Enrichment Of Tumor Cells Research Articles

EPCO-02. HIGH CELLULAR PLASTICITY STATE OF HUMAN MEDULLOBLASTOMA LOCAL RECURRENCE AND DISTANT DISSEMINATION

Abstract Medulloblastoma (MB) is a heterogeneous pediatric brain tumor with variable clinical outcomes. Since current treatments facing limitations due to tumor resistance, less than 10% of relapsed patients survive beyond five years, with poorly understood recurrence mechanisms. To obtain cellular diversity and genetic characters in primary and recurrent MB, we conducted comprehensive analyses utilizing the single-cell/nucleus RNA sequencing (sc/snRNA-seq), snATAC-seq and spatial transcriptomics, complemented with bulk RNA sequencing and matched primary-recurrent whole exon profiles. SHH and Group_3 subgroups revealed distinct cellular subsets, including cycling, differentiated and quiescent tumor cells. Recurrent or disseminated MB displayed a dramatically different cellular ecosystem compared to primary tumors, with varying proportions of shared cell types. Local recurrence exhibited a higher enrichment of cycling tumor cells, while differentiated subset was notably present in disseminated lesions, indicating that local recurrences harbored the stronger proliferative capacity, whereas disseminations relatively well differentiated. Additionally, chromosomal alterations were assessed, reflecting distinct genetic subclones, such as chr7q gain and chr11 loss in Group_3 disseminations. Notably, the emergence of a subpopulation termed “high cellular plasticity (HCP)” during MB progression was noted, characterized by dynamic adaptability and stemness properties. As validated, HCP state was associated with increased chromatin accessibility, contributing to tumor recurrence and immune microenvironment remodeling. Functionally, inhibiting markers associated with HCP cells, such as PTPRZ1, efficiently suppressed MB metastasis in preclinical models. In conclusion, our findings fill the critical knowledge gaps in understanding the cellular diversity, chromosomal alterations and biological dynamics in MB recurrence and dissemination, offering potential therapeutic interventions.

Research on the Method of Detecting TPN-Labeled Tumor Cells in Pleural Effusion Based on the Microfluidic Chip.

The clinical diagnosis of a malignant pleural effusion (MPE) is still based on the detection of tumor cells in the pleural effusion. The question of how to improve the efficiency and accuracy of detecting an MPE still remains. This study explores the use of microfluidic technology to concentrate cells in an MPE and achieved the detection of the cell marker TPN in the microarray capture area. TPN is a mitochondria-specific bio-probe that can identify tumor cells on the basis of differences in the mitochondrial potential. First, we designed a microfluidic chip to analyze its performance. The results show that when the total flow rate of the injected chip was 12 mL/h and the volume ratio of cell separation liquid to cell suspension was 1:1, the target cells (A549, MCF-7, and Hela) were enriched and the purity was improved to 98.7-99.3%. Finally, an MPE from cancer patients was used to detect the chip's ability to isolate and enrich tumor cells. Furthermore, the fluorescent identification of the TPN within the tumor cells was simultaneously achieved on the microfluidic chip. In conclusion, the potential to improve the efficiency of the clinical diagnosis of MPEs is provided by the chip structure and analysis conditions explored in this study.

Tumor cell enrichment by tissue suspension improves sensitivity to copy number variation in diffuse gastric cancer with low tumor content

The detection of copy number variations (CNVs) and somatic mutations in cancer is important for the selection of specific drugs for patients with cancer. In cancers with sporadic tumor cells, low tumor content prevents the accurate detection of somatic alterations using targeted sequencing. To efficiently identify CNVs, we performed tumor cell enrichment using tissue suspensions of formalin-fixed paraffin-embedded (FFPE) tissue sections with low tumor cell content. Tumor-enriched and residual fractions were separated from FFPE tissue suspensions of intestinal and diffuse-type gastric cancers containing sporadic tumor cells, and targeted sequencing was performed on 225 cancer-related genes. Sequencing of a targeted panel of cancer-related genes using tumor-enriched fractions increased the number of detectable CNVs and the copy number of amplified genes. Furthermore, CNV analysis using the normal cell-enriched residual fraction as a reference for CNV scoring allowed targeted sequencing to detect CNV characteristics of diffuse-type gastric cancer with low tumor content. Our approach improves the CNV detection rate in targeted sequencing with tumor enrichment and the accuracy of CNV detection in archival samples without paired blood.

Novel Isolating Approaches to Circulating Tumor Cell Enrichment Based on Microfluidics: A Review.

Circulating tumor cells (CTCs), derived from the primary tumor and carrying genetic information, contribute significantly to the process of tumor metastasis. The analysis and detection of CTCs can be used to assess the prognosis and treatment response in patients with tumors, as well as to help study the metastatic mechanisms of tumors and the development of new drugs. Since CTCs are very rare in the blood, it is a challenging problem to enrich CTCs efficiently. In this paper, we provide a comprehensive overview of microfluidics-based enrichment devices for CTCs in recent years. We explore in detail the methods of enrichment based on the physical or biological properties of CTCs; among them, physical properties cover factors such as size, density, and dielectric properties, while biological properties are mainly related to tumor-specific markers on the surface of CTCs. In addition, we provide an in-depth description of the methods for enrichment of single CTCs and illustrate the importance of single CTCs for performing tumor analyses. Future research will focus on aspects such as improving the separation efficiency, reducing costs, and increasing the detection sensitivity and accuracy.

A Microfluidic, Multi-Antibody Cell Capture Method to Evaluate Tumor Cells in Cerebrospinal Fluid in Patients With Suspected Leptomeningeal Metastases.

Leptomeningeal disease (LMD) is a clinical sequela of central nervous system metastasis involving the cerebrospinal fluid (CSF), often seen in late-stage solid tumors. It has a grave prognosis without urgent treatment. Standard of care methodologies to diagnose LMD include CSF cytology, magnetic resonance imaging, and clinical evaluation. These methods offer limited sensitivity and specificity for the evaluation of LMD. Here, we describe the analytic performance characteristics of a microfluidic-based tumor cell enrichment and detection assay optimized to detect epithelial cells in CSF using both contrived samples as well as CSF from patients having suspected or confirmed LMD from carcinomas. To demonstrate the feasibility of using a microfluidic, multi-antibody cell capture assay to identify and quantify tumor cells in CSF. An artificial CSF solution was spiked with 34 different human carcinoma cell lines at different concentrations and assayed for the ability to detect tumor cells to assess analytic accuracy. Two cell lines were selected to assess linearity, intra-assay precision, interinstrument precision, and sample stability. Clinical verification was performed on 65 CSF specimens from patients. Parameters assessed included the number of tumor cells, coefficient of variation percentage, and percentage of tumor cell capture (TCC). Among contrived samples, average tumor cell capture ranged from 50% to 82% (261 of 522; 436 of 531), and coefficients of variation ranged from 7% to 67%. The cell capture assay demonstrated a sensitivity of 92% and a specificity of 95% among clinical samples. This assay demonstrated the ability to detect and enumerate epithelial cells in contrived and clinical specimens in an accurate and reproducible fashion. The use of cell capture assays in CSF may be useful as a sensitive test for the diagnosis and longitudinal monitoring of LMD from solid tumors.

Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection

BackgroundBreast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors.MethodsWe retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers.ResultsWe observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA–protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors.ConclusionsThis study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Abstract PO1-15-09: Vitamin A Metabolism Induces Ferroptosis to Enhance Immune Therapy Efficacy in HR+/HER2- Breast Cancer

Abstract Background: Immune checkpoint blockade (ICB) has substantially improved patient overall and progression-free survival in triple-negative breast cancer (TNBC), but its efficacy remains to be elucidated in the hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) breast cancer, which is characterized by generally impotent lymphocytic infiltration. In the I-SPY2 trial (NCT01042379), pembrolizumab- or durvalumab-containing neoadjuvant therapy increased the pathologic complete response of HR+/HER2- patients. However, pembrolizumab-containing therapy failed to improve the survival of metastatic HR+/HER2- breast cancer patients (NCT03051659). These inconsistent results underscored the necessity of the investigation of predictive biomarkers to pinpoint potential ICB responders in HR+/HER2- breast cancer. Method: In this study, we collected pretreatment tumor samples from 16 patients in two respective clinical trials (NCT: 04215003 and 04355858) and profiled droplet-based single-cell sequencing, bulk RNA-Seq. Patients were treated with camrelizumab (PD-1 inhibitor)-containing therapy, and five of them achieved partial relief after an 8-week course. We also leveraged the bulk RNA-Seq data from the I-SPY2 trial and Fudan University Shanghai Cancer Center (FUSCC) TNBC ICB trials (NCT04418154 and NCT04613674) for external validation. Another FUSCC transcriptome dataset consisting of 933 treatment-naïve HR+/HER2- or TNBC breast cancer patients was used to detect the abundance of cell types. In vitro and in vivo experiments were used to explore mechanisms of anti-tumor immune response. Results: Based on the single-cell sequencing results, we comprehensively delineated the microenvironmental landscape of HR+/HER2- breast cancer. We discovered responders of immunotherapy showed a higher presence of CXCL9/10+ M1-like macrophages and exhibited enrichment of tumor cells with ferroptosis. We further identified the upregulation of vitamin A metabolism and the vitamin A metabolic gene CRABP1 in tumor cells with ferroptosis. We verified the vitamin A metabolism could induce ferroptosis in HR+/HER2- breast cancers rather than TNBC by CRABP1-ERK axis, which could further recruit CXCL9/10+ M1-like macrophages and promote anti-tumor immune response in HR+/HER2- breast cancer. Combining anti-PD1 with enhancing vitamin A metabolism via retinoid acid possessed greater therapeutic efficacy than monotherapy in HR+/HER2- breast cancers. Finally, we developed an immune therapy response score and validated its reliability in predicting the immunotherapy efficacy for HR+/HER2-breast cancers through both internal and external cohorts. Conclusion: Our study represents the earliest efforts to decipher the microenvironmental landscape and the mechanisms underlying the response to ICB in HR+/HER2- breast cancer at single-cell resolution. We unraveled a HR+/HER2- breast cancer-specific Vitamin A-ferroptosis axis that mediated immunotherapy response and constructed a robust immunotherapy efficacy prediction score based on this Vitamin A-ferroptosis axis. Citation Format: Yi-Fan Zhou, Hu-Yun-Long Zhang, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. Vitamin A Metabolism Induces Ferroptosis to Enhance Immune Therapy Efficacy in HR+/HER2- Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-09.

PRKDC-Mediated NHEJ May Play a Crucial Role in Aneuploidy of Chromosome 8-Driven Progression of Ovarian Cancer.

High malignancy is a prominent characteristic of epithelial ovarian cancer (EOC), emphasizing the necessity for further elucidation of the potential mechanisms underlying cancer progression. Aneuploidy and copy number variation (CNV) partially contribute to the heightened malignancy observed in EOC; however, the precise features of aneuploidy and their underlying molecular patterns, as well as the relationship between CNV and aneuploidy in EOC, remain unclear. In this study, we employed single-cell sequencing data along with The Cancer Genome Atlas (TCGA) to investigate aneuploidy and CNV in EOC. The technique of fluorescence in situ hybridization (FISH) was employed using specific probes. The copy number variation within the genomic region of chromosome 8 (42754568-47889815) was assessed and utilized as a representative measure for the ploidy status of individual cells in chromosome 8. Differential expression analysis was performed between different subgroups based on chromosome 8 ploidy. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI), and hub-gene analyses were subsequently utilized to identify crucial genes involved. By classifying enriched tumor cells into distinct subtypes based on chromosome 8 ploidy combined with TCGA data integration, we identified key genes driving chromosome 8 aneuploidy in EOC, revealing that PRKDC gene involvement through the mediated non-homologous end-joining pathway may play a pivotal role in disease progression. Further validation through analysis of the GEO and TCGA database and survival assessment, considering both mRNA expression levels and CNV status of PRKDC, has confirmed its involvement in the progression of EOC. Further functional analysis revealed an upregulation of PRKDC in both ovarian EOC cells and tissues, with its expression showing a significant correlation with the extent of copy number variation (CNV) on chromosome 8. Taken together, CNV amplification and aneuploidy of chromosome 8 are important characteristics of EOC. PRKDC and the mediated NHEJ pathway may play a crucial role in driving aneuploidy on chromosome 8 during the progression of EOC.

Abstract 3706: Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system

Abstract Introduction: Liquid biopsy-based testing has increasingly been adopted by oncologists for biomarker-driven diagnostics, therapy selection, and patient monitoring. Unlike tissue-based approaches, the target analytes in blood specimens are often rare events that must be discriminated from a background of non-tumor derived material. Droplet digital PCR (ddPCR) is an ideal platform for detection of rare nucleic acid markers in the clinic, because it is highly sensitive and specific, robust, and quantitative. Additionally, the workflow is simple to adopt and execute. The QX600 System is a new ddPCR instrument designed for advanced multiplexing which enables simultaneous detection of up to 6 unique fluorophores in a single well. This capability can decrease the sub-sampling needed for detection of multiple analytes. The objective of our study was to develop multiplexed ddPCR assays for detection of cell-based and cell-free circulating biomarkers from a single tube of whole blood. Methods: Tumor cells were enriched from the buffy coat fraction of whole blood collected in fixative-containing cfDNA BCT based on size and morphology using a microfluidics-based system. Enriched tumor cells were enumerated by immunofluorescence (DAPI+/Pan-CK+/CD45-), and gene expression was measured using the QX600 for a panel of cytokeratins (CK) and PD-L1 in CTC negative, NSCLC donor specimens (n=8). Results were compared to CTC positive, contrived specimens (whole blood spiked with breast tumor cell line MCF7 cells; n=5). For ctDNA analysis of the plasma fraction, a multiplexed ddPCR assay was designed to test for five actionable mutations prevalent in NSCLC, including EGFR del19, L858R, T790M, KRAS G12C, BRAF V600E, and wildtype EGFR. To evaluate performance of the multiplexed assay, concordance evaluation was conducted using cfDNA from 8 mutation-positive NSCLC donors on the QX600 and compared to single assays run on the QX200 system. Results: CK8 and CK19 were differentially expressed in the cells enriched from the CTC negative NSCLC cohort as compared to the contrived positives (p=0.0058, and p=0.0049, respectively). All the NSCLC cfDNA samples were positive for the reference variant, and linear regression analysis of the minor variant frequency for ctDNA biomarkers showed good concordance between QX systems (R2=0.8847). Conclusions: These studies demonstrate proof of concept for single cell and cell-free analysis from a single blood draw into a cfDNA BCT using the QX600 system. Liquid biopsy analysis of CTCs and ctDNA can enable minimally invasive, comprehensive decision making in the diagnostic workup of patients. Future studies will focus on further optimizing the cell enrichment methodology from cfDNA BCT and development of additional multiplexed ddPCR assays for NSCLC-relevant biomarkers on the QX600. Citation Format: Leisa Jackson, Colin Cochran, Claire Gould, Sarah Kreston, Jazmin Bello, Brittany D’Alessio, Janice Riley, Alisa Tubbs, Gary A. Pestano. Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3706.

Proteogenomic analysis of enriched HGSOC tumor epithelium identifies prognostic signatures and therapeutic vulnerabilities

We performed a deep proteogenomic analysis of bulk tumor and laser microdissection enriched tumor cell populations from high-grade serous ovarian cancer (HGSOC) tissue specimens spanning a broad spectrum of purity. We identified patients with longer progression-free survival had increased immune-related signatures and validated proteins correlating with tumor-infiltrating lymphocytes in 65 tumors from an independent cohort of HGSOC patients, as well as with overall survival in an additional 126 HGSOC patient cohort. We identified that homologous recombination deficient (HRD) tumors are enriched in pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells are uniquely sensitive to BMI-1 inhibition.

Evaluating circulating tumour cell enrichment techniques to establish an appropriate method for clinical application in glioblastomas.

Brain tumours reduce life expectancy for an average of 20 years per patient, the highest of any cancer. A third of brain tumour patients visit their GP at least five times before diagnosis and many of those are diagnosed late through emergency departments. A possible solution to this challenge is to utilise a "liquid biopsy" blood test designed for circulating tumour cells (CTCs). Such a test could be applied at a primary healthcare centre, contributing to informed decision making for diagnostic imaging referrals. Furthermore, it could also be applied at secondary health care centres for the ongoing monitoring of disease recurrence. There is increased interest in CTC enrichment methods as a potential approach for faster diagnosis and monitoring of disease progression. The aim of this review to compare four CTC enrichment methods - OncoQuick®, Screen Cell®, pluriBead® and Cell Search® - with the objective of identifying a suitable method for application in the clinical setting for the isolation of CTCs from glioblastomas.

Abstract A007: Representative sampling of invasive breast carcinomas: Interim report from a prospective study (HOLST-F)

Abstract Background: Although a range of therapeutic options are available in the management of invasive breast carcinomas, spatial segregation of tumour subclones has hampered biomarker identification in single-region samples. Representative sampling (RS) overcomes spatial bias by sampling from a homogenized and well-mixed cancer specimen. The Homogenization of Leftover Surgical Tissue Feasibility Study (HoLST-F - NCT03832062) is a prospective trial aiming to assess the feasibility of RS in tumor tissue leftover after pathology sampling. An interim analysis of the breast cancer cohort is presented. Methods: In the context of the HoLST-F study and pre-specified end-points, representative samples derived from 75 leftover invasive breast carcinoma specimens underwent flow cytometric analysis of CK8/18, CD3, Ki67 and DAPI. Tumor cell enrichment by CK8/18 positivity and ploidy (in aneuploid carcinomas) was performed for downstream DNA extraction and whole exome sequencing. Somatic variants were determined using a bespoke pipeline to remove artefacts associated with fixation. Variant oncogenicity and therapeutic evidence levels were assigned by OncoKB variant annotation. Quantitative image analysis was applied to tissue sections from diagnostic FFPE blocks stained with Ki67 immunohistochemistry (n=78) and H&E for lymphocytic infiltration scores (n=175) to correlate with flow cytometry. Results: In enriched representative samples, oncogenic mutations were commonly identified at high variant allele frequency (VAF) in known breast carcinoma driver genes including PIK3CA, CDH1, TP53, KMT2C and GATA3. Other clinically relevant oncogenic mutations were identified in ESR1, PTEN, ERBB2, RB1, AKT1, BRCA1, NF1 and FOXA1 which have been associated with resistance to various anti-cancer therapies. These mutations occurred at low and high VAFs indicative of both of clonal and sub-clonal resistance mechanisms. Recurrent variants in ESR1 (e.g. D538G) and PIK3CA (e.g. H1047R/L) were associated with Level 1 evidence for use as predictive biomarkers, while variants in twelve other genes across the cohort were associated with Level 2-4 evidence. Lymphocyte infiltration scores and Ki67 expression varied by tumor region, however RS by flow cytometry showed strong correlation with a weighted average across multiregional quantification of Ki67 expression (R=0.81, p=2.8 × 10-8) and lymphocyte infiltration (R=0.61, p<2.2 × 10-16). Conclusions: RS of invasive breast carcinoma in the HoLST-F trial has recapitulated the expected genomic driver landscape in breast cancer, in addition to identifying both clonal and subclonal genomic mechanisms of therapy resistance. Many of these mutations are either current or emerging therapeutic targets. Flow quantification of cells expressing phenotypic biomarkers (Ki67 and CD3) is feasible through RS and early analysis indicates that RS correlates with a weighted average across multiple regions. Leftover surgical tissue is an underutilized resource for biomarker assessment in breast carcinomas and can be examined by RS. Citation Format: Brian Hanley, Lisa Gallegos, Lavinia Spain, Husayn Pallikonda, Zayd Tippu, Samantha Hill, Aoune Barhoumi, Fiona Byrne, Yulia Dogva, Ashley Gilchrist, Glenn Noel-Storr, Hannah Veloz, Stacey Stanislaw, Harold Sansano, Kim Edmonds, Eleanor Carlyle, Nicholas Turner, James Larkin, Nelson Alexander, Samra Turajlic. Representative sampling of invasive breast carcinomas: Interim report from a prospective study (HOLST-F) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A007.

Defining an Optimized Workflow for Enriching and Analyzing Residual Tumor Populations Using Intracellular Markers

Tumor relapse is well-recognized to arise from treatment-resistant residual populations. Strategies enriching such populations for in-depth downstream analyses focus on tumor-specific surface markers; however, enrichment using intracellular biomarkers remains challenging. Using B-cell lymphoma as an exemplar, we demonstrate feasibility to enrich BCL2high populations, a surrogate marker for t(14;18)+ lymphomas, for use in downstream applications. Different fixation protocols were assessed for impact on antibody expression and RNA integrity; glyoxal fixation demonstrated superior results regarding minimal effects on surface and intracellular expression, and RNA quality, compared to alternative fixatives evaluated. Furthermore, t(14;18)+ B-cells were effectively detected using intracellular BCL2 over-expression to facilitate tumor cell enrichment. Tumor cell populations were enriched using the cellenONE F1.4 single cell sorting platform, which detected and dispensed BCL2high-expressing cells directly into library preparation reagents for transcriptome analyses. Sorted glyoxal-fixed cells generated good quality sequencing libraries, with high concordance between live and fixed single cell transcriptomic profiles, discriminating cell populations predominantly on B-cell biology. Overall, we successfully develop a proof-of-concept workflow employing a robust cell preparation protocol for intracellular markers combined with cell enrichment using the cellenONE platform, providing an alternative to droplet-based technologies when cellular input is low or requires prior enrichment to detect rare populations. This workflow has wider prognostic and therapeutic potential to study residual cells in a pan-cancer setting.

High-Throughput Sorting and Single-Cell Mechanotyping by Hydrodynamic Sorting-Mechanotyping Cytometry.

The existence of many background blood cells hinders the accurate identification of circulating tumor cells (CTCs) in the blood of cancer patients. To unlock this limitation, a hydrodynamic sorting-mechanotyping cytometry (HSMC) integrated with a sorting-concentration chip and a detection chip is proposed for simultaneously achieving the high-throughput cell sorting and the multi-parameter mechanotyping of the sorted tumor cells. The HSMC adopts the spiral inertial microfluidics for label-free sorting of cells in a high-throughput manner, allowing the efficient enrichment of tumor cells from the large background blood cells. Then, the sorted cells are concentrated by the concentration unit and finally passed through the detection unit for hydrodynamic deformation. The HSMC has a high throughput for sorting and detection and can successfully reveal the differences in the cellular mechanical properties. After characterizing and optimizing the single chips, the identification of white blood cells (WBCs) and three types of tumor cells (A549, MCF-7, and MDA-MB-231 cells) is successfully achieved. The identification accuracies for WBCs and different tumor cells are all larger than 94%, while the highest identification accuracy is up to 99.2%. This study envisions that the HSMC will offer an avenue for the analysis of single cell intrinsic mechanics in clinical medicine.

Using the photon isoeffective dose formalism to compare and combine BNCT and CIRT in a head and neck tumour

Boron Neutron Capture Therapy (BNCT) is a radiotherapy technique based on the enrichment of tumour cells with suitable 10-boron concentration and on subsequent neutron irradiation. Low-energy neutron irradiation produces a localized deposition of radiation dose caused by boron neutron capture reactions. Boron is vehiculated into tumour cells via proper borated formulations, able to accumulate in the malignancy more than in normal tissues. The neutron capture releases two high-LET charged particles (i.e., an alpha particle and a lithium ion), losing their energy in a distance comparable to the average dimension of one cell. Thus BNCT is selective at the cell level and characterized by high biological effectiveness. As the radiation field is due to the interaction of neutrons with the components of biological tissues and with boron, the dosimetry requires a formalism to express the absorbed dose into photon-equivalent units. This work analyzes a clinical case of an adenoid cystic carcinoma treated with carbon-ion radiotherapy (CIRT), located close to optic nerve and deep-seated as a practical example of how to apply the formalism of BNCT photon isoeffective dose and how to evaluate the BNCT dose distribution against CIRT. The example allows presenting different dosimetrical and radiobiological quantities and drawing conclusions on the potential of BNCT stemming on the clinical result of the CIRT. The patient received CIRT with a dose constraint on the optic nerve, affecting the peripheral part of the Planning Target Volume (PTV). After the treatment, the tumour recurred in this low-dose region. BNCT was simulated for the primary tumour, with the goal to calculate the dose distribution in isoeffective units and a Tumour Control Probability (TCP) to be compared with the one of the original treatment. BNCT was then evaluated for the recurrence in the underdosed region which was not optimally covered by charged particles due to the proximity of the optic nerve. Finally, a combined treatment consisting in BNCT and carbon ion therapy was considered to show the consistency and the potential of the model. For the primary tumour, the photon isoeffective dose distribution due to BNCT was evaluated and the resulted TCP was higher than that obtained for the CIRT. The formalism produced values that are consistent with those of carbon-ion. For the recurrence, BNCT dosimetry produces a similar TCP than that of primary tumour. A combined treatment was finally simulated, showing a TCP comparable to the BNCT-alone with overall dosimetric advantage in the most peripheral parts of the treatment volume. Isoeffective dose formalism is a robust tool to analyze BNCT dosimetry and to compare it with the photon-equivalent dose calculated for carbon-ion treatment. This study introduces for the first time the possibility to combine the dosimetry obtained by two different treatment modalities, showing the potential of exploiting the cellular targeting of BNCT combined with the precision of charged particles in delivering an homogeneous dose distribution in deep-seated tumours.

Generally applicable circulating tumor cell enrichment and identification through a membrane glycoprotein-targeting strategy combining magnetic isolation and biological orthogonality labeling.

Although the importance of circulating tumor cells (CTCs) has been widely recognized, it is still a challenge to realize high-efficiency and accurate enrichment and identification of highly heterogeneous CTCs derived from various types of tumors in complex cancer processes. Currently, the most widely used methods follow the general idea of sequential immunoaffinitive capture and immunostaining to achieve the abovementioned goal. However, different organ/tissue origins as well as the inherent heterogeneity of CTCs would lead to the missed detection of certain CTC subtypes using such methods. Further, immunocytochemistry (ICC) immunostaining disrupts the physiological structure of cells, severely limiting the detection and application scenarios that require the participation of live cells. To address these limitations, we have developed a generally applicable strategy for the isolation and labeling of CTCs. This strategy focuses on targeting the universal characteristics of all tumor cells, specifically the abnormally expressed cell membrane glycoproteins, such as the transferrin receptor and sialic acid. Strategically, transferrin-functionalized magnetic beads (TMBs) were applied to enrich CTCs, and azide-based bioorthogonal chemistry was employed to label target CTCs. Accordingly, the membrane glycoprotein-targeting strategy achieved unbiased enrichment and labeling of broad-spectrum CTCs that were both epithelial and non-epithelial phenotypic populations with varied organ/tissue origins (MCF-7, HepG2, A549, Jurkat, and B16), with a capture efficiency of >95% and a detection limit as low as 5 cells per mL in artificial blood. In particular, our developed strategy displayed excellent specificity, and the CTCs under capture and fluorescence labelling remained with good viability and could be further cultivated and analyzed. Finally, the membrane glycoprotein-targeting strategy successfully detected and identified 33-223 CTCs from 1 mL patient blood samples.

Circulating Tumor Cell Enrichment and Single-Cell Isolation Combining the CellSearch® and DEPArray™ Systems.

The analysis of circulating tumor cells (CTCs) has shown potential for detection of cancer spread, prognosis, therapeutic target selection, and monitoring of treatment response. CTCs can be obtained repeatedly by simple blood draws as so-called "liquid biopsy." Thus, they can serve as a surrogate material for primary or metastatic tissue biopsies. In addition, isolation of CTCs provides the possibility to investigate those cells which may hold the (molecular) traits responsible for metastatic progression and ultimately patient death. As such, CTCs represent a target of utmost importance in cancer research and therapy. In this chapter, we describe a workflow for the enrichment of CTCs with the FDA-cleared CellSearch® system followed by the isolation of single CTCs using the DEPArray™ technology enabling further molecular single-cell analyses.

Translational development of a novel BAFF-R CAR-T therapy targeting B-cell lymphoid malignancies.

Several CD19-targeting CAR-T cells are used to treat leukemias and lymphomas; however, relapsed and/or refractory (R/R) disease is still observed in a significant number of patients. Additionally, the success of CD19-CAR-T cell therapies is not uniform across hematological malignancies, particularly in chronic lymphocytic leukemia (CLL). In this study, we present the development of a novel CAR-T cell therapy targeting B-cell activating factor receptor (BAFF-R), a key regulator of B-cell proliferation and maturation. A new monoclonal antibody against BAFF-R was generated from a hybridoma clone and used to create a novel MC10029 CAR construct. Through a series of in vitro and in vivo models using the Nalm-6 cell line for leukemia and the Z138 cell line for lymphoma, we demonstrated the antigen-specific cytotoxicity of MC10029 CAR-T cells against tumor cells. Additionally, MC10029 CAR-T cells exhibited potent antitumor effects against CD19 knockout tumor cells, mimicking CD19-negative R/R disease. MC10029 CAR-T cells were specifically targeted to CLL, in which BAFF-R is nearly always expressed. The cytotoxicity of MC10029 CAR-T cells was first shown in the MEC-1 CLL cell line, before we turned our efforts to subject-derived samples. Using healthy donor-engineered MC10029 CAR-T cells against enriched primary tumor cells, followed by subject-derived MC10029 CAR-T cells against autologous tumor cells, we showed the efficacy of MC10029 CAR-T cells against CLL subject samples. With these robust data, we have advanced to the production of MC10029 CAR-T cells, using GMP lentivirus, and obtained an IND approval in preparation for a Phase 1 clinical trial.

348. IDENTIFYING NOVEL MOLECULAR SUBTYPES OF ESOPHAGEAL ADENOCARCINOMA USING LASER CAPTURE MICRODISSECTED RNA-SEQ SAMPLES

Abstract Background Treatment options for esophageal adenocarcinoma (EAC) are limited by a lack of disease stratification methods. In other cancers, gene expression profiling has been successfully used to identify prognostically-relevant and treatment-susceptible molecular subtypes. Previous efforts to subtype EAC have been hindered by low biopsy and resection tumour cellularity. We hypothesize that laser-capture microdissection (LCM) tumour cell enrichment will permit the classification EAC tumours into multiple molecular subtypes that will be important for future therapeutic strategies. Methods Treatment naïve patient samples (N = 52) were collected from primary biopsies (N = 37), resections (N = 10) and metastatic biopsies (N = 5). Samples were laser-capture microdissected to enrich tumour cells followed by total RNA-seq. Gene expression was quantified using salmon and non-negative matrix factorization with 10 components was used to identify gene expression programs. The components identified were validated on a publicly available normal tissue cohort (N = 13 esophagus, gastric, intestinal, Barrett’s samples) and the TCGA EAC cohort (N = 80). We applied NMF with 4 components to the normal tissue (K = cohort) to identify normal tissue gene signatures to validate our RNA-seq data. Results We verified that our RNA-seq samples were depleted for normal tissue using the four NMF signatures from normal tissue cohort and, as expected, our samples were depleted for gene expression from the gastric and esophagus tissues compared to TCGA. Next, we explored the gene expression programs from our tumour samples and identified seven tumour-intrinsic components and three tumour microenvironmental components. The top 50 genes from each tumour intrinsic components were used with consensus cluster plus to identify K = 6 subtypes (Figure 1). Interestingly, two of these subtypes would be difficult to separate from normal tissue contamination without the use of LCM. Conclusion We have successfully used LCM to deplete normal tissue gene expression from our esophageal adenocarcinoma cohort and identified six molecular subtypes. We are currently evaluating the treatment and clinical implications of these subtypes and aiming to accumulate an additional (N = 50) EAC RNA-seq samples.

Floating Immunomagnetic Microspheres for Highly Efficient Circulating Tumor Cell Isolation under Facile Magnetic Manipulation.

Among circulating tumor cell enrichment strategies, immunomagnetic beads (IMBs) have received great attention due to their excellent performance. However, traditional strategies using IMBs normally require an additional mechanical stirring device to fully mix the IMBs and specimens, and this step may cause mechanical cellular damage. In this study, by changing the architecture and motion trajectory control strategy of the IMBs, floating immunomagnetic microspheres (FIMMs) and their matching rotary magnetic manipulation device were proposed to achieve highly efficient CTC capture under a cell-friendly condition. Generally, the FIMMs were prepared through layer-by-layer assembly of the individual functional components, and their stress state governed by either buoyancy or magnetic force was tuned by the rotary magnetic manipulation device. Consequently, recognition of FIMMs and target cells as well as CTC recovery can be simply realized through external magnetic manipulation. Accordingly, satisfactory enrichment efficiencies for CTCs with varied epithelial expression levels were obtained as 92.93 ± 3.23% for MCF-7, 79.93 ± 3.31% for A549, and 92.57 ± 5.22% for HepG2. Besides, an extremely low detection limitation of 5 cells mL-1 can be achieved from complex sample conditions, even the whole blood. In addition, FIMMs successfully enriched 23-56 CTCs from 1.5 mL of blood samples from cancer patients.