Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry.

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or more fluorescent reagents, each functionalized with a different fluorescent molecule (fluorophore) that binds to cells selectively based on their phenotypic characteristics, such as cell surface antigen expression. The intensity of fluorescence from each reagent bound to cells can be measured on the flow cytometer using channels that detect a specified range of wavelengths. When multiple fluorophores are used, the light from individual fluorophores often spills over into undesired detection channels, which requires a correction to the fluorescence intensity data in a process called compensation. Compensation control particles, typically polymer beads bound to a single fluorophore, are needed for each fluorophore used in a cell labeling experiment. Data from compensation particles from the flow cytometer are used to apply a correction to the fluorescence intensity measurements. This protocol describes the preparation and purification of polystyrene compensation beads covalently functionalized with the fluorescent reagent meso-tetra(4-carboxyphenyl) porphine (TCPP) and their application in flow cytometry compensation. In this work, amine-functionalized polystyrene beads were treated with TCPP and the amide coupling reagent EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) at pH 6 and at room temperature for 16 h with agitation. The TCPP beads were isolated by centrifugation and resuspended in a pH 7 buffer for storage. TCPP-related particulates were observed as a byproduct. The number of these particulates could be reduced using an optional filtration protocol. The resultant TCPP beads were successfully used on a flow cytometer for compensation in experiments with human sputum cells labeled with multiple fluorophores. The TCPP beads proved stable following storage in a refrigerator for 300 days.

Detection of early-stage lung cancer in sputum using automated flow cytometry and machine learning.

BACKGROUND: Low-dose spiral computed tomography (LDCT) may not lead to a clear treatment path when small to intermediate-sized lung nodules are identified. We have combined flow cytometry and machine learning to develop a sputum-based test (CyPath Lung) that can assist physicians in decision-making in such cases. METHODS: Single cell suspensions prepared from induced sputum samples collected over three consecutive days were labeled with a viability dye to exclude dead cells, antibodies to distinguish cell types, and a porphyrin to label cancer-associated cells. The labeled cell suspension was run on a flow cytometer and the data collected. An analysis pipeline combining automated flow cytometry data processing with machine learning was developed to distinguish cancer from non-cancer samples from 150 patients at high risk of whom 28 had lung cancer. Flow data and patient features were evaluated to identify predictors of lung cancer. Random training and test sets were chosen to evaluate predictive variables iteratively until a robust model was identified. The final model was tested on a second, independent group of 32 samples, including six samples from patients diagnosed with lung cancer. RESULTS: Automated analysis combined with machine learning resulted in a predictive model that achieved an area under the ROC curve (AUC) of 0.89 (95% CI 0.83-0.89). The sensitivity and specificity were 82% and 88%, respectively, and the negative and positive predictive values 96% and 61%, respectively. Importantly, the test was 92% sensitive and 87% specific in cases when nodules were < 20 mm (AUC of 0.94; 95% CI 0.89-0.99). Testing of the model on an independent second set of samples showed an AUC of 0.85 (95% CI 0.71-0.98) with an 83% sensitivity, 77% specificity, 95% negative predictive value and 45% positive predictive value. The model is robust to differences in sample processing and disease state. CONCLUSION: CyPath Lung correctly classifies samples as cancer or non-cancer with high accuracy, including from participants at different disease stages and with nodules < 20 mm in diameter. This test is intended for use after lung cancer screening to improve early-stage lung cancer diagnosis. Trial registration ClinicalTrials.gov ID: NCT03457415; March 7, 2018.

Sputum analysis by flow cytometry; an effective platform to analyze the lung environment.

Low dose computed tomography (LDCT) is the standard of care for lung cancer screening in the United States (US). LDCT has a sensitivity of 93.8% but its specificity of 73.4% leads to potentially harmful follow-up procedures in patients without lung cancer. Thus, there is a need for additional assays with high accuracy that can be used as an adjunct to LDCT to diagnose lung cancer. Sputum is a biological fluid that can be obtained non-invasively and can be dissociated to release its cellular contents, providing a snapshot of the lung environment. We obtained sputum from current and former smokers with a 30+ pack-year smoking history and who were either confirmed to have lung cancer or at high risk of developing the disease. Dissociated sputum cells were counted, viability determined, and labeled with a panel of markers to separate leukocytes from non-leukocytes. After excluding debris and dead cells, including squamous epithelial cells, we identified reproducible population signatures and confirmed the samples' lung origin. In addition to leukocyte and epithelial-specific fluorescent antibodies, we used the highly fluorescent meso-tetra(4-carboxyphenyl) porphyrin (TCPP), known to preferentially stain cancer (associated) cells. We looked for differences in cell characteristics, population size and fluorescence intensity that could be useful in distinguishing cancer samples from high-risk samples. We present our data demonstrating the feasibility of a flow cytometry platform to analyze sputum in a high-throughput and standardized matter for the diagnosis of lung cancer.

Quality-Controlled Sputum Analysis by Flow Cytometry.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs. The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

Human, mouse, and dog bone marrow show similar mesenchymal stromal cells within a distinctive microenvironment.

Bone marrow stromal cells (BMSCs) are a key part of the hematopoietic niche. Mouse and human BMSCs are recognized by different markers (LepR and NGFR/CD271, respectively). However, there has not been a detailed in situ comparison of both populations within the hematopoietic microenvironment. Moreover, dog BMSCs have not been characterized in situ by any of those markers. We conducted a systematic histopathological comparison of mouse, human, and dog BMSCs within their bone marrow architecture and microenvironment. Human and dog CD271+ BMSCs had a morphology, frequency, and distribution within trabecular bone marrow similar to those of mouse LepR+ BMSCs. However, mouse bone marrow had higher cellularity and megakaryocyte content. In conclusion, highly comparable bone marrow mesenchymal stromal cell distribution among the three species establishes the validity of using mouse and dog as a surrogate experimental model of hematopoietic stem cell-BMSC interactions. However, the distinct differences in adipocyte and megakaryocyte microenvironment content of mouse bone marrow and how they might influence hematopoietic stem cell interactions as compared with humans require further study.

Identification of a novel mechanism for meso-tetra (4-carboxyphenyl) porphyrin (TCPP) uptake in cancer cells.

Porphyrins are used for cancer diagnostic and therapeutic applications, but the mechanism of how porphyrins accumulate in cancer cells remains elusive. Knowledge of how porphyrins enter cancer cells can aid the development of more accurate cancer diagnostics and therapeutics. To gain insight into porphyrin uptake mechanisms in cancer cells, we developed a flow cytometry assay to quantify cellular uptake of meso-tetra (4-carboxyphenyl) porphyrin (TCPP), a porphyrin that is currently being developed for cancer diagnostics. We found that TCPP enters cancer cells through clathrin-mediated endocytosis. The LDL receptor, previously implicated in the cellular uptake of other porphyrins, only contributes modestly to uptake. We report that TCPP instead binds strongly ( KD=42nM ) to CD320, the cellular receptor for cobalamin/transcobalamin II (Cbl/TCN2). Additionally, TCPP competes with Cbl/TCN2 for CD320 binding, suggesting that CD320 is a novel receptor for TCPP. Knockdown of CD320 inhibits TCPP uptake by up to 40% in multiple cancer cell lines, including lung, breast, and prostate cell lines, which supports our hypothesis that CD320 both binds to and transports TCPP into cancer cells. Our findings provide some novel insights into why porphyrins concentrate in cancer cells. Additionally, our study describes a novel function for the CD320 receptor which has been reported to transport only Cbl/TCN2 complexes.

Context Matters: Distinct Disease Outcomes as a Result of Crebbp Hemizygosity in Different Mouse Bone Marrow Compartments.

Perturbations in CREB binding protein (CREBBP) are associated with hematopoietic malignancies, including myelodysplastic syndrome (MDS). Mice hemizygous for Crebbp develop myelodysplasia with proliferative features, reminiscent of human MDS/myeloproliferative neoplasm-unclassifiable (MDS/MPN-U), and a proportion goes on to develop acute myeloid leukemia (AML). We have also shown that the Crebbp+/- non-hematopoietic bone marrow microenvironment induces excessive myeloproliferation of wild-type cells. We now report that transplantation of unfractionated Crebbp+/- bone marrow into wild-type recipients resulted in either early-onset AML or late-onset MDS and MDS/MPN-U. In contrast, purified Lin-Sca-1+c-Kit++ cells primarily gave rise to MDS with occasional transformation to AML. Furthermore, Crebbp+/- common myeloid progenitors and granulocyte/macrophage progenitors could trigger skewed myelopoiesis, myelodysplasia and late-onset AML. Surprisingly, the phenotypically abnormal cells were all of wild-type origin. MDS, MPN and AML can thus all be transferred from Crebbp+/- BM to wild-type hosts but fractionated bone marrow does not recapitulate the full disease spectrum of whole bone marrow, indicating that not only mutational status but also cellular context contribute to disease outcome. This has important consequences for structuring and interpreting future investigations into the underlying mechanisms of myeloid malignancies as well as for their treatment.

DNMT3A Loss Drives Enhancer Hypomethylation in FLT3-ITD-Associated Leukemias.

DNMT3A, the gene encoding the de novo DNA methyltransferase 3A, is among the most frequently mutated genes in hematologic malignancies. However, the mechanisms through which DNMT3A normally suppresses malignancy development are unknown. Here, we show that DNMT3A loss synergizes with the FLT3 internal tandem duplication in a dose-influenced fashion to generate rapid lethal lymphoid or myeloid leukemias similar to their human counterparts. Loss of DNMT3A leads to reduced DNA methylation, predominantly at hematopoietic enhancer regions in both mouse and human samples. Myeloid and lymphoid diseases arise from transformed murine hematopoietic stem cells. Broadly, our findings support a role for DNMT3A as a guardian of the epigenetic state at enhancer regions, critical for inhibition of leukemic transformation.

A mechanism for 1,4-Benzoquinone-induced genotoxicity.

Benzene is a common environmental toxin and its metabolite, 1-4-Benzoquinone (BQ) causes hematopoietic cancers like myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). BQ has not been comprehensively assessed for its impact on genome maintenance, limiting our understanding of the true health risks associated with benzene exposure and our ability to identify people with increased sensitivity to this genotoxin. Here we analyze the impact BQ exposure has on wild type and DNA repair-defective mouse embryonic stem (ES) cells and wild type human cells. We find that double strand break (DSB) repair and replication fork maintenance pathways including homologous recombination (HR) and Fanconi anemia (FA) suppress BQ toxicity. BQ-induced damage efficiently stalls replication forks, yet poorly induces ATR/DNA-PKCS responses. Furthermore, the pattern of BQ-induced γH2AX and 53BP1foci is consistent with the formation of poly(ADP-ribose) polymerase 1 (PARP1)-stabilized regressed replication forks. At a biochemical level, BQ inhibited topoisomerase 1 (topo1)-mediated DNA ligation and nicking in vitro; thus providing mechanism for the cellular phenotype. These data are consistent with a model that proposes BQ interferes with type I topoisomerase's ability to maintain replication fork restart and progression leading to chromosomal instability that has the potential to cause hematopoietic cancers like MDS and AML.

Early Detection of Lung Cancer with Meso Tetra (4-Carboxyphenyl) Porphyrin-Labeled Sputum.

INTRODUCTION: Early detection of lung cancer in high-risk individuals reduces mortality. Low-dose spiral computed tomography (LDCT) is the current standard but suffers from an exceedingly high false-positive rate (>96%) leading to unnecessary and potentially dangerous procedures. We, therefore, set out to develop a simple, noninvasive, and quantitative assay to detect lung cancer. METHODS: This proof-of-concept study evaluated the sensitivity/specificity of the CyPath Early Lung Cancer Detection Assay to correctly classify LDCT-confirmed cohorts of high-risk control (n = 102) and cancer (n = 26) subjects. Fluorescence intensity parameters of red fluorescent cells (RFCs) from tetra (4-carboxyphenyl) porphyrin (TCPP)-labeled lung sputum samples and subjects' baseline characteristics were assessed for their predictive power by multivariable logistic regression. A receiver operating characteristic curve was constructed to evaluate the sensitivity/specificity of the CyPath assay. RESULTS: RFCs were detectable in cancer subjects more often than in high-risk ones (p = 0.015), and their characteristics differed between cohorts. Two independent predictors of cancer were the mean of RFC average fluorescence intensity/area per subject (p < 0.001) and years smoked (p = 0.003). The CyPath-based classifier had an overall accuracy of 81% in the test population; false-positive rate of 40% and negative predictive value of 83%. CONCLUSIONS: The tetra (4-carboxyphenyl) porphyrin -based CyPath assay correctly classified study participants into cancer or high-risk cohorts with considerable accuracy. Optimizing sputum collection, sample reading, and refining the classifier should improve sensitivity and specificity. The CyPath assay thus has the potential to complement LDCT screening or serve as a stand-alone approach for early lung cancer detection.

Revisiting the case for genetically engineered mouse models in human myelodysplastic syndrome research.

Much-needed attention has been given of late to diseases specifically associated with an expanding elderly population. Myelodysplastic syndrome (MDS), a hematopoietic stem cell-based blood disease, is one of these. The lack of clear understanding of the molecular mechanisms underlying the pathogenesis of this disease has hampered the development of efficacious therapies, especially in the presence of comorbidities. Mouse models could potentially provide new insights into this disease, although primary human MDS cells grow poorly in xenografted mice. This makes genetically engineered murine models a more attractive proposition, although this approach is not without complications. In particular, it is unclear if or how myelodysplasia (abnormal blood cell morphology), a key MDS feature in humans, presents in murine cells. Here, we evaluate the histopathologic features of wild-type mice and 23 mouse models with verified myelodysplasia. We find that certain features indicative of myelodysplasia in humans, such as Howell-Jolly bodies and low neutrophilic granularity, are commonplace in healthy mice, whereas other features are similarly abnormal in humans and mice. Quantitative hematopoietic parameters, such as blood cell counts, are required to distinguish between MDS and related diseases. We provide data that mouse models of MDS can be genetically engineered and faithfully recapitulate human disease.

Potential relationship between inadequate response to DNA damage and development of myelodysplastic syndrome.

Hematopoietic stem cells (HSCs) are responsible for the continuous regeneration of all types of blood cells, including themselves. To ensure the functional and genomic integrity of blood tissue, a network of regulatory pathways tightly controls the proliferative status of HSCs. Nevertheless, normal HSC aging is associated with a noticeable decline in regenerative potential and possible changes in other functions. Myelodysplastic syndrome (MDS) is an age-associated hematopoietic malignancy, characterized by abnormal blood cell maturation and a high propensity for leukemic transformation. It is furthermore thought to originate in a HSC and to be associated with the accrual of multiple genetic and epigenetic aberrations. This raises the question whether MDS is, in part, related to an inability to adequately cope with DNA damage. Here we discuss the various components of the cellular response to DNA damage. For each component, we evaluate related studies that may shed light on a potential relationship between MDS development and aberrant DNA damage response/repair.

Dnmt3a loss predisposes murine hematopoietic stem cells to malignant transformation.

DNA methyltransferase 3A (DNMT3A) is mutated in hematologic malignancies affecting myeloid, mixed, and lymphoid lineages, and these mutations are associated with poor prognosis. Past studies in mice revealed Dnmt3a-knockout (KO)hematopoietic stem cells (HSCs) had increased self-renewal, but no leukemia was observed. Here, all lethally irradiated mice transplanted with Dnmt3a-deleted HSCs died within 1 year. Animals were diagnosed with a spectrum of malignancies similar to those seen in patients with DNMT3A mutations, including myelodysplastic syndrome, acute myeloid leukemia, primary myelofibrosis, and T- and B-cell acute lymphocytic leukemia. In some cases, acquired malignancies exhibited secondary mutations similar to those identified in patients. Loss of Dnmt3a led to disturbed methylation patterns that were distinct in lymphoid and myeloid disease, suggesting lineage-specific methylation aberrations promoted by Dnmt3a loss. Global hypomethylation was observed in all of the malignancies, but lymphoid malignancies also exhibited hypermethylation, particularly at promoter regions. This mouse model underscores the important role of Dnmt3a in normal hematopoietic development and demonstrates that Dnmt3a loss of function confers a preleukemic phenotype on murine HSCs. This model may serve as a tool to study DNMT3A mutation associated malignancies and for developing targeted strategies for eliminating preleukemic cells for prevention and treatment of hematologic malignancies in the future.

Identifying DNA mutations in purified hematopoietic stem/progenitor cells.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases. LacI transgenic mice carry a recoverable λ phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of ß-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation. The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin(-)IL7R(-)Sca-1(+)cKit(++)(LSK) cells and other subpopulations of the hematopoietic system.

Acquired mutations that affect pre-mRNA splicing in hematologic malignancies and solid tumors.

The application of next-generation sequencing technologies to interrogate the genome of human hematologic malignancies is providing promising insights into their molecular etiology and into the pathogenesis of seemingly unrelated malignancies. Among the somatic mutations identified by this approach are ones that target components of the spliceosome, a ribonucleoprotein complex responsible for the posttranscriptional processing of primary transcripts to form mature messenger RNA species. These mutations were initially detected in patients with chronic lymphocytic leukemia or a myelodysplastic syndrome, but can also occur at relatively high frequency in some solid tumors, including uveal malignant melanoma, adenocarcinoma of the lung, and estrogen receptor-positive breast cancers. Their presence in a variety of malignancies suggests that the spliceosomal mutations may play a fundamental role in defining the malignant phenotype. The development and testing of drugs that eliminate cells bearing a spliceosomal mutation, or normalize their altered transcript splicing patterns, are therefore a priority. Here, we summarize the effects of spliceosome-associated mutations on transcript processing in vitro and in vivo, and their impact on disease initiation and/or progression and patient outcome. Moreover, we discuss the therapeutic potential of compounds already known to target splicing factor 3B subunit 1 (SF3B1), an essential component of the spliceosome that is frequently mutated.

Myelodysplastic syndrome: an inability to appropriately respond to damaged DNA?

Myelodysplastic syndrome (MDS) is considered a hematopoietic stem cell disease that is characterized by abnormal hematopoietic differentiation and a high propensity to develop acute myeloid leukemia. It is mostly associated with advanced age, but also with prior cancer therapy and inherited syndromes related to abnormalities in DNA repair. Recent technologic advances have led to the identification of a myriad of frequently occurring genomic perturbations associated with MDS. These observations suggest that MDS and its progression to acute myeloid leukemia is a genomic instability disorder, resulting from a stepwise accumulation of genetic abnormalities. The notion is now emerging that the underlying mechanism of this disease could be a defect in one or more pathways that are involved in responding to or repairing damaged DNA. In this review, we discuss these pathways in relationship to a large number of studies performed with MDS patient samples and MDS mouse models. Moreover, in view of our current understanding of how DNA damage response and repair pathways are affected by age in hematopoietic stem cells, we also explore how this might relate to MDS development.

Defining a genotoxic profile with mouse embryonic stem cells.

Many genotoxins are found in the environment from synthetic to natural, yet very few have been studied in depth. This means we fail to understand many molecules that damage DNA, we do not understand the type of damage they cause and the repair pathways required to correct their lesions. It is surprising so little is known about the vast majority of genotoxins since they have potential to cause disease from developmental defects to cancer to degenerative ailments. By contrast, some of these molecules have commercial and medical potential and some can be weaponized. Therefore, we need a systematic method to efficiently generate a genotoxic profile for these agents. A genotoxic profile would include the type of damage the genotoxin causes, the pathways used to repair the damage and the resultant mutations if repair fails. Mouse embryonic stem (ES) cells are well suited for identifying pathways and mutations. Mouse ES cells are genetically tractable and many DNA repair mutant cells are available. ES cells have a high mitotic index and form colonies so experiments can be completed quickly and easily. Furthermore, ES cells have robust DNA repair pathways to minimize genetic mutations at a particularly vulnerable time in life, early development when a mutation in a single cell could ultimately contribute to a large fraction of the individual. After an initial screen, other types of cells and mouse models can be used to complement the analysis. This review discusses the merging field of genotoxic screens in mouse ES cells that can be used to discover and study potential genotoxic activity for chemicals commonly found in our environment.

Mammospheres from murine mammary stem cell-enriched basal cells: clonal characteristics and repopulating potential.

Identification of murine mammary stem cells (MaSCs) has been attempted with various in vitro and in vivo assays. While, the in vivo repopulation assay remains as the most definitive assay for MaSC detection, it is expensive, time-consuming, and technically challenging. The in vitro mammosphere assay was considered unreliable because of major concerns about its clonal origin. In the current study, co-culture experiments with mammary cells from fluorescent protein transgenic mice and time-lapse video microscopy revealed that >90% mammospheres formed from sorted basal epithelial-enriched cells were of clonal origin in terms of stem cell. These basal-cell derived mammospheres were further distinguished morphologically in a 3-dimensional extracellular matrix culture and functionally in the in vivo repopulation assay. Transplant of single mammospheres or the resultant 3-dimensional solid structures into gland-free mammary fat pads yielded a 70% success rate of multilineage mammary gland reconstitution. Thus, this in vitro sphere formation and differentiation assay is a reliable alternative to the in vivo repopulation assay for the study of MaSCs.

JAK2 and genomic instability in the myeloproliferative neoplasms: a case of the chicken or the egg?

The myeloproliferative neoplasms (MPNs) are a particularly useful model for studying mutation accumulation in neoplastic cells, and the mechanisms underlying their acquisition. This review summarizes our current understanding of the molecular defects present in patients with an MPN, and the effects of mutations targeting Janus kinase 2 (JAK2)-mediated intracellular signaling on DNA damage and on the elimination of mutation-bearing cells by programmed cell death. Moreover, we discuss findings that suggest that the acquisition of disease-initiating mutations in hematopoietic stem cells of some MPN patients may be the consequence of an inherent genomic instability that was not previously appreciated.