Individual Tumor Cells Research Articles

The potential of circulating tumor DNA to use as a molecular marker to screen and diagnose hepatocellular carcinoma: A systematic review

Now a day's molecular characterization of individual patients' tumor cells is becoming instantly important for early detection and effective treatment of the disease. The idea of applying liquid biopsy technologies for early diagnosis of cancer through the specific and sensitive determination of circulating tumor DNA (ctDNA) among circulating free DNA (cfDNA) in plasma is a relatively recent approach with considerable promise, but also presented with great challenges. Ongoing advancement in the field has shown that ctDNA has huge potential to serve as a biomarker for early detection and precision treatment as well as prognosis of hepatocellular carcinoma (HCC). As ctDNA in HCC patients harbors the molecular characteristics of HCC tumor cells, ctDNA analysis in the blood of HCC patients might be an adequate and non-intrusive approach for locating tumors, disease prediction, and treatment. In the sight of this fact, this review tried to sum up and discuss the surveillance of HCC, the origins and molecular characteristics of molecular markers of hepatocellular carcinoma, the current status, and the potentials of ctDNA as a marker for HCC surveillance and early detection. Moreover, this review also describes the major tumor-specific genetic modifications in ctDNA, such as DNA methylation, microsatellite alterations, point mutations, chromosomal rearrangements. Finally, the challenges associated with the clinical use of ctDNA for HCC detection are also discussed.

PATH-07. QUALITY ASSURANCE IN CEREBROSPINAL FLUID CYTOLOGY ASSESSMENT FOR MEDULLOBLASTOMA STAGING LEADS TO POTENTIAL IMPROVED RISK-GROUP ASSESSMENT IN THE PROSPECTIVE MULTICENTER HIT-2000 TRIAL

BACKGROUNDCerebrospinal fluid (CSF) dissemination of medulloblastoma (M1 stage) is a high-risk prognostic factor. However, because diagnostic criteria for M1 staging are missing we specified process-related and cytomorphological parameters influencing the predictive value of the CSF status.PATIENTS AND METHODSCSF samples and cytology reports from 405 medulloblastoma patients of the prospective multicenter trial HIT-2000 were reviewed and related to 5-year progression free survival (5y-PFS).RESULTSTumor cells were detected in 237/1073 CSF cytospins. M1-patients and M2/3 patients with radiologically detected metastases showed a worse 5y-PFS than M0 patients (54% and 52% vs. 76%; p=0.01 and p<0.001). Lumbar sampling was more sensitive than ventricular sampling. M0 diagnosed specimens containing >50% lytic cells and/or less than 10 nucleated cells showed a decreased 5y-PFS (61%). Further investigation of cytological parameters revealed a poor outcome for cases harboring > 3 tumor cell clusters and individual tumor cells (5y-PFS 33%) vs. cases with ≥ 2 individual tumor cells but no clusters (5y-PFS 61%). In bi-variable Cox-regression, ≥ 2 vs. 0 or 1 tumor cells were associated with a Hazard Ratio (HR) of 0.52 (95%-Confidence Interval (CI): 0.12, 2.30; p=0.39), whereas > 3 vs. no tumor cell clusters were associated with a HR of 8.94 (95%-CI: 1.66, 48.22; p=0.01).CONCLUSIONSCSF staging in medulloblastoma should comprise lumbar specimens with <50% lytic cells and a minimum of 10 nucleated cells. The predictive value of CSF cytology in M1 cases may predominantly depend on tumor cell clusters. The latter finding needs to be confirmed in prospective trials.

Discovery and Validation of a Metastasis-Related Prognostic and Diagnostic Biomarker for Melanoma Based on Single Cell and Gene Expression Datasets.

BackgroundSingle cell sequencing can provide comprehensive information about gene expression in individual tumor cells, which can allow exploration of heterogeneity of malignant melanoma cells and identification of new anticancer therapeutic targets.MethodsSingle cell sequencing of 31 melanoma patients in GSE115978 was downloaded from the Gene Expression Omniniub (GEO) database. First, the limma package in R software was used to identify the differentially expressed metastasis related genes (MRGs). Next, we developed a prognostic MRGs biomarker in the cancer genome atlas (TCGA) by combining univariate cox analysis and the least absolute shrinkage and selection operator (LASSO) method and was further validated in another two independent datasets. The efficiency of MRGs biomarker in diagnosis of melanoma was also evaluated in multiple datasets. The pattern of somatic tumor mutation, immune infiltration, and underlying pathways were further explored. Furthermore, nomograms were constructed and decision curve analyses were also performed to evaluate the clinical usefulness of the nomograms.ResultsIn total, 41 MRGs were screened out from 1958 malignant melanoma cell samples in GSE115978. Next, a 5-MRGs prognostic marker was constructed and validated, which show more effective performance for the diagnosis and prognosis of melanoma patients. The nomogram showed good accuracies in predicting 3 and 5 years survival, and the decision curve of nomogram model manifested a higher net benefit than tumor stage and clark level. In addition, melanoma patients can be divided into high and low risk subgroups, which owned differential mutation, immune infiltration, and clinical features. The low risk subgroup suffered from a higher tumor mutation burden (TMB), and higher levels of T cells infiltrating have a significantly longer survival time than the high risk subgroup. Gene Set Enrichment Analysis (GSEA) revealed that the extracellular matrix (ECM) receptor interaction and epithelial mesenchymal transition (EMT) were the most significant upregulated pathways in the high risk group.ConclusionsWe identified a robust MRGs marker based on single cell sequencing and validated in multiple independent cohort studies. Our finding provides a new clinical application for prognostic and diagnostic prediction and finds some potential targets against metastasis of melanoma.

Abstract IA-02: Pancreatic cancer heterogeneity and plasticity: The mix of seed and soil

Abstract A series of transcriptomic analyses of human pancreatic ductal adenocarcinoma (PDAC) have consistently demonstrated multiple cancer subtypes with different functional behavior in preclinical models and patient outcomes in clinical studies. The two major molecular subtypes described in PDAC are the classical epithelial (E) and the quasi-mesenchymal (QM) subtypes, otherwise known as the basal-like or squamous subtype. Although a model of E and QM subtypes to dichotomize PDAC tumor behavior affords the simplicity of use in biomarker and functional studies, there has been increasing evidence of epithelial mesenchymal transition (EMT) transcriptional plasticity of PDAC cells in model systems as well as evidence from single cell RNA-seq studies. This suggests that the E and QM states are on a continuum that can result in interconverting cell types. We have utilized single cell technologies, functional model systems, and human tumor analysis to characterize the landscape of PDAC heterogeneity and the factors involved with cellular plasticity between E and QM states. Initial single cell RNA-seq studies in PDAC mouse models revealed that partial EMT is enriched in circulating tumor cells (CTCs) compared to primary tumor cells (Ting DT et al. Cell Reports 2014). These CTCs were also highly enriched for extracellular matrix (ECM) gene expression that were shared with stromal fibroblasts in the primary tumor indicating that the “seeds” of metastasis produce their own “soil”. Functional studies in patient derived PDAC cell lines and cancer associated fibroblasts (CAFs) revealed EMT heterogeneity was partially driven by the relative amounts of stromal CAFs (Ligorio M*, Sil S* et al. Cell 2019). Using RNA in situ hybridization (RNA-ISH) in human primary PDAC tumors and high content digital image analysis, we classified individual tumor cells for EMT and proliferative (PRO) phenotypes that were linked with relative stromal content and characterize the significant inter- and intra-tumoral heterogeneity of these cell types within distinct tumor glands. In addition, analysis of PDAC tumorspheres revealed a significant induction of EMT changes in response to FOLFIRINOX, which was also found in patient tumors that were resected after neoadjuvant FOLFIRINOX (Porter RL et al. PNAS 2019). Finally, we have recently characterized human PDAC CTCs that has identified 4 different stem cell genes (WNT5A/KLF4, LGALS3, LIN28B) that are enriched in these metastatic precursors and cluster separately between patients (Franses JW*, Philipp J* et al. Nature Communications 2020). Each of these CTC phenotypes are all associated with EMT features suggestive of different pathways that can achieve similar metastatic behavior. Altogether, single cell heterogeneity of PDAC is functionally linked with tumor stromal interactions, response to chemotherapy, and the ability to seed distant metastases. Citation Format: David T. Ting. Pancreatic cancer heterogeneity and plasticity: The mix of seed and soil [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr IA-02.

An extracellular vesicle-related gene expression signature identifies high-risk patients in medulloblastoma.

Medulloblastoma (MB) is a malignant brain tumor in childhood. It comprises 4 subgroups with different clinical behaviors. The aim of this study was to characterize the transcriptomic landscape of MB, both at the level of individual tumors as well as in large patient cohorts. We used a combination of single-cell transcriptomics, cell culture models and biophysical methods such as nanoparticle tracking analysis and electron microscopy to investigate intercellular communication in the MB tumor niche. Tumor cells of the sonic hedgehog (SHH)-MB subgroup show a differentiation blockade. These cells undergo extensive metabolic reprogramming. The gene expression profiles of individual tumor cells show a partial convergence with those of tumor-associated glial and immune cells. One possible cause is the transfer of extracellular vesicles (EVs) between cells in the tumor niche. We were able to detect EVs in co-culture models of MB tumor cells and oligodendrocytes. We also identified a gene expression signature, EVS, which shows overlap with the proteome profile of large oncosomes from prostate cancer cells. This signature is also present in MB patient samples. A high EVS expression is one common characteristic of tumors that occur in high-risk patients from different MB subgroups or subtypes. With EVS, our study uncovered a novel gene expression signature that has a high prognostic significance across MB subgroups.

Abstract PO-123: Distinct subpopulations of osteosarcoma cells cooperate to establish lung metastases

Abstract Four decades of intense multinational efforts to improve outcomes for children and teenagers with osteosarcoma have failed. Today, we still treat patients with a regimen first developed in 1982. The same 35-40% of young people diagnosed with osteosarcoma die from their disease, almost always from complications related to lung metastasis. Believing that therapies targeting lung metastasis could dramatically improve outcomes, we have sought to understand the mechanisms that drive lung colonization in this disease. We previously found that interactions with lung epithelium cause osteosarcoma cells to produce IL6 and CXCL8 and that this activity is necessary for metastasis. Inhibiting IL6 and CXCL8 signaling prevents metastasis entirely in 85% of mice with osteosarcoma. While conducting these studies, we learned that individual tumor cells react differently to signals from lung epithelium. Nearly all the IL6 and CXCL8 produced by osteosarcoma cells come from a small subset of cells. Interactions with pulmonary epithelium cause this discrete subset of tumor cells to produce large quantities of inflammatory cytokines, become strongly p21+, and stop proliferating. Early metastatic lesions consist almost entirely of cells with this phenotype, positioning them to orchestrate the development of the metastatic niche. Thus, we call these hypo-proliferative, cytokine-producing cells that survive early attrition "anchor cells." In the initial stages of metastasis, anchor cells alter the surrounding lung tissue in ways that facilitate subsequent colonization by tumor cells with proliferating phenotypes ("growth cells"). Growth cells then divide to create expanding, clinically-relevant metastasis. Importantly, one can leverage the biology of these anchor cells to develop rational targeting strategies. Anchor cells exhibit profound sensitivity to known senolytic agents. Combining these with conventional therapies (which effectively target growth cells) can eradicate established metastatic lesions and prevent new metastases from forming. These novel findings fundamentally alter our understanding of osteosarcoma and metastasis biology and have direct implications for approaches to treatment. Citation Format: James B. Reinecke, John M. Hinckley, Amy C. Gross, Maren Cam, Sanjana Rajan, Ryan D. Roberts. Distinct subpopulations of osteosarcoma cells cooperate to establish lung metastases [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-123.

SERRS multiplexing with multivalent nanostructures for the identification and enumeration of epithelial and mesenchymal cells

Liquid biopsy represents a new frontier of cancer diagnosis and prognosis, which allows the isolation of tumor cells released in the blood stream. The extremely low abundance of these cells needs appropriate methodologies for their identification and enumeration. Herein we present a new protocol based on surface enhanced resonance Raman scattering (SERRS) gold multivalent nanostructures to identify and enumerate tumor cells with epithelial and mesenchimal markers. The validation of the protocol is obtained with spiked samples of peripheral blood mononuclear cells (PBMC). Gold nanostructures are functionalized with SERRS labels and with antibodies to link the tumor cells. Three types of such nanosystems were simultaneously used and the protocol allows obtaining the identification of all individual tumor cells with the help of a Random Forest ensemble learning method.

Immunomagnetic Capture and Multiplexed Surface Marker Detection of Circulating Tumor Cells with Magnetic Multicolor Surface-Enhanced Raman Scattering Nanotags.

Circulating tumor cells (CTCs) have substantial clinical implications in cancer diagnosis and monitoring. Although significant progress has been made in developing technologies for CTC detection and counting, the ability to quantitatively detect multiple surface protein markers on individual tumor cells remains very limited. In this work, we report a multiplexed method that uses magnetic multicolor surface-enhanced Raman scattering (SERS) nanotags in conjunction with a chip-based immunomagnetic separation to quantitatively and simultaneously detect four surface protein markers on individual tumor cells in whole blood. Four-color SERS nanotags were prepared using magnetic-optical iron oxide-gold core-shell nanoparticles with different Raman reporters to recognize four different cancer markers with respective antibodies. A microfluidic device was fabricated to magnetically capture the nanoparticle-bound tumor cells and to perform online negative staining and single-cell optical detection. The level of each targeted protein was obtained by signal deconvolution of the mixed SERS signals from individual tumor cells using the classic least squares regression method. The method was tested with spiked tumor cells in human whole blood with three different breast cancer cell lines and compared with the results of purified cancer cells suspended in a phosphate buffer solution. The method, with either spiked cancer cells in blood or purified cancer cells, showed a strong correlation with purified cancer cells by enzyme-linked immunosorbent assay, suggesting the potential of our method for the reliable detection of multiple surface markers on CTCs. Combining immunomagnetic enrichment with high specificity, multiplexed targeting for the capture of CTC subpopulations, multicolor SERS detection with high sensitivity and specificity, microfluidics for handling rare cells and magnetic-plasmonic nanoparticles for dual enrichment and detection, our method provides an integrated, yet a simple and an efficient platform that has the potential to more sensitively detect and monitor cancer metastasis.

The Role of the ECM in Lung Cancer Dormancy and Outgrowth.

The dissemination of tumor cells to local and distant sites presents a significant challenge in the clinical management of many solid tumors. These cells may remain dormant for months or years before overt metastases are re-awakened. The components of the extracellular matrix, their posttranslational modifications and their associated factors provide mechanical, physical and chemical cues to these disseminated tumor cells. These cues regulate the proliferative and survival capacity of these cells and lay the foundation for their engraftment and colonization. Crosstalk between tumor cells, stromal and immune cells within primary and secondary sites is fundamental to extracellular matrix remodeling that feeds back to regulate tumor cell dormancy and outgrowth. This review will examine the role of the extracellular matrix and its associated factors in establishing a fertile soil from which individual tumor cells and micrometastases establish primary and secondary tumors. We will focus on the role of the lung extracellular matrix in providing the architectural support for local metastases in lung cancer, and distant metastases in many solid tumors. This review will define how the matrix and matrix associated components are collectively regulated by lung epithelial cells, fibroblasts and resident immune cells to orchestrate tumor dormancy and outgrowth in the lung. Recent advances in targeting these lung-resident tumor cell subpopulations to prevent metastatic disease will be discussed. The development of novel matrix-targeted strategies have the potential to significantly reduce the burden of metastatic disease in lung and other solid tumors and significantly improve patient outcome in these diseases.

Solid pseudopapillary tumour of pancreas

Solid Pseudopapillary tumor (SPT) is a tumor with low malignant potential and comprises around 1-2% of all tumors occurring in Pancreas. Pseudocyst of Pancreas, Microcystic adenoma, neuroendocrine tumor and mucinous cystic neoplasm are considered as challenging differentials of SPT. High suspicion followed by accurate diagnosis is key to management because SPT is known for its good prognosis and successive cure rate on surgical resection. 21-year-old female patient presented with complaints of abdominal pain and dyspnoea. Imaging studies revealed a retroperitoneal tumor. Surgical resection carried out. Gross examination of the specimen showed circumscribed lobular brown mass weighing around 468 gm with a measurement of 15x12x10 cm. On sectioning, the tumor had a variegated appearance containing multiple cysts varying from 1 - 3 cm in size. Microscopic examination revealed an encapsulated cellular neoplasm with individual tumor cells arranged in solid, pseudopapillae and cystic patterns. Tumor cells were round to oval with no distinct nucleoli. Mitotic figures were very few in number. Hyaline globules were noted. Mucinous changes were seen focally. Occasional areas exhibited clear cell change of tumor cells. Cystic areas were filled with heamorrhage. Vascular areas were unremarkable. Normal Pancreatic parenchyma was noted at the tumor edge. Morphology of SPTs are varied ranging from solid to cystic appearance. No definitive morphology is identified to differentiate benign from malignant SPT. SPT are known to mimic a wide range of pancreatic and retroperitoneal lesions that may pose a challenge to diagnostic pathologists as seen in this case.

Abstract 2093: DeepRePath: Finding prognostic features of tumor cell on the histopathologic image of lung cancer using explainable deep convolutional neural network

Abstract Introduction Histopathological images in high-powered contain quantitative information for tumor cell morphology, but humans could not sufficiently extract prognostic features of tumor cell morphology associated with tumor recurrence of patient. In this study, we developed and trained the DeepRePath model, a deep convolutional neural network (CNN), on histopathological images to predict the recurrence of resected lung adenocarcinoma (LUAD). Methods A total of 244 haematoxylin and eosin stained histopathology slides of resected LUAD patients were used to train and validate the DeepRePath model. We manually segmented a cancer region in a LUAD tissue slide and captured to an image. All images were captured at x400 magnification and split into 13,329 image patches. We trained the DeepRePath model by transfer learning using 13,329 image patches from 244 LUAD patients. Results 95 out of 244 patients developed recurrence after surgical resection. The DeepRePath model showed an average area under the curve (AUC) of 0.78 in 5-fold cross validation on the training cohort. We plan to employ external validation to evaluate the proposed model. In addition, using gradient-weighted class activation mapping (Grad-CAM), we confirmed that DeepRePath identified and used individual tumor cells with atypia to predict the recurrence of the patient. Conclusions DeepRePath model using transfer learning and CNN can accurately predict recurrence after curative resection of LUAD based on atypia of tumor cell. Citation Format: Yoon Ho Ko, Soon Auck Hong, Tae-Jung Kim, Ji Hyung Hong, Sang Hoon Chun, Seoree Kim, Ho Jung An, Sungsoo Park, Won Sang Shim. DeepRePath: Finding prognostic features of tumor cell on the histopathologic image of lung cancer using explainable deep convolutional neural network [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2093.

Abstract 351: Alterations in immune cell signatures during breast cancer metastasis at single cell resolution

Abstract Metastasis is the major cause of cancer-related deaths due to insufficient therapeutic effects of conventional treatment. Individual tumor cells exhibit heterogeneous features and growth potentials within the same tumor. This intra-tumor heterogeneity influences the ability of the tumor to form metastasis. The underlying mechanisms of why some tumor cells can give rise to metastases, whereas others cannot are remarkably poorly understood. Tumor cells acquire complex properties in order to invade into surrounding tissue, intravasate into the circulation, survive the journey to the distant site, where they extravasate into the new tissue and give rise to a secondary tumor. During this multistep process, tumor cells adapt their phenotype to various microenvironments that are distinct from their original site and are shaped by different immune and stromal cell populations. Metastatic tumor cells are heterogeneous and different from the primary tumor cells and may depend on the microenvironment at the distant site. Our study aims to better understand how the tumor heterogeneity influences the tumor-immune cell axis and how, in turn, immune cells support tumor cell plasticity and metastasis formation. We established patient-derived xenograft models of breast cancer with different metastatic potential and preserved tumor heterogeneity. We analyzed the gene expression of individual tumor and metastatic cells and their associated immune cell signature using a novel multiplexing approach for high-throughput single-cell RNA sequencing (MULTI-seq). In our study, we characterize the heterogeneity of different myeloid cells and their involvement in metastatic progression. Our data show that primary tumors from different individuals share a similar immune cell signature. However, metastasis-associated immune cells show distinct expression patterns related to the metastatic potential of the primary tumor and metastatic progression. In addition, prior to metastatic seeding, we identify an altered immune cell signature in the tissue distant from the primary breast tumor. Additionally, our data reveal that the plasticity of tumor cells is beneficial for the formation of metastasis. Tumors with higher levels of plastic tumor cells that undergo epithelial-mesenchymal-transition (EMT), have a greater ability to form metastasis. Our data indicate that subsets of immune cells of the myeloid lineage could promote this tumor plasticity and are enriched in tumors with higher metastatic potential. Thus, these innate immune cells provide a potential target for immune cell therapy, which may inhibit tumor cell plasticity and thereby the development of metastasis. Citation Format: Juliane Winkler, Weilun Tan, Christopher McGinnis, Zev Gartner, Spyros Darmanis, Zena Werb. Alterations in immune cell signatures during breast cancer metastasis at single cell resolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 351.

Abstract 1111: Verteporfin inhibits GBM growth and migration and confers survival benefit in xenograft models

Abstract The diffusely infiltrative growth and spread, complex molecular and oncogenic signaling aberrations, and inter-and intra-tumoral heterogeneity in glioblastoma (GBM) impedes gross-total resection and chemoradiation and highlight the need for improved tumor-specific brain-penetrant therapies that inhibit cell migration in addition to proliferation. Invasive GBM cells undergo an epithelial-mesenchymal transition (EMT) phenotypic switch, expressing higher levels of genes involved in EMT remodeling, survival, and immune response compared to non-invasive GBM tumor cells. Recently, our lab defined a regulatory chromatin accessibility signature centered around the TEAD transcriptional family, which relates specifically to tumor migration in uncultured, patient-derived GBM stem cell populations, and we functionally validated TEAD1 as a driver of GBM migration and EMT, both in vitro and in vivo. The TEAD family of transcription factors, along with their co-activators YAP/TAZ, are the main downstream effectors of the Hippo pathway, a regulator of tissue growth and cell fate whose dysregulation has been implicated in tumor invasion, metastasis, and chemoresistance in other solid tumors. Here we explored the therapeutic efficacy of Verteporfin (VP), an FDA-approved macular degeneration therapy, and a small-molecule inhibitor of the YAP/TEAD complex, in patient-derived GBM cells and orthotopic xenotransplant mouse models (PDX), assessing VP's impact on GBM proliferation and migration both in vitro and in vivo. VP treatment across three different cell lines inhibited not only glioma growth but also significantly impaired tumor migration in three different in vitro assays (live cell tracking, transwell invasion, and spheroid dispersion) in a dose-dependent manner. At the protein and whole transcriptomic levels, VP-treated cells showed dose-dependent downregulation of TEAD-target activity and significant downregulation of EMT and cell migration functional gene sets, compared to vehicle. In several independent in vivo experiments, intraperitoneal administration of VP in mice with aggressive PDX glioblastoma showed consistent drug penetration into the brain parenchyma without systemic toxicity, resulted in lower tumor burden both at the tumor core and the leading infiltrative edge (n=22 VP, n=21 VEH, p=0.02, each), showed decreased number of individual distal migratory tumor cells (n=6 VP, n=5 VEH, p=0.0001 / n=6 VP, n=5 VEH, p=0.04), and, notably, conferred a survival benefit in combination with TMZ and radiation (n=10 per condition, p=0.02). Furthermore, in PDX mice transplanted with a more typical GBM, daily administration of VP for up to 280 days conferred survival benefit even as monotherapy (n=8 per condition, p=0.02), in the absence of systemic toxicity. The inhibitory effect of Verteporfin on downstream YAP/TEAD signaling and GBM migration, and its brain penetrance at non-toxic levels, underscore potential future therapeutic value and repurposing of this drug in GBM patients. Citation Format: Anne Marie Barrette, Alexandros Bouras, German Nudelman, Dominique Bozec, Noshin Shakawat, Elena Zaslavsky, Constantinos Hadjipanayis, Marc R. Birtwistle, Nadejda M. Tsankova. Verteporfin inhibits GBM growth and migration and confers survival benefit in xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1111.

Rare osteosarcoma cell subpopulation protein array and profiling using imaging mass cytometry and bioinformatics analysis

BackgroundSingle rare cell characterization represents a new scientific front in personalized therapy. Imaging mass cytometry (IMC) may be able to address all these questions by combining the power of MS-CyTOF and microscopy.MethodsWe have investigated this IMC method using < 100 to up to 1000 cells from human sarcoma tumor cell lines by incorporating bioinformatics-based t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis of highly multiplexed IMC imaging data. We tested this process on osteosarcoma cell lines TC71, OHS as well as osteosarcoma patient-derived xenograft (PDX) cell lines M31, M36, and M60. We also validated our analysis using sarcoma patient-derived CTCs.ResultsWe successfully identified heterogeneity within individual tumor cell lines, the same PDX cells, and the CTCs from the same patient by detecting multiple protein targets and protein localization. Overall, these data reveal that our t-SNE-based approach can not only identify rare cells within the same cell line or cell population, but also discriminate amongst varied groups to detect similarities and differences.ConclusionsThis method helps us make greater inroads towards generating patient-specific CTC fingerprinting that could provide an accurate tumor status from a minimally-invasive liquid biopsy.

A lineage-tracing tool to map the fate of hypoxic tumour cells.

ABSTRACTIntratumoural hypoxia is a common characteristic of malignant treatment-resistant cancers. However, hypoxia-modification strategies for the clinic remain elusive. To date, little is known on the behaviour of individual hypoxic tumour cells in their microenvironment. To explore this issue in a spatial and temporally controlled manner, we developed a genetically encoded sensor by fusing the O2-labile hypoxia-inducible factor 1α (HIF-1α) protein to eGFP and a tamoxifen-regulated Cre recombinase. Under normoxic conditions, HIF-1α is degraded but, under hypoxia, the HIF-1α-GFP-Cre-ERT2 fusion protein is stabilised and in the presence of tamoxifen activates a tdTomato reporter gene that is constitutively expressed in hypoxic progeny. We visualise the random distribution of hypoxic tumour cells from hypoxic or necrotic regions and vascularised areas using immunofluorescence and intravital microscopy. Once tdTomato expression is induced, it is stable for at least 4 weeks. Using this system, we could show in vivo that the post-hypoxic cells were more proliferative than non-labelled cells. Our results demonstrate that single-cell lineage tracing of hypoxic tumour cells can allow visualisation of their behaviour in living tumours using intravital microscopy. This tool should prove valuable for the study of dissemination and treatment response of post-hypoxic tumour cells in vivo at single-cell resolution.This article has an associated First Person interview with the joint first authors of the paper.

PTPmu-targeted nanoparticles label invasive pediatric and adult glioblastoma

Poor prognosis for glioblastoma (GBM) is a consequence of the aggressive and infiltrative nature of gliomas where individual cells migrate away from the main tumor to distant sites, making complete surgical resection and treatment difficult. In this manuscript, we characterize an invasive pediatric glioma model and determine if nanoparticles linked to a peptide recognizing the GBM tumor biomarker PTPmu can specifically target both the main tumor and invasive cancer cells in adult and pediatric glioma models. Using both iron and lipid-based nanoparticles, we demonstrate by magnetic resonance imaging, optical imaging, histology, and iron quantification that PTPmu-targeted nanoparticles effectively label adult gliomas. Using PTPmu-targeted nanoparticles in a newly characterized orthotopic pediatric SJ-GBM2 model, we demonstrate individual tumor cell labeling both within the solid tumor margins and at invasive and dispersive sites.

Acinic cell carcinoma of ascending ramus mandible: a rare case report

Acini cell carcinoma are low grade tumors of the salivary glands with a rare occurrence in the axial or appendicular skeleton. The parotid, submandibular and the minor salivary glands are sites of involvement in that order. Painless swelling is the usual presentation with a lytic appearance on radiology and imaging, CT or MRI, FNAC can to some extent differentiate between a benign or malignant lesion thereby dictating the likely extent of excision. On histopathological examination of resected specimens, acinic cell carcinoma shows tumor cells arranged as papillary clusters, acinar pattern and dispersed population with individual tumor cells displaying moderate nuclear pleomorphism, anisonucleosis, round nuclei and moderate amount of ill-defined granular cytoplasm. A unique intraosseous presentation of acinic cell carcinoma, in an elderly male, necessitated a segmental partial mandibulectomy and a comprehensive neck dissection. The tumour area was widely excised from the second premolar region to the coronoid process, and radical neck dissection was performed.

Drug-Induced Resistance in Micrometastases: Analysis of Spatio-Temporal Cell Lineages

Resistance to anti-cancer drugs is a major cause of treatment failure. While several intracellular mechanisms of resistance have been postulated, the role of extrinsic factors in the development of resistance in individual tumor cells is still not fully understood. Here we used a hybrid agent-based model to investigate how sensitive tumor cells develop drug resistance in the heterogeneous tumor microenvironment. We characterized the spatio-temporal evolution of lineages of the resistant cells and examined how resistance at the single-cell level contributes to the overall tumor resistance. We also developed new methods to track tumor cell adaptation, to trace cell viability trajectories and to examine the three-dimensional spatio-temporal lineage trees. Our findings indicate that drug-induced resistance can result from cells adaptation to the changes in drug distribution. Two modes of cell adaptation were identified that coincide with microenvironmental niches—areas sheltered by cell micro-communities (protectorates) or regions with limited drug penetration (refuga or sanctuaries). We also recognized that certain cells gave rise to lineages of resistant cells (precursors of resistance) and pinpointed three temporal periods and spatial locations at which such cells emerged. This supports the hypothesis that tumor micrometastases do not need to harbor cell populations with pre-existing resistance, but that individual tumor cells can adapt and develop resistance induced by the drug during the treatment.

Single-cell EMT-related transcriptional analysis revealed intra-cluster heterogeneity of tumor cell clusters in epithelial ovarian cancer ascites.

Malignant ascites of epithelial ovarian cancer is a metastatic tumor microenvironment in which large amounts of disseminated single cells (DSCs) and disseminated tumor cell clusters (DTCCs) are commonly observed. The tumor cell clusters are known to be more aggressive than individual tumor cells in cancer metastasis; however, little is known about the mechanism. Applying single-cell epithelial-to-mesenchymal transition (EMT)-related transcriptional analysis in 120 DSCs and 195 intra-cluster cells from 27 DTCCs, we demonstrated that DTCCs were heterogeneous cellular units comprised of epithelial tumor cells, leukocytes, and cancer-associated fibroblasts (CAFs). Through the analysis of intra-DTCC heterogeneity, we identified that CAFs induced EMT of tumor cells via TGFβ signaling within the DTCC microenvironment. The activation of EMT program, in particular the upregulation of ZEB2, enabled the acquisition of additional chemoresistance and metastasis abilities of the intra-DTCC tumor cells, which resulted in the aggressiveness of DTCCs.

Abstract B65: Mathematical modeling studies on spatial profiles of tumor-infiltrating T cells

Abstract Activated cytotoxic T lymphocytes have been demonstrated to be able to kill antigen-specific cancer cells via various mechanisms. Not surprisingly, stronger infiltration of cytotoxic T cells into tumor/tumor-cell clusters generally associates with better prognosis, which has been demonstrated in various cancers. There have been efforts on quantifying the distribution of cytotoxic T cells on the whole tumor level. On the other hand, a solid tumor is usually composed of many tumor-cell clusters as well as stroma in gaps between those clusters. It has been noticed that T cells can be mostly localized in the stromal regions of a solid tumor. Therefore, it is also important to quantify the spatial pattern of T cells on the tumor-cell cluster level and further investigate the mechanism underlying the observed limited infiltration. In our work, we quantified the spatial distribution of CD8+ T cells with respect to their distance to the boundary of individual tumor-cell clusters. Generally, we observed that: i) patients differ in infiltration at the cluster level; ii) for most samples, T cells mainly accumulate outside the tumor-cell clusters; iii) for some samples T cells can effectively infiltrate the tumor-cell clusters; and iv) for other samples, the T-cell infiltration profile is intermediate. This last possibility reveals a non-monotonic distribution of T cells, i.e., a drop of T-cell density at the boundary coupled with a second accumulation of T cells at the center of tumor-cell clusters. Based on the quantified CD8+ T-cell profiles on the tumor-cell cluster level, we constructed mathematical models to test two hypothesized contributors affecting the spatial distribution of CD8+ T cells: a physical motility barrier set up by the ECM fibers in the stroma and a biochemical inhibitor ultimately due to the cancer cells inside the tumor-cell clusters. Mathematical models that only include physical barrier effects can qualitatively capture some (but not all) spatial features of the T-cell profiles. However, there is one significant shortcoming: the physical barrier scenario predicts that the profiles observed should be transient and hence eventually T cells should infiltrate all tumors. This appears inconsistent with simple time-scale estimates. A biochemical model focusing on T-cell repulsion can give rise to the observed spatial profiles as steady-state solutions; the different patterns correspond to different properties of cancer cells in different patients. We therefore favor this modeling framework. Of course, a definitive test would require experiments that would enable us to study the infiltration of T cells in a time-dependent manner or alternatively perturb possible mechanisms with drugs to directly test their effects on the infiltration pattern of T cells. Citation Format: Tina Gruosso, Morag Park, Herbert Levine, Xuefei Li. Mathematical modeling studies on spatial profiles of tumor-infiltrating T cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B65.