Single Tumor Cells Research Articles

High‐Throughput, Living Single‐Cell, Multiple Secreted Biomarker Profiling Using Microfluidic Chip and Machine Learning for Tumor Cell Classification

Secreted proteins provide abundant functional information on living cells and can be used as important tumor diagnostic markers, of which profiling at the single-cell level is helpful for accurate tumor cell classification. Currently, achieving living single-cell multi-index, high-sensitivity, and quantitative secretion biomarker profiling remains a great challenge. Here, a high-throughput living single-cell multi-index secreted biomarker profiling platform is proposed, combined with machine learning, to achieve accurate tumor cell classification. A single-cell culture microfluidic chip with self-assembled graphene oxide quantum dots (GOQDs) enables high-activity single-cell culture, ensuring normal secretion of biomarkers and high-throughput single-cell separation, providing sufficient statistical data for machine learning. At the same time, the antibody barcode chip with self-assembled GOQDs performs multi-index, highly sensitive, and quantitative detection of secreted biomarkers, in which each cell culture chamber covers a whole barcode array. Importantly, by combining the K-means strategy with machine learning, thousands of single tumor cell secretion data are analyzed, enabling tumor cell classification with a recognition accuracy of 95.0%. In addition, further profiling of the grouping results reveals the unique secretion characteristics of subgroups. This work provides an intelligent platform for high-throughput living single-cell multiple secretion biomarker profiling, which has broad implications for cancer investigation and biomedical research.

PD58-04 A COMPREHENSIVE LIQUID BIOPSY TO DETECT PRIMARY UPPER TRACT UROTHELIAL CARCINOMA: A PILOT STUDY

You have accessJournal of UrologyCME1 May 2022PD58-04 A COMPREHENSIVE LIQUID BIOPSY TO DETECT PRIMARY UPPER TRACT UROTHELIAL CARCINOMA: A PILOT STUDY Alireza Ghoreifi, Stephanie Shishido, Jeremy Mason, George Courcoubetis, Manju Aron, Inderbir Gill, Monish Aron, Mihir Desai, Siamak Daneshmand, Peter Kuhn, and Hooman Djaladat Alireza GhoreifiAlireza Ghoreifi More articles by this author , Stephanie ShishidoStephanie Shishido More articles by this author , Jeremy MasonJeremy Mason More articles by this author , George CourcoubetisGeorge Courcoubetis More articles by this author , Manju AronManju Aron More articles by this author , Inderbir GillInderbir Gill More articles by this author , Monish AronMonish Aron More articles by this author , Mihir DesaiMihir Desai More articles by this author , Siamak DaneshmandSiamak Daneshmand More articles by this author , Peter KuhnPeter Kuhn More articles by this author , and Hooman DjaladatHooman Djaladat More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002643.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Diagnosis and preoperative risk stratification of patients with upper tract urothelial carcinoma (UTUC) present distinct challenges given the limitations of current available diagnostic tools, such as imaging and endoscopic biopsy. Blood-based liquid biopsy has emerged as a novel diagnostic and prognostic biomarker in cancer patients, yet the possibility of this test in UTUC patients is unknown. The aim of this study is to explore the feasibility of a liquid biopsy to detect UTUC. METHODS: Under an IRB-approved protocol, blood samples were collected from primary UTUC patients before radical nephroureterectomy between May and October 2021. Patients with concomitant bladder cancer or those with positive bladder biopsies within the past 3 months were excluded. The samples were analyzed using third generation of comprehensive high-definition single cell assay (HDSCA3.0) to detect single cell circulatory tumor cells (CTCs). The findings were compared to the blood samples of normal blood donors (NBDs) who had no known pathology. RESULTS: A total of 5 UTUC patients and 5 NBDs were included in the pilot analysis. Demographic and pathological features of the UTUC patients are shown in Table 1. All patients showed papillary UTUC with no variant histology. HDSCA3.0 analysis indicated that rare cells are detectable in the peripheral blood collected from UTUC patients with high heterogeneity (Figure 1-A). The total rare cell count detected in UTUC patient samples was significantly higher than that from the NBDs (p-value = 0.009). Similar trend was seen in total CK+ cells (p=0.009), DAPI|CK (p=0.009), DAPI|Vim (0.028), DAPI|CK|Vim|CD45/CD31 (p=0.009), and DAPI only (p=0.009) (Figure 1-B). CONCLUSIONS: In this pilot study, the proposed liquid biopsy is feasible in UTUC patients. UTUC samples showed significantly higher rare cell count compared to the normal blood donors. A larger sample size study, including follow-up blood samples, to validate the accuracy of this test is the next step. Source of Funding: USC Urology Pilot Research Program © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e1012 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alireza Ghoreifi More articles by this author Stephanie Shishido More articles by this author Jeremy Mason More articles by this author George Courcoubetis More articles by this author Manju Aron More articles by this author Inderbir Gill More articles by this author Monish Aron More articles by this author Mihir Desai More articles by this author Siamak Daneshmand More articles by this author Peter Kuhn More articles by this author Hooman Djaladat More articles by this author Expand All Advertisement PDF DownloadLoading ...

Tie2 Receptor in Tumor-Infiltrating Macrophages Is Dispensable for Tumor Angiogenesis and Tumor Relapse after Chemotherapy.

Multiple preclinical tumor models, cell stimulation experiments, and meta-analysis of published tumor single cell RNA sequencing data challenge the reported role of Tie2-positive macrophages for tumor angiogenesis, metastasis, and relapse after chemotherapy. See related commentary by Zhang and Brekken, p. 1172.

DualGCN: a dual graph convolutional network model to predict cancer drug response

BackgroundDrug resistance is a critical obstacle in cancer therapy. Discovering cancer drug response is important to improve anti-cancer drug treatment and guide anti-cancer drug design. Abundant genomic and drug response resources of cancer cell lines provide unprecedented opportunities for such study. However, cancer cell lines cannot fully reflect heterogeneous tumor microenvironments. Transferring knowledge studied from in vitro cell lines to single-cell and clinical data will be a promising direction to better understand drug resistance. Most current studies include single nucleotide variants (SNV) as features and focus on improving predictive ability of cancer drug response on cell lines. However, obtaining accurate SNVs from clinical tumor samples and single-cell data is not reliable. This makes it difficult to generalize such SNV-based models to clinical tumor data or single-cell level studies in the future.ResultsWe present a new method, DualGCN, a unified Dual Graph Convolutional Network model to predict cancer drug response. DualGCN encodes both chemical structures of drugs and omics data of biological samples using graph convolutional networks. Then the two embeddings are fed into a multilayer perceptron to predict drug response. DualGCN incorporates prior knowledge on cancer-related genes and protein–protein interactions, and outperforms most state-of-the-art methods while avoiding using large-scale SNV data.ConclusionsThe proposed method outperforms most state-of-the-art methods in predicting cancer drug response without the use of large-scale SNV data. These favorable results indicate its potential to be extended to clinical and single-cell tumor samples and advancements in precision medicine.

Fibroblast activation protein identifies Consensus Molecular Subtype 4 in colorectal cancer and allows its detection by 68Ga-FAPI-PET imaging

BackgroundIn colorectal cancer (CRC), the consensus molecular subtype 4 (CMS4) is associated with therapy resistance and poor prognosis. Clinical diagnosis of CMS4 is hampered by locoregional and temporal variables influencing CMS classification. Diagnostic tools that comprehensively detect CMS4 are therefore urgently needed.MethodsTo identify targets for molecular CMS4 imaging, RNA sequencing data of 3232 primary CRC patients were explored. Heterogeneity of marker expression in relation to CMS4 status was assessed by analysing 3–5 tumour regions and 91.103 single-tumour cells (7 and 29 tumours, respectively). Candidate marker expression was validated in CMS4 peritoneal metastases (PM; n = 59). Molecular imaging was performed using the 68Ga-DOTA-FAPI-46 PET tracer.ResultsFibroblast activation protein (FAP) mRNA identified CMS4 with very high sensitivity and specificity (AUROC > 0.91), and was associated with significantly shorter relapse-free survival (P = 0.0038). Heterogeneous expression of FAP among and within tumour lesions correlated with CMS4 heterogeneity (AUROC = 1.00). FAP expression was homogeneously high in PM, a near-homogeneous CMS4 entity. FAPI-PET identified focal and diffuse PM that were missed using conventional imaging. Extra-peritoneal metastases displayed extensive heterogeneity of tracer uptake.ConclusionFAP expression identifies CMS4 CRC. FAPI-PET may have value in the comprehensive detection of CMS4 tumours in CRC. This is especially relevant in patients with PM, for whom effective imaging tools are currently lacking.

Expression profile of the microenvironment of morphological structures in luminal breast cancer

In breast cancer, the predictive value of tumor microenvironment parameters differs depending on the molecular subtype. Multidirectional significance may be reflection of heterogeneity of invasive breast carcinoma and requires further study. Significance of the tumor microenvironment in the formation and determination of the properties of various morphological structures is still not fully understood. The aim of the study is to determine the differences in the expression profile of the microenvironment of various morphological structures of breast cancer. 84 patients with breast cancer (T1-3N0-3M0) were included in the study of tumor-infiltrating lymphocytes level in microenvironment of different morphological structures. Microdissected samples of the microenvironment of different morphological structures sequenced using NextSeq500 (Illumina). CIBERSORT analysis was performed to deciphered the cellular composition of the microenvironment of each type of the structures. The microenvironment of all morphological structures was quite diverse in terms of cellular composition. The microenvironment of the single tumor cells exhibits pronounced pro-tumor properties, including expression of the IL1B gene, activation of inflammatory signaling pathways, increased signature of M2 macrophages. The results shown the role of the microenvironment of single tumor cells in breast cancer distant metastasis.

Tumor spheroid invasion in epidermal growth factor gradients revealed by a 3D microfluidic device

Epidermal growth factor (EGF), a potent cytokine, is known to promote tumor invasion both in vivo and in vitro. Previously, we observed that single breast tumor cells (MDA-MB-231 cell line) embedded within a 3D collagen matrix displayed enhanced motility but no discernible chemotaxis in the presence of linear EGF gradients using a microfluidic platform. Inspired by a recent theoretical development that clustered mammalian cells respond differently to chemical gradients than single cells, we studied tumor spheroid invasion within a 3D extracellular matrix (ECM) in the presence of EGF gradients. We found that EGF gradients promoted tumor cell detachment from the spheroid core, and the position of the tumor spheroid core showed a mild chemotactic response towards the EGF gradients. For those tumor cells detached from the spheroids, they showed an enhanced motility response in contrast to previous experimental results using single cells embedded within an ECM. No discernible chemotactic response towards the EGF gradients was found for the cells outside the spheroid core. This work demonstrates that a cluster of tumor cells responds differently than single tumor cells towards EGF gradients and highlights the importance of a tumor spheroid platform for tumor invasion studies.

An Integrated Analysis of the Identified PRPF19 as an Onco-immunological Biomarker Encompassing the Tumor Microenvironment, Disease Progression, and Prognoses in Hepatocellular Carcinoma.

Background: Targeting the mRNA splicing process has been identified as a therapeutic strategy for human cancer. PRPF19 is an RNA binding protein that is involved in pre-mRNA processing and repairing DNA damage; the aberrant expression of PRPF19 is potentially associated with carcinogenesis. However, the biological role of PRPF19 in hepatocellular carcinoma (HCC) is still elusive. Methods: Data obtained from TCGA, Oncomine, and GEO were used to investigate the PRPF19 expression level and its role in tumor immune infiltration, prognosis, and the tumor progression of cohorts from HCC. Using various databases and tools (UALCAN, TIMER, TISMO, and PathCards), we presented the potential mechanisms of PFPF19 upregulation, PRPF19-related pathways, and its biological functions in liver cancer. Results: For HCC, PRPF19 expression was found upregulated both in single tumor cells and tissues. Furthermore, the increased expression of PRPF19 was significantly correlated to clinical characteristics: advanced stage, vascular invasion, high AFP, and poor prognosis of HCC. According to the tumor-immunological analysis, we found that PRPF19 is positively correlated with infiltrating myeloid-derived suppressor cells (MDSCs). Moreover, the microenvironment of HCC tissues with high expression of PRPF19 is highly immunosuppressive (lower T-lymphocytes, multiple immune checkpoints upregulated). Patients with high expression of PRPF19 and high MDSCs had a worse survival prognosis as well. TP53 mutation may have a positive effect on PRPF19 expression via decreased promoter methylation of PRPF19. By TF-mRNA network analysis, key transcription factors (TFs) in TC-NER and PCS pathways (PRPF19 involved) were identified. Conclusion: This work implied that PRPF19 is associated with tumor immune evasion and progression, and serves as a prognostic marker for worse clinical outcomes with HCC. Thus, this critical regulator could serve as a potential therapeutic target of HCC.

Abstract P1-05-07: Spatially-resolved single-cell tumor heterogeneity captured by TumorScope biophysical modeling software using MR Imaging

Abstract Background: Dysregulated cellular metabolism is a hallmark of breast cancer, and targeting it has promising implications for improving care and patient outcomes. Specifically, heterogeneity in tumor metabolism is thought to play a role in determining chemotherapy response, the development of resistance, and promoting metastastasis. Despite this, metabolic tumor heterogeneity for individual breast cancer patients has not been characterized completely. Methods: In this study, we used state-of-the-art techniques to characterize metabolic heterogeneity within individual patient tumors by integrating single cell RNA-seq data with genome-scale metabolic modeling. Using SimBioSys’ TumorScope - a commercially available biophysical modeling platform, we compared intra-tumoral metabolic heterogeneity from experimental single cell RNA-seq data to simulated intra tumoral heterogeneity. Results: Using single cell RNA-seq data, we found that intra-tumoral gradients in nutrient availability are widely present within patient tumors (for a single luminal A patient, glucose import flux ranged from 0.19 - 1.25 g/gDW/day, while glutathione import ranged from 0.004 - 0.054 g/gDW/day). We also found that these gradients lead to cellular growth rate gradients within individual tumors (for our representative patient, median SGR = 0.62 %/day +/- 0.33 %/day stdev). Using TumorScope, we found this same gradient behavior within patient tumors. Selecting a similarly growing luminal A patient from our TumorScope simulations resulted in gradients in glucose import (range = 0.17 - 1.26 g/gDW/day), glutathione import (range = 0.024 - 0.058 g/gDW/day), and tumor SGR (median = 0.40 %/day, stdev = 0.42 %/day), which closely match metabolism from single cells (comparing maximum-scaled SGR distributions between single cells and TumorScope yielded a p-value = 0.10). We next examined which nutrients govern heterogeneity in tumor SGR. We found that glucose availability with the tumor microenvironment is more limiting to cell growth than oxygen availability, and this result was consistent between metabolic profiles from both single cell RNA-seq data and TumorScope simulations. TumorScope’s spatially resolved simulations offered the additional insight that gradients in nutrient availability are caused by heterogeneity in the distribution of macro- and micro-vasculature and the composition of the tumor microenvironment. We then used data reduction techniques to compare populations of single cells with differing metabolic phenotypes to identify molecular behavior at the single cells in higher molecular resolution. We found that single cells collected from the clinic co-cluster with single cells from TumorScope simulations, suggesting that a significant amount of intra-tumoral metabolic heterogeneity observed in patients is captured by TumorScope simulations. Conclusion: Accessing tumor heterogeneity has traditionally required specialized equipment, analytic expertise, and invasive procedures, largely limiting its study to large, academic hospitals. Currently, metabolic heterogeneity is only understood in 2D and for few markers (using pathology slides) or in relatively few cells with little or no spatial resolution (for single cell RNA-seq). TumorScope provides a novel approach to simulate metabolic heterogeneity at the single cell scale in 3D across a whole tumor. TumorScope democratizes the study of tumor heterogeneity by making it accessible to clinicians and researchers from MRI data alone. TumorScope has the capability to capture tumor metabolic heterogeneity at a higher scale than previously achievable which will allow for a dramatic increase in our understanding of tumor biology and ultimately improve clinical decision making. Citation Format: Daniel J Cook, John Whitman, Nicole Liadis, John Cole. Spatially-resolved single-cell tumor heterogeneity captured by TumorScope biophysical modeling software using MR Imaging [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-07.

Refining M1 stage in medulloblastoma: criteria for cerebrospinal fluid cytology and implications for improved risk stratification from the HIT-2000 trial

BackgroundMedulloblastoma is the most common malignant paediatric brain tumour, and cerebrospinal fluid (CSF) dissemination (M1 stage) is a high-risk prognostic factor. Criteria for CSF evaluation and for differentiating M0 from M1 stage are not clearly defined, and the prognostic significance of M1 stage in this context is unknown. Patients and methodsCSF investigations from 405 patients with medulloblastoma of the prospective multicenter trial HIT-2000 (HIirnTumor-2000) were reviewed. Data from 213 patients aged ≥4 years were related to 5-year progression-free (5y-PFS) and overall survival. ResultsPatients with cytological tumour dissemination only (M1 stage only) aged ≥4 years (n = 18) and patients with radiologically detected metastases (M2/3, n = 85) showed a worse 5y-PFS than M0 patients (n = 110) without signs of metastatic disease (5y-PFS 61.1% and 59.6% vs 80.7%; p < 0.02 and p < 0.01, log rank). Patients with positive samples drawn early after surgery who turned negative within 14 days postoperatively (n = 9) and patients with atypical cells (n = 6) showed a 5y-PFS similar to M0 patients. No tumour cells were detected in samples containing <10 nucleated cells. Analysis of cytological criteria showed a better predictive value for tumour cell clusters than ≥2 individual tumour cells. ConclusionBased on our results, we suggest that CSF medulloblastoma staging should be performed 14 days postoperatively by lumbar puncture, and specimens should contain at least 10 nucleated cells. Cytological tumour dissemination alone (M1 stage only) appears a high-risk prognostic factor associated with an outcome comparable to M2/M3 stage. Tumour cell clusters seem to have a greater impact on prognosis than single tumour cells. This should be validated further.

Functional visualization of NK cell-mediated killing of metastatic single tumor cells.

Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here, we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hr of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hr after tumor cell arrival, but after 24 hr of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24-hr period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing, the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung.

Low-grade oncocytic tumor of kidney harboring TSC/MTOR mutation: clinicopathologic, immunohistochemical and molecular characteristics support a distinct entity.

Low-grade oncocytic tumor (LOT) has recently been described as a distinct renal tumor. LOT shows consistent morphologic features and a CK7-positive/CD117-negative immunophenotype. To examine the clinicopathological, immunohistochemical, and molecular features of LOT, we searched our institutional archives and identified seven cases of LOT. All patients were female, with a mean age of 66years (range 44-79years). The average tumor size was 3.2cm (range 1.6-5.5cm). Macroscopically, the tumors showed tan-brown and solid cut surfaces. Microscopically, the tumors showed compact nested to solid growth pattern, three cases with areas of edematous stroma containing loosely connected small clusters, cords or dispersed single tumor cells. The tumor cells had uniformly round to oval nuclei with eosinophilic cytoplasm, and showed perinuclear halos. Two cases focally had nuclear irregularities and binucleated cells were occasionally seen in three cases. Immunohistochemically, diffuse positivity for CK7 and lack of CD117 expression were present in all cases. All of the tumors were negative for CD10, CK20, vimentin, CA9, TFE3, TFEB, HMB45, and Melan-A. All tumors were positive for MTOR and negative for Cathepsin-K. FH and SDHB were retained. Next generation sequencing identified genetic variations in the MTOR pathway related genes: TSC1 (4/7), TSC2 (5/7), and MTOR (1/7). All patients were alive and without disease progression, after a mean follow-up of 43months (range 6-89months). LOT is an uncommon eosinophilic renal neoplasm with unique morphological and characteristic immunophenotypic features, and may represent an emerging separate renal entity characterized by mutations in the TSC/MTOR pathway.

Single-Cell Genomics-Based Molecular Algorithm for Early Cancer Detection.

As one of the prime applications of liquid biopsy, the detection of tumor-derived whole cells and molecular markers is enabled in a noninvasive means before symptoms or hints from imaging procedures used for cancer screening. However, liquid biopsy is not a diagnostic test of malignant diseases per se because it fails to establish a definitive cancer diagnosis. Although single-cell genomics provides a genome-wide genetic alternation landscape, it is technologically challenging to confirm cell malignancy of a suspicious cell in body fluids due to unknown technical noise of single-cell sequencing and genomic variation among cancer cells, especially when tumor tissues are unavailable for sequencing as the reference. To address this challenge, we report a molecular algorithm, named scCancerDx, for confirming cell malignancy based on single-cell copy number alternation profiles of suspicious cells from body fluids, leading to a definitive cancer diagnosis. The scCancerDx algorithm has been trained with normal cells and cancer cell lines and validated with single tumor cells disassociated from clinical samples. The established scCancerDx algorithm then validates hexokinase 2 (HK2) as an efficient metabolic function-associated marker of identifying disseminated tumor cells in different body fluids across many cancer types. The HK2-based test, together with scCancerDx, has been investigated for the early detection of bladder cancer (BC) at a preclinical phase by detecting high glycolytic HK2high tumor cells in urine. Early BC detection improves patient prognosis and avoids radical resection for enhancing life quality.

Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray\u2122\u2013HER2-FISH workflow

BackgroundIn breast cancer (BC), overexpression of HER2 on the primary tumor (PT) is determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) to stratify samples as negative, equivocal and positive to identify patients (pts) for anti-HER2 therapy. CAP/ASCO guidelines recommend FISH for analyzing HER2/neu (ERBB2) gene amplification and for resolving equivocal HER2 IHC results. However, pre-analytical and analytical aspects are often confounded by sample related limitations and tumor heterogeneity and HER2 expression may differ between the PT and circulating tumor cells (CTCs), the precursors of metastasis. We used a validation cohort of BC patients to establish a new DEPArray™-PT-HER2-FISH workflow for further application in a development cohort, characterized as PT-HER2-negative but CTC-HER2/neu-positive, to identify patients with PT-HER2 amplified cells not detected by routine pathology.Methods50 µm FFPE tumor curls from the validation cohort (n = 49) and the development cohort (n = 25) underwent cutting, deparaffinization and antigen retrieval followed by dissociation into a single-cell suspension. After staining for cytokeratin, vimentin, DAPI and separation via DEPArray™, single cells were processed for HER2-FISH analysis to assess the number of chromosome 17 and HER2 loci signals for comparison, either with available IHC or conventional tissue section FISH. CTC-HER2/neu status was determined using the AdnaTest BreastCancer (QIAGEN, Hilden, Germany).ResultsApplying CAP/ASCO guidelines for HER2 evaluation of single PT cells, the comparison of routine pathology and DEPArray™-HER2-FISH analysis resulted in a concordance rate of 81.6% (40/49 pts) in the validation cohort and 84% (21/25 pts) in the development cohort, respectively. In the latter one, 4/25 patients had single HER2-positive tumor cells with 2/25 BC patients proven to be HER2-positive, despite being HER2-negative in routine pathology. The two other patients showed an equivocal HER2 status in the DEPArray™-HER2-FISH workflow but a negative result in routine pathology. Whereas all four patients with discordant HER2 results had already died, 17/21 patients with concordant HER2 results are still alive.ConclusionsThe DEPArray™ system allows pure tumor cell recovery for subsequent HER2/neu FISH analysis and is highly concordant with conventional pathology. For PT-HER2-negative patients, harboring HER2/neu-positive CTCs, this approach might allow caregivers to more effectively offer anti-HER2 treatment.

A case of solitary plasmacytoma of bone showing co-expression of both immunoglobulin light chains

BackgroundSolitary plasmacytoma of bone (SPB) is a rare plasma cell neoplasm. It arises in bone as a single locus in the absence of any plasma cell myeloma lesions. Plasma cell neoplasms intrinsically express only one immunoglobulin light chain (IgL)—kappa or lambda—and using this fact, kappa/lambda deviation is the decisive factor for diagnosis. Co-expression of both IgLs in a single tumor cell is extremely rare.Case presentationWe report a case of SPB that arose in the vertebra of a 52-year-old Japanese woman. Histologically, the resected mass showed diffuse plasma cell proliferation. Dual IgL expression was detected by flow cytometry, immunohistochemistry, and in situ hybridization (ISH) targeting IgL mRNA.ConclusionWe have presented an extremely rare case of SPB showing dual expression of kappa and lambda IgLs. This unusual case of plasma cell neoplasia might represent a possible exceptional example of failure of “IgL isotypic exclusion.”

Immunohistochemical features of gastrointestinal stromal tumors and their role for differential diagnosis and prognosis

The aim. To clarify all most important immunohistochemical features of gastrointestinal stromal tumors with different histological patterns and analyze the role of expression of Ki-67, MMP-9, VEGF and p16ink4A as a predictive markers of tumor progression.&#x0D; Materials and methods. The study is based on analysis of 100 primary GISTs for description of their morphological features and 36 GISTs taken from this 100 for study of prognostic markers.&#x0D; Results. All spindle cell GISTs have shown diffuse expression of CD117 in tumor cells. The levels of CD117 expression varied from strong expression (3+) until mild expression (1+). Strong expression were seen in 75,8 % of spindle cell GISTs. Epithelioid GISTs demonstrated heterognous moderate or mild expression of CD117. All primary epithelioid GISTs from patients that had relapse of tumor in period from 1 till 3 years demonstrated focal mild expression of CD 117 in tumor cells. Expression of DOG-1 were seen in all 100 cases of GISTs, that were included in our study. The strong expression of DOG-1 (3+) were seen in all 45 GISTs that had low mitotic rate (≤5 mitoses per 50HPF) and not associated with their histological pattern. GISTs with high mitotic rate demonstrated heterogeneous expression of DOG-1 in tumors: moderate expression (2+) with patchy areas of strong expression (3+). Expression of CD56 was not found in spindle cell GISTs, but single tumor cells of epithelioid GISTs that had high mitotic rate demonstrated expression of this marker. The average expression of p16ink4A were higher in tumors that gave relapses compared with tumors without relapses (50,3 % versus 5,7 % respectively, U-test=16.5; p≤0,01).The average expression of MMP-9 also were significantly higher in GISTs that gave relapses: 63,2 % compared with 13,4 % in GISTs without relapse (U-test=16; p≤0 ,01).The strong VEGF expression was found in 66,7 % of GISTs that had relapses and only in 8,3 % of GISTs without relapses. 50 % of GISTs without relapses was negative for VEGF. Finally, the average expression of Ki-67 were 13,4 % in GISTs with relapses and 8,7 % in GISTs without them (U-test=16; p≤0,01).&#x0D; Conclusion. We highly recommend using DOG-1 for epithelioid GISTs. Additionally in epithelioid GISTs can be used CD56 that can give focal positive reaction in some tumour cells. The following minimal panel of markers for differential diagnosis of spindled GISTs from other mesenchymal tumors of gastrointestinal tract is proposed: CD117, DOG-1 and SMA, where the first too markers will demonstrated the moderate or strong diffuse expression and SMA can be occasionally positive in some tumor cells. p16ink4A, ki-67, VEGF and MMP-9 can be used as additional prognostic markers in GISTs.

Phage-Based Profiling of Rare Single Cells Using Nanoparticle-Directed Capture.

Advances in single-cell level profiling of the proteome require quantitative and versatile platforms, especially for rare cell analyses such as circulating tumor cell (CTC) profiling. Here we demonstrate an integrated microfluidic chip that uses magnetic nanoparticles to capture single tumor cells with high efficiency, permits on-chip incubation, and facilitates in situ cell-surface protein expression analysis. Combined with phage-based barcoding and next-generation sequencing technology, we were able to monitor changes in the expression of multiple surface markers stimulated in response to CTC adherence. Interestingly, we found fluctuations in the expression of Frizzled2 (FZD2) that reflected the microenvironment of the single cells. This platform has a high potential for in-depth screening of multiple surface antigens simultaneously in rare cells with single-cell resolution, which will provide further insights regarding biological heterogeneity and human disease.

Spatial and temporal dynamics of RhoA activities of single breast tumor cells in a 3D environment revealed by a machine learning-assisted FRET technique

One of the hallmarks of cancer cells is their exceptional ability to migrate within the extracellular matrix (ECM) for gaining access to the circulatory system, a critical step of cancer metastasis. RhoA, a small GTPase, is known to be a key molecular switch that toggles between actomyosin contractility and lamellipodial protrusion during cell migration. Current understanding of RhoA activity in cell migration has been largely derived from studies of cells plated on a two-dimensional (2D) substrate using a FRET biosensor. There has been increasing evidence that cells behave differently in a more physiologically relevant three-dimensional (3D) environment. However, studies of RhoA activities in 3D have been hindered by low signal-to-noise ratio in fluorescence imaging. In this paper, we present a a machine learning-assisted FRET technique to follow the spatiotemporal dynamics of RhoA activities of single breast tumor cells (MDA-MB-231) migrating in a 3D as well as a 2D environment. We found that RhoA activity is more polarized along the long axis of the cell for single cells migrating on 2D fibronectin-coated glass versus those embedded in 3D collagen matrices. In particular, RhoA activities of cells in 2D exhibit a distinct front-to-back and back-to-front movement during migration in contrast to those in 3D. Finally, regardless of dimensionality, RhoA polarization is found to be moderately correlated with cell shape.

Effect of energy requirements in the growth of brain tumor: a theoretical approach

A malignant tumor is an uncontrolled growth of tissues receiving energy in form of the nutrients provided by the microvascular networks. It is proposed that the supplied energy to a tumor is used for three purposes: the creation of new cells, maintenance of tumor cells, and tumor volume expansion by overcoming external pressure. A mathematical model studying the effects of energy required for maintenance and overcoming external pressure, the energy required creating a single cell, death rate, and tumor cell density on tumor development has been formulated. Including a term, residual energy for tumor growth in the tumor growth equation, the well-known logistic equation has been re-derived for tumors. Analytical solutions have been developed, and numerical analysis for the growth in brain tumors with the variation of parameters related to energy supply, the energy required for maintenance, and expansion of tumor has been performed. Expressions for the tumor growth rate(r) and carrying capacity(C) of the tumor are formulated in terms of the parameters used in the model. The range of ‘r’, estimated using our model is found within the ranges of tumor growth rates in gliomas reported by the other researchers. Selecting the model parameters precisely for a particular individual, the tumor growth rate and carrying capacity could be estimated accurately. Our study indicates that the actual growth rate and carrying capacity of a tumor reduce and tumor saturation time increases with the increase of death rate, the energy required for a single cell division, and energy requirement for the tumor cell maintenance.

Genetic analysis of single disseminated tumor cells in the lymph nodes and bone marrow of patients with head and neck squamous cell carcinoma.

Considering the limited information on the biology and molecular characteristics of disseminated tumor cells (DTCs) in head and neck squamous cell carcinoma (HNSCC), we examined the genomic alterations in DTCs from HNSCCs and their potential clinical relevance. To analyze both the lymphatic and hematogenous routes of tumor cell dissemination, we investigated samples from lymph nodes (LNs) and bone marrow (BM) of 49 patients using immunofluorescence double staining for epithelial cells expressing cytokeratin 18 (KRT18) and/or epithelial cell adhesion molecules (EpCAM, CD326). The identified marker‐positive cells were isolated by micromanipulation followed by single‐cell whole‐genome amplification and metaphase‐based comparative genomic hybridization (mCGH) to determine genome‐wide copy number alterations. The findings were correlated with clinical parameters and follow‐up data. We detected chromosomal aberrations in KRT18‐ and EpCAM‐positive cells from both compartments; BM‐derived cells showed a significantly higher percentage of aberrant genome (PAG) per cell than cells detected in LNs. No significant association was found between DTC data and clinical follow‐up. Genomic profiling of BM‐DTCs revealed genomic alterations typical for HNSCC, suggesting hematogenous dissemination of subclones around the time of surgery. In contrast, DTC data in LNs revealed that several marker‐positive cells were not of malignant origin, indicating the presence of epithelial glandular inclusions in parts of the processed neck LN samples. Therefore, DTC detection of LNs in the neck based only on epithelial markers is not advisable and requires detection of chromosomal instability (CIN), gene mutations, or additional markers, which have yet to be identified. Nevertheless, our investigation paves the way for larger studies to focus on HNSCC BM‐DTCs with high‐resolution methods to gain deeper insights into the biology of hematogenous metastasis in this cancer.