Individual Tumor Cells Research Articles

Abstract 3116: Spatial single-cell atlas of stage III colorectal cancer

Abstract Background: Spatial heterogeneity and multicellular interactions within the tumor microenvironment drive tumor progression and response to therapy, yet, tumor molecular profiling often requires dissociation of cells, losing the spatial context. Spatial proteomic technologies enable capturing single-cell information with the spatial context allowing us to link cancer-causing mutations to outcomes in cell signaling, responses and survival that will aid in diagnosis, prognostication, and treatment of cancer. We aim to capture single cells in intact tissue sections of stage III colorectal tumors from 52 patients to spatially characterize cell types, interactions, and organization of the tumor microenvironment in relation to genetic alterations and clinical outcomes. Methods: Using Hyperion Imaging Mass Cytometry (IMC), we profiled 16 protein markers across 170 tissue regions, capturing molecular signatures of tissue architecture, tumor cells, and immune cells. We developed an analysis pipeline to quantify single-cell protein expression and identify cell phenotypes. We preserved spatial information to characterize the interactions between neighboring cells and identify multicellular communities. We also generated whole exome sequencing data, RNAseq data and histopathological images from sections of the same tissue blocks. We compared cellular and spatial features between tumors harboring prognostic genomic evens such as TP53 deletion and MSI and between patients stratified by clinical outcomes such as survival and recurrence. Results: We spatially profiled over 800,000 cells to identify 10 tumor, immune and stromal cell phenotypes, and validated identified cell types against matched histopathology and CIBERSORTx deconvoluted RNAseq. To quantify cell-cell interactions, we measured the distance between neighboring cells and performed neighborhood enrichment analysis. We characterized the multicellular organization of the tumor microenvironment to identify 7 consistent cell neighborhoods of up to 10 adjacent cells. We quantified p53 expression in individual tumor cells and found that TP53 deletion (all heterozygous) had no significant association with p53 expression or p53+ tumor cell abundance, suggesting spatial proteomics reveals additional information absent in traditional sequencing. We find that MSI is exclusive of TP53 deletion and that MSI positive tumors had few p53+ tumor cells, increased CD8+ T cell composition and B cells that were further away from p53+ tumor cells compared to MSI negative tumors. Conclusions: Using Hyperion IMC to acquire a spatial single-cell atlas of stage III colorectal cancer enabled us to characterize the tumor microenvironment by defining cell types, quantifying cell interactions, and identifying multicellular communities. These features enable the linkage of prognostic genetic alternations and clinical outcomes with changes in the tumor microenvironment. Citation Format: Andrew Su, Minh Tran, HoJoon Lee, Anuja Sathe, Xiangqi Bai, Richard Cruz, Lance Pflieger, Quan Nguyen, Hanlee P. Ji, Terence Rhodes. Spatial single-cell atlas of stage III colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3116.

Abstract 4621: Distinct spatial and molecular maps in the tumour microenvironment of resistant melanoma patients treated with checkpoint inhibitor immunotherapy

Abstract Background: Checkpoint-based immunotherapies can achieve durable clinical responses in a variety of advanced cancers, including melanoma. However, clinical responses are not universal and there is still a substantial gap in understanding the mechanistic insights of immunotherapy resistance. This study aims to better understand patients’ resistance to immune checkpoint therapies by dissecting and tracing individual tumour and immune cell clones. Methods: Melanoma tumour dissociates and lesion matched formalin-fixed paraffin-embedded tissue samples were collected from patients with advanced melanoma receiving ipilimumab in combination with nivolumab; 2 patients were long-term responders, 2 had innate resistance and 1 acquired resistance. The molecular characteristics and spatial cellular relations associated with tumour and immune subsets in response to treatment were identified using single-cell indexing of transcriptomes and epitopes by sequencing and CODEX (CO-Detection by indEXing) multiplexed tissue imaging. Integration of spatial and molecular information was performed by combining reference-based phenotyping, Louvain clustering, and a mutual nearest neighbours-based transfer learning algorithm. Results: Pre-treatment melanoma biopsies from 5 patients (3 responding and 2 non-responding) along with a subsequent acquired resistance biopsy from an initially responding patient were analysed. Comparisons of cellular phenotypes identified higher proportions of gamma delta T cells (1.98% versus 0.2%) and enriched memory T cell profiles (CD3E, CD44, CD45RO, CD8) in responding compared to non-responding pre-treatment biopsies. Fibroblast from non-responding lesions overexpressed stromal related cytokine, IL1A (log2 fold change (FC) = 3.3) and CXCL1 (log2 FC = 1.4), which were enrichment at the tumour margin compared to responding lesions. Comparisons of a patient’s pre-treatment biopsy that initially responded to a subsequent progressing biopsy with acquired resistance had altered expression of transcript factor, ZFX (log2 FC = -1.59; adjusted p = 0.0119), and MBD2 (log2 FC = -1.5; adjusted p = 0.0225) within the patient’s memory T-cells. Conclusion: The combination of high dimensional single cell transcriptomics and spatial tissue imaging are effective tools to identify specific changes in cell phenotypes, intracellular signalling, and spatial location. This analysis identified reduced memory T cells, increased fibroblast, and altered cellular signalling were associated with resistance to immunotherapies. Citation Format: Camelia Quek, Aditya Pratapa, Ines Pires da Silva, Ghamdan Al-Eryani, Aaron Mayer, Nenad Bartonicek, Kate Harvey, Oliver Braubach, Robyn P.M. Saw, Jonathan Stretch, Kerwin F. Shannon, Alexander M. Menzies, Richard A. Scolyer, Georgina V. Long, Alexander Swarbrick, James S. Wilmott. Distinct spatial and molecular maps in the tumour microenvironment of resistant melanoma patients treated with checkpoint inhibitor immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4621.

Abstract 860: The dynamics of LINE-1 retrotransposition in cellular populations

Abstract Retrotransposons are genetic elements that have the ability to copy and paste themselves in the genome via the proteins they encode. Since the rapid growth of a retrotransposon can be highly deleterious at the organismal level, many of these genetic parasites are suppressed or have been inactivated over evolutionary time. However, the human genome still hosts the active retrotransposon LINE-1, whose aberrant expression is associated with various diseases, especially epithelial cancers. Despite these associations, a quantitative understanding of the dynamics and diversity of LINE-1 insertions in somatic tissue is lacking. In order to address this gap, we present a population dynamic model of LINE-1 retrotransposition in individual cells. Using the model, we show that at demographic equilibrium, the LINE-1 copy number distribution is broader than would be expected from classical population genetic models. We demonstrate how this broadening distorts the frequency spectrum of insertions. We compare the model's predictions to data from whole genome sequencing of individual tumor cells. Citation Format: Stephen Martis, Alexander Solovyov, Jayon Lihm, Hao Li, Benjamin Greenbaum. The dynamics of LINE-1 retrotransposition in cellular populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 860.

Abstract 5388: A unified computational pathology method to quantify HER2 expression from raw IHC and IF images in breast cancer

Abstract Background: The development of automated quantitative methods to measure biomarker expression such as HER2 aims at reducing the subjective variability of pathologist-performed biomarker assessment of immunohistochemically (IHC) stained tissue slides. As an example, the deep learning-based Quantitative Continuous Scoring (QCS) algorithm [1] enables the objective measurement of biomarker expression based on the detection of individual tumor cells in the tumor regions and - for each cell, on the instance segmentation of its nucleus, cytoplasm, and membrane compartments. Methods: In order to extend the QCS image analysis to similarly analyze tissue slides stained with immunofluorescence (IF), we re-trained the fully supervised deep-learning models, adjusted the normalization of images on tissue controls to account for variability between different staining batches, and finally used the normalized signal in IF instead of the Optical Density (OD) signal in IHC as a 8-bit grayscale image on which the biomarker expression is estimated. We performed QCS on HER2 IF images (HER2 clone 29D8 [CST], imaged with Vectra [Akoya] in parallel with HER2 IHC clone 4B5 [Roche Tissue Diagnostics]) performed on 26 primary and metastatic breast cancer samples representing the full range of HER2 expression, from null to highly overexpressed. Results: Our analysis demonstrated that the QCS-based scoring on IHC-HER2 images correlates with the QCS-based scoring on IF-HER2 images. We observed a Pearson correlation of R=0.92 between the median membrane OD in IHC and the median normalized membrane signal in IF. Defining a positive cell as having an estimated membrane expression higher than a given so-called positivity threshold, we found a median Pearson correlation of R=0.85 between the percentage of positive cells detected in IHC and the percentage of positive cells detected in IF for increasing values of positivity thresholds. Correlation of the QCS median normalized membrane signal in IF was R=0.91 with mRNA (ERBB2 transcript levels [Nano String]) and R=0.88 with IHC-based H-scores, against R=0.83 and R=0.87 respectively for the QCS median membrane OD signal in IHC. Conclusion: We describe the extension of a computational pathology-based approach for biomarker quantification in IHC to IF stained tissue slides. The consistency of the image analysis method translates into the consistency of the measurements in the two imaging methods. The use of IF could enable the improved quantification of expression, co-localization and spatial distribution of multiple proteins on the tissue sample.

Abstract 5947: Tumor cells escape cell death after radiotherapy by senescence - a mechanism that can be counteracted with zinc oxide nanoparticles

Abstract Radiotherapy is still an important pillar for the therapy of head and neck squamous cell carcinoma (HNSCC); however, local recurrences arise in 15-50% of patients. During radiotherapy, there is a risk that individual tumor cells survive and proliferate again after a period of dormancy. Growing evidence suggests that senescence may play a key role in this context. We investigated in vitro radiotherapy-induced senescence and found that quiescent tumor cells survived treatment with 16 Gy. Those cells exhibited characteristics of senescence, yet were able to regain their proliferative capacity, and senescence was associated with radioresistance. During radiotherapy, the cells of the tumor stroma are inevitably irradiated as well. It is already known that cancer-associated fibroblasts (CAFs) can promote tumor progression. However, their contribution to the survival of senescent tumor cells after radiotherapy is unknown so far. Furthermore, the association between radiotherapy-induced senescence and recurrences raises the question of how residual, senescent tumor cells can be eliminated to prevent the development of a recurrence at an early stage. These aspects were the subject of the study presented here. To study the interaction between tumor cells and CAFs during their response to irradiation, both cell types were irradiated with 16 Gy in vitro individually and in co-culture. Subsequently, proliferation, cell death, morphological characteristics, and the secretome were analyzed. Additionally, we investigated whether senescent tumor cells can be targeted by treatment with zinc oxide nanoparticles. We could show that not only the tumor cells but also the CAFs go into a senescent state in response to irradiation, seen by loss of proliferation, increased senescence-associated β-galactosidase activity, and typical morphological characteristics. The secretome analysis revealed that the co-culture of tumor cells with CAFs significantly altered the repertoire of cytokines released upon irradiation, indicating intense communication between the cell types during their response to irradiation. Therefore, it is important for future studies on radiotherapy-induced senescence to include the tumor stroma as well. Finally, we demonstrated that senescent tumor cells were vulnerable to cell death induced by treatment with zinc oxide nanoparticles, which thus could potentially contribute to eliminating residual tumor cells after radiotherapy. This study revealed that radiotherapy-induced senescence in tumor cells and associated fibroblasts could well be a therapeutic problem, as senescent tumor cells are not targetable by radiotherapy anymore. In the future, zinc oxide nanoparticles may help to counteract this escape mechanism to prevent recurrences. Citation Format: Nadine Wiesmann, Rita Gieringer, Johannes Kupka, Jonas Eckrich, Sebahat Kaya, Peer Kaemmerer, Bilal Al-Nawas, Juergen Brieger. Tumor cells escape cell death after radiotherapy by senescence - a mechanism that can be counteracted with zinc oxide nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5947.

Single Cell Analysis of Inertial Migration by Circulating Tumor Cells and Clusters

Single-cell analysis provides a wealth of information regarding the molecular landscape of the tumor cells responding to extracellular stimulations, which has greatly advanced the research in cancer biology. In this work, we adapt such a concept for the analysis of inertial migration of cells and clusters, which is promising for cancer liquid biopsy, by isolation and detection of circulating tumor cells (CTCs) and CTC clusters. Using high-speed camera tracking live individual tumor cells and cell clusters, the behavior of inertial migration was profiled in unprecedented detail. We found that inertial migration is heterogeneous spatially, depending on the initial cross-sectional location. The lateral migration velocity peaks at about 25% of the channel width away from the sidewalls for both single cells and clusters. More importantly, while the doublets of the cell clusters migrate significantly faster than single cells (~two times faster), cell triplets unexpectedly have similar migration velocities to doublets, which seemingly disagrees with the size-dependent nature of inertial migration. Further analysis indicates that the cluster shape or format (for example, triplets can be in string format or triangle format) plays a significant role in the migration of more complex cell clusters. We found that the migration velocity of a string triplet is statistically comparable to that of a single cell while the triangle triplets can migrate slightly faster than doublets, suggesting that size-based sorting of cells and clusters can be challenging depending on the cluster format. Undoubtedly, these new findings need to be considered in the translation of inertial microfluidic technology for CTC cluster detection.

Borrelia Infected Neoplastic Glial Cells in Five Patients With Glioblastoma Multiformae

Glioblastoma multiformae (GBM) is a primary malignancy of brain astrocytes. Transcranial stereotactic biopsy specimens from five deceased and anonymous patients with GBM were retrieved from archival storage two years after the death of the patients. All patients were residents of the Long Island, New York metropolitan region where borrelia burgdorferi and borrelia miyamotoi infections are hyperendemic. Fluorescence in situ hybridization (FISH) using two DNA probes specific for genes of burgdorferi borrelia and an additional FISH method study with two DNA probes specific for miyamotoi borrelia each and individually produced high signal borrelia DNA hybridizations inside malignant cells in brain biopsies from all five decedents. Bright signal bacterial DNA hybridizations inside live borrelia spirochetes were found at all levels in serial sections of tumor biopsy fragments in low magnification imaging and outright invasion of the nuclei of individual tumor cells in high resolution imaging. Herein is the first report of a cluster of GBM patients with universal borrelia microbe invasion of neoplastic glial cells.

Additive Integrative Methods in The Treatment of Patients with Breast Cancer

An increasingly integrative cancer treatment is worth striving for. There is more and more evidence for this in the literature. Liquid biopsy with drug testing can provide a basis. We have tried to make an optimisation with the help of ranking lists of on the one hand chemical and on the other hand natural remedies. The most frequently positive and thus effective complementary remedies are listed. Some of them are well-known, others are relatively new or even controversial. However, if efficiency on individual tumor cells has been demonstrated in the laboratory, this should be taken into account. Patients can receive a benefit from this.

Abstract PD4-08: PD4-08 A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors

Abstract A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors Austin Whitman, Hyunsoo Kim, Kamila Wisniewska, Rasha Kakati, Susana Garcia Recio, Hector Franco, Charles Perou, Philip Spanheimer Background: Resistance to endocrine therapy is a primary cause of treatment failure and death in patients with estrogen receptor (ER)-positive breast cancer. Intratumor heterogeneity is associated with resistance to therapy across tumors, and specifically in ER+/HER2- breast cancer, heterogeneity in ER and PR expression is associated with a worse response to endocrine therapy. We hypothesize that subpopulations within and across ER+/HER2- human breast tumors have distinct responses to tamoxifen and that discerning heterogeneity in response will improve understanding of inherent and emerging resistance to endocrine therapy. Methods: We developed an operating room-to-laboratory pipeline immediately after surgical resection for studies using alive tissue. Tissue samples were obtained and single cell suspensions created using physical and enzymatic dissociation. Cells were treated with tamoxifen (10 M) or control media for 12 hours in suspension and single cell RNA libraries generated using the 10X Genomics droplet-based kit and sequenced using the Illumina NextSeq2000. Results: We obtained normal breast tissue from 2 women undergoing reduction mammoplasty and tumor tissue from 10 women with ER+/HER2- invasive breast carcinoma. In tamoxifen treated and control matched pairs, a total of 22,195 cells from normal breast and 94,558 cells from tumor samples were sequenced. Computational analysis using consensus clustering was performed and cell types assigned using canonical correlation. Both tumor and normal samples identified clustering by cell type and not by patient revealing significant variability in cell type abundance between samples. In the normal breast samples, we performed differentially expressed genes (DEG) analysis comparing tamoxifen treatment to control for each cell type (Immune cells, fibroblasts, basal epithelial cells, luminal progenitor cells, and mature luminal cells) and enrichment analysis of up- and down-regulated genes performed. Strong depletion of estrogen induced genes was observed in tamoxifen-treated normal luminal progenitor and mature luminal cells, but not in basal epithelial cells or fibroblasts, demonstrating distinct, subpopulation-specific response to tamoxifen. In the 10 tumor matched pairs, 4 had a high epithelial proportion and tumor cells identified using inferred copy number variation. Tumor cells in 3 of these 4 samples showed significant down regulation of estrogen response genes with tamoxifen treatment. Using scBCSubtype to assign PAM50 subtype to individual tumor cells, the 3 responsive tumors were comprised primarily of LumA cells while the unresponsive tumor was predominantly LumB. Finally, we developed a novel score to quantify responsiveness at the single cell level based on downregulation of estrogen response genes with tamoxifen treatment relative to matched cluster-specific untreated expression. This analysis demonstrated heterogeneity in response to tamoxifen in tumor cells and identified distinct subpopulations of responsive and unresponsive tumor cells to tamoxifen treatment. Conclusion: We developed a novel ex vivo model to determine heterogeneity in therapeutic response to tamoxifen in normal human breast tissue and primary human breast tumors. We demonstrate differences in tamoxifen response by cell type and identify distinctly responsive and resistant subpopulations within human tumors. This provides a foundation to define features of responsive and resistant populations on the individual cell and specimen basis, and should allow us to develop precise, single cell-based predictors of response to endocrine therapy, and to identify genes and pathways driving resistance to therapy. Citation Format: Hyunsoo Kim, Austin Whitman, Kamila Wisniewska, Susana Garcia-Recio, Rasha Kakati, Hector L. Franco, Charles M. Perou, Philip Spanheimer. PD4-08 A Novel Single Cell Model of Tamoxifen Response in Primary Human Breast Tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-08.

Abstract P3-08-06: Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model

Abstract Background: Lymphovascular invasion (LVI) is a major route of metastatic dissemination and recent studies indicate its value as an independent prognostic indicator for advanced breast, colorectal, squamous cell, prostate, brain cancers. LVI is a clinicopathological hallmark of inflammatory breast cancer (IBC), an understudied and most lethal breast cancer. IBC is often misdiagnosed due to an absence of a solid mass and its unique presentation of diffuse tumor cell clusters/emboli in the dermal lymphatics. Widely used mammary tumor implantation models coupled with bioluminescence or fluorescence imaging to monitor tumor growth kinetics are ineffective for evaluating spatial and temporal changes in growth and migration patterns of individual tumor cells and clusters within their microenvironmental context. The goal of this study was to develop a murine model to simulate the unique clinicopathological features of IBC patients and to assess both qualitatively and quantitatively local tumor growth, motility, and LVI. Methods: To specifically facilitate visualization of lymphatic and endothelial vessels along with tumor-vessel interactions, we generated a transgenic nude mice model (ProxTom RFP Nu/Nu) wherein, the mice exhibit red, fluorescent lymphatics [tdTomato fluorophore under control of a Prox1 promoter, which encodes a transcription factor (prospero-related homeobox 1) necessary for the formation and maintenance of lymphatic vessels]. Next, we employed a surgical technique, wherein a window chamber is placed on the dorsal skinfold of mice, which allows for microscopic examination of implanted tumor cells and ability to track dynamic changes of the tumor in its local microenvironment from the time of implantation up to 10 days. Patient-derived IBC or PDX stably transfected to express green, red fluorescent and/or dual tagged with luciferase reporters were transplanted in mice bearing window chambers. Intravital fluorescence microscopy and IVIS imaging were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0-140h. Results: Multichannel optical imaging of the window chamber in the ProxTom RFP Nu/Nu mice demonstrated co-localization of IBC tumor cells and lymphatics. Diffuse tumor cells were observed along regions of lymphatic vessels both proximal and distal to the primary tumor site. However, measurement of blood and lymph vessel density showed no significant change over time. Next, these datasets were used for quantitative analysis by setting the tumor cell channel (GFP) at a threshold to count any clusters greater than 50 pixels2 (~0.0013mm2) and with greater than the mean + 2 standard deviations of the background signal while avoiding noise/artifacts from very small regions (<~0.001mm2). This allowed for computation of the total tumor area including average area of each cluster. In addition, the area moment, which describes the basic directional growth pattern of the tumor cells, was quantified by multiplying a cluster distance from the center of mass by its area. Conclusions: A key novel finding from structured illumination imaging data was the observation of LVI occurring early, similar with the clinical presentation in IBC patients. This model was able to effectively track tumor cluster migration. This approach of short-term longitudinal imaging time frame in studying transient or dynamic events of diffuse, collectively migrating tumor cells in the local environment and quantitative analysis of the tumor area, motility and vessel characteristics is an innovation that can be used to investigate other cancer cell types exhibiting LVI. Funding in part by DoD W81XWH-17-1-0297; W81XWH201053 (GRD); ACS Mission Boost grant MBG-20-141-01 (GRD) and NIH grant P30-CA014236 (Imaging Core/GMP) Citation Format: Gayathri R Devi, Dorababu Sannareddy, Alexandra Bennion, Ashlyn Rickard, Douglas C Rouse, Mark W Dewhirst, Gregory M Palmer. Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-06.

Abstract A007: Development of a fibrinolytic platelet system to dissociate circulating tumor cell clusters

Abstract Circulating tumor cell (CTC) clusters play a significant role in cancer metastasis. During metastasis, CTCs can travel in the bloodstream as individual cells or as clusters associated with fibrin and platelets (Palumbo et al., Blood 2000; Karpatkin and Pearlstein, Annals of Internal Medicine, 1981). The CTC clusters have a higher metastatic potential than individual tumor cells (Aceto, Cell 2014) due to the increased viability of tumor cells in clusters, the increased chance of large clusters arresting in small vessels, and the increased ability of clusters to form a metastatic niche at a distal location. As such, there is a need to develop methods to disperse CTC clusters. Previous work with urokinase-type plasminogen activator has shown initial success in reducing metastasis by dissociating cancer cell clusters (Choi et al., Cancer Res. 2015). However, there is another key component of this metastatic challenge, the interaction of CTCs with platelets. Platelets bind to the CTC surface which protects the CTC clusters from shear stress and immune detection, increasing their metastatic potential (Morris, Schnoor, and Papa, Biochim. Biophys. Acta BBA - Rev. Cancer 2022). However, these interactions also provide an approach to target CTC clusters. As such, the objective of this project is to develop a fibrinolytic platelet system to dissociate CTC clusters while also disrupting CTC-platelet interactions. For this approach, TPA was loaded onto two modified platelet systems: platelet decoys (Papa et al., Sci. Transl. Med. 2019) and lyophilized platelets (Schnoor and Papa, Front. Bioeng. Biotechnol. 2022). Each system was assessed to determine the surface receptor characteristics, drug loading, and interaction with tumor cells in vitro. The fibrinolytic activity of the systems was also characterized using a FRET based assay and the angiogenic effects of the systems were measured using a HUVEC tube formation assay. Furthermore, the ability of the system to dissociate cancer cell clusters in vitro was assessed for the first time using light transmission aggregometry. The results demonstrate that the TPA was successfully loaded onto both platelet decoys and lyophilized platelets while maintaining the fibrinolytic activity of the platelets. Furthermore, a high percentage of cancer cells interacted with the TPA-loaded carriers, which corresponds to the maintenance of key surface receptors such as GP IIb/IIIa. The data also demonstrates that the fibrinolytic platelet systems can successfully dissociate cancer cells in vitro. Currently, the effectiveness of these fibrinolytic platelet systems for preventing cancer cell metastasis is being assessed in vivo. Based on these results, this fibrinolytic platelet approach is a promising method to leverage platelet-CTC interactions to disperse CTC clusters and reduce metastasis. Citation Format: Brian Schnoor, Anne-Laure Papa. Development of a fibrinolytic platelet system to dissociate circulating tumor cell clusters [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A007.

Micrometastasis detection using modified papanicolaou stain in nodal tissues of oral squamous cell carcinoma - A histological study.

Cervical lymph node metastasis is the most important prognostic factor in Squamous Cell Carcinoma of Head and Neck (SCCHN). Detection and evaluation of micro-metastasis forms the basis for diagnosis, staging, treatment options and prognosis. Lymph node prognostic factors are extremely important for the survival and recurrence in the patient. Assessing lymph node metastasis in the absence of clinical enlargement is challenging. To evaluate micrometastasis and individual tumor cells (ITC) in regional lymph nodes of oral squamous cell carcinoma (OSCC) by modified papanicolaou (PAP) stain and re-evaluate the tumor staging. The retrospective study was executed at MS Ramaiah University of Applied Sciences. The current study constituted a total of 40 lymph nodes from OSCC patients, metastatic (n=20) and non-metastatic lymph nodes (n=20). All sections were stained with H & E followed by modified PAP stain. Modified PAP was used for identification of micrometastasis deposits. The Chi square test was employed to analyze significance. Modified PAP stain proved to be more accurate (p = 0.006) than H and E stain in detecting micrometastasis which accounted for 15% of non-metastatic lymph node sections used in our study. Special stain like modified PAP stain is valuable and sensitive in detecting micro-metastasis over H and E stain. Detection of micrometastasis in OSCC patients is advantageous for the patient as it influences staging, it modifies the treatment plan in terms of both radiotherapy and chemotherapy.

Epithelioid trophoblastic tumor: A case series.

An epithelioid trophoblastic tumor (ETT) is an extremely rare gestational trophoblastic tumor. Cases of ETT present with abnormal vaginal bleeding in women of reproductive age group with marginally elevated beta human chorionic gonadotrophin (B-hCG) levels. Here, we describe a series of four patients (all were females) including histomorphology, immunoprofiles, and diagnostic difficulty of this rare entity. All cases were in their reproductive age group. The mean pre-treatment hCG level was 665.24 (mIU/mL). Microscopically, all cases had a tumor showing an epithelioid appearance arranged in large nests and sheets. Individual tumor cells were round to polygonal with abundant eosinophilic cytoplasm, with central vesicular nuclei and prominent nucleoli. Areas of hemorrhage, necrosis, and intercellular hyaline-like material deposition were identified in all cases (100%). Immunohistochemically, tumor cells in all cases showed diffuse positivity for AE1/AE3 and p63 (100%). GATA3 was available in one case (25%), which was positive in the tumor cells. In one case (25%), hPL was focally positive, and in one case (25%), it was negative. SALL4 was performed in two cases (50%) and was negative in tumor cells. The mean Ki67 labeling index was 19.2 (range 10-30%). All four patients underwent surgical intervention and were treated with hysterectomy. The mean follow-up in this series was 39.4 months (range 6-70), and all patients are alive to date with a mean survival of 32.8 months (range, 4-67).

Nuclear NPM-ALK Protects Myc from Proteosomal Degradation and Contributes to High Protein Expression in Cancer Stem-like Cells in ALK-Positive Anaplastic Large Cell Lymphoma

While Myc is one of the oncoproteins most frequently deregulated in human cancers, its expression and significance in ALK-positive anaplastic large cell lymphoma (ALK+ALCL) has not been extensively studied. Previous studies from our group have shown that a high level of Myc proteins is a characteristic feature of a small subset of ALK+ALCL cells that carry cancer stem-like features (labeled RR cells), and shRNA knockdown or pharmacological inhibition of Myc in these cells significantly decreased their cancer stemness and phenotypically converted them into 'bulk' cancer cells (labeled RU cells). In this study, we hypothesized that NPM-ALK, the key oncogenic driver in ALK+ALCL, is implicated in the differential protein expression of Myc between RU and RR cells, even though cells from both cell subsets are known to express a similar level of NPM-ALK proteins. We first tested how NPM-ALK may impact the proteasomal degradation of Myc (or PD-Myc) in HEK293 cells. We found that transfection of NPM-ALK effectively diminished PD-Myc and dramatically increased the Myc protein level. Since NPM-ALK is known to heterodimerize with full-length NPM1, a protein that carries a Myc-binding domain and functions as a key component of the PD-Myc pathway in other cell types, we speculated that the suppression of PD-Myc by NPM-ALK is mediated via the heterodimers formed by NPM-ALK and full-length NPM1 (i.e. NPM-ALK:NPM1). In keeping with this hypothetical model, while shRNA knockdown of full-length NPM1 (by targeting its C-terminal) in HEK293 cells increased the Myc protein level, the same treatment of NPM-ALK-transfected HEK293 cells showed the opposite effects. To further support and validate this model, we extended our studies to purified RU and RR cells derived from SupM2, an ALK+ALCL cell line. In keeping with our model, the degradation half-life of Myc was found to be substantially longer in RR cells as compared to RU cells. Similar to NPM-ALK-transfected HEK293 cells, RR cells showed a dramatic decrease in the Myc protein level upon shRNA knockdown of the full-length NPM1, again suggesting the importance of the NPM-ALK:NPM1 heterodimers in this context. Similarly, siRNA knockdown of ALK in RR cells resulted in substantial decrease in Myc. Immunoprecipitation experiments showed that full-length NPM1 proteins in RU cells form protein complexes that are normal components of PD-Myc, whereas full-length NPM1 in RR cells pulled down a large amount of NPM-ALK, indicating the relative abundance of the NPM-ALK:NPM1 heterodimers in these cells. To validate this model in ALK+ALCL patient samples, we performed immunofluorescence triple staining for full-length NPM1, ALK and Myc, and images of 24 individual ALK+ALCL cells per sample were analyzed using confocal microscopy and the ImageJ software. We found that the pixel density of the fusion signals of NPM-ALK and full-length NPM1 significantly correlated with that of the Myc signals (Pearson correlation analysis, r=0.81; p<0.0001). In conclusion, our findings have provided evidence in support of a novel oncogenic function of NPM-ALK exerted in the nucleus. Specifically, in the small subset of cancer stem-like cells (i.e. RR cells), nuclear NPM-ALK proteins often heterodimerize with full-length NPM1, thereby protecting Myc away from the PD-Myc machinery and inhibiting its degradation. In contrast, in the 'bulk' RU cells, NPM-ALK:NPM1 heterodimers are less abundant, and Myc proteins are unprotected and subjected to degradation. Thus, the control of the NPM-ALK:NPM1 heterodimer formation in the nucleus regulates the Myc protein levels and the degree of cancer stemness in individual tumor cells.

ANGI-02. EPIGENETIC REGULATION OF GLIOMA INVASION BY CHITINASE-3-LIKE PROTEIN 1 (CHI3L1) CPG PROMOTER METHYLATION

Abstract Epigenetically controlled processes play an important role in development but also in the formation and progression of many common diseases. Molecular signatures are increasingly incorporated into cancer classifications and DNA methylation-based analyses of brain tumors including glioma is currently the gold standard. Glioblastoma (GBM), CNS WHO grade 4, is characterized by a very diffuse infiltrative growth into the adjacent brain parenchyma. Due to these highly invasive but also genetically heterogeneous and plastic cellular phenotypes, standard treatment, including maximum resection, radio- and chemotherapy, has limited efficacy. It can be assumed that intertumoral or molecular heterogeneity of gliomas at the transcriptome level is caused by altered epigenetic changes within the individual tumor cells. Considering this, we hypothesize that tumor cell invasion being one of the major hallmarks of highly malignant gliomas can be associated with altered DNA methylation and therefore transcript expression. To analyze this, we isolated fractions of glioma cells with increased invasive properties and performed Illumina® array-based methylation profiling as well as RNA sequencing analysis to identify methylation-driven, invasion-promoting genes. Among other, we focused on Chitinase-3-like protein 1 (CHI3L1) as a candidate gene with significantly decreased promoter methylation in invasive cell fractions and overall strong transcript expression in high-grade gliomas. Differential expression and methylation correlation of TCGA data combined with our own dataset revealed that CHI3L1 expression is dependent on the methylation status of at least three CpG sites within the gene promoter region. We validated these findings using several in vitro experimental settings and propose an upstream regulatory mechanism in which single CpG DNA demethylation within the CHI3L1 promoter facilitates transcription factor binding resulting in gene expression.

EPCO-27. REVEALING TUMOR HETEROGENEITY AND IMMUNE MICROENVIRONMENT IN NF-1 GLIOMA BY SINGLE-CELL GENE EXPRESSION PROFILING

Abstract Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF-1), the most common human genetic cancer predisposition syndrome. NF1 patients are prone to develop peripheral and central nervous system tumors, with glioma occurring in 15-20% of patients. The molecular profiles of glioma from patients with NF-1 have been previously explored by large scale DNA and RNA bulk analyses, providing the first comprehensive genetic landscape of NF-1 glioma. However, the intra-tumoral architecture and the microenvironment organization remained largely concealed within the global bulk profile. Here, to characterize the complexity of molecular states of both malignant and non-malignant cells, we profiled NF-1 glioma gene expression at individual cell resolution. We performed single-nuclei RNA sequencing (Nucseq) on frozen NF-1 glioma specimens, including high-grade and low-grade tumors. The functional classification of the transcriptional programs activated in the individual tumor cells identified three main molecular states that partially overlapped with the cellular subtypes of sporadic glioblastoma. Tumor cells of low-grade NF-1 glioma exhibited marked enrichment of neuronal functions, suggesting that the gliomagenesis process in the context of NF1 inactivation might be supported by specialized neuronal activities. Conversely, the proliferative state characterized high-grade NF-1 glioma, indicating that cell cycle-associated pathways are drivers of tumor progression. The single-cell deconvolution of immune microenvironment in NF-1 low grade glioma revealed infiltration of CD8+ T lymphocytes and the active crosstalk of these cells with pro-inflammatory macrophages and mature dendritic cells, supporting a possible anti-tumor cell-mediated response. The identification of divergent tumor cell states as well as the characterization of the immune infiltrates provide new insights for accurate stratification of NF-1 glioma patients and to inform personalized therapeutics.

Expression of Vascular Endothelial Growth Factor (VEGF) in Colorectal Adenoma and Carcinoma in a Tertiary Care Center.

Colorectal cancer is one of the malignancies in which angiogenesis has been implicated and its importance at different stages of malignant disease. Neovascularization begins when the angiogenic switch is turned on and when angiogenesis activators outweigh angiogenic inhibitors. The process of blood vessel formation is regulated through several growth factor systems. The vascular endothelial growth factor (VEGF) system is one of the important ones and may consequently be a key system in relation to different aspects of colorectal carcinoma treatment.Because of the well-defined steps in its progression (adenoma - Tis - T1 invasive cancer- T2 advanced cancer with metastases), colorectal cancer (CRC) represents a model for investigating the effects of angiogenesis throughout tumor development.The aim of this study is to determine the role of VEGF expression by immunohistochemistry (IHC) in normal epithelium, dysplasia, carcinoma of colorectal specimens and to correlate the same with tumor grade, stage, nodal status, and metastasis. This is a retrospective study on paraffin blocks of 50 colon cancer specimens, 40 adenoma specimens, and 10 normal colonic mucosa specimens. Immunohistochemical stain for VEGF was done on the sections along with controls. Monoclonal antibody detected against VEGF antigen was observed in the cytoplasm of the tumor cells and the intensity ofVEGF expression in individual tumor cells was scored on a scale of 0 (no staining) to 3 (strong intensity), and the percentage of cells with VEGF staining at each intensity was estimated from 0 to 100. Pearson's Chi-squared test and Mann-Whitney test were used to determine significant clinicopathological differences between VEGF expression in positive and negative tumors. In normal epithelium, VEGF immunoreactivity was seen in all 10 cases with high intensity. Among adenomas, VEGF expression was seen in 26 (65%) of the 40 cases. Out of which in tubular adenomas VEGF expression was seen in 13 cases (60%) and negative in eight cases (40%). In tubulo villous adenoma, VEGF expression was seen in nine cases (60%) and negative in six cases (40%). Villous adenomas showed VEGF expression in all four cases (100%). In adenocarcinoma, VEGF expression was seen to be expressed in 42 cases (84%) and negative in eight cases (16%) and expression washigher in low-grade carcinomas (70%) compared to high-grade carcinomas. A significant difference in the expression ofVEGF among adenomas and carcinomas was observedwith higher intensity present in adenoma when compared to carcinoma. Expression ofVEGF could be considered as an early carcinogenic factor incolorectal carcinomas as it is expressed in higher intensity in the precancerous lesion and low-grade and stage 1 adenocarcinoma. Hence, we infer that early colorectal carcinomas are an important model for targeted therapy with antiangiogenic factors for VEGF.

Clinicopathological observation of 10 cases of salivary secretory carcinoma

Objective: To investigate the clinical and pathological features of salivary secretory carcinoma (SSC). Methods: Ten cases of SSC confirmed in the Department of Pathology,Capital Medical University School of Stomatology from January 2014 to December 2021 were retrospectively included, including 5 males and 5 females, with a median age of 46.5 years. The microscopic morphology, immunophenotype, special staining and clinical follow-up of 10 cases of salivary secretory carcinoma were observed. Ten patients were tested with S-100, vimentin, mammaglobin, Dog-1, p63 and Ki-67, 9 cases with cytokeratin (CK) 8/18, 8 with CK7, 6 with calponin, 5 with smooth muscle actin (SMA) and GCDFP15, 4 with CK5/6 and 1 with SOX10. The ETV6-NTRK3 fusion gene was detected by fluorescence in situ hybridization. Results: Seven of the 10 SSC were located in the parotid gland and 3 were located in the cheeks. Histomorphology showed solid, papillary-cystic, follicular, microcystic, and macrocystic types. In 7 cases, tumor cells were dominated by single arrangement type, while certain mixed arrangements existed in some areas. The cytoplasm of the tumor cells was rich in eosinophilic, fine granular or vacuolar shapes, and clear cytoplasm was seen in 2 cases. The nuclei were mostly oval-shaped vesicular nuclei, with nucleoli in the center. Immunohistochemistry showed CK7 (8/8) positive, CK8/18 (9/9) positive, S-100 (10/10) positive, vimentin (5/10) positive, (4/10) partially positive and (1/10) less partially positive, mammaglobin (7/10) positive, (1/10) partially positive and (2/10) some individual cells positive, Dog-1 (10/10) negative, CK5/6 (4/4) negative, p63 (7/10) negative and (3/10) partially positive, SMA (5/5) negative, calponin (6/6) negative, and Ki-67 index was 5%-20%. Secretions of 5 cases showed periodic acid-Schiff (PAS) and PAS with diastase (PAS-D) staining positive. All 10 cases showed ETV6-NTRK3 fusion positive. Six cases were successfully followed up for 32-91 months, of which 2 cases recurred after 28 and 74 months and underwent surgical resection again. All cases followed up are alive and disease-free. Conclusions: The salivary secretory carcinoma is a rare low-grade malignant tumor. In certain cases, morphology is atypical and mammaglobin is immunohistochemically positive in only individual tumor cells. Therefore, the diagnosis should be supported with morphology, immunohistochemical staining, and molecular feature preferably.

Loss of endothelial EMCN drives tumor lung metastasis through the premetastatic niche

BackgroundMetastasis is the primary cause of cancer-related mortality. Metastasis involves a complex multistep process during which individual tumor cells spread primarily through destruction of the endothelial barrier, entering the circulatory system to colonize distant organs. However, the role of the endothelial barrier as the rate-limiting process in tumor metastasis and how these processes affect the regulation of the host microenvironment at the molecular level are poorly understood.MethodsHere, we analyzed differentially expressed genes in breast cancer and lung adenocarcinoma, including metastatic and recurrent specimens, using TCGA dataset. The effects of EMCN on endothelial cells in vitro and in vivo were analyzed by assessing angiogenesis and vascular permeability, respectively. We established a syngeneic mouse model of endothelial cell-specific knockout of EMCN (EMCNecko) to study the role of EMCN in tumor growth and metastasis. Transcriptome sequencing, Western blotting, qPCR and immunofluorescence confirmed important factors in the premetastatic niche. A mouse model of allograft tumor resection with lung metastasis was established to confirm the therapeutic effect of a notch inhibitor combined with an anti-TGF-β antibody.ResultsWe found a strong correlation of EMCN deficiency with tumor recurrence and metastasis. Comparative experiments in WT and EMCNecko mice revealed that endothelial EMCN deficiency did not affect primary tumor growth significantly but strongly promoted spontaneous metastasis. EMCN deficiency was associated with gene profiles that regulate cell junctions in vitro and enhance vascular permeability in vivo. Mechanistically, EMCN deficiency mainly affected the host microenvironment and led to the formation of a lung premetastatic niche by recruiting Ly6G+ neutrophils and upregulating MMP9, S100A8/A9 and TGF-β expression. Anti-TGF-β antibody effectively eliminated TGF-β-induced neutrophil polarization, thereby reducing lung metastasis. Notably, the combination of a Notch inhibitor and an anti-TGF-β antibody effectively inhibited tumor growth and lung metastasis and prolonged the survival time of mice.ConclusionsWe present a new translational strategy of EMCN as a new key player in tumor lung metastasis by affecting the host microenvironment. These findings could provide a sound theoretical basis for clinical treatment.

Predicting the true extent of glioblastoma based on probabilistic tractography

Glioblastoma is the most frequent type of primary brain tumors. Despite the advanced therapy, most of the patients die within 2 years after the diagnosis. The tumor has a typical appearance on MRI: a central hypointensity surrounded by an inhomogeneous, ring-shaped contrast enhancement along its border. Too small to be recognized by MRI, detached individual tumor cells migrate along white matter fiber tracts several centimeters away from the edge of the tumor. Usually these cells are the source of tumor recurrence. If the infiltrated brain areas could be identified, longer survival time could be achieved through supratotal resection and individually planned radiation therapy. Probabilistic tractography is an advanced imaging method that can potentially be used to identify infiltrated pathways, thus the real extent of the glioblastoma. Our study consisted of twenty high grade glioma patients. Probabilistic tractography was started from the tumor. The location of tumor recurrence on follow-up MRI was considered as the primary infiltrated white matter tracts. The results of probabilistic tractography were evaluated at thirteen different thresholds. The overlap with the tumor recurrence of each threshold level was then defined to calculate the sensitivity and specificity. In the group level, sensitivity (81%) and specificity (90%) were the most reliable at 5% threshold level. There were two outliers in the study group, both with high specificity and very low sensitivity. According to our results, probabilistic tractography can help to define the true extent of the glioblastoma at the time of diagnosis with high sensitivity and specificity. Individually planned surgery and irradiation could provide a better chance of survival in these patients.