Individual Tumor Cells Research Articles

Calcium Carbonate Nanoparticles Can Activate the Epithelial⁻Mesenchymal Transition in an Experimental Gastric Cancer Model.

Previously, we have shown the possibility of intramucosal gastric carcinoma induction by the intragastric administration of a mixture of formaldehyde and hydrogen peroxide in rats. In this study, we report a sizable increase in carcinogenic properties of the mixture when a suspension containing calcium carbonate nanoparticles was added to it. This technique allowed us to reduce both the number of the carcinogen administrations from twelve to two and the time to the cancer induction from six to four months. Although the induced tumors were represented by the intramucosal carcinomas, they were characterized by the extensive invasion of individual tumor cells and their clusters into the muscle layer and serosa as well as into the omentum and blood vessels. Considering that the invasive tumor cells were positive for vimentin, Snail and TGF-β2, we concluded that their invasion was the result of the activation of epithelial–mesenchymal transition (EMT) mechanisms. Thus, taking into account the data obtained, it can be assumed that under the conditions of inflammation or carcinogenesis, the calcium carbonate nanoparticles may affect the activation of EMT mechanisms.

Ultrastructural comparative analysis of neuroendocrine tumors of pancreas

Detection of ultrastructural distinctions of tumor cells and cell-to-cell cooperation in insulinomas and nonfunctioning neuroendocrine tumors of pancreas. Ultrastructural study of 38 insulinomas and 35 nonfunctioning neuroendocrine tumors of pancreas of the patients treated at Vishnevskiy Surgery Institute from 2010 to 2015. Insulinomas are characterized by high differentiation of intracellular organelles and specific secretory granules. In insulinomas the number of immature endocrine granules exceeded the number of mature granules. The tumor cells were often joined together and held with desmosomes ensuring very tight connection. Desmosome complexes were characterized by their extended length. Such ultrastructural changes were not followed by destruction of tumor cells. There was formation of specific syncytium-like structures consisting of cells connected by cytoplasmic channels with the common cytoplasm with cell organelles. In nonfunctioning tumors the most of the granules were distinguished by polymorphism and had no specific ultrastructure indicating the synthesized hormone. In large areas there were loss of plasmolemma integrity, up to its complete absence, which led to unification of cytoplasmic contents of the cells. Common junctions dominated in nonfunctioning tumors. Desmosomes were rare, poorly developed, and in the form of immature and reduced junctions. Complex cellular junctions are well developed in the cells, ensuring tight connection of tumor cells, reducing their invasive and metastatic potential. Nonfunctioning neuroendocrine tumors on the contrary are characterized by underdevelopment of cellular organelles and immaturity of secretory granules. Predominance of weak direct cell junctions, rupture of membranes of neighboring cells with simultaneous reduction of desmosomes facilitate the migration of individual tumor cells and formation of metastases.

Extracellular Vesicles as Transmitters of Hypoxia Tolerance in Solid Cancers.

Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after treatment. This is mainly due to increased resistance of hypoxic cells to radio- and chemotherapy and the association of hypoxic cells with increased metastasis development. It is therefore not surprising that an increased hypoxic tumour fraction is associated with poor patient survival. The extent of hypoxia within a tumour is influenced by the tolerance of individual tumor cells to hypoxia, a feature that differs considerably between tumors. High numbers of hypoxic cells may, therefore, be a direct consequence of enhanced cellular capability inactivation of hypoxia tolerance mechanisms. These include HIF-1α signaling, the unfolded protein response (UPR) and autophagy to prevent hypoxia-induced cell death. Recent evidence shows hypoxia tolerance can be modulated by distant cells that have experienced episodes of hypoxia and is mediated by the systemic release of factors, such as extracellular vesicles (EV). In this review, the evidence for transfer of a hypoxia tolerance phenotype between tumour cells via EV is discussed. In particular, proteins, mRNA and microRNA enriched in EV, derived from hypoxic cells, that impact HIF-1α-, UPR-, angiogenesis- and autophagy signalling cascades are listed.

Coordination microparticle vaccines engineered from tumor cell templates.

A method for encapsulation of individual tumor cells with an epigallocatechin-3-gallate (EGCG)-Al(iii) coordination layer, which can be used as potential tumor vaccines, was developed. The interaction between microparticle vaccines and dendritic cells was investigated in vitro. The microparticles (∼10 μm) were efficiently internalized by DCs with enhanced uptake efficiency via actin polymerization and clathrin-mediated endocytosis.

Homophilic CD44 Interactions Mediate Tumor Cell Aggregation and Polyclonal Metastasis in Patient-Derived Breast Cancer Models.

Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. The intercellular CD44-CD44 homophilic interactions directed multicellular aggregation, requiring its N-terminal domain, and initiated CD44-PAK2 interactions for further activation of FAK signaling. Our studies highlight that CD44+ CTC clusters, whose presence is correlated with a poor prognosis of patients with breast cancer, can serve as novel therapeutic targets of polyclonal metastasis. SIGNIFICANCE: CTCs not only serve as important biomarkers for liquid biopsies, but also mediate devastating metastases. CD44 homophilic interactions and subsequent CD44-PAK2 interactions mediate tumor cluster aggregation. This will lead to innovative biomarker applications to predict prognosis, facilitate development of new targeting strategies to block polyclonal metastasis, and improve clinical outcomes.See related commentary by Rodrigues and Vanharanta, p. 22.This article is highlighted in the In This Issue feature, p. 1.

BIOPSY OF SIGNAL LYMPHATIC NODULES IN PATIENTS WITH BREAST CANCER AFTER NEOAVARIANT CHEMOTHERAPY

One of the advantages of systemic cytotoxic therapy is the «transformation» of positive axillary nodes into negative nodes (cN+ → cN-); a similar concept is used in randomized clinical trials with «post-neoadjuvant» sentinel lymph node - pNsn. In studies of ACOSOG Z1071, SENTINA, SN FNAC, was evaluated the frequency of a false-negative result with a biopsy of the sentinel lymph node (BSLU) after neodjuvant chemotherapy. It was proved that there was no need for immunohistochemical examination of lymph nodes using the BSLU technique followed by adjuvant therapy, since the detected micrometastases did not worsen overall survival. As for patients with biopsy of signaling lymph nodes after neodjuvant chemotherapy, nowadays particular interest of prognostic in significance for micrometastases and individual tumor cells in the lymph nodes, as well as an estimate of the frequency of false-negative result.

Single-cell mutation identification via phylogenetic inference.

Reconstructing the evolution of tumors is a key aspect towards the identification of appropriate cancer therapies. The task is challenging because tumors evolve as heterogeneous cell populations. Single-cell sequencing holds the promise of resolving the heterogeneity of tumors; however, it has its own challenges including elevated error rates, allelic drop-out, and uneven coverage. Here, we develop a new approach to mutation detection in individual tumor cells by leveraging the evolutionary relationship among cells. Our method, called SCIΦ, jointly calls mutations in individual cells and estimates the tumor phylogeny among these cells. Employing a Markov Chain Monte Carlo scheme enables us to reliably call mutations in each single cell even in experiments with high drop-out rates and missing data. We show that SCIΦ outperforms existing methods on simulated data and applied it to different real-world datasets, namely a whole exome breast cancer as well as a panel acute lymphoblastic leukemia dataset.

P18 - Social Behavior of Breast Cancer Stem Cell in Metastasis

My early work demonstrated that Rb tumor suppressor prevent the chemotherapeutic agent cisplatin-induced ROS stress and cell death by maintaining ATP and NAD levels as well as PARP-1 activity. Rb and Trp53 have been shown by the Sawyers and Goodrich groups in limiting cancer lineage plasticity through inhibiting Sox2 and Ezh2 pathways. My laboratory has focused on identifying gene and microRNA regulators of breast cancer stem cells in driving chemoresistance and metastasis, such as miR-30c, miR-138, and miR-206. While miR-138 targets EZH2 in breast CSCs to promote differentiation and inhibit tumor invasion, both miR-30c and miR-206 target cytoskeleton genes such as TWF1 to inhibit cancer stemness, chemoresistance and metastasis. Recently, we have identified a new social behavior of CSCs in circulating tumor cell cluster formation which greatly enhanced lung colonization and polyclonal metastasis of breast cancer. CTC clusters were less frequently detected but more metastatic than single CTCs of triple negative breast cancer patients and representative patient-derived-xenograft (PDX) models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding.

Leveraging digital pathology for molecular testing.

Leveraging digital pathology for molecular testing.

P1.09-26 A Case Report of Discordant Markers of Lung Cancer Tumor Cells: An Unusual Immunophenotype of Uncertain Significance

Introduction Non Small Cell Lung Cancer (NSCLC) is the most frequent type of lung tumor, with two major histological subtypes: adenocarcinomas and squamous cell carcinomas. Tissue transcription factor-1 (TTF-1) and Napsin A are expressed in up to 75%- 80% of primary lung adenocarcinomas respectively and the sensitivity of both p40 and p63 for squamous cell carcinoma is 92%. Here, we report an unusual case of NSCLC with coexisting histology within the same tumor cells. Case A 65-year-old male with 50 pack year smoking history underwent screening CT chest and was found to have a right upper lobe mass measuring 10 cm x 8.6 cm x 6.9 cm with narrowing of the right upper lobe bronchus in the posterior segment. Bronchoscopy and EBUS guided biopsy of the right paratracheal node revealed NSCLC, poorly differentiated, with TTF-1, Napsin A, P40 and P63 staining positive (70-80%) for both adenocarcinoma and squamous cell carcinoma within the same tumor cells. Subsequent Brain MRI and PET did not reveal any evidence of metastatic disease and the patient was classified as T4 N1 stage IIIB disease. Discussion The line between lung adenocarcinoma and squamous cell carcinoma has already been shaken by an uncommon adenosquamous variant, but our case represents a new histologic subtype that expands on the understanding of lung cancer. Adenosquamous carcinoma (ADSQC) of the lung is a rare classification of NSCLC with a reported incidence of 0.4-4%. Histology defined by the World Health Organization defines ADSQC as a mixed type of tumor, with each component representing greater than 10% of the entire tumor. Based on studies by Naunheim et al, the median survival of patients with stage III ADSQC was 5.0 months compared to 9.0 months with adenocarcinoma and 7.8 months with squamous cell carcinoma. However, our case is different, as immunohistochemistry was positive for TTF-1, Napsin A, p40 and p63 biomarkers within the same individual tumor cells, thus diagnosing an entirely different pathology. After review of the literature, this is the third case with such co-expression. This case is important as it promotes awareness of this rare pathology. Both clinicians and pathologists need to be aware of its existence, in order to prevent misdiagnosis and treatment delay in what may be hypothesized as an aggressive tumor. As more cases emerge, further studies can determine incidence, prognosis, presence of driver mutations, and treatment implications. Section not applicable Section not applicable Section not applicable

The Auricular VX2 Carcinoma Is a Suitable Animal Model for Identifying Biomarkers for HNSCC Therapy Response

The rabbit auricular VX2 carcinoma is an established animal model for human head and neck squamous cell carcinoma (HNSCC). Previously, we observed that intraperitoneal oxidative (O3/O2) stress induced tumor remission. Our aim was to evaluate candidate genes associated with tumor regression. For identification of tumor remission-related genes, microarray analysis was performed with subsequent validation by polymerase chain reaction (PCR), in situ hybridization, immunohistochemistry and western blot analysis. Microarray analysis indicated a prominent reduction of epidermal growth factor receptor (Egfr, Erbb1) expression levels in regressing tumors. Quantitative PCR confirmed a significant (p<0.005) down-regulation of Erbb1-3 mRNA in regressing VX2 tumors. Histological localization of Erbb1-3 mRNA transcript and protein indicated reduced Erbb gene expression occurring at the level of individual VX2 tumor cells rather than solely being an effect of tumor shrinkage. This study highlights changes in the Erbb gene signature of regressing VX2 carcinomas as a predictor for therapy response. The VX2 carcinoma animal model, therefore, appears suitable for the identification and evaluation of new diagnostic, prognostic and therapeutic biomarkers prior to their application in patients with HNSCC.

Leucine-rich repeat-containing G-protein-coupled receptor 5 expression and clinicopathological features of colorectal neuroendocrine neoplasms.

LGR5 is expressed in various tumors and has been identified as a putative intestinal stem cell marker. Here we investigated LGR5 expression in colorectal neuroendocrine neoplasms and analyzed the correlation with pathological characteristics. We evaluated the clinicopathological features of 8 neuroendocrine tumor (NET) grade 1 (NET G1), 4 NET Grade 2 (NET G2), and 8 NET Grade 3 (NET G3; also termed neuroendocrine carcinoma, or NEC) cases. We examined LGR5 expression using an RNAscope, a newly developed RNA in situ hybridization technique, with a tissue microarray of the neuroendocrine neoplasm samples. LGR5 staining in individual tumor cells was semi-quantitatively scored using an H-score scale. We also performed a combination of LGR5 RNA in situ hybridization and synaptophysin immunohistochemistry. All cases contained tumor cells with some LGR5-positive dots. For all cases, H-scores showed a positive correlation with nuclear beta-catenin expression. In the NEC group, there was a strong positive correlation between H-score and beta-catenin expression. Our findings suggest that LGR5 may serve as a stem cell marker in NEC, as is the case in colon adenocarcinoma. The positive correlation between H-score and beta-catenin expression suggests that LGR5 expression might be affected by beta-catenin expression in neuroendocrine neoplasms and especially in NEC.

Abstract 90: Metastatic melanoma and cervical tumor cell clusters can exit blood vessels through angiopellosis augmenting tumor formation ability

Abstract Introduction Accumulating data suggests metastatic primary tumor cells can travel through the circulation and form secondary tumors as multicellular clusters. However, whether these clusters must first dissociate into single cells prior to exiting of blood vessels remains largely unknown. Of late, researchers have identified a mechanism of cell extravasation (exiting blood vessel), termed angiopellosis (Allen 2016). However, whether tumor cell clusters possess the ability to exit vessles using the angiopellosis method is unstudied. We hypothesized that circulating tumor cell (CTC) clusters could exit blood vessels while maintaining a multicellular phenotype, through angiopellosis. Methodology To test this, we used an in vivo zebrafish larvae model using the tg(fli1a:egfp) line, in which blood vessels exclusively fluoresce. First, we infused fluorescent human melanoma (A375), mouse melanoma (B16F10), and human cervical (HeLa) cells into the circulation of the larvae. Next, we used lightsheet microscopy to observe the method the tumor cells used to exit blood vessels. Additionally, mouse melanoma (B16F10) cells were intravenously infused into the circulation of C57BL/6J mice as either tumor clusters or individual cells. The mice were imaged for metastatic lung foci, to determine cluster extravasation potential. Experimental results The intravital lightsheet microscopy allowed us to determine that tumor cells can utilize angiopellosis to exit blood vessels in the zebrafish model, both as individual CTCs and CTC clusters. Extravasating CTCs exited blood vessels through angiopellosis at 94%, with only 6% exiting through the diapedesis method (n=30). CTCs which exited as a cluster through angiopellosis were observed to form tumor masses at an 88% higher rate compared to individual tumor cells in vivo, over the course of 96 hours. In the murine model, CTCs clusters which were infused via tail vein (n=4) showed significantly higher metastatic lung foci in comparison to individual CTCs that were infused (n=3). Following isolation and analysis of metastatic CTCs, multiple genes were shown to be differentially expressed in CTC clusters in both the zebrafish and murine models. Discussion Here, we show circulating tumor cell clusters possess the ability to exit blood vessels through the recently identified angiopellosis. We determined tumor cell clusters exhibit an augmented ability to proliferate in vivo, while individually tumor cells are more prone to dormancy. Our results challenge the belief that circulating tumor cell clusters must first disassociate to exit the circulation, and posits an alternative model. We propose tumor cells both travel and exit the circulation while maintaining a multicellular phenotype, using angiopellosis, stimulating proliferation-augmenting genetic/molecular alterations. This has been termed the Cancer Exodus Hypothesis. Citation Format: Tyler Allen, Emmanuel Amu, Dana Asad, Ke Cheng. Metastatic melanoma and cervical tumor cell clusters can exit blood vessels through angiopellosis augmenting tumor formation ability [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 90.

Abstract 715: Expression of tumor cell-associated immune checkpoint molecules in multiple human solid tumors

Abstract Cancer immunotherapy using monoclonal antibodies targeting inhibitory immune checkpoint molecules is being established as a new paradigm in cancer treatment. This crucial strategy involves inhibition of the interaction between immune checkpoint receptors, expressed on the immune cells, and their respective ligands, expressed on antigen-presenting cells. While this receptor-ligand concept is well accepted in infection immunology, there have been numerous reports on the expression of immune checkpoint molecules in tumor cells. In addition to well-characterized tumor-intrinsic expression of PD11 and PD-L12, many other checkpoint molecules are associated with tumor cells3, conferring important impact on tumor biology and clinical consequences. In this study, we evaluated in situ single-cell expression profiles of immune checkpoint molecules in the tumor microenvironment (TME) of archived FFPE tissues from multiple solid cancers, including breast, kidney, ovarian, stomach, colorectal, and non-small cell lung cancer. Applying the RNAscope® assay, a highly specific and sensitive in situ hybridization (ISH) technology, tissue microarrays consisting of 20-35 FFPE tumors per each cancer type were stained for key therapeutic targets including PD1, PD-L1, TIM3, and LAG3. We observed that, in addition to PD-L1, all evaluated immune checkpoint molecules were expressed in the tumor cells of multiple tumor types though at a lower level than in immune cells. While there was notable heterogeneity of expression in different tumor regions in some samples, the single-cell expression pattern of each checkpoint molecule across all tumor cells in each region suggested a potential tumor-intrinsic mechanism for the expression of these molecules. In selected lung and ovarian cancers, fluorescence multiplex ISH-IHC staining confirmed that multiple different checkpoint molecules were co-expressed together in individual tumor cells. These findings add to the growing evidence that immune checkpoint genes can be expressed in tumor cells in addition to immune cells, which will be missed by gene expression analysis techniques using bulk tissue. The RNAscope® ISH platform is especially well suited for this type of analysis since it allows for assessing cell type-specific expression of any gene within the native tissue architecture even when it is expressed at very low levels. The approach illustrated in this study may help to better understand the anti-tumor activities of immune checkpoint inhibitors and provide new insights into better predictive biomarkers and therapeutic strategies.

Abstract 2184: Enrichment and isolation of tumor cells using a unique flow-sorting approach to decrypt intratumoral heterogeneity in matched samples of squamous cell carcinoma of the lung

Abstract Background: Genomic intratumoral heterogeneity in cancers is a driving force for progression or recurrence. Availability of pure tumor cell material is critical for studying clonal evolution. In general, tumors are highly mixed with benign cells with a mean tumor cell purity of only 49%1 shown in lung squamous cell carcinoma (LSCC). Using a unique flow-sorting based approach enables us to overcome this limitation by highly enriching for tumor cells. Here, we aimed at precisely identifying different tumor populations in primary LSCC and matched distant metastases from 18 patients. Methods: Nuclei from snap-frozen and FFPE tumor specimens were extracted and subjected to flow-sorting using DNA content/ploidy as a parameter. Pan-cytokeratin (pCK) was used to differentiate diploid tumor cell populations from benign diploid stromal cells. DNA has been extracted from these individual tumor cell populations and the mutational profile and copy number status are currently being analyzed using whole exome seqeuencing (WES). Results: We successfully enriched for tumor cells with a purity of higher than 95% using the multi parameter flow-sorting approach. Besides the aneuploid tumors with higher ploidies, we discovered near-diploid tumor populations after enrichment with the pCK antibody. The pCK negative fraction consisting of stromal cells can serve as a germ line control in further downstream analysis as WES. Conclusion: The present study illustrates a unique method that allows high enrichment for tumor cells. Avoiding the dilution of DNA by benign stromal cells, together with the purity of aneuploid tumor cells, the precision of genomic analysis highly increases. At the same time, the diploid and pCK negative stromal cells can serve as a germ line control. Comprehensive genomic profiling of flow-sorted aneuploid and pCK positive diploid tumor populations represent a powerful technique to study intratumoral heterogeneity.

A descriptive subgroup analysis of individual tumor cells from 4 patients with ovarian cancer

A descriptive subgroup analysis of individual tumor cells from 4 patients with ovarian cancer

Single cell sequencing: A descriptive subgroup analysis of individual tumor cells from 4 patients with ovarian cancer

Single cell sequencing: A descriptive subgroup analysis of individual tumor cells from 4 patients with ovarian cancer

Facile Tumor Spheroids Formation in Large Quantity with Controllable Size and High Uniformity

A facile method for generation of tumor spheroids in large quantity with controllable size and high uniformity is presented. HCT-116 cells are used as a model cell line. Individual tumor cells are sparsely seeded onto petri-dishes. After a few days of growth, separated cellular islets are formed and then detached by dispase while maintaining their sheet shape. These detached cell sheets are transferred to dispase-doped media under orbital shaking conditions. Assisted by the shear flow under shaking and inhibition of cell-to-extracellular matrix junctions by dispase, the cell sheets curl up and eventually tumor spheroids are formed. The average size of the spheroids can be controlled by tuning the cell sheet culturing period and spheroid shaking period. The uniformity can be controlled by a set of sieves which were home-made using stainless steel meshes. Since this method is based on simple petri-dish cell culturing and shaking, it is rather facile for forming tumor spheroids with no theoretical quantity limit. This method has been used to form HeLa, A431 and U87 MG tumor spheroids and application of the formed tumor spheroids in drug screening is also demonstrated. The viability, 3D structure, and necrosis of the spheroids are characterized.

Relapse pathway of glioblastoma revealed by single-cell molecular analysis.

Glioblastoma multiforme (GBM) remains an incurable brain tumor. The highly malignant behavior of GBM may, in part, be attributed to its intraclonal genetic and phenotypic diversity (subclonal evolution). Identifying the molecular pathways driving GBM relapse may provide novel, actionable targets for personalized diagnosis, characterization of prognosis and improvement of precision therapy. We screened single-cell transcriptomes, namely RNA-seq data of primary and relapsed GBM tumors from a patient, to define the molecular profile of relapse. Characterization of hundreds of individual tumor cells identified three mutated genes within single cells, involved in the RAS/GEF GTP-dependent signaling pathway. The identified molecular pathway was further verified by meta-analysis of RNA-seq data from more than 3000 patients. This study showed that single-cell molecular analysis overcomes the inherent heterogeneity of bulk tumors with respect to defining tumor subclonal evolution relevant to GBM relapse.

Sensitivity to sequencing depth in single-cell cancer genomics

BackgroundQuerying cancer genomes at single-cell resolution is expected to provide a powerful framework to understand in detail the dynamics of cancer evolution. However, given the high costs currently associated with single-cell sequencing, together with the inevitable technical noise arising from single-cell genome amplification, cost-effective strategies that maximize the quality of single-cell data are critically needed. Taking advantage of previously published single-cell whole-genome and whole-exome cancer datasets, we studied the impact of sequencing depth and sampling effort towards single-cell variant detection.MethodsFive single-cell whole-genome and whole-exome cancer datasets were independently downscaled to 25, 10, 5, and 1× sequencing depth. For each depth level, ten technical replicates were generated, resulting in a total of 6280 single-cell BAM files. The sensitivity of variant detection, including structural and driver mutations, genotyping, clonal inference, and phylogenetic reconstruction to sequencing depth was evaluated using recent tools specifically designed for single-cell data.ResultsAltogether, our results suggest that for relatively large sample sizes (25 or more cells) sequencing single tumor cells at depths > 5× does not drastically improve somatic variant discovery, characterization of clonal genotypes, or estimation of single-cell phylogenies.ConclusionsWe suggest that sequencing multiple individual tumor cells at a modest depth represents an effective alternative to explore the mutational landscape and clonal evolutionary patterns of cancer genomes.