionisation (MALDI) and Desorption Electrospray Ionisation (DESI) MSI were applied. Molecular profiles were obtained and analysed by multivariate statistical approaches, providing insight into the biochemical and biological differences/similarities within the patient sample cohort (n = 15). Methods Enucleations were collected over 6 years and subjected to the standard fixation and paraffin embedding protocols. In preparation for MS, 5 μm sections were produced with removal of the paraffin followed by heat induced antigen retrieval. MALDI MSI tissue sections were prepared by applying a matrix solution onto the tissue sections to help ionization of molecules directly from the tissue. All MSI experiments (MALDI and DESI) were carried out using a SYNAPT mass spectrometer (Waters Corporation, Manchester, UK). Multivariate analyses were performed using MATLAB (MathWorks, Inc., Natick, MA, US) and the Eigenvector PLS_Toolbox. Results Initial MALDI MSI images acquired with a spatial resolution of 100 μm x 100 μm in positive ion mode showed distinct spatial distribution of many molecular species throughout the choroid, cornea, retina, lens and UM tumor regions. Further MALDI MSI experiments using consecutive tissue sections at 50 μm x 50 μm spatial resolution show substantial variation in the spatial distribution of species within the low molecular mass range. DESI MSI experiments were carried out at 200 μm x 200 μm in negative ionization mode. Deprotonated molecular ions were detected from a variety of lipid related species, localized to specific regions within the eye including the tumor region. Multivariate analysis classified UM samples (good vs. poor prognosis). Using unsupervised PCA, an unbiased representation of the data was generated, with clear sample grouping and differentiation observed based upon tumor status. Use of the supervised PLS-DA technique provided even clearer separation between the selected samples, with tumor profiles displaying dominant discriminatory peaks. These peaks could be identified as sphingolipids and Lyso-phosphocholine, a phosphatidylcholine degradation product. Conclusions It was possible to analyze clinical research FFPE tissue sections by MALDI and DESI MSI, illustrating that specific small molecular species remained localized to certain tissue types including the UM tumor. Initial correlation with tumor status was determined from the statistical analysis of the MALDI MSI datasets. Citation Format: Laura M. Cole, Hardeep S. Mudhar, Karen Sisley, Andrew Peck, Mike Batey, Emmanuelle Claude, Malcolm Clench. Analysis of FFPE treated clinical tissue sections obtained from human intraocular malignancy, uveal melanoma by mass spectrometry imaging (MSI). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4231.

The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in up to 70% of human breast cancer cases. Our previous studies demonstrated that Apc mutation advances the MMTV-Polyoma Middle T Antigen (PyMT) model of breast cancer, with an increase in signaling downstream of focal adhesion kinase (FAK)/Src/JNK activation in MMTV-PyMT;ApcMin/+ tumors. In addition, the majority of tumors that arose in the MMTV-PyMT;ApcMin/+ animals were classified as adenosquamous carcinoma as compared to tumors from MMTV-PyMT;Apc+/+ animals, which were solid carcinomas. Given the heterogeneous nature of these tumors, laser capture microdissection (LCM) of the adenosquamous carcinoma in MMTV-PyMT;ApcMin/+ tissues followed by quantitative real-time RT-PCR was performed to identify gene expression changes responsible for the altered tumor phenotype. In parallel studies, gene expression microarrays were performed using triplicate samples of the MMTV-PyMT;Apc+/+ and MMTV-PyMT;ApcMin/+ cell lines. RNA samples were subjected submitted to quality control via the Agilent Bioanalyzer and were hybridized to Affymetrix Mouse Genome 430 2.0 microarrays. Data analysis was performed using the Gene-Go software, and significant changes were confirmed using qRT-PCR. Multiple changes consistent with the phenotypic and proliferative changes downstream of APC loss were identified, including changes in expression of cyclooxygenase-2 (COX-2). The findings presented here indicate that APC-mediated gene expression changes can be used to predict tumor phenotype and downstream therapeutic targets. Citation Format: Andjela Pehar, Jennifer M. Cole, Jenifer R. Prosperi. Gene expression changes downstream of APC loss predict tumor phenotype. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5385. doi:10.1158/1538-7445.AM2013-5385 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The alkylating agent cyclophosphamide is a prodrug which is metabolized in vivo to produce both therapeutic and toxic effects. Cyclophosphamide metabolism was investigated in 36 children with various malignancies. Concentrations of cyclophosphamide and its principal metabolites were measured in plasma and urine using a quantitative high-performance TLC method. The results indicated a high degree of inter-patient variation in metabolism. In contrast to previous adult studies on urinary metabolites, plasma carboxyphosphamide concentrations did not support the existence of polymorphic metabolism. Plasma concentrations of dechlorethylcyclophosphamide and carboxyphosphamide were correlated in individual patients, suggesting that the activity of both aldehyde dehydrogenase and cytochrome P450 enzyme(s) determine carboxyphosphamide production in vivo. The presence of ketocyclophosphamide in plasma was strongly associated with dexamethasone pretreatment and was also accompanied by a high clearance of the parent drug. Interpatient differences in metabolism reflect individual levels of enzyme expression and may contribute to variation in clinical effect.

The nitric oxide synthase inhibitor NG-nitro-L-arginine (NOARG) was examined for its ability to alter energy metabolism in three murine tumors using 31P magnetic resonance spectroscopy. NOARG (10 mg/kg, i.v.) increased the inorganic phosphate:total phosphate ratio (Pi:total) 2-3-fold in the KHT, RIF-1, and SCCVII/Ha intradermal back tumors from 30 min to 6 h after injection, but the 31P magnetic resonance spectrum from normal tissue on the mouse back was unchanged after this treatment. NOARG (10 mg/kg, i.v.) injected 30 min before X-rays increased tumor cell survival 3-5-fold in SCCVII/Ha and 50-200-fold in RIF-1, measured using an in vivo/in vitro clonogenic assay. These effects were equivalent to those obtained from clamped tumors, indicating full radiobiological hypoxia. In KHT, only a 2-fold increase in radioresistance was observed after NOARG, which was less than the response of clamped tumors. In RIF-1 tumors, NOARG induced full radiobiological hypoxia when given from 30 min to 6 h prior to X-rays, consistent with the time course for the increase in Pi:total, measured by 31P magnetic resonance spectroscopy. Pi:total after NOARG doses of 0.1-10 mg/kg, i.v., increased in a dose-dependent manner in this tumor. Increased RIF-1 tumor radioresistance was similarly dependent on NOARG dose. The combination of the bioreductive agent RB6145 (300 mg/kg, i.p.) 15 min prior to NOARG (10 mg/kg, i.v.) produced greater than 5 decades of KHT tumor cell killing at 24 h after treatment. This combination also increased Pi:total 4.5-fold over the control value at 24 h in the KHT tumor. Histological examination of tumors at this time indicated extensive necrosis.

Expression of the main classes (pi, mu, and alpha) of glutathione S-transferase (GST) was assessed in the blasts of children presenting with acute lymphoblastic leukemia using an immunohistochemical technique. Bone marrow trephine biopsies obtained at presentation from 71 cases were studied (42 boys, 29 girls; age range, 6 months-14 years; median age, 4 years) and expression was correlated with event-free survival. The period of follow-up was 12-108 months, during which time 21 patients (30%) relapsed. All the samples examined were negative for alpha class GST. Samples from 8 patients, all of whom remained in remission at the time of analysis, were found to be negative for pi class GST at presentation. Samples from 44 (patients were negative for mu class GST (62%); of these, 36 patients (82%) remained in remission. In comparison, of the 27 patients who were positive for mu class GST, only 14 (52%) remained in remission. Analysis of event-free survival demonstrated that expression of mu class GST predicts a 3-fold increased risk of relapse (95% confidence interval, 1.25-7.26). This risk factor appears to be independent of other recognized prognostic factors.

Pharmacokinetic studies of etoposide administered at 100-200 mg/m2 to 33 children are described. Twenty-seven studies were performed in children aged10 years. Repeat studies were performed in 11 patients. Median pharmacokinetic parameters were as follows: plasma clearance, 26 ml/min/m2; volume of distribution, 4.9 liters/m2; area under the etoposide plasma concentration-time curve (AUC), 3.9 mg/ml x min per 100 mg/m2. Interindividual variability in pharmacokinetic parameters was large (coefficient of variation (CV) = 30, 28, and 27%, respectively) in comparison with intraindividual variability (CV = 12, 14, and 12%, respectively). Variability in AUC was much greater in those patients treated with 150-200 mg/m2 etoposide than with 100 mg/m2 (CV, 35 versus 13%) and was related to variability in renal function and prior exposure to cisplatin. Data from the first 20 studies were used to develop pharmacokinetic monitoring equations which were validated in a further 13 patients. The most accurate equation relies upon the elimination constant of 51Cr-EDTA and a single blood specimen taken at the end of the etoposide infusion. [formula: see text] where K = 51Cr-EDTA elimination rate constant. This equation showed no significant bias, and the predictive error was small with respect to AUC calculated according to a two-compartment model. Predictive error did not increase with increasing AUC, whereas a marked increase in predictive error was seen for dosing according to body surface area. Dosing according to body surface area alone led to marked over- or underexposure to etoposide in 8 patients. Pharmacokinetic monitoring using the equation described would have identified these patients and permitted dose modification. This approach provides an accurate means of monitoring etoposide AUC for administration times of 1-4 h without the need for detailed pharmacokinetic sampling. It will allow a significant reduction in the variability of exposure seen with surface area-based dosing.