Single Tumor Cells Research Articles

Abstract 2147: Decoding the natural biology of triple-negative breast cancer and response to chemotherapy by single-cell transcriptomics

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks the expression of estrogen receptor (ER), progesterone receptor (PR) and HER2 and therefore have limited hormonal treatment options. Neoadjuvant chemotherapy (NAC) is backbone of treatment for TNBC, and about 50% of patients respond well leading to pathological complete response (pCR). However, the remaining patients develop resistance to NAC and progress to metastatic disease and poor survival in 1-2 years after the initial treatment. Previous studies have performed bulk RNA expression profiling of TNBC patients and identified 5-6 subgroups of patients, however these studies could not resolve expression programs at single cell resolution to distinguish between the tumor cells and different components of the tumor microenvironment (TME). Here we performed scRNA-seq of pre-treatment fresh core biopsy tissue samples from TNBC patients in the ARTEMIS clinical trial and compared these data between pCR and non-pCR patients to identify programs associated with response to NAC. We also compared these data to scRNA-seq data from patients with disease-free breast tissue to understand the basic biology of TNBC and identify cell types that are reprogrammed in malignant disease. Using the single cell tumor cell data, we identified 4 archetypes of TNBC which represent patient-level intertumor expression programs: luminal secretory-like (LS), basal/luminal-like (BL), immunoregulatory (IM), and luminal androgen receptor (LAR). Notably, the archetype BL was associated with non-pCR, while IM was associated with pCR. We further identified 13 metatraits, which are unique intratumoral expression programs that are shared across patients. Across the cancer cells, we identified 13 metatraits such as cell cycling, stress, hypoxia, interferon response, HLA, partial epithelial-mesenchymal transition, and endoplasmic reticulum stress, many of which corresponded to NAC response. In the immune compartment, we found 15 myeloid cell states, 14 T/NK cell states, and 6 B cell states, several of which corresponded to pCR/non-pCR. Similarly, in the stromal compartment, there were 4 fibroblast cell states, 4 pericyte cell states, and 7 endothelial cell subtypes, of which several cell states were associated with NAC response. Overall, these data report the natural biology of TNBC patients and malignant cell states that are reprogrammed in malignant disease, as well as their correspondence to NAC response, providing new data to predict which TNBC patients are likely to respond to chemotherapy. Citation Format: Tapsi Kumar, Yiyun Lin, Yun Yan, Shanshan Bai, Jianzhuo Li, Tuan Tran, Min Hu, Elizabeth Ravenberg, Maia Rauch, Alyson Clayborn, Alastair Thompson, Lei Huo, Stacy Moulder, Clinton Yam, Nicholas Navin. Decoding the natural biology of triple-negative breast cancer and response to chemotherapy by single-cell transcriptomics [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 2147.

Abstract 4055: CD40L stimulates melanoma infiltrating B cells and enhances ex vivo TIL expansion

Abstract Adoptive transfer of tumor infiltrating lymphocytes (TIL) is a feasible and effective therapy for melanoma and lung cancer[1,2]. Multiple factors may determine the quality of the TIL product including components of the tumor microenvironment. In this work, we analyzed the role of melanoma infiltrating B cells in the context of TIL expansion based on their documented association with response to other types of immunotherapies[3]. We stimulated melanoma infiltrating B cells using human recombinant CD40L on the first day of ex-vivo TIL expansion. Samples were expanded from cryopreserved melanoma tumor single cell suspensions, in high dose IL-2 alone (standard protocol), or in high dose IL-2 plus CD40L. After 48h, analysis of activation markers on the CD40-expressing cells by flow cytometry was performed. For further investigation of the changes induced by CD40L stimulation, TIL expansion cultures (+/- CD40L) were analyzed using scRNA-seq (10X Genomics Chromium NextGEM Single Cell 5’ v2 and V(D)J Reagent kits; Illumina NovaSeq 6000 instrument with S4 sequencing flow cell) at 48h of culture (n=7 patients). The TIL expansion success rate was 68% with the CD40L treatment condition compared to 36% with the standard protocol. TILs cultured in the presence of CD40L expanded to on average three times more than with the standard protocol (P ≤ 0.01). Treatment with CD40L increased the percentage of CD39- CD69- T cells (P ≤ 0.05). Within the tumor digests, a higher percentage of B cells, including switched memory B cells (CD27+ IgD− ), was associated with successful TIL expansion (P=0.04). scRNA-seq analysis demonstrated different clustering patterns within the B cell compartment based on culture conditions. No clear partition was observed for other cell types, including the myeloid compartment. B cells displayed 126 DEGs associated to CD40L addition, CCL22, CD83, EBI3 and CD58 were among the upregulated genes in the CD40L-treated B cells. Other cell types experienced minimal to no change in transcriptomic profiles. B cell clusters were sub-classified based on CD27 and IgD expression[4], showing a predominance of naïve and switched memory B cells. Our results show that higher presence of B cells within tumors is associated with better TIL expansion, suggesting an interplay between T and B cells, and providing rationale for the design of improved TIL expansion protocols based on B cell stimulation with CD40L. This work has been supported in part by the Flow Cytometry, Genomics and Biostatistics and Bioinformatics Core Facilities at Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292). We acknowledge Moffitt’s Melanoma Center of Excellence and the Mark Foundation for the financial support. [1] Sarnaik, A.A. et al. JCO 39, 2656-2666 (2021). [2] Creelan, B. et al. Nat Med 27, 1410-1418 (2021). [3] Cabrita, R. et al. Nature 577, 561-565 (2020). [4] Sanz, I. et al. Front Immunol 10, 2458 (2019). Citation Format: Renata Ariza Marques Rossetti, Leticia Tordesillas, Matthew Beatty, Dongliang Du, Yian Ann Chen, Amod Sarnaik, Shari Pilon-Thomas, Daniel Abate-Daga. CD40L stimulates melanoma infiltrating B cells and enhances ex vivo TIL expansion. [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 4055.

Abstract 5785: A pan-cancer single-cell tumor micro-environment atlas predictive of immunotherapy response

Abstract The tumor microenvironment (TME) shapes response to immune checkpoint blockade (ICB). Several pan-cancer single-cell RNA-sequencing (scRNA-seq) analyses have reported how TME heterogeneity profoundly differs between cancer types. These studies mainly focused on one cell type (e.g., T-cells) and combined different technologies and bioinformatics pipelines with data being collected from both published and newly generated datasets. A comprehensive pan-cancer scRNA-seq map of the TME involving all cell types is therefore still lacking. We obtained scRNA-seq (10x Genomics) on 234 fresh tissue samples from 161 treatment-naïve patients. Samples were collected from 9 cancer types and subjected to a uniform in-house optimized protocol of tissue dissociation, sequencing and bioinformatics analysis. Abundancies of cell types and subtypes were correlated with each other and a tumor-reactive T-cell signature. From 683,184 high-quality single cells, we identified 9 cell types and 71 subtypes of T-cells, B-cells, dendritic cells (DCs), monocytes/macrophages and endothelial-cells (ECs), shared between cancer types. PDCD1 (PD1) was expressed by differentiated T-cells subtypes (e.g., CD8+ exhausted and CD4+ T-helper-1 subclusters), while CD274 and PDCD1LG (PDL-1/PDL-2) were mainly expressed by regulatory B-cells, immune-regulatory DCs, CXCL10+ and CCL2+ monocyte-derived macrophages as well as inflammatory ECs. Pairwise analyses showed positive correlations between PD1-expressing T-cell subclusters, CD4+ T-regulatory cells, plasma B-cells (plasmablasts, IgA and IgG plasma cells), immune-regulatory DCs, CXCL10+ and CCL2+ macrophages and lymphatic ECs. On the other hand, negative correlations were observed with naïve T- and B-cells, conventional DC2 (cDC2), monocytes, CX3CR1+ macrophages, as well as arterial and capillary ECs. When ranking individual tumors based on a tumor-reactive T-cell signature, we found reactive tumors to correlate with positively interacting subtypes, while they anti-correlated with subclusters negatively interacting with PD1-expressing differentiated T-cells. Upon deconvolution of bulk RNA-seq data using gene signatures derived from each subcluster, these correlations were replicated in TCGA datasets across cancers. These signature scores correlated with tumor mutation burden (TMB) and other immunological features, while they were also predictive of response in clinical trials involving ICB. We provide insights into the TME complexity at unprecedented level, identifying numerous subclusters enriched in immune-reactive (hot) or -suppressive (cold) tumors across cancer types. We validate these observations in TCGA by deconvolution of bulk RNA-seq data, and show that already pre-treatment several TME subclusters predict response to ICB. Citation Format: Francesca Lodi, Sam Vanmassenhove, Elena Donders, Pierre Van Mol, Amelie Franken, Sarah Cappuyns, Ayse Bassez, Siel Olbrecht, Liselore Loverix, Michel Bila, Hanne Vos, Joanna Pozniak, Kevin Punie, Diether Lambrechts. A pan-cancer single-cell tumor micro-environment atlas predictive of immunotherapy response. [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 5785.

Pagetoid Spread in Basal Cell Carcinoma: Potential for Misdiagnosis.

Basal cell carcinomas are one of the most common cutaneous carcinomas and show classical histologic features of basaloid nests with peripheral palisading. Pagetoid and intraepidermal spread has not been described in basal cell carcinoma to the best of our knowledge. We report 5 cases of basal cell carcinoma with classic histologic patterns and overlying basaloid nests and single intraepidermal tumor cells. A panel of immunostains were performed that included CK7, MOC31, CEA-m, EMA, androgen receptor, and Bcl2. Most of our cases were positive for both MOC31 and CK7, and all cases were negative for CEA-m and EMA excluding extramammary Paget disease, one of the most common differential diagnoses. These cases expand the spectrum of findings that can be seen in basal cell carcinoma and can help prevent misdiagnoses of basal cell carcinomas as more aggressive tumors.

Semi-Supervised Learning to Automate Tumor Bud Detection in Cytokeratin-Stained Whole-Slide Images of Colorectal Cancer

Tumor budding is a histopathological biomarker associated with metastases and adverse survival outcomes in colorectal carcinoma (CRC) patients. It is characterized by the presence of single tumor cells or small clusters of cells within the tumor or at the tumor-invasion front. In order to obtain a tumor budding score for a patient, the region with the highest tumor bud density must first be visually identified by a pathologist, after which buds will be counted in the chosen hotspot field. The automation of this process will expectedly increase efficiency and reproducibility. Here, we present a deep learning convolutional neural network model that automates the above procedure. For model training, we used a semi-supervised learning method, to maximize the detection performance despite the limited amount of labeled training data. The model was tested on an independent dataset in which human- and machine-selected hotspots were mapped in relation to each other and manual and machine detected tumor bud numbers in the manually selected fields were compared. We report the results of the proposed method in comparison with visual assessment by pathologists. We show that the automated tumor bud count achieves a prognostic value comparable with visual estimation, while based on an objective and reproducible quantification. We also explore novel metrics to quantify buds such as density and dispersion and report their prognostic value. We have made the model available for research use on the grand-challenge platform.

Spatial landscape of the tumor immune microenvironment

Two recent studies published in Nature by Karimi et al. and Sorin et al. applied multiplexed imaging mass cytometry (IMC) to characterize the single-cell tumor immune landscapes covering millions of cells in brain tumors and lung adenocarcinomas (LUAD). They identified specific cell subtypes and cell–cell interactions that correlate with distinct clinical outcomes, thus providing valuable insights into tumor biology and prognostic prediction based on spatial architecture.

Abstract P6-14-05: Regulation of cellular identity and spatial organization during collective breast cancer invasion

Abstract An early step in breast cancer progression is invasion of tumor cells into surrounding tissues. In many breast cancers, particularly ductal carcinomas, this invasion is accomplished by tumor cells migrating as a cohesive group. This often involves cells that take on heterogeneous roles as either leader or follower cells. While studies in common mouse and human breast cancer models have established that leader cells express high levels of keratin-14 (K14) and other basal epithelial markers, the molecular mechanisms regulating K14+ leader cell identity remain obscure. Here we performed time-sampled single cell RNA-sequencing in 3D type I collagen-embedded tumor organoids isolated from the MMTV-PyMT luminal B model of breast cancer. 11 distinct cellular transcriptional states were identified and correlated with K14 expression and invasive strand formation. Having identified the leader cell state we next asked what transcription factors were enriched, reasoning that transcription factors could be master regulators of leader cell fate. 30 different shRNAs targeting 10 genes were systematically evaluated for their effects on collective invasion. From this screen, suppression of Hes1, the downstream target of Notch signaling, yielded a marked switch from collective to single cell invasion. Disseminating single tumor cells maintained high expression of K14 in Hes1 knockdown organoids which was phenocopied by gamma-secretase inhibition in a human TNBC PDX model. Because K14+ tumor cells highly express the Notch ligand Jag1, these results support a model in which Notch signaling, specifically through activation of Hes1, dictates leader cell identity and spatial organization during collective invasion. Studies are ongoing investigating the impact of Hes1 dynamics on leader cell adhesion, hybrid EMT state, and preference for single versus collective metastasis. Because Notch suppression induces leader cell dissemination, we propose that Notch targeted therapy should be combined with therapies eradicating leader cells. Citation Format: Andrea E. Doak, Kevin J. Cheung. Regulation of cellular identity and spatial organization during collective breast cancer invasion. [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 P6-14-05.

VIOLET: A phase I/II trial evaluation of radioligand treatment in men with metastatic castration-resistant prostate cancer with [161Tb]Tb-PSMA-I&T.

TPS281 Background: [177Lu]Lu-PSMA is an effective class of therapy for men with metastatic castration-resistant prostate cancer (mCRPC); however, progression is inevitable. The limited durability of response is partially explained by the presence of micrometastatic deposits. Single tumor cells and micrometastases are energy-sheltered deposits receiving low absorbed radiation, due to the ~0.7mm mean path-length of Lutetium-177 (177Lu). Terbium-161 (161Tb) has abundant emission of Auger electrons that deposit a higher concentration of radiation over a shorter path, particularly to single tumor cells and micrometastases. 161Tb has shown superior in-vitro and in-vivo results in comparison with 177Lu. We hypothesize that [161Tb]Tb-PSMA-I&T will deliver effective radiation to sites of metastatic prostate cancer with an acceptable safety profile. Methods: This single-center, single-arm, phase I/II trial will recruit 30 to 36 men with progressive mCRPC. The phase I dose-escalation is designed with a 3+3 model to establish the safest dose of [161Tb]Tb-PSMA-I&T (dose levels: 4.4, 5.5 and 7.4 GBq). The maximum tolerated dose (MTD) will be defined as the highest dose level at which a dose-limiting toxicity occurs in less than 1/3 or 2/6 participants. The phase II dose-expansion will include 24 participants at the MTD. Up to six cycles of [161Tb]Tb-PSMA-I&T will be administered intravenously every six weeks, with each subsequent dose for each patient reduced by 0.4GBq. Key eligibility criteria include a diagnosis of mCRPC with progression after at least one line of taxane chemotherapy and a second-generation anti-androgen, PSMA-positive disease on PSMA PET/CT (SUVmax ≥20), no sites of discordance on FDG PET/CT, adequate bone marrow, hepatic and renal function, ECOG performance status ≤2, and no prior treatment with another radioisotope. The co-primary objectives are to establish the MTD of [161Tb]Tb-PSMA-I&T, and safety profile [CTCAE v5.0]. Secondary objectives include measuring absorbed radiation dose [Gray], evaluating anti-tumour activity [PSA 50% response rate, radiographic and PSA progression-free survival, overall survival, objective response rate], and evaluation of pain [BPI-SF] and health-related quality of life [FACT-P and FACT-RNT] over the first 12 months after treatment commences. Exploratory objectives include ctDNA analysis at baseline, during treatment and at progression, and optional tissue biopsies, to determine biomarkers of treatment response and resistance. Patient enrolment began in October 2022, with recruitment expected to continue for 24 months. Clinical trial information: NCT05521412 .

Assessment of complete pathologic response after neoadjuvant immunotherapy for MSI-H gastrointestinal cancers.

43 Background: Immunotherapy (IO) has shown remarkable efficacy in gastrointestinal (GI) cancers with high microsatellite instability (MSI-H). We evaluated real world outcomes of patients treated with neoadjuvant IO for MSI-H colorectal (CRC) and gastroesophageal (GE) cancers. Methods: We queried diagnoses, pathology reports, and clinic notes of patients receiving IO at Massachusetts General Hospital from October 2014 to March 2022 using the Research Patient Data Registry and MATLAB. 1140 patients were identified with esophageal, gastric, colon, or rectal primaries. Of these, 56 were MSI-H and seven received neoadjuvant IO with curative intent. Results: Of the seven patients who received neoadjuvant IO for MSI-H CRC or GE cancers, three patients achieved complete pathologic response (pCR). Three patients had partial responses; one had a single residual lymph node tumor cell, one had residual T1b tumor and negative nodes, and one had residual T3 tumor and positive nodes. All patients with pCR had non-metastatic disease. Three of four patients with partial responses or progression had metastatic disease. Five patients had no recurrence by median follow-up of 10 months post-resection. One patient's cancer recurred 15 months post-resection, and one patient had progressive disease on neoadjuvant IO so did not undergo primary resection. Conclusions: Neoadjuvant IO shows promise for MSI-H GI cancers. Three of seven patients (43%) had pCR and three others (43%) had notable partial responses. There is a need to understand IO-refractory primary tumors and the differing effects of IO in local versus metastatic disease. While our data is limited by sample size, larger clinical trials can establish the safety and utility of neoadjuvant IO, potentially sparing many patients from surgery. We will collaborate with other centers for a larger scale analysis on neoadjuvant IO in MSI-H GI cancers. [Table: see text]

Abstract IA017: An oncogenic histone reader promotes metaplastic TNBC

Abstract Human carcinosarcomas or metaplastic breast cancers (MpBC) are a rare, chemorefractory subclass of triple-negative breast cancers (TNBC). Conditional overexpression of histone reader Tripartite motif containing protein 24 (TRIM24) in mouse mammary epithelia (Trim24COE) led to spontaneous development of mammary carcinosarcoma tumors, lacking ER, PR and HER2, comprising 70% of all mammary tumors that developed with 40% penetrance. We saw strong correlation of Trim24COE carcinosarcoma tumors with human MpBC tumors and derived a conserved Trim24COE gene signature of Glycolysis, EMT and mTORC1 signaling pathways. Global and single-cell tumor profiling revealed Met as a direct oncogenic target of TRIM24, leading to aberrant PI3K/mTOR activation. To assess TRIM24-dependent metabolic alterations, we developed Trim24COE carcinosarcoma spheroid cultures. Tumor-derived Trim24COE carcinosarcoma spheroids exhibit TRIM24-dependent EMT and repressed cellular and mitochondrial ROS levels. We found that TRIM24 expression increased c-myc, NADPH and NRF2 levels to drive ROS repression and TRIM24-dependent cell survival under oxidative stress. TRIM24 mediates NRF2 activation at the level of chromatin structure activation, as shown by ATAC-seq of Trim24COE and shTrim24 carcinosarcoma spheroids. These in vivo and ex vivo models of metaplastic TNBC offer a platform for mechanistic discovery and nomination of potential therapeutic avenues for this highly malignant TNBC subtype. Citation Format: Vrutant V. Shah, Shucheng Miao, Patrick M. Krause, Sabrina A. Stratton, Lalit R. Patel, Michelle C. Barton. An oncogenic histone reader promotes metaplastic TNBC [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr IA017.

Immunological characteristics of primary tumor and bone marrow in patients with breast cancer

Introduction. The Implementation of immunotherapy in cancer requires a thorough analysis of the biological characteristics of the tumor. Therefore, the study of tumor immunophenotype is the leading scientific direction. Molecules of the major histocompatibility complex are considered as promising markers for evaluating the possibility of using immunotherapy. Objective. To study the immunophenotype of the primary tumor in molecular subtypes of breast cancer and analyze it depending on the frequency of bone marrow damage. Materials and methods. The study included 99 patients with breast cancer. Tumor tissue and bone marrow samples were studied. Stages: T1 (51.5%), T2 (44.4%), T3 (2.0%). Metastatic involvement of lymph nodes (N+) was noted in 39.4% (n = 39) of cases. Immunophenotyping of the tumor was carried out on cryostat sections by immunofluorescence. Antibodies to HLA-I, HLA-II, KL1, FITC-labeled F(ab2) antiserum fragments (Becton Dickinson, USA) were used. The bone marrow was studied by morphological (Zeiss microscope, Axioskop, Germany) and immunological methods (flow cytometry, FACS Canto II, USA), data analysis – Kaluza Analysis v2.1 software. Used monoclonal antibodies: CD45, EPCAM (Becton Dickinson, USA). Statistical data processing was performed using the IBM-SPSS Statistics 21 package. Results . The HLA-I antigen is absent or expressed by single tumor cells in 50.8% of cases of luminal breast cancer. Pronounced or partial expression was noted in 36.9% of cases. With the Erb-B2 subtype, all samples had the expression of antigens of the HLA-I class. Loss of HLA-I antigen or weak expression was observed in 30% (3/10) of cases of the triple-negative subtype, in the same percentage of cases the expression of HLA-II molecules was noted. There were no significant differences in the expression of HLA molecules between subtypes. Using flow cytometry, bone marrow involvement was detected in 40% (26/65) of cases. There were no significant relationships between bone marrow damage and the size of the primary tumor, stage, grade of breast cancer, or expression of HLA molecules. Conclusions. The expression of HLA molecules does not differ in significant differences depending on the biological subtype. The frequency of bone marrow damage did not depend on the expression of HLA-I, class II molecules.

Cathodic Electrochemiluminesence Microscopy for Imaging of Single Carbon Nanotube and Nucleolin at Single Tumor Cell.

Cathodic electrochemiluminesence (ECL) microscopy based on luminol analog L012 was originally established to implement the imaging of a single nanotube and nucleolin on a single tumor cell. This microscopy utilizes multiwalled carbon nanotubes (MWCNTs) as advanced coreactant accelerators to efficiently convert dissolved oxygen (O2) and H2O2 into reactive oxygen species (ROS) due to excellent electrocatalytic properties. The produced ROS could oxide L012 into an excited state of L012 leading to a bright cathodic ECL illumination, thereby promoting ECL imaging of MWCNTs at a low triggering potential. After being modified with AS1411 aptamers, MWCNTs@AS1411 probes were incubated with tumor cells for specific ECL imaging of nucleolin on the plasma membrane, which permits cathodic ECL microscopy for label-free bioassays without ECL tags. The L012-based cathodic ECL microscopy with a moderate operating potential and label-free characteristics provides a universal approach in single nanomaterial and single-cell imaging and analyses.

Modeling cell clusters and their near-wall dynamics in shear flow

The studies that compare the metastatic potential of tumor cell clusters in microcirculation to that of single tumor cells show that the clusters contribute significantly to metastasizing. The metastatic potential is conditioned by the presence of the cancer cells near vessel walls. Detailed understanding of dynamical behavior of clusters near the vessel walls can thus elucidate the process of adhesion. We have developed a biomechanical model of cell clusters capable of simulating both strong and weak adhesion among the cells in the cluster in various spatial configurations. We have validated the model on data from cell separation experiments. The developed model has been used to study near-wall dynamics in shear flow with focus on cluster–wall contact. To quantify the presence of cells near walls, we have evaluated metrics involving time of contact and contact area of clusters tumbling and rolling near the wall. The computational results suggest two trends: First, more elastic clusters and clusters of weakly adhesive cells have decreased cluster–wall contact to the walls than rigid clusters or clusters composed of strongly adhesive cells. Second, more spherical cluster shapes tend to drift away from the walls, thus decreasing the wall contact time.

Histomorphological Evaluation of Desmoplastic Tumor Stroma in Malignant Ovarian Surface Epithelial Tumors.

Ovarian cancer is the 8th most common cancer in women worldwide. Tumor budding is defined as a type of invasive growth in carcinomas with either a single tumor cell or a cluster of up to four cells at the invasive tumor front and is associated with epithelial-mesenchymal transition. A reactive stroma rich in cancer-associated fibroblasts is associated with higher tumor grade and poorer prognosis in breast, colorectal, and oral cancers. The present study was conducted to highlight the prognostic significance of tumor budding and fibrotic cancer stroma in malignant ovarian surface epithelial tumors with known prognostic parameters. This was a retrospective cross-sectional study conducted over a 2-year period, in which all histologically diagnosed cases of malignant ovarian surface epithelial tumors who underwent surgery were included. The fibrotic stroma was classified into three distinct categories - mature, intermediate, and immature. The number of tumor buds was counted at the invasive front of the tumor and graded based on the number of buds - 0-5, 5-9, and ≥10 buds. Among the 50 cases, 32% (16 cases) had mature stroma, whereas 30% (15 cases) and 38% (19 cases) had intermediate and immature stroma, respectively. Although a significant association could not be established between tumor budding and stroma grade, a fair agreement was established between them. A significant association could be established between histological grade with both tumor budding (P = 0.03) and fibrotic stroma grade (P = 0.02). The study highlighted the role of stromal response in malignant surface epithelial tumors of the ovary since a higher-grade tumor was associated with an immature stroma, whereas a lower-grade tumor was associated with a mature stroma.

Advent of Single Tumor Cell Analysis in Oral Squamous Cell Carcinoma – An Insight

Oral squamous cell carcinoma OSCC has a complex portfolio comprised of genetic and epigenetic instability. Gene expression studies are necessary for molecular typing of oral cancer. The conventional studies on gene expression obtained from whole cell populations average the differences between individual cells. The evolving tools and techniques using single tumor cell analysis STCA allows identification and analysis of individual cell type within a system and enables better characterization of cancer and for prognostication. The present paper intends to highlight the available evidence on STCA in OSCC. STCA has been carried out in carcinomas of the breast prostate pancreas colon and lung. However much needs to be explored in the region of head and neck especially OSCC. STCA enables better understanding of tumor heterogeneity and its microenvironment. Further it helps in identification and detection of cancer stem cells and circulating tumor cells. STCA has been remarkable in understanding clonal evolution of cancer stem cells tumor specific neo-antigens and evaluation of drug resistance mechanisms. Few limitations of STCA could be artefactual errors procedural difficulties and such others. STCA when combined with other technologies could facilitate the integration of multidimensional datasets having a significant impact in the analysis of risk stratification diagnosis and treatment of patients with OSCC worldwide.

IL-3 DISCONNECTING BRAIN TUMOR NETWORKS FOR IMPROVED THERAPIES

Abstract Integration into communicating tumor cell networks is a way how the entire brain tumor organism can progress in the brain, and resist therapies. It started with our discovery of ultra-long membrane protrusions, tumor microtube (TM), in tumor cells from glioblastomas and other incurable adult and pediatric glioma entities (Osswald et al., Nature 2015). These TMs are used for very effective brain invasion, and later interconnect single tumor cells to communicating and resistant multicellular networks, but also glioma cells to neurons in the brain, with neuron-glioma synapses stimulating glioma growth(Venkataramani et al., Nature 2019) and invasion (Venkataramani et al., Cell 2022). Increasing formation of these homotypic and heterotypic networks is seen with increasing glioma malignancy: lowest in low-grade oligodendrogliomas, and highest in glioblastomas and K27M mutated gliomas. The genetic reduction of GAP-43, a crucial neurodevelopmental factor that drives TM formation and function, not only deprived glioblastoma cells from the ability to form functional tumor networks, but also made radiotherapy much more effective, by and large eradicating experimental tumors from the mouse brain, which is typically not possible with radiotherapy alone. Consistent with the concept of tumor cell resilience by network integration, radiotherapy and also chemotherapy (Weil et al., Neuro-Oncology 2017) preferentially killed the unconnected glioblastoma cells, leaving the tumor cell networks largely intact. Damage to the tumor network by surgical measures induced a self-healing response. Taken together, integration into multicellular, highly functional, communicating and resilient networks is not only a feature of the nervous system, but also of incurable, most aggressive brain tumors. In this talk, clinical concepts that originate from these discoveries are presented, including a network disconnection strategy currently tested in the German MecMeth trial (EudraCT2021-000708-39), and the AMPAR inhibitor perampanel as a potential inhibitor of neuron-glioma synapses. Furthermore, new basic and translational developments in the field of brain tumor networks are presented and discussed.

A numerical study of tumor cell arrest in microvessels qualifying for mechanical entrapment

During hematogenous metastasis, the arrest of tumor cells in the microvasculature is a prerequisite for extravasation from the circulation to a distant host organ. To reveal such arrest behavior, we implement three-dimensional numerical simulations on the motion of a single tumor cell in microvessels at the cellular scale and mainly investigate the interactions among mechanical entrapment, adhesion, and cell stiffness, and their effects on the tumor cell arrest. Two types of vascular configurations qualifying for mechanical entrapment are considered, the constriction and bifurcation structures that are comparable in diameter with the tumor cell. The main results indicate that in the constriction tube, as the constriction radius is increased, the tendency that number of adhesion bonds increases with increasing shear modulus becomes more and more obvious. However, the adhesion behavior has little effect on the tumor cell arrest in the constriction region, regardless of the number of adhesion bonds. The mechanical entrapment plays a more important role than the cell stiffness in the tumor cell arrest in the constriction tube. In the bifurcated tube, the tumor cell is more likely to be arrested in the bifurcation region with a small bifurcation angle. Moreover, as the bifurcation angle or shear modulus is decreased, the effect of adhesion behavior on the tumor cell arrest becomes increasingly obvious. These results are helpful in understanding the biomechanism of tumor metastasis.

NIMG-51. 18KDA TRANSLOCATOR PROTEIN (TSPO) DECLINES IN TUMOR AND IMMUNE CELLS DURING PROGRESSION OF EXPERIMENTAL GLIOBLASTOMA

Abstract Glioblastoma combines a lack of immunogenicity with a highly immunosuppressive tumor microenvironment (TME), including both tumor and immune cells. However, biomarkers that allow monitoring of the immune phenotype are still lacking. Hence, we investigated the 18kDa translocator protein (TSPO) during tumor progression in an experiential glioblastoma mouse model (SB28) mimicking human TME. We used TSPO-PET imaging ([18F]GE-180) and in vivo measures of single cell tracer uptake between days 6 and 18 after inoculation to study alterations and dependence of TSPO in tumor and peripheral organs in SB28 mice (n= 27) in comparison to sham controls (n= 11). CSF1R inhibition was applied to deplete tumor associated microglia/macrophages (TAM) followed by withdrawal to induce immune cell rebound (PLX5622 day -20 to day 6, n= 3). Compared to sham, TSPO-PET signals were distinctly elevated in tumor (+83%, p< 0.001), heart (+35%, p< 0.05), lung (+42%, p< 0.01) and bone (+26%, p< 0.05) of SB28 mice at day 6. TSPO-PET increases were lower at day 18 (tumor: +52%, p< 0.05; all organs < +15%, n.s.). The tumor TSPO-PET signal was strongly coupled with TSPO-PET signal in peripheral organs (all R≥ 0.88, all p< 0.001). Single tumor cells and TAM showed strong early increases of TSPO tracer uptake at day 6 (tumor: 32-fold, TAM: 8.5-fold) and a decline of these increases at day 18 (tumor: 2.4-fold, TAM: 1.7-fold) when compared to sham microglia. Immune cell rebound restored TSPO tracer uptake in TAM (+120%, p< 0.01) but not in tumor cells (-24%, n.s.) when compared to therapy-naïve SB28 mice. TSPO declines in tumor cells and TAM during progression of experimental glioblastoma. TSPO in peripheral organs could serve as a supportive indicator of declining immune response in glioblastoma. CSF1R inhibition and reinitiation yields specific restoring of TSPO in TAM and could serve as immunomodulatory therapy strategy.

NPInter v5.0: ncRNA interaction database in a new era.

Noncoding RNAs (ncRNAs) play key regulatory roles in biological processes by interacting with other biomolecules. With the development of high-throughput sequencing and experimental technologies, extensive ncRNA interactions have been accumulated. Therefore, we updated the NPInter database to a fifth version to document these interactions. ncRNA interaction entries were doubled from 1 100 618 to 2 596 695 by manual literature mining and high-throughput data processing. We integrated global RNA-DNA interactions from iMARGI, ChAR-seq and GRID-seq, greatly expanding the number of RNA-DNA interactions (from 888 915 to 8 329 382). In addition, we collected different types of RNA interaction between SARS-CoV-2 virus and its host from recently published studies. Long noncoding RNA (lncRNA) expression specificity in different cell types from tumor single cell RNA-seq(scRNA-seq)data were also integrated to provide a cell-type level view of interactions. A new module named RBP was built to display the interactions of RNA-binding proteins with annotations of localization, binding domains and functions. In conclusion, NPInter v5.0 (http://bigdata.ibp.ac.cn/npinter5/) provides informative and valuable ncRNA interactions for biological researchers.

Circulating caspase-cleaved cytokeratin 18 correlates with tumour burden and response to therapy in patients with colorectal cancer liver metastasis

Background and aimsSystemic chemotherapy followed by hepatic resection is the treatment of choice for patients with colorectal cancer liver metastasis (CRCLM) but reliable biomarkers predicting response to therapy are needed. Spontaneous apoptosis of single tumour cells is common in CRCLM. We explored the potential of circulating apoptosis markers to predict treatment response. Materials and methodsFifty-eight patients with CRCLM or hepatocellular carcinoma (HCC) were included in this study. Tumour tissue and blood samples were obtained before and after initiation of chemotherapy. Immunohistochemistry and ELISA assays were utilized to quantify the apoptosis marker caspase-cleaved cytokeratin 18 (M30) in tissue and circulation. ResultsCRCLM tissues showed more apoptotic tumour cells than HCC, or healthy liver. This was associated with elevated levels of circulating M30 (median = 244 U/l vs. 37 U/l in healthy controls, p = 0.009) which correlated with tumour volume (r2 = 0.92). Patients with progressive disease during chemotherapy showed higher M30 levels before therapy than responders (745 U/l vs. 136 U/l, p = 0.016). The predictive potential of M30 was higher than that of the tumour markers CA19-9 or CEA (AUC: 0.93, 0.63, and 0.78, respectively). ConclusionsApoptotic tumour cells release cellular debris into the circulation, which provides information about tumour size and vitality.