Individual Tumor Cells Research Articles

Characteristics of stem sings of EpCAM-negative and EpCAM-positive tumor cells in primary tumor and 2D and 3D cultures in breast cancer

One of the predictors of adverse prognosis in breast cancer is the overexpression of the EpCAM protein. However, a signifcant part of tumor cells have low or no EpCAM expression. The molecular features and the ability to grow in 2D and 3D cultures, indirectly refecting metastatic potential of tumor cells without EpCAM expression, have not been sufficiently studied.The aim of the study was to compare phenotypic variants of stem cells and EMT depending on the EpCAM expression in the primary tumor of breast cancer and 2D and 3D cultures.Material and Methods. The study included 7 patients with invasive breast carcinoma of no special type. Luminal A subtype was found in 2/7 patients (29 %), and luminal B HER2-negative molecular subtype was found in 5/7 patients (71 %). The patients didn’t have neoadjuvant chemotherapy. Cells from the primary tumor were cultured in 2D and 3D. The primary tumor cells, 2D and 3D cultures were phenotyped using fow cytometry with antibodies targeting: CD45, EpCAM (CD326), CD44, CD24, N-cadherin (CD325), and CD133, CK7/8, EpCAM (CD326).Results. No significant differences were found in the frequency of detection and the number of cells exhibiting stem cell features among EpCAM- and EpCAM+ tumor cells, 2D and 3D cultures. All phenotypic variants of co-expression of stemness markers CD44, CD24, CD133, and ALDH were present both in the primary tumor and 2D/3D cultures. Stem cells in mammospheres had features of both epithelial and hybrid EMT phenotypes. In primary tumors and 2D/3D cultures, the smallest proportion consisted of stem cells with the CD44+CD24- phenotype and cells co-expressing CD44+CD24- with other stemness markers, while the largest proportion comprised stem cells with ALDH+ and ALDH+CD133+ phenotypes. Marked intra- and inter-tumor heterogeneity in the phenotypic composition of primary tumors, 2D, and 3D cultures was noted.Conclusion. The results indicate that EpCAM-negative and EpCAM-positive tumor cells of luminal molecular subtype of breast cancer are capable of exhibiting various phenotypic variants of stemness and EMT in the primary tumor and 2D and 3D cultures. In most cases, individual tumor cells show co-expression of several stemness markers. The phenotypic composition of primary tumors, 2D and 3D cultures is characterized by pronounced intra- and inter-tumor heterogeneity.

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

BackgroundSomatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.MethodsA landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.ResultsWe observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.ConclusionssPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.

Cancer cell migration depends on adjacent ASC and adipose spheroids in a 3D bioprinted breast cancer model

Breast cancer develops in close proximity to mammary adipose tissue and interactions with the local adipose environment have been shown to drive tumor progression. The specific role, however, of this complex tumor microenvironment in cancer cell migration still needs to be elucidated. Therefore, in this study, a 3D bioprinted breast cancer model was developed that allows for a comprehensive analysis of individual tumor cell migration parameters in dependence of adjacent adipose stroma. In this co-culture model, a breast cancer compartment with MDA-MB-231 breast cancer cells embedded in collagen is surrounded by an adipose tissue compartment consisting of adipose-derived stromal cell (ASC) or adipose spheroids in a printable bioink based on thiolated hyaluronic acid. Printing parameters were optimized for adipose spheroids to ensure viability and integrity of the fragile lipid-laden cells. Preservation of the adipogenic phenotype after printing was demonstrated by quantification of lipid content, expression of adipogenic marker genes, the presence of a coherent adipo-specific extracellular matrix, and cytokine secretion. The migration of tumor cells as a function of paracrine signaling of the surrounding adipose compartment was then analyzed using live-cell imaging. The presence of ASC or adipose spheroids substantially increased key migration parameters of MDA-MB-231 cells, namely motile fraction, persistence, invasion distance, and speed. These findings shed new light on the role of adipose tissue in cancer cell migration. They highlight the potential of our 3D printed breast cancer-stroma model to elucidate mechanisms of stroma-induced cancer cell migration and to serve as a screening platform for novel anti-cancer drugs targeting cancer cell dissemination.

Label-free fluorescence lifetime imaging for the assessment of cell viability in living tumor fragments.

To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM). Using murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death. We use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays. Through a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected. These data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments.

The Balance Between Shear Flow and Extracellular Matrix in Ovarian Cancer-on-Chip.

Ovarian cancer is the most lethal gynecologic cancer in developed countries. In the tumor microenvironment, the extracellular matrix (ECM) and flow shear stress are key players in directing ovarian cancer cells invasion. Artificial ECM models based only on ECM proteins are used to build an ovarian tumor-on-chip to decipher the crosstalk between ECM and shear stress on the migratory behavior and cellular heterogeneity of ovarian tumor cells. This work shows that in the shear stress regime of the peritoneal cavity, the ECM plays a major role in driving individual or collective ovarian tumor cells migration. In the presence of basement membrane proteins, migration is more collective than on type I collagen regardless of shear stress. With increasing shear stress, individual cell migration is enhanced; while, no significant impact on collective migration is measured. This highlights the central position that ECM and flow shear stress should hold in in vitro ovarian cancer models to deepen understanding of cellular responses and improve development of ovarian cancer therapeutic platforms. In this frame, adding flow provides significant improvement in biological relevance over the authors' previous work. Further steps for enhanced clinical relevance require not only multiple cell lines but also patient-derived cells and sera.

Computational analysis of cancer cell adhesion in curved vessels affected by wall shear stress for prediction of metastatic spreading.

Introduction: The dynamics of circulating tumor cells (CTCs) within blood vessels play a pivotal role in predicting metastatic spreading of cancer within the body. However, the limited understanding and method to quantitatively investigate the influence of vascular architecture on CTC dynamics hinders our ability to predict metastatic process effectively. To address this limitation, the present study was conducted to investigate the influence of blood vessel tortuosity on the behaviour of CTCs, focusing specifically on establishing methods and examining the role of shear stress in CTC-vessel wall interactions and its subsequent impact on metastasis. Methods: We computationally simulated CTC behaviour under various shear stress conditions induced by vessel tortuosity. Our computational model, based on the lattice Boltzmann method (LBM) and a coarse-grained spectrin-link membrane model, efficiently simulates blood plasma dynamics and CTC deformability. The model incorporates fluid-structure interactions and receptor-ligand interactions crucial for CTC adhesion using the immersed boundary method (IBM). Results: Our findings reveal that uniform shear stress in straight vessels leads to predictable CTC-vessel interactions, whereas in curved vessels, asymmetrical flow patterns and altered shear stress create distinct adhesion dynamics, potentially influencing CTC extravasation. Quantitative analysis shows a 25% decrease in the wall shear stress in low-shear regions and a 58.5% increase in the high-shear region. We observed high-shear regions in curved vessels to be potential sites for increased CTC adhesion and extravasation, facilitated by elevated endothelial expression of adhesion molecules. This phenomenon correlates with the increased number of adhesion bonds, which rises to approximately 40 in high-shear regions, compared to around 12 for straight vessels and approximately 5-6 in low-shear regions. The findings also indicate an optimal cellular stiffness necessary for successful CTC extravasation in curved vessels. Discussion: By the quantitative assessment of the risk of CTC extravasation as a function of vessel tortuosity, our study offers a novel tool for the prediction of metastasis risk to support the development of personalized therapeutic interventions based on individual vascular characteristics and tumor cell properties.

Drug screening on digital microfluidics for cancer precision medicine

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

Primary omental smooth muscle tumor in an adult male: a diagnostic dilemma for leiomyoma: a case report

BackgroundThe greater omentum comprises peritoneal, adipose, vascular, and lymphoid tissues. Most omental malignancies are metastatic tumors, and the incidence of primary tumors is rare. We report on a prior omental smooth muscle tumor case in an adult male patient.Case presentationA 54-year-old Japanese male patient with no relevant medical history was diagnosed with an abdominal mass during a routine medical checkup. Subsequent contrast-enhanced computed tomography revealed a mass of approximately 3 cm in size in the greater omentum, and a laparotomy was performed. A 27 × 25 × 20 mm raised lesion was found in the omentum. Microscopically, spindle cells were observed and arranged in whorls and fascicles. Individual tumor cells had short spindle-shaped nuclei with slightly increased chromatin and were characterized by a slightly eosinophilic, spindle-shaped cytoplasm. The mitotic count was less than 1 per 50 high-power fields. The tumor cells showed positive immunoreactivity for α smooth muscle actin, HHF35, and desmin on immunohistochemical examination. The Ki-67 labeling index using the average method was 1.76% (261/14806). No immunoreactivity was observed for any of the other tested markers. We considered leiomyoma owing to a lack of malignant findings. However, primary omental leiomyoma has rarely been reported, and it can be difficult to completely rule out the malignant potential of smooth muscle tumors in soft tissues. Our patient was decisively diagnosed with a primary omental smooth muscle tumor considering leiomyoma. Consequently, the patient did not undergo additional adjuvant therapy and was followed up. The patient was satisfied with treatment and showed neither recurrence nor metastasis at the 13-month postoperative follow-up.Discussion and conclusionWe encountered a primary smooth muscle tumor of the greater omentum with no histological findings suggestive of malignancy in an adult male patient. However, omental smooth muscle tumors are extremely difficult to define as benign, requiring careful diagnosis. Further case reports with long-term follow-up and case series are required to determine whether a true omental benign smooth muscle tumor (leiomyoma) exists. In addition, proper interpretation of the Ki-67 labeling index should be established. This case study is a foundation for future research.

Abstract PO3-08-08: Basal-Luminal (BL) Cells in Histologically Normal and Malignant Breast Tissue from BRCA1 and BRCA2 Carriers

Abstract Background Single-cell studies of normal breast tissue in BRCA mutation carriers have led to the identification of a candidate cancer precursor cell, the basal-luminal (BL) cell. The localization of BL cells within normal, pre-malignant and malignant breast tissue architecture is not well characterized. Here, using cyclic immunofluorescence (CyCIF), we quantify and localize BL cells within normal and malignant breast tissues from BRCA1 and BRCA2 carriers. Methods Formalin-fixed paraffin-embedded breast tissue sections from 9 BRCA1 and 9 BRCA2 consented patients treated at Dana Farber Cancer Institute (2001-2019) were retrieved from the clinical archives and subjected to CyCIF for luminal and basal markers including CK5, CK14 and CK19. Using one section/case, cells co-expressing basal and luminal cytokeratins (CK14/CK19 and/or CK5/CK19) were visually identified in invasive carcinoma, ductal carcinoma in situ (DCIS) and in adjacent normal breast tissue. In histologically normal tissue, regions of interest (ROIs) with co-expressing cells were classified as terminal duct lobular units (TDLUs) or isolated ducts. Within each ROI, the quantity of BL cells was scored as low (1-5 cells), moderate (6-10 cells), or high ( >10 cells). Results The 9 BRCA1 tumors included 8 invasive ductal carcinomas/no special type and 1 tubular carcinoma. The 9 BRCA2 tumors included 4 invasive ductal carcinomas/no special type and 5 invasive carcinomas with ductal and lobular features. 496 ROIs were evaluated in histologically normal tissue: 297 from BRCA1 cases and 199 from BRCA2 cases. BL cells co-expressing CK14/CK19 and/or CK5/CK19 were identified in normal TDLUs and isolated ducts in both BRCA1 and BRCA2 cases. BL cells were more frequently identified in BRCA2 cases, with at least one BL cell present in 97/297 ROIs (33%) in BRCA1 cases and in 133/199 ROIs (67%) in BRCA2 cases (p< 0.01). 55% of BRCA2 ROIs had >10 BL cells each versus 42% of BRCA1 ROIs; however, this difference was not significant. Four BRCA1 cases showed a diffuse BL cell phenotype within the invasive carcinoma with either a diffuse or scattered BL cell phenotype within the DCIS. The BL cell phenotype was not seen diffusely in invasive carcinoma or DCIS in the other 14 cases; however individual tumor cells or small cell clusters with the BL cell phenotype were occasionally found. Conclusions BL cells are found in histologically normal TDLUs and isolated ducts in breast tissue from BRCA1 and BRCA2 carriers, with greater prevalence in BRCA2 carriers. The BL cell phenotype is present diffusely within tumor cells in almost half of BRCA1-associated cancers and none of the BRCA2-associated cancers in this small cohort. Further study of this interesting cell population is warranted in efforts to understand early changes in epithelium at high risk for the development of cancer. Citation Format: Rodrigo Fonseca Abreu, Tabata Alves Domingos, Roberto Bonfim Pimenta Peixoto, Ashka Patel, Krishan Taneja, McKenna Moore, Alicia Pollaci, Judy Garber, Nomeda Girnius, Deborah Dillon. Basal-Luminal (BL) Cells in Histologically Normal and Malignant Breast Tissue from BRCA1 and BRCA2 Carriers [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 PO3-08-08.

The impact of tumor budding and single-cell invasion on survival in patients with stage III/IV locally advanced oral squamous cell carcinoma- results from a prospective cohort study.

Tumor budding (TB) refers to the presence of small clusters of tumor cells at the invasive front of a malignant tumor. Single tumor cell invasion (SCI) is an extreme variant of TB, in which individual loose tumor cells are present at the invasive front. Both TB and SCI are important histomorphologic risk factors postulated to indicate loss of cellular cohesion. In this study, we investigated the influence of TB and SCI on different survival outcomes in patients with locally advanced oral squamous cell carcinoma (OSCC). We included 129 patients with locally advanced OSCC (pT3-4) from a single-center, prospectively maintained cohort. We examined the association of TB and SCI with the presence of occult lymph node metastasis using a logistic regression model. Survival probabilities were estimated using the Kaplan-Meier method and cumulative incidence functions. The association of TB and SCI on overall survival (OS), oral cancer-specific survival (OCSS), and local recurrence-free survival (LRFS) was investigated using Cox's proportional hazards regression models. TB was detected in 98 (76%) of the tumors, while SCI was observed in 66 (51%) patients. There was a significant association between TB and the occurrence of occult lymph node metastasis (OR=3.33, CI: 1.21-10.0). On multivariate analysis, TB had no detectable impact on survival outcomes. However, SCI showed a higher risk for local recurrence (Hazards ratio (HR): 3.33, CI: 1.19 - 9.27). This study demonstrates that TB and SCI in locally advanced OSCC function as an independent risk factor for occult lymph node metastases, as well as local recurrences. Both histomorphologic risk factors could serve as an additional parameter for stratifying therapy and escalating multimodal treatment approaches.

LPCAT2 inhibits colorectal cancer progression via the PRMT1/SLC7A11 axis

Colorectal cancer (CRC) has a high degree of heterogeneity and identifying the genetic information of individual tumor cells could help enhance our understanding of tumor biology and uncover potential therapeutic targets for CRC. In this study, we identified LPCAT2+ tumor cell populations with less malignancy than LPCAT2- tumor cells in human and mouse CRC tissues using scRNA-seq. Combining in vitro and in vivo experiments, we found that LPCAT2 could inhibit the proliferation of CRC cells by inducing ferroptosis. Mechanistically, LPCAT2 arrested PRMT1 in cytoplasm of CRC cells via regulating acetylation of PRMT1 at the K145 site. In turn, PRMT1 enhanced SLC7A11 promoter activity. Thus, LPCAT2 attenuated the positive regulatory effect of PRMT1 on SLC7A11 promoter. Notably, SLC7A11 acts as a ferroptosis regulator. Furthermore, in LPCAT2 knockout mice (LPCAT2−/−) colon cancer model, we found that LPCAT2−/− mice exhibited more severe lesions, while PRMT1 or SLC7A11 inhibitors delayed the progression. Altogether, we elucidated that LPCAT2 suppresses SLC7A11 expression by inhibiting PRMT1 nuclear translocation, thereby inducing ferroptosis in CRC cells. Moreover, inhibitors of the PRMT1/SLC7A11 axis could delay tumor progression in CRC with low LPCAT2 expression, making it a potentially effective treatment for CRC.

Computational analysis of super-resolved in situ sequencing data reveals genes modified by immune-tumor contact events.

Cancer cells can manipulate immune cells and escape from the immune system response. Quantifying the molecular changes that occur when an immune cell touches a tumor cell can increase our understanding of the underlying mechanisms. Recently, it became possible to perform such measurements in situ-for example, using expansion sequencing, which enabled in situ sequencing of genes with super-resolution. We systematically examined whether individual immune cells from specific cell types express genes differently when in physical proximity to individual tumor cells. First, we demonstrated that a dense mapping of genes in situ can be used for the segmentation of cell bodies in 3D, thus improving our ability to detect likely touching cells. Next, we used three different computational approaches to detect the molecular changes that are triggered by proximity: differential expression analysis, tree-based machine learning classifiers, and matrix factorization analysis. This systematic analysis revealed tens of genes, in specific cell types, whose expression separates immune cells that are proximal to tumor cells from those that are not proximal, with a significant overlap between the different detection methods. Remarkably, an order of magnitude more genes are triggered by proximity to tumor cells in CD8 T cells compared to CD4 T cells, in line with the ability of CD8 T cells to directly bind major histocompatibility complex (MHC) class I on tumor cells. Thus, in situ sequencing of an individual biopsy can be used to detect genes likely involved in immune-tumor cell-cell interactions. The data used in this manuscript and the code of the InSituSeg, machine learning, cNMF, and Moran's I methods are publicly available at doi:10.5281/zenodo.7497981.

Extraocular Sebaceous Carcinoma in the Lower Back: Rare Presentation of a Great Masquerader

Abstract Malignant adnexal tumors of the skin are rare. Sebaceous carcinoma (SC) is an aggressive malignant tumor of the sebaceous glands. It is traditionally classified into periocular and extraocular types. We present a rare case of extraocular SC of a 38-year-old female who presented with nontender swelling over the right flank for 1 year. Clinically, it was diagnosed with a sebaceous cyst. The tumor was excised and sent for histopathological examination. Sections showed sheets and lobules of tumor cells separated by fibrovascular stroma. Individual tumor cells were atypical basaloid to clear cells with prominent pleomorphism. Mitosis was brisk with areas of necrosis. On immunohistochemistry, the tumor cells were focally positive for epithelial membrane antigen and negative for S100 and PAX8 with a high Ki-67 index of 80%. Her postoperative period and follow-up for 1 year have been uneventful. This case is being reported for its rarity.

Nuclear morphological atypia in biopsy accurately reflects the prognosis of myxoid liposarcoma.

Currently, it is difficult to predict the prognosis of myxoid liposarcoma (MLS) in biopsy specimens. In this study, we determined whether nuclear morphology may be used to predict the prognosis of MLS in primary biopsy specimens. Two pathologists evaluated nuclear morphology using the modified WHO/ISUP and Fuhrman grades. Survival analyses were performed by grouping nuclear high- and low-grades. We examined 53 MLS cases, which included 29 (54.7%) male and 24 (45.3%) female patients with a median age of 46years (interquartile range, 37 - 60). In total, 7 (13.2%) and 16 (30.2%) cases were assigned to the high nuclear grade group based on the modified WHO/ISUP and Fuhrman gradings, respectively. Survival analyses revealed a significantly worse disease-free survival in the high-grade group (hazard ratio (HR), 7.51; 95% confidence interval (CI), 2.67-21.1, p < 0.001 by the modified WHO/ISUP grading; HR, 4.45; 95% CI, 1.63-12.1, p = 0.001 by the modified Fuhrman grading). Moreover, the modified WHO/ISUP grade showed a significantly worse overall survival in the high-grade group (HR, 4.39; 95% CI, 1.04-18.6, p = 0.028), and the modified Fuhrman grade exhibited a similar, but not significant, trend. Our results indicate that nuclear morphology grading is a good predictor of patient prognosis at the time of biopsy in MLS. Even when cell density is sparse, treatment strategies should be carefully considered when individual tumor cells exhibit atypical nuclei.

Single-Cell Sequencing Illuminates Thymic Development: An Updated Framework for Understanding Thymic Epithelial Tumors.

Thymic epithelial tumors (TETs) are rare tumors for which treatment options are limited. The ongoing need for improved systemic therapies reflects a limited understanding of tumor biology as well as the normal thymus. The essential role of the thymus in adaptive immunity is largely effected by its epithelial compartment, which directs thymocyte (T-cell) differentiation and immunologic self-tolerance. With aging, the thymus undergoes involution whereby epithelial tissue is replaced by adipose and other connective tissue, decreasing immature T-cell production. Against this natural drive toward involution, a fraction of thymuses will instead undergo oncologic transformation, leading to the formation of TETs, including thymoma and thymic carcinoma. The rarity of these tumors restricts investigation of the mechanisms of tumorigenesis and development of rational treatment options. To this end, the development of technologies which allow deep molecular profiling of individual tumor cells permits a new window through which to view normal thymic development and contrast the malignant changes that result in oncogenic transformation. In this review, we describe the findings of recent illuminating studies on the diversity of cell types within the epithelial compartment through thymic differentiation and aging. We contextualize these findings around important unanswered questions regarding the spectrum of known somatic tumor alterations, cell of origin, and tumor heterogeneity. The perspectives informed by single-cell molecular profiling offer new approaches to clinical and basic investigation of thymic epithelial tumors, with the potential to accelerate development of improved therapeutic strategies to address ongoing unmet needs in these rare tumors.

Abstract 5863: Double-negative prostate cancer emerges through a mixed basal, club, and hillock cell identity driven by KLF5

Abstract The purpose of this study was to characterize the phenotypes and drivers of therapy-induced lineage plasticity during the development of castration resistant prostate cancer (CRPC). While the majority of localized prostate cancer can be cured with surgery or radiation, metastatic disease is lethal. Therapies targeting the androgen receptor (AR) are initially effective, but eventually men will develop CRPC. About 70-75% of CRPC displays restored AR signaling (ARPC), while the remaining 25-30% display lineage plasticity evidenced by the emergence of AR-negative subtypes including those that display neuroendocrine (NE) markers, or double negative CRPC (DNPC) lacking both AR and NE markers. AR independent manifestations of CRPC represent a clinical challenge because no effective therapies are available and therapeutic targets are largely unknown. We analyzed publicly available bulk and single-cell RNA-seq datasets derived from clinical CRPC specimens to characterize the cellular identity of AR-negative tumors. Using a database of all known transcriptional regulators, we identified potential drivers of these phenotypes. We performed siRNA-mediated knockdown experiments in a model of DNPC to explore the extent to which the candidate regulators maintain AR-negative cell identity and sustain growth of DNPC prostate cancer cells. We identified a subset of CRPC tumors that co-express gene sets defining basal, club, and hillock cells in the benign prostate. This data supports the existence of epithelial cell lineage plasticity manifesting within CRPC tumors, as well as individual CRPC tumor cells, wherein AR-positive tumor cells transform to a phenotype encompassing basal, club, and hillock cell identities. We nominated the stem cell transcription factor KLF5 as a regulator of basal, club, and hillock cell identities in CRPC. In cell line models of DNPC, knock down of KLF5 inhibited cell growth and reduced the expression of genes defining basal, club, and hillock cell identities. This work links a prevalent and poorly-defined subtype of AR-negative CRPC to AR-negative epithelial cell types in benign prostate tissue. Inhibition of KLF5 and its downstream effectors could represent a therapeutic strategy to prevent or delay lineage plasticity and thereby extend patient response to AR-targeted therapies. Citation Format: Samuel Pitzen, Yinjie Qiu, Sarah Munro, Scott Dehm. Double-negative prostate cancer emerges through a mixed basal, club, and hillock cell identity driven by KLF5 [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 5863.

Abstract 4020: Donor-dependent anti-tumoral efficacy of human CD19 CAR T cells in a leukemic xenograft mouse model

Abstract The recent advances in cellular immunotherapies have revolutionized the treatment options for hematological malignancies. Among others, genetically engineered T cells to express chimeric antigen receptors targeting CD19 have shown clinical success in patients with B cell malignancies. However, depending on the study design only one to two thirds of the patients experience a complete response upon CD19 CAR T cells treatment. Antigen escape, immune suppressive microenvironment, CAR T cell dysfunction as well as lack of CAR T cell persistence have been discussed to be reasons for the lack of sustained therapy response. Well established models for the preclinical evaluation of cellular therapies are needed for the evaluation of next generation CAR T cells as well as novel combination therapies. Here, we describe a xenograft animal model of human acute lymphoblastic leukemia (NALM-6 cells) with a suboptimal treatment response to human CD19 CAR T cells from three different donors. First, the activation status and memory phenotype of the CAR T cells from the different donors was characterized by flow cytometry and the anti-tumoral efficacy of the CD19 CAR T cells was evaluated in an in vitro co-culture killing assay using luciferase expressing NALM-6 cells. Next, CD19 CAR T cells from three donors were injected in NALM-6_luc tumor bearing animals. The tumor growth was monitored by bioluminescence imaging and the phenotype of the transferred CAR T cells was checked by flow cytometry. The frequency of memory T cells differed between the donors whereas the T cells from all donors exhibited all low expression of activation markers. In vitro, all three donors showed high and comparable anti-tumoral killing efficacy independent of their memory status. In vivo, CD19 CAR T cells from all three donors delayed the tumor growth significantly although none of the animals had a complete remission. In line with the individual memory status of the CAR T cells prior to infusion, there were slight differences in the kinetics of the anti-tumoral response. The flow cytometric analysis revealed that the transferred CAR T cells were almost absent in the peripheral blood and spleen whereas for individual animals tumor cells and highly activated CAR T cells were detectable in the bone marrow of the animals. In summary, the NALM-6_luc xenograft model is a value tool for evaluating next generation cellular therapies and novel combination strategies to overcome the current limitations of cellular therapies. Citation Format: Philipp Metzger, Carla N. Castro, Jonas F. Hummel, Maria Silvia Roman Azcona, Dinu Antony, Claudio Mussolino, Daniel Zurr, Swetlana Adamsky, Holger Weber. Donor-dependent anti-tumoral efficacy of human CD19 CAR T cells in a leukemic xenograft mouse model [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 4020.

Abstract 2810: Intracellular tumor microbiota promotes metastasis and drives an immunosuppressive phenotype in ovarian cancer

Abstract Background: The tumor microenvironment has immense potential to change a patient’s prognosis and therapeutic response. One of the emerging components of interest in the tumor microenvironment is the presence of intracellular microbes. Numerous studies have focused on colon and pancreatic cancer because of their proximity to the commensals in the gut. The female reproductive system is another site in our body where the rich vaginal microbiome has the potential to affect nearby organs and impact diseases. However, there is still little investigation done to understand the impact of microbes on Ovarian Cancer (OC). Objective: To understand the role of the microbiome in OC metastasis, to develop new therapies to target the tumor microbiome Methods: We used a co-culture study, the parameters of which were optimized to study the infection of microbes in different cell lines. Using a wide array proteome profiler, we looked for cytokines and chemokines to understand changes in signaling post-infection. Protein levels in the conditioned media using ELISA. Changes in actin, paxillin, and LC3B (autophagy marker) were monitored using immunofluorescence microscopy and western blots. Migration was studied by using transwell assay and spheroid cultures. Results: We have identified two species of bacteria preferentially enriched in human ovarian tumors based on recent findings in the literature. We have demonstrated that these microbes are better at infecting OC cells in vitro over non-specific bacteria, can reside intracellularly, and are metabolically active. Upon infection, this intracellular uptake drives the release of pro-inflammatory cytokines and chemokine signaling by cancer and normal ovarian surface cells. We have also observed increased autophagy, changes in the actin cytoskeleton and reduced Paxillin foci on cancer cells infected with OC-associated microbes. This has led to increased migration in them; however, inactivated microbes did not change their migratory behavior. The infection also caused individual tumor cells to form more numerous and larger colonies when seeded into soft agar, suggesting an effect on cancer cell migration. Furthermore, we have shown that these microbes also elicit a more robust reaction in immune cells like Dendritic cells and macrophages, causing them to secrete chemokines like CCL 2,3,4,5, which also cause the cancer cells to migrate and have the potential to recruit neutrophils to drive the tumor microenvironment towards an immunosuppressive phenotype. Conclusion: Most ovarian cancer patients are diagnosed at Stage III/IV, where cancer has metastasized, and is unresponsive to immunotherapy as the cancer is considered “cold”. Through functional studies, we were able to gain insights into the role of the microbiome, which is associated with the clinical outcomes of OC, hoping to potentiate the existing therapies by probing the tumor microbiome. Citation Format: Ashutosh Kumar, Neelkanth Manoj Bardhan, Angela M. Belcher. Intracellular tumor microbiota promotes metastasis and drives an immunosuppressive phenotype in ovarian cancer [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 2810.

BEHAV3D: a 3D live imaging platform for comprehensive analysis of engineered T cell behavior and tumor response.

Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond.

“Patchiness” in mechanical stiffness across a tumor as an early-stage marker for malignancy

Mechanical phenotyping of tumors, either at an individual cell level or tumor cell population level is gaining traction as a diagnostic tool. However, the extent of diagnostic and prognostic information that can be gained through these measurements is still unclear. In this work, we focus on the heterogeneity in mechanical properties of cells obtained from a single source such as a tissue or tumor as a potential novel biomarker. We believe that this heterogeneity is a conventionally overlooked source of information in mechanical phenotyping data. We use mechanics-based in-silico models of cell-cell interactions and cell population dynamics within 3D environments to probe how heterogeneity in cell mechanics drives tissue and tumor dynamics. Our simulations show that the initial heterogeneity in the mechanical properties of individual cells and the arrangement of these heterogenous sub-populations within the environment can dictate overall cell population dynamics and cause a shift towards the growth of malignant cell phenotypes within healthy tissue environments. The overall heterogeneity in the cellular mechanotype and their spatial distributions is quantified by a “patchiness” index, which is the ratio of the global to local heterogeneity in cell populations. We observe that there exists a threshold value of the patchiness index beyond which an overall healthy population of cells will show a steady shift towards a more malignant phenotype. Based on these results, we propose that the “patchiness” of a tumor or tissue sample, can be an early indicator for malignant transformation and cancer occurrence in benign tumors or healthy tissues. Additionally, we suggest that tissue patchiness, measured either by biochemical or biophysical markers, can become an important metric in predicting tissue health and disease likelihood just as landscape patchiness is an important metric in ecology.