Small Cell Clusters Research Articles

Break-Up of Plant Cell Structures in High Pressure Homogenizers – Prospects and Challenges for Processing of Plant-Based Beverages

Abstract For more than a century, the dairy industry has used high-pressure homogenization for size reduction of fat globules. The prevailing break-up mechanism, turbulence, has been thoroughly investigated and the equipment continuously optimized thereafter. However, the high-pressure homogenizer is also used in size reduction of plant cell structures, for example in production lines of plant-based beverages, fruit and vegetable juices and ketchup. This review will provide a scientific basis for homogenization of plant-based materials with focus on break-up mechanisms. A cross-study comparison shows that different raw materials break in different ways, e.g. individual cells breaking into cell wall fragments and cell clusters breaking into smaller cell clusters. In general, raw materials which after intense premixing exist as cell clusters are more difficult to break than raw materials existing as individual cells. The resistance to break-up also appears to follow ‘raw material hardness’, where harder raw materials, e.g., parsnip and almond, are more difficult to break than softer raw materials, e.g., strawberry and orange. It can also be concluded that the initial particle size is of large importance for the size after high pressure homogenization. It is concluded that little is known about the break-up mechanism(s). Much does, however, point towards the mechanism being different from that of emulsion drop break-up. Suggestions for future studies, both regarding fundamental understanding (e.g., cell strength and breakup, HPH mechanistic studies and break up visualisations) and industrial applications (e.g., energy optimal operation, device design and wear) are provided.

CellStudio: a Modular, Tunable and Accessible Platform for Analysis of Growth Factors Secretions in Cell Cultures.

Traditional cell culture methods face significant limitations in monitoring cell secretions with spatial and temporal precision. Advanced microsystems incorporating biosensors have been developed to address these challenges, but they tend to lack versatility, and their complexity, along with the requirement for specialized equipment, limits their broader adoption. CellStudio offers an innovative, user-friendly solution that exploits Printing and Vacuum Lithography combined with bead-based assays to create modular and tunable cell patterns surrounded by biosensors. This platform allows for high-resolution, spatially resolved analysis of secreted proteins, such as VEGF and FGF-2, while being easily implementable in standard laboratory settings. CellStudio's design is compatible with conventional laboratory equipment, facilitating its integration into existing workflows without the need for extensive training or specialized tools. Validation experiments using mesenchymal stem cells and HeLa cells demonstrated that CellStudio can detect small secretion levels from small cell clusters with high sensitivity and analyze diffusion profiles, remarking the possibilities for studying cell behavior. By offering a standardized, cost-effective approach to detailed cellular analysis, CellStudio significantly enhances the capabilities of traditional cell culture techniques with broad applications across biological and biomedical research.

Cytological features of oncocytic pleomorphic adenoma of the salivary gland: Using the Milan classification system to report 3 patients.

Pleomorphic adenoma (PA) is the most common salivary gland tumour. Pre-operative fine-needle aspiration (FNA) is currently one of the most widely used cytological examination techniques for the diagnosis of salivary gland tumours. Because PA exhibits characteristic cytological features, cytological diagnosis is straightforward in most cases. However, limitations have emerged in certain cases, specifically in cases of oncocytic metaplasia (rare in PA), characterised by rich eosinophilic granular cytoplasm and relatively large nuclei, which can make cytological diagnosis challenging. To date, only two cytological reports of oncocytic PA have been published. The present study retrospectively analysed patients with oncocytic PA of the salivary gland who underwent preoperative FNA to describe the clinicopathological features of oncocytic PA. In addition, the application of the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) was discussed. The study cohort included 3 patients with parotid gland tumours. The cytological specimens had small and/or large clusters of oncocytic cells containing rich granular cytoplasm, relatively large nuclei, and occasional nucleoli in a myxoid (2 patients) and clear (1 patient) background, with no necrotic material. A review of the cytological features of the presented cases, as well as previously reported cases, indicated that oncocytic PA may be overdiagnosed as carcinoma, especially carcinoma ex pleomorphic adenoma (CXPA) because these cells have relatively large nuclei. The cytological features of oncocytic cells in PA resemble those of salivary duct carcinoma, the most common carcinoma component of CXPA. The absence of necrotic material and high-grade nuclear atypia are important diagnostic features. Furthermore, cytological specimens with atypical oncocytic cells in the PA should be classified as salivary neoplasms in the uncertain malignant potential category of the MSRSGC.

Hypoxia drives the formation of lung micropapillary adenocarcinoma-like structure through hypoxia-inducible factor-1α

Micropapillary adenocarcinoma (MPC) is an aggressive histological subtype of lung adenocarcinoma (LUAD). MPC is composed of small clusters of cancer cells exhibiting inverted polarity. However, the mechanism underlying its formation is poorly understood. Here we show that hypoxia is involved in MPC formation. Hypoxia induced the formation of MPC-like structures (MLSs) in a three-dimensional culture system using A549 human LUAD cells, and HIF-1α was indispensable for MLS formation. RNA sequencing analysis demonstrated that A549 cells forming MLSs exhibited a gene expression signature similar to that of lung MPC. Moreover, MLS formation enhanced the resistance of A549 cells to natural killer cell cytotoxicity. Our findings suggest that hypoxia drives lung MPC formation through HIF-1α and that immune escape from natural killer cells might underlie the aggressiveness of MPC.

First European records of Puccinia modiolae and P. platyspora, two native South American rust fungi, and new observations on their life cycle and morphology

ABSTRACT This paper reports the South American rust fungi Puccinia modiolae and P. platyspora (Pucciniales/Uredinales) as new alien species of the European rust funga. Puccinia modiolae is presently known from Switzerland and Germany, P. platyspora from Switzerland, Germany, and France. The records of P. platyspora are the first ones from outside South America. The specimens were identified by teliospore characters and sequences of the nuclear ribosomal DNA (internal transcribed spacer 2 and domains D1–D2 of the nuclear ribosomal large subunit) and the mitochondrial CO3 (cytochrome c oxidase III) gene. Puccinia modiolae and P. platyspora have been recorded so far in Europe on members of the genera Alcea, predominantly on Alcea rosea, Althaea, and Malva of the Malvaceae, subfam. Malvoideae. Alcea rosea is host of both species and shared also with the common mallow rust, P. malvacearum, allowing for mixed infections. The plant is commonly grown as an ornamental and may play a major role for the spread of the alien Malvaceae rust fungi. It was observed for the first time that P. platyspora can produce spermogonia and aecidium-type aecia, suggesting phenotypic plasticity regarding the formation of spore states. The observed spermogonia mainly remained closed and did not liberate spermatia. They produced telio- and aeciospores besides spermatia in their cavity and eventually converted entirely into telia or, rarely, into aecidium-like sori. Small clusters of aeciospores and peridial cells were commonly found hidden in the telial plectenchyma, and well-developed aecidium-type aecia provided with a peridium developed rarely in the center of mature telia. Spermogonia belonging to group V type 4 were found in P. malvacearum, which is generally supposed to lack spermogonia. Some spermogonia produced only spermatia in their cavity; others formed spermatia and teliospores, and some eventually converted into telia.

Exploring prognostic biomarkers in pathological images of colorectal cancer patients via deep learning.

Hematoxylin and eosin (H&E) whole slide images provide valuable information for predicting prognostic outcomes in colorectal cancer (CRC) patients. However, extracting prognostic indicators from pathological images is challenging due to the subtle complexities of phenotypic information. We trained a weakly supervised deep learning model on data from 640 CRC patients in the prostate, lung, colorectal, and ovarian (PLCO) cancer screening trial dataset and validated it using data from 522 CRC patients in the cancer genome atlas (TCGA) dataset. We created the colorectal cancer risk score (CRCRS) to assess patient prognosis, visualized the pathological phenotype of the risk score using Grad-CAM, and employed multiomics data from the TCGA CRC cohort to investigate the potential biological mechanisms underlying the risk score. The overall survival analysis revealed that the CRCRS served as an independent prognostic indicator for both the PLCO cohort (p < 0.001) and the TCGA cohort (p < 0.001), with its predictive efficacy remaining unaffected by the clinical staging system. Additionally, satisfactory chemotherapeutic benefits were observed in stage II/III CRC patients with high CRCRS but not in those with low CRCRS. A pathomics nomogram constructed by integrating the CRCRS with the tumor-node-metastasis (TNM) staging system enhanced prognostic prediction accuracy compared with using the TNM staging system alone. Noteworthy features of the risk score were identified, such as immature tumor mesenchyme, disorganized gland structures, small clusters of cancer cells associated with unfavorable prognosis, and infiltrating inflammatory cells associated with favorable prognosis. The TCGA multiomics data revealed potential correlations between the CRCRS and the activation of energy production and metabolic pathways, the tumor immune microenvironment, and genetic mutations in APC, SMAD2, EEF1AKMT4, EPG5, and TANC1. In summary, our deep learning algorithm identified the CRCRS as a prognostic indicator in CRC, providing a significant approach for prognostic risk stratification and tailoring precise treatment strategies for individual patients.

Growth pattern of de novo small clusters of colorectal cancer is regulated by Notch signaling at detachment.

Cancer cell clusters have a higher capacity for metastasis than single cells, suggesting cancer cell clusters have biological properties different from those of single cells. The nature of de novo cancer cell clusters that are newly formed from tumor masses is largely unknown. Herein, we generated small cell clusters from colorectal cancer organoids and tracked the growth patterns of the clusters up to four cells. Growth patterns were classified into actively growing and poorly growing spheroids (PG). Notch signaling was robustly activated in small clusters immediately after dissociation, and Notch signaling inhibition markedly increased the proportion of PG spheroids. Only a limited number of PG spheroids grew under growth-permissive conditions invitro, but xenograft tumors derived from Notch inhibited clusters showed growth rates comparable to those of untreated spheroids. Thus, de novo clusters are composed of cells with interchangeable growth fates, which are regulated in a context-dependent manner by Notch signaling.

Abstract PR-12: The circadian rhythm gene Dec2 regulates multiple components of the antigen presentation pathway to promote pancreatic cancer dormancy by immune evasion

Abstract Metastatic recurrence following curative-intent pancreatic surgery is a major clinical problem as nearly all die of this at both early and latent periods following surgery. While latent recurrences are due to the reactivation of disseminated dormant tumor cells, the mechanisms that regulate dormancy remain unclear. Using a novel model of early-stage resectable pancreatic cancer that mimics early and latent outcomes in patients, we identified Dec2 (bhlh41) a transcriptional repressor and circadian regulator, as upregulated in dormant tumor cells harvested from the liver. Disseminated tumor cells harvest from the livers of latent recurrent mice were Dec2 hi/Ki67lo indicating that these cells are dormant. We confirmed the relationship of Dec2 and Ki67 in human pancreatic cancer using liver tissue specimens from patients with metastatic disease whereby small clusters of metastatic cells were Dec2hi/Ki67lo and larger metastatic tumors were Dec2lo/Ki67hi. To understand the mechanism of Dec2 in dormancy we deleted Dec2 from the tumor cells evaluated them in the resection model and observed a substantial increase in survival after resection due to an immune mediated mechanism as the survival benefit was abrogated in immunodeficient conditions indicating that Dec2 promotes tumor progression through immune mediated mechanism. Dec2 promotes immune evasion by downregulating multiple components of the antigen presentation pathway including proteosome and immunoproteosome genes, MHCI and MHCI chaperones. As Dec2 is a circadian regulator, we evaluated several components of the antigen presentation pathway including proteosome and surface MHCI and found these to display oscillation that was not only anti-phase with Dec2 but resulted in differential T-cell mediated tumor cell killing depending on the time of day. These studies elucidate a new mechanism by which Dec2 regulates cancer dormancy through immune evasion and identifies several novel therapeutic strategies to restore anti-tumor immunity. They also provide the first evidence that antigen presentation is under circadian regulation in pancreatic cancer. Citation Format: Chris Harris, Crissy Dudgeon, Lan Wang, Orjola Prela, Juliana Cazarin de Menezes, Brian Altman, Brian Haab, Chongfeng Gao, Zachary Klamer, Anna Repesh, Subhajyoti De, Darren R Carpizo. The circadian rhythm gene Dec2 regulates multiple components of the antigen presentation pathway to promote pancreatic cancer dormancy by immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-12.

Exploring the Usefulness of Tumor Budding as a Histopathological Marker Compared to Tumor-Node-Metastasis (TNM) Staging in Breast Cancer.

Tumor budding, defined as small clusters of tumor cells at the invasive front of carcinomas, has gained attention as a potential prognostic marker in various cancers. This study aimed to evaluate the utility of tumor budding as a histopathological marker in breast cancer and compare it to traditional prognostic markers such as histological grading, tumor-node-metastasis (TNM) staging, and molecular subtypes. A prospective, cross-sectional study was conducted over two years (June 2022 to May 2024) in the Department of Pathology at Jawaharlal Nehru Medical College, Wardha. Seventy-two female patients diagnosed with breast carcinoma who underwent modified radical mastectomy were included. Tumor budding was assessed through histopathological analysis and categorized as high or low. Statistical correlations were established between tumor budding and tumor size, histological grade, lymph node involvement, vascular invasion, and molecular subtypes (estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and triple-negative breast cancer, TNBC). The chi-square test and multiple regression analyses were applied to assess significance. High tumor budding was observed in 68.1% of patients and was significantly associated with larger tumor size (p = 0.040), higher histological grade (p = 0.009), lymph node metastasis (p = 0.002), and vascular invasion (p = 0.003). The postmenopausal age group (>55 years) demonstrated a higher prevalence of high budding (p = 0.010). No significant correlation was found between tumor budding and molecular subtypes (p = 0.39), although high budding was more frequent in TNBCcases. Tumor budding is significantly associated with more aggressive tumor characteristics in breast cancer. Incorporating tumor budding into routine pathological assessments alongside TNM staging and histological grading may enhance the ability to identify high-risk patients and guide treatment strategies. Further large-scale, multicenter studies are warranted to confirm its prognostic value.

The Locus Coeruleus in Chronic Pain.

Pain perception is the consequence of a complex interplay between activation and inhibition. Noradrenergic pain modulation inhibits nociceptive transmission and pain perception. The main source of norepinephrine (NE) in the central nervous system is the Locus Coeruleus (LC), a small but complex cluster of cells in the pons. The aim of this study is to review the literature on the LC-NE inhibitory system, its influence on chronic pain pathways and its frequent comorbidities. The literature research showed that pain perception is the consequence of nociceptive and environmental processing and is modulated by the LC-NE system. If perpetuated in time, nociceptive inputs can generate neuroplastic changes in the central nervous system that reduce the inhibitory effects of the LC-NE complex and facilitate the development of chronic pain and frequent comorbidities, such as anxiety, depression or sleeping disturbances. The exact mechanisms involved in the LC functional shift remain unknown, but there is some evidence that they occur through plastic changes in the medial and lateral pathways and their brain projections. Additionally, there are other influencing factors, like developmental issues, neuroinflammatory glial changes, NE receptor affinity and changes in LC neuronal firing rates.

Cytomorphological characteristics of low-grade papillary urothelial carcinoma in voided urine samples: Distinction from benign and high-grade papillary urothelial carcinoma.

Given its frequent recurrence and the potential for high-grade transformation, accurate diagnosis of low-grade papillary urothelial carcinoma (LGPUC) in urine cytology is clinically important. We attempted to identify cytomorphologic features in urine samples, which could be helpful for the identification of LGPUC. We conducted a retrospective review of voided urine specimens collected from patients with histopathologic diagnoses of LGPUC. Their cytomorphological features were compared with those from patients with benign conditions and high-grade papillary urothelial carcinoma (HGPUC). A total of 115 voided urine specimens were evaluated, including 30 benign, 41 LGPUC, and 44 HGPUC cases. In LGPUC, 18 cases (44%) were diagnosed as atypical, a proportion significantly higher than that observed in benign cases (4 cases, 13%), while the remaining 23 cases (56%) were diagnosed as negative. LGPUC urine samples tended to have higher cellularity than benign cases, but the difference was not statistically significant. Three cytological features, namely nuclear enlargement, higher nuclear-to-cytoplasmic (N/C) ratio, and presence of small cell clusters, were statistically more prevalent in LGPUC compared to benign cases, although the changes were relatively subtle. In contrast, cytomorphological distinction between LGPUC and HGPUC was evident, as high cellularity, nuclear enlargement, hyperchromasia, high N/C ratio, irregular nuclear membrane, and apoptosis were significantly more prevalent in HGPUC cases. Several cytomorphologic features in voided urine samples were more prevalent in cases with LGPUC, albeit not observed in all instances. Since these alterations were relatively subtle, meticulous attention to these cytomorphologic details is crucial to suggest the possibility of LGPUC.

Adolescent BCG revaccination induces a phenotypic shift in CD4+ T cell responses to Mycobacterium tuberculosis

A recent clinical trial demonstrated that Bacille Calmette-Guérin (BCG) revaccination of adolescents reduced the risk of sustained infection with Mycobacterium tuberculosis (M.tb). In a companion phase 1b trial, HVTN 602/Aeras A-042, we characterize in-depth the cellular responses to BCG revaccination or to a H4:IC31 vaccine boost to identify T cell subsets that could be responsible for the protection observed. High-dimensional clustering analysis of cells profiled using a 26-color flow cytometric panel show marked increases in five effector memory CD4+ T cell subpopulations (TEM) after BCG revaccination, two of which are highly polyfunctional. CITE-Seq single-cell analysis shows that the activated subsets include an abundant cluster of Th1 cells with migratory potential. Additionally, a small cluster of Th17 TEM cells induced by BCG revaccination expresses high levels of CD103; these may represent recirculating tissue-resident memory cells that could provide pulmonary immune protection. Together, these results identify unique populations of CD4+ T cells with potential to be immune correlates of protection conferred by BCG revaccination.

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 ( &amp;gt;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&amp;lt; 0.01). 55% of BRCA2 ROIs had &amp;gt;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.

An Assessment of the Mechanophysical and Hormonal Impact on Human Endometrial Epithelium Mechanics and Receptivity.

The endometrial epithelium and underlying stroma undergo profound changes to support and limit embryo adhesion and invasion, which occur in the secretory phase of the menstrual cycle during the window of implantation. This coincides with a peak in progesterone and estradiol production. We hypothesized that the interplay between hormone-induced changes in the mechanical properties of the endometrial epithelium and stroma supports this process. To study it, we used hormone-responsive endometrial adenocarcinoma-derived Ishikawa cells growing on substrates of different stiffness. We showed that Ishikawa monolayers on soft substrates are more tightly clustered and uniform than on stiff substrates. Probing for mechanical alterations, we found accelerated stress-relaxation after apical nanoindentation in hormone-stimulated monolayers on stiff substrates. Traction force microscopy furthermore revealed an increased number of foci with high traction in the presence of estradiol and progesterone on soft substrates. The detection of single cells and small cell clusters positive for the intermediate filament protein vimentin and the progesterone receptor further underscored monolayer heterogeneity. Finally, adhesion assays with trophoblast-derived AC-1M-88 spheroids were used to examine the effects of substrate stiffness and steroid hormones on endometrial receptivity. We conclude that the extracellular matrix and hormones act together to determine mechanical properties and, ultimately, embryo implantation.

Abstract 2281: A tumor-intrinsic signature involving immunosuppression via MIF-CD74 signaling is associated with overall survival in ICT-treated lung adenocarcinoma

Abstract Introduction: Immune checkpoint therapies (ICT) have changed cancer care, yielding robust and durable responses in a subset of patients. Identifying patients who are likely to respond to ICT remains an ongoing challenge. In addition, only a portion of patients with clinical biomarkers respond to therapy. Signatures of RNA expression have been developed to predict response, the majority of which focus on T-cell and cytotoxicity markers, yet have been unable to substantially improve outcome predictions. Here, we present a RNA signature that instead describes tumor-intrinsic immune resistance and a potential mechanism of immunosuppression via tumor signaling on macrophages, derived from single-cell RNA-sequencing (scRNA-seq). Methods: We performed dimensionality reduction using a Variational Autoencoder (VAE) on 30 scRNA-seq samples from 15 lung adenocarcinoma (LUAD) patients, comprising a total of 183,873 cells from the Cell Ranger pipeline. The VAE model was trained on each sample for 250 iterations, yielding 20 signatures from each patient, for a total of 300 signatures. The relationship of each signature with real-world overall survival (rwOS) across 1,983 bulk RNA-sequenced LUAD patients treated with an FDA approved ICT was assessed via a Cox proportional hazards model with risk set adjustment, using TMB and line of therapy as covariates. The NATMI python package was used to identify putative active ligand-receptor interactions between tumor cells and the immune environment. Results: Among the VAE encodings, we identified an immune resistance signature significantly associated with decreased rwOS across the patient cohort (HR = 4.2 [2.8-6.3], adjusted p = 4.3e-10). This signature was derived from a small cluster of neoplastic cells (4.4% of cells) in a patient sample that was otherwise dominated by immune cells, including a substantial fraction of cytotoxic CD8 T cells (24.5%). The strongest predicted ligand-receptor interactions were found between the neoplastic cells and macrophages, via the MIF-CD74 interaction, an interaction we found upregulated in multiple single-cell tumor samples. Further, MIF RNA expression alone was significantly associated with rwOS across the 1,983 patient LUAD cohort (HR = 1.5 [1.1-2.1], p = 0.004). Discussion: MIF-CD74 is a known immunosuppressive interaction and MIF signaling from tumor cells on macrophages has been previously shown to have immunosuppressive effects in a mouse model of melanoma that were largely reversible via MIF-CD74 blockade. Taken together, these results identify a signature of tumor intrinsic immune suppression that can indicate patients likely to experience reduced benefit from ICT. In addition, this signature provides evidence to support blockade of the MIF-CD74 axis as a means to enhance anti-tumor immune responses in LUAD. Citation Format: Rossin Erbe, Michelle M. Stein, Tim A. Rand, Justin Guinney. A tumor-intrinsic signature involving immunosuppression via MIF-CD74 signaling is associated with overall survival in ICT-treated lung adenocarcinoma [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 2281.

Abstract 3764: Spatially resolved immune landscape revealed sex-biased T-cell infiltration in glioblastoma

Abstract Glioblastoma (GBM) multiforme is the most aggressive type of brain cancer. These lethal brain tumors are characterized by the inter- and intra-tumor molecular heterogeneity and demonstrated cell plasticity. A male:female distribution of 1.6:1; survival outcome from treatment (debulking, radiation, and temozolomide) benefits females nearly twice that for males. Through spatial transcriptomics, we interrogated the immune landscape of GBM, seeking to tease out whether distinct biological and therapeutic niches in GBM were evident, and to understand how the tumor microenvironments contributed to the heterogeneity of GBM. We designed a set of RNA hybridization probes specifically for portraying the cell identities and cell functional states within GBM tumor. The probe set was used to profile six human GBM samples from male and female cohorts on the Vizgen MERSCOPE platform. An integrated analysis of 505,614 cells from the six samples yielded a rich and varied spatial cell atlas of glioblastoma. Our preliminary findings depict distinct spatial niches of different tumor cell subpopulations that are associated with local environmental factors such as hypoxia. We identified a small cluster of cells that are in neuronal-like state, which could potentially drive invasion. We found spatial proximity of CD4+ cells and cells that express HIF1A, suggesting the dynamic interactions between tumor cells, regulatory T cells and tumor microenvironment. We also observed significant difference in the degree of CD8+ T-cell infiltration in female samples versus male samples. The average CD4+/CD8+ T-cell ratio in males is twice as high as that of females, suggesting intrinsic sex-based differences in immune response to tumor. Expansion of these studies in additional specimens will further illuminate microenvironmental influences on glioma progression and may instruct specific interventions leading to more effective and predictable immune-oncology of this disease. Citation Format: Yue Hao, George Reid, Angela Baker, Karen L. Fink, George J. Snipes, Bruce E. Mickey, Andrew E. Sloan, Jill S. Barnholtz-Sloan, Michael Berens. Spatially resolved immune landscape revealed sex-biased T-cell infiltration in glioblastoma [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 3764.

Renewable microgeneration cooperation with base station sleeping-mode strategy for energy-efficient operation of 5G infrastructures

The energy consumption of the mobile network is becoming a growing concern for mobile network operators and it is expected to rise further with operational costs and carbon footprints due to the massive deployment of small cell base stations in 5G and beyond 5G networks. Renewable energy harvesting has proved its extraordinary potential in green mobile communication to reduce energy costs and carbon footprints. However, the stochastic behavior of renewable energy generation and traffic load makes the reliable operation of mobile networks challenging. Therefore, this paper proposes an energy-sustainable framework of cooperative microgeneration energy power supplies for nearby clusters of small cells to maximize the utilization of sustainable energy by intra-microgrid and inter-microgrid energy cooperation. In addition, a resource-on-demand approach of small cells based on advanced sleep modes is integrated with energy cooperation to optimize energy saving and the techno-economic feasibility of microgrids. The proposed solution of microgeneration energy cooperation framework with a resource-on-demand strategy optimally shares surplus energy between microgrids via physically deployed resistive transmission lines under a practical energy loss model and restrains the mismatch between traffic arrival and energy production. Exhaustive simulation is performed to examine the optimal system performance, carbon emissions performance, energy savings, and cost assessment. Results suggest that the proposed solution performs effectively in terms of cost, emissions, and energy savings.

Lymph Node Isolated Tumor Cells in Patients With Endometrial Cancer

Isolated tumor cells (ITCs) are the histopathological finding of small clusters of cancer cells no greater than 0.2 mm in diameter in the regional lymph nodes. For endometrial cancer, the prognostic significance of ITCs is uncertain. To assess clinico-pathological characteristics and oncologic outcomes associated with ITCs in endometrial cancer. This retrospective cohort study using the National Cancer Database included patients with endometrial cancer who had primary hysterectomy and nodal evaluation from 2018 to 2020. Patients with microscopic and macroscopic nodal metastases and distant metastases were excluded. Data were analyzed from June to September 2023. Regional nodal status with ITCs (N0[i+] classification) or no nodal metastasis (N0 classification). (1) Clinical and tumor characteristics associated with ITCs, assessed with multivariable binary logistic regression model, and (2) overall survival (OS) associated with ITCs, evaluated by nonproportional hazard analysis with restricted mean survival time at 36 months. A total of 56 527 patients were included, with a median (IQR) age of 64 (57-70) years. The majority had T1a lesion (37 836 [66.9%]) and grade 1 or 2 endometrioid tumors (40 589 [71.8%]). ITCs were seen in 1462 cases (2.6%). In a multivariable analysis, ITCs were associated with higher T classification, larger tumor size, lymphovascular space invasion (LVSI), and malignant peritoneal cytology. Of those tumor factors, LVSI had the largest association with ITCs (7.9% vs 1.4%; adjusted odds ratio [aOR], 4.37; 95% CI, 3.87-4.93), followed by T1b classification (5.3% vs 1.3%; aOR, 2.62; 95% CI, 2.30-2.99). At the cohort level, 24-month OS rates were 94.3% (95% CI, 92.4%-95.7%) for the ITC group and 96.1% (95% CI, 95.9%-96.3%) for the node-negative group, and the between-group difference in expected mean OS time at 36 months was 0.35 (SE, 0.19) months, but it was not statistically significant (P = .06). There was a statistically significant difference in OS when the low-risk group (stage IA, grade 1-2 endometrioid tumors with no LVSI) was assessed per nodal status and adjuvant therapy use (P < .001): (1) among the cases treated with surgical therapy alone, 24-month OS rates were 95.9% (95% CI, 89.5%-98.5%) for the ITC group and 98.8% (95% CI, 98.6%-99.0%) for the node-negative group, and the between-group mean OS time difference at 36 months was 0.61 (SE, 0.43) months (P = .16); and (2) among the cases with ITCs, adjuvant therapy (radiotherapy alone, systemic chemotherapy alone, or both) was associated with improved survival compared with no adjuvant therapy (24-month OS rates, 100% vs 95.9%; between-group mean OS time difference at 36 months, 0.95 [SE, 0.43] months; P = .03). In this cohort study of patients with surgically staged endometrial cancer, the results of exploratory analysis suggested that presence of ITCs in the regional lymph node may be associated with OS in the low-risk group. While adjuvant therapy was associated with improved OS in the low-risk group with ITCs, careful interpretation is necessary given the favorable outcomes regardless of adjuvant therapy use. This hypothesis-generating observation in patients with low-risk endometrial cancer warrants further investigation, especially with prospective setting.

MED15::TFE3 fusion renal cell carcinoma with extensive cystic change: A clinicopathologic and molecular genetic study of 2 cases, with an emphasis on differential diagnosis.

TFE3-rearranged renal cell carcinomas (RCCs) harbor gene fusions between TFE3 and 1 of many partner genes. MED15::TFE3 fusion RCC is rare, often cystic, and easily misdiagnosed. This study aimed to characterize 2 cases of MED15::TFE3 fusion RCC with extensive cystic change using fluorescence in situ hybridization and targeted RNA sequencing. Both patients were young adult women aged 29 and 35 years. Radiologically, both presented with a cystic Bosniak category II renal lesion. The cysts measured 9.3 cm and 4.8 cm in greatest dimension. Both patients underwent cyst enucleation, and neither had tumor recurrence or metastasis at 26 and 6 months of follow-up, respectively. Microscopically, both tumors were entirely cystic, with thick, fibrous cystic walls lined by small clusters of cells with clear to eosinophilic cytoplasm and uniform, round nuclei with inconspicuous nucleoli. There were also small aggregations of similar clear cells within the cystic walls. Foci of basement membrane-like material depositions were noted in 1 case; calcifications were observed in both cases. Both cases demonstrated nuclear positivity for PAX8 and TFE3 and cytoplasmic staining for Melan-A; HMB45, CAIX, and CK7 were negative. Fluorescence in situ hybridization revealed that both tumors were positive for TFE3 rearrangements. RNA sequencing identified MED15::TFE3 gene fusions in both cases. The main differential diagnosis of MED15::TFE3 fusion RCC includes multilocular cystic renal neoplasm of low malignant potential and atypical renal cysts. Molecular confirmation of TFE3 fusion is essential for establishing the correct diagnosis.