Quantitative polymerase chain reaction is the current "golden standard" for quantification of nucleic acids; however, its utility is constrained by an inability to easily and reliably detect multiple targets in a single reaction. We have successfully overcome this problem with a novel combination of two widely used approaches: target-specific multiplex amplification with 15 cycles of polymerase chain reaction (PCR), followed by single-molecule detection of amplicons with atomic force microscopy (AFM). In test experiments comparing the relative expression of ten transcripts in two different human total RNA samples, we find good agreement between our single reaction, multiplexed PCR/AFM data, and data from 20 individual singleplex quantitative PCR reactions. This technique can be applied to virtually any analytical problem requiring sensitive measurement concentrations of multiple nucleic acid targets.
Abstract Hematopoietic cell transplantation (HCT) conditioned using myeloablative conditioning (MAC) is complicated by end organ injury due to endothelial dysfunction and graft versus host disease. Mucositis and oxidant injury results in micronutrient deficiency. Ascorbic acid (AA) levels were measured in 15 patients undergoing HCT conditioned with MAC (11 allogeneic and 4 autologous HCT). Ascorbate levels declined post conditioning to 27.3 (±14.1) by day 0 (p <0.05 compared with baseline), reaching a nadir level of 21.5 (±13.8) on day 14 (p <0.05) post-transplant. Patients undergoing allogeneic HCT continued to have low AA levels to day 60 post transplant, whereas recipients of autologous HCT recovered plasma AA levels to normal. The role of AA in maintaining endothelial function and hematopoietic as well as T cell recovery is provided, developing the rationale for repletion of vitamin C following HCT.
DNA length polymorphisms are found in many serious diseases, and assessment of their length and abundance is often critical for accurate diagnosis. However, measuring their length and frequency in a mostly wild-type background, as occurs in many situations, remains challenging due to their variable and repetitive nature. To overcome these hurdles, we combined two powerful techniques, digital polymerase chain reaction (dPCR) and high-speed atomic force microscopy (HSAFM), to create a simple, rapid, and flexible method for quantifying both the size and proportion of DNA length polymorphisms. In our approach, individual amplicons from each dPCR partition are imaged and sized directly. We focused on internal tandem duplications (ITDs) located within the FLT3 gene, which are associated with acute myeloid leukemia and often indicative of a poor prognosis. In an analysis of over 1.5 million HSAFM-imaged amplicons from cell line and clinical samples containing FLT3-ITDs, dPCR–HSAFM returned the expected variant length and variant allele frequency, down to 5% variant samples. As a high-throughput method with single-molecule resolution, dPCR–HSAFM thus represents an advance in HSAFM analysis and a powerful tool for the diagnosis of length polymorphisms.
Prompt and repeated assessments of tumor sensitivity to available therapeutics could reduce patient morbidity and mortality by quickly identifying therapeutic resistance and optimizing treatment regimens. Analysis of changes in cancer cell biomass has shown promise in assessing drug sensitivity and fulfilling these requirements. However, a major limitation of previous studies in solid tumors, which comprise 90% of cancers, is the use of cancer cell lines rather than freshly isolated tumor material. As a result, existing biomass protocols are not obviously extensible to real patient tumors owing to potential artifacts that would be generated by the removal of cells from their microenvironment and the deleterious effects of excision and purification. In this present work, we show that simple excision of human triple-negative breast cancer (TNBC) tumors growing in immunodeficient mouse, patient-derived xenograft (PDX) models, followed by enzymatic disaggregation into single cell suspension, is enabling for rapid and accurate biomass accumulation-based predictions of in vivo sensitivity to the chemotherapeutic drug carboplatin. We successfully correlate in vitro biomass results with in vivo treatment results in three TNBC PDX models that have differential sensitivity to this drug. With a maximum turnaround time of 40 h from tumor excision to useable results and a fully-automated analysis pipeline, the assay described here has significant potential for translation to clinical practice.
Abstract Melanoma is the most aggressive type of skin cancer. Tumor heterogeneity and drug resistance are significant obstacles to survival benefits from mutation-targeted therapy. Over 50% of metastatic melanomas harbor V600BRAF mutations. Targeting of MUTBRAF melanoma with BRAF inhibitor monotherapy or the combination of BRAF and MEK inhibitors leads to a high rate of initial responses, but adaptive and acquired resistance frequently leads to clinical relapse. Although the presence of V600BRAF mutations guides the selection of therapy with MAPK inhibitors, methods to track preexisting and adaptive resistance and thereby predict response patterns, or to use this new information to modify therapy, are still lacking. Here, we aimed to develop a rapid, massively parallel tumor cell profiling method, based on Live Cell Interferometry (LCI), to quantify heterogeneous single tumor cell responses and emergent drug resistance. LCI is an ex vivo imaging approach that quantifies changes in total cell biomass, biomass motion, or cell stiffness over time. We report a novel high-throughput screening version of the LCI platform, HSLCI, which rapidly profiles changes in biomass in BRAF inhibitor (BRAFi)-sensitive, parental melanoma cell lines and their isogenic, BRAFi-resistant sub-lines. We show reproducible results from two different HSLCI platforms at two institutions and generate biomass kinetic signatures capable of discriminating between BRAFi-sensitive and -resistant melanoma cells within 24 hours. Our measurements require no fluorescence or dye labeling and are faster than field-standard growth inhibition assays. The accuracy and speed of HSLCI in profiling tumor cell heterogeneity and therapy resistance are promising features of potential tools to guide patient therapeutic selections. Citation Format: Kevin A. Leslie, Dian Huang, Graeme Murray, Daniel Guest, Irena J. Roy, Marco Piva, Gatien Moriceau, Roger S. Lo, Michael A. Teitell, Jason C. Reed. Quantifying melanoma drug resistance and tumor heterogeneity by live cell interferometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1180.
Abstract Hematopoietic cell transplantation (HCT) conditioned using myeloablative conditioning (MAC) is complicated by end organ injury due to endothelial dysfunction and graft versus host disease. Mucositis and oxidant injury results in micronutrient deficiency. Ascorbic acid (AA) levels were measured in 15 patients undergoing HCT conditioned with MAC (11 allogeneic and four autologous HCT). Ascorbate levels declined postconditioning to 27.3 μMol/L (±14.1) by day 0 ( P = .03 compared with pretransplant baseline), reaching a nadir level of 21.5 (±13.8) on day 14 ( P = .003) post‐transplant. Patients undergoing allogeneic HCT continued to have low AA levels to day 60 post‐transplant. The role of AA in maintaining endothelial function and hematopoietic as well as T‐cell recovery is provided, developing the rationale for repletion of vitamin C following HCT.
In this study, we used a rapid, highly-sensitive, single-cell biomass measurement method, Live Cell Interferometry (LCI), to measure biomass in populations of CD3 + T cells isolated from hematopoietic stem cell transplant (SCT) patients at various times pre- and post-transplant (days 0-100). CD3 + T cell 'mass spectra' were obtained from five autologous and 20 allogenic transplant recipients. We found a pronounced rise in median T cell biomass (+25%; p <0.001) shortly after transplant (day 14), which moderated by day 60. Further, the inter-patient and intra-patient cell masses were most variable at days 14 and 30 post-transplant. T cell biomass trends were similar in both autologous and allogenic transplant recipients. These data suggest that T cell biomass changes are associated with immune reconstitution occurring in the first few weeks post-transplant. To our knowledge, this is the first time single-cell biomass measurements have been studied in human clinical trials. With refinement, these data may prove useful in guiding the withdrawal of immunosuppression following SCT, reducing the likelihood of Graft-Versus-Host Disease or cancer relapse occurring.
Progress in whole-genome sequencing using short-read (e.g., <150 bp), next-generation sequencing technologies has reinvigorated interest in high-resolution physical mapping to fill technical gaps that are not well addressed by sequencing. Here, we report two technical advances in DNA nanotechnology and single-molecule genomics: (1) we describe a labeling technique (CRISPR-Cas9 nanoparticles) for high-speed AFM-based physical mapping of DNA and (2) the first successful demonstration of using DVD optics to image DNA molecules with high-speed AFM. As a proof of principle, we used this new "nanomapping" method to detect and map precisely BCL2-IGH translocations present in lymph node biopsies of follicular lymphoma patents. This HS-AFM "nanomapping" technique can be complementary to both sequencing and other physical mapping approaches.
Abstract Objective: The novel severe acute respiratory coronavirus virus 2 (SARS-CoV-2) was first reported in Wuhan, China, in December 2019 and is notable for being highly contagious and potentially lethal; and SARS-CoV-2 is mainly spread by droplet transmission. The US healthcare system’s response to the COVID-19 pandemic has been challenged by a shortage of personal protective equipment (PPE), especially N95 respirators. Restricted use, reuse, and sanitation of PPE have been widely adopted to provide protection for frontline healthcare workers caring for often critically ill and highly contagious patients. Here, we describe our validated process for N95 respirator sanitation. Design: Process development, validation, and implementation. Setting: Level 1, urban, academic, medical center. Methods: A multidisciplinary team developed a novel evidence-based process for N95 respirator reprocessing and sanitation using ultraviolet (UV) light. Dose measurement, structural integrity, moisture content, particle filtration, fit testing, and environmental testing were performed for both quality control and validation of the process. Results: The process achieved UV light dosing for sanitation while maintaining the functional and structural integrity of the N95 respirators, with a daily potential throughput capacity of ∼12,000 masks. This process has supported our health system to provide respiratory PPE to all frontline team members. Conclusions: This novel method of N95 respirator sanitation can safely enable reuse of the N95 respirators essential for healthcare workers caring for patients with COVID-19. Our high-throughput process can extend local supplies of this critical PPE until the national supply is replenished.
