Cells In Microenvironment Research Articles

Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer.

Inducing ferroptosis in tumor cells is emerging as a strategy for treating malignancies that are refractory to traditional treatment modalities. However, the consequences of ferroptosis of immune cells in the tumor microenvironment (TME) need to be better understood in order to realize the potential of this approach. In this study, we discovered that neutrophils in chemoresistant breast cancer are highly sensitive to ferroptosis. Reduction of the acyltransferase MBOAT1 in chemoresistance-associated neutrophils induced phospholipid reprogramming, switching the preference from monounsaturated fatty acids to polyunsaturated fatty acids, which increased their susceptibility to ferroptosis. Ferroptotic neutrophils secreted PGE2, IDO and oxidized lipids that suppressed the proliferation and cytotoxicity of antitumor CD8+ T cells. Furthermore, neutrophil ferroptosis was closely related to a distinct subset of IL-1beta+CXCL3+CD4+ (Fer-CD4) T lymphocytes, which were enriched in chemoresistant tumors. Fer-CD4 T cells orchestrated neutrophil ferroptosis by modulating MBOAT1 expression via IL-1beta/IL-1R1/NF-kappaB signaling. Moreover, Fer-CD4 T cells secreted CXCL3, IL-8 and S100A9 to replenish the neutrophil pool in the TME. Ferroptotic neutrophils in turn fostered Fer-CD4 T cell differentiation. In spontaneous tumorigenesis mouse models, targeting IL-1beta+ CD4+ T cells or IL-1R1+ neutrophils broke the crosstalk, restraining neutrophil ferroptosis, enhancing antitumor immunity, and overcoming chemoresistance. Overall, these findings uncover the role of neutrophil ferroptosis in shaping the immune landscape and propose appealing targets for restoring immunosurveillance and chemosensitivity in breast cancer.

Targeting of tumoral NAC1 mitigates myeloid-derived suppressor cell-mediated immunosuppression and potentiates anti-PD-1 therapy in ovarian cancer.

Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer with a low rate of response to immunotherapy such as immune checkpoint blockade (ICB) therapy. Here, we report that nucleus accumbens-associated protein 1 (NAC1), a putative driver of EOC, has a critical role in immune evasion. We showed in murine ovarian cancer models that depleting or inhibiting tumoral NAC1 reduced the recruitment and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME), led to significant increases of cytotoxic tumor-infiltrating CD8+ T cells, and promoted antitumor immunity and suppressed tumor progression. We further showed that tumoral NAC1 directly enhanced the transcription of CXCL16, by binding to CXCR6, thereby promoting MDSC recruitment to the tumor. Moreover, lipid C20:1T produced by NAC1-expressing tumor cells fueled oxidative metabolism of MDSCs and promoted their immune-suppressive function. We also showed that NIC3, a small molecule inhibitor of NAC1, was able to sensitize mice-bearing NAC1-expressing ovarian tumors to anti-PD-1 therapy. Our study reveals a critical role for NAC1 in controlling tumor infiltration of MDSCs and in modulating the efficacy of ICB therapy. Thus, targeting of NAC1 may be exploited to sensitize ovarian cancer to immunotherapy.

ScDrugAct: a comprehensive database to dissect tumor microenvironment cell heterogeneity contributing to drug action and resistance across human cancers.

The transcriptional heterogeneity of tumor microenvironment (TME) cells is a crucial factor driving the diversity of cellular response to drug treatment and resistance. Therefore, characterizing the cells associated with drug treatment and resistance will help us understand therapeutic mechanisms, discover new therapeutic targets and facilitate precision medicine. Here, we describe a database, scDrugAct (http://bio-bigdata.hrbmu.edu.cn/scDrugAct/), which aims to establish connections among drugs, genes and cells and dissect the impact of TME cellular heterogeneity on drug action and resistance at single-cell resolution. ScDrugAct is curated with drug-cell connections between 3838 223 cells across 34 cancer types and 13 857 drugs and identifies 17 274 drug perturbation/resistance-related genes and 276 559 associations between>10 000 drugs and 53 cell types. ScDrugAct also provides multiple flexible tools to retrieve and analyze connections among drugs, genes and cells; the distribution and developmental trajectories of drug-associated cells within the TME; functional features affecting the heterogeneity of cellular responses to drug perturbation and drug resistance; the cell-specific drug-related gene network; and drug-drug similarities. ScDrugAct serves as an important resource for investigating the impact of the cellular heterogeneity of the TME on drug therapies and can help researchers understand the mechanisms of action and resistance of drugs, as well as discover therapeutic targets.

TMIC-48. THREE DIMENSIONAL CANCER CELL-MONOCYTE CO-CULTURE SPHEROIDS RECAPITULATE IMMUNOSUPPRESSIVE AND PRO-TUMORIGENIC MICROENVIRONMENTS IN BRAIN METASTATIC BREAST CANCER

Abstract Brain metastasis occurs in 15 % of breast cancer patients and is one of the major causes of breast cancer related morbidity. Brain metastatic breast cancer (BMBC) microenvironment is a heterogeneous niche of cellular and acellular components of which the innate immune cells, microglia, and macrophages, comprise the major fraction. However, the role of these innate immune cells in the tumor microenvironment (TME) in mediating cancer progression and response to therapy is not well understood. To address this need, herein, we developed three dimensional (3D) co-culture spheroids using brain metastatic triple negative breast cancer (TNBC) cell line MDA-MB-231Br or patient derived brain metastatic TNBC cell line F2-7 with THP1 monocytes. Under co-culture conditions MDA-MB-231Br co-culture spheroids assumed highly protrusive morphology with enhanced mesenchymal and proliferative characteristics. Additionally, phenotypic determination of cocultured macrophages revealed elevated levels of markers associated with alternatively activated macrophages i.e. CD163, CD206, and c-Myc. Furthermore, we found that under coculture conditions immunosuppressive and evasive markers PD1, PDL1, CD47 and CD24 were significantly upregulated. Similarly, F2-7 and monocyte cocultures also led to an immunosuppressive environment with elevated levels of M2 type markers and associated anti-inflammatory cytokines. We further found that the presence of M2 type macrophages in the microenvironment of BMBC spheroids led to acquired resistance against Paclitaxel, a clinically relevant chemotherapeutic drug against BMBC. In sum, in our coculture conditions, brain metastatic TNBC cells polarized monocytes into immunosuppressive and pro-tumorigenic tumor associated macrophages leading to a pro-tumoral microenvironment. Thus, 3D co-culture spheroids of BMBC cells and monocytes can serve as robust platform for studying cancer cell and innate immune cell interactions occurring in the BMBC environment.

CNSC-27. INTRATUMORAL CANCER CELL NETWORKS IN BRAIN METASTASES

Abstract Recently, we have shown that primary brain tumors form highly organized, small-world scale-free networks, reminiscent of early networks in neurodevelopment and highly resistant to therapy. Such putative cancer-cell intrinsic neural mechanisms are becoming an increasing focus in the field of Cancer Neuroscience, but it has not yet been shown whether tumors of non-CNS origin also have the ability to form multicellular networks which sustain proliferative signaling and increase resistance to therapy. We observed synchronized calcium activity using longitudinal microscopy both in vivo and in vitro that were reminiscent of gap junction coupled networks. Indeed, dye transfer experiments verified functional coupling of brain metastases cells in vitro. Similarly, inhibition of gap junctions with different pharmacological agents significantly reduced calcium oscillations and tumour proliferation in vitro. Furthermore, gap junctions are significantly upregulated in brain-tropic sublines compared to parental cells. To understand the functional relevance of these gap-junction coupled tumor cell networks, we performed bulk RNA-Sequencing of two melanoma metastasis models in monoculture under gap junction inhibition and control: We observed reduced calcium communication and a concordant downregulation of neurodevelopmental and synaptic pathways within the tumor cells upon network disconnection, highlighting a potential recapitulation of neural-like features in metastases to the brain. To investigate the therapeutic potential of targeting these networks, we treated mice with brain metastases with two brain-penetrant gap junction blockers and observed significantly reduced tumor burden. Finally, we investigated the presence of heterotypic networks between brain metastases cells and the brain microenvironment with our SR101 dye transfer pipeline. Contrary to our findings in primary brain tumors, no gap junction connections to cells of the brain microenvironment in those models with reduced tumor growth upon network inhibition could be detected, supporting a cancer cell intrinsic gap junction mediated network and highlighting a selectivity of these homogenous tumor networks and potential therapeutic vulnerability.

CNSC-44. TARGETING OF THE YAP/TAZ-TEAD AXIS DIMINISHES VIABILITY AND DIFFERENTIATION POTENTIAL IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract Pediatric high-grade gliomas (pHGGs) are invasive and fast-growing brain malignancies. They account for up to 40% of all brain tumor-related deaths in childhood. Genetic alterations that characterize these gliomas include H3 onco-histone mutations and genetic alterations in receptor-tyrosine kinases (RTKs), including amplification, mutation, and gene fusions in RTKs. However, direct targeting with tyrosine kinase inhibitors (TKIs) has shown at best partial responses in patients due to the molecular heterogeneity within these tumors and the lack of pharmaceutical penetration into the central nervous system (CNS). Thus, alternative therapeutic approaches to treat pHGGs are urgently needed. The YAP/TAZ transcription factors, which cooperate with TEAD co-factors to promote target gene expression, orchestrate stem-cell fate decisions and proliferation downstream of growth factor signaling pathways, such as RTKs. We observed that YAP expression is upregulated in RTK mutant and H3 mutant pHGGs. Depletion of YAP via RNAi methods decreased proliferation, inhibited pHGG stem cell self-renewal, and induced apoptosis of pHGG cells. Use of Verteporfin (VP), a FDA-approved YAP/TAZ small molecule inhibitor, mimicked the effects seen by YAP genetic depletion. Using an in vitro human cortical organoid-tumor model in which tumor cells are engrafted to human brain cortical organoids, we screened for responses in tumor cells and in non-tumor cells in the neural microenvironment upon VP treatment, using single-cell RNA-sequencing and immunofluorescence to visualize responses. These organoid models offer improved models for modeling how the neural CNS microenvironment shapes responses of tumor cells to YAP/TAZ inhibition and VP treatments. Results from our work showing that YAP/TAZ-TEAD inhibition is a promising therapeutic avenue for management of pHGGs will be presented. Further pre-clinical work will follow to correlate observation and understand the therapeutic efficacy of YAP/TAZ-TEAD inhibition for pHGG treatment.

EXTH-11. PEDIATRIC GLIOMA IMMUNE PROFILING IDENTIFIES TIM3 AS A THERAPEUTIC TARGET IN BRAF-FUSION PILOCYTIC ASTROCYTOMA

Abstract Despite being the leading cause of childhood mortality, pediatric gliomas have been relatively understudied. Repurposing of adult brain tumor immunotherapies in pediatric patients has not been successful, highlighting the need for pediatric-specific immunotherapies. Pilocytic astrocytomas (PA) are the most common pediatric glioma. While surgical resection of PA is the first-line treatment, the ten-year progression-free survival rate for patients with residual tumors is <50%. Notably, some PAs undergo spontaneous regression after partial resection, suggesting potential baseline tumor immunoreactivity. Whole transcriptome sequencing performed on a commercial platform of pediatric gliomas (n=250) showed that MAPK-driven gliomas have higher interferon signatures compared to other molecular subgroups. Single-cell sequencing identified a unique activated and cytotoxic microglia population designated MG-Act in BRAF-fused MAPK-activated PA (n=13; p < 0.05), but not in pediatric high-grade gliomas (n=5) or normal brain (n=3). TIM3 was expressed on the luminal side of endothelial cells and MG-Act, but not in the surrounding normal brain based on sequential multiplex imaging. Flow cytometry showed that TIM3 intensity is upregulated on immune cells in the PA tumor microenvironment (TME) relative to matched peripheral blood immune cells (n=6; p < 0.01). Anti-TIM3 reprogrammed fresh ex vivo immune cells from the TME of human PAs (n=3) to a pro-inflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK-driven low-grade gliomas, median survival (MS) was 252 days in mice treated with anti-TIM3 (n=21; p<0.01) relative to IgG control (MS: 110 days; n=20) or anti-PD-1 (MS: 134 days; n=20). The therapeutic activity of anti-TIM3 was abrogated in the CX3CR1 microglia knockout background. ScRNA sequencing data during the therapeutic window of anti-TIM3 in wild-type mice demonstrated enrichment of the MG-Act population within low-grade gliomas at two different longitudinal time points but not in IgG-treated controls. Together, these data indicate that anti-TIM3 should be considered for clinical trials in pediatric low-grade MAPK-driven gliomas.

EXTH-76. REDIRECTING STROMAL CELLS TO IGNITE ANTI-TUMOR IMMUNITY AGAINST GLIOBLASTOMA

Abstract BACKGROUND The dismal prognosis of high-grade gliomas demands novel treatment approaches. Through the development of a novel mRNA lipid particle aggregate (RNA-LPA) vaccine, our lab has shown anti-tumor efficacy in preclinical murine studies and promising immunological responses in canine glioma models and human studies (Cell. 2024 May 9;187(10):2521-2535.e21). The translational potential necessitates further investigating the immune activation mechanism to allow greater applicability of this therapy in the future. OBJECTIVE We sought to unravel mechanistic cross-talk for immune initiation following intravenous administration of RNA-LPAs. METHODS We used Ai14 reporter mice to determine RNA-LPA transfected cellular subsets and dual reporter mice to assess cellular infiltration into murine glioma models. Murine gliomas were generated via implantation of K2 or KR158b-luc cell lines. RESULTS Following intravenous administration, we found fibroblastic reticular cells in secondary lymphoid organs take up RNA-LPAs. We show that FRCs have direct contact with antigen presenting cells and mediate release of full-length proteins following RNA-LPA transfection. Similar to the periphery, there is transfection of stromal cells in the brain tumor microenvironment (TME) and recruitment of infiltrating leukocytes. In response to RNA-LPAs, transfected stromal cells release type I interferon (IFN-I), which is requisite for anti-tumor efficacy. IFN-I mediates upregulation of CD69 on lymphocytes leading to sequestration following co-localization with RNA-LPAs. IFN-I is necessary for LFA-I expression on T cells, which is important for T cell trafficking across the blood-brain barrier. In preclinical models to track antigen specific immunity, we find systemic RNA administration elicits an increase in tumor reactive T cells with decreased S1P1 expression suggestive of a ‘locked-in’ response within the TME. CONCLUSION While stromal cells are regulatory niches in glioblastoma ecosystems, we show they can be leveraged for immunologic cross-talk that facilitates T cell recruitment, trafficking and sequestration in the TME critical for immunologic reprogramming and long-lasting immunotherapy.

TMOD-20. RAPID PROCESSING AND BIOBANKING OF RESECTED BRAIN TUMOR TISSUE TO GENERATE PATIENT-DERIVED BRAIN TUMOR EXPLANT ORGANOIDS (GBOS) FOR PRECLINICAL STUDIES

Abstract Improved preclinical tools are urgently needed to translate new brain cancer treatments. Patient-derived brain tumor explant organoids (GBOs) offer promise for studying tumor cells in a relevant human tumor microenvironment and predicting patient responses to therapy. However, generating GBOs is time-consuming, costly, and technically challenging, hindering the creation of comprehensive biobanks covering the spectrum of brain tumor heterogeneity. This work presents a semi-automated method for producing GBOs, involving tumor tissue processing, size selection, and same-day cryopreservation, reducing generation time to less than 1 hour. We established a biobank of 24 GBOs from 33 samples (11 males and 13 females) with primary (n=13, GBO Yield (GY)>90%) and recurrent (n=5, GY>90%) glioblastomas, high-grade gliomas (HGG, n=2, GY≤50%), and low-grade gliomas (LGG, n=2 with GY>90%, n=2 with GY≤50%). Using GBO size and propidium iodide as readouts, we used this biobank to compare anticancer activities of novel idronoxil-conjugated benzopyran compounds (NX786, NX904/904E1) against Bortezomib (100% cell death reference). NX786 and NX904/904E1 reduced GBO growth (≥50%) in all primary glioblastoma and one (of two) recurrent glioblastoma GBOs with milder effects in LGG GBOs. Two (of four) primary glioblastoma, one (of two) recurrent glioblastoma, and one LGG GBOs exhibited significant cell death (>60%) in response to NX904/904E1. In contrast, NX786 induced cell death in one (of three) primary glioblastoma (34% cell death) and one (of two) recurrent glioblastoma (71% cell death) GBOs but not in LGG GBOs, together showcasing varied responses across different brain tumors. Highly-passaged GBOs lacking the non-malignant tumor microenvironment were more susceptible to these treatments, underlining the tumor microenvironment’s critical role in responses to anticancer agents. We provide a new method for efficient processing and cryopreserving GBOs, enabling the establishment of large biobanks for patient-specific preclinical drug testing across brain tumor subgroups in a clinically relevant human 3D model.

TMIC-73. SEX DIFFERENCES IN INTERFERON-GAMMA RESPONSES IN GLIOMA MICROENVIRONMENT

Abstract IFNγ is a cytokine with both pro- and anti-tumorigenic effects and its role in glioma immunotherapy resistance is not well understood. In the tumor microenvironment, T and NK cells produce IFNγ, which affects cellular phenotypes in most of the cancer and stromal cells since IFNγ receptor is ubiquitously expressed. IFNγ induces expression of molecules that confer resistance to immunotherapies, such as PDL1, PDL2, IDO1, and iNOS. The goal of this study is to elucidate cell type-specific responses to IFNγ in immune competent glioma models we developed that are resistant to immunotherapy. We intracranially injected male and female murine glioma cells into B6 and Ifng-/- honst mice to measure overall survival and perform immune phenotyping and single cell RNA-sequencing analyses. Ifnγ-/- hosts exhibited a shorter survival, and the proportion of GBM cells is higher in Ifng-/- hosts compared to wild type, indicating that the net effect of IFNγ signaling in glioma is anti-tumorigenic. In a recent study, we published that IFNγ-response pathway is enriched in the mesenchymal-like (MES-like) subtype of human GBM cells, compared to neutral-progenitor-like (NPC-like) and oligodendrocyte-progenitor-like (OPC-like) cells (Abdelfattah et al., 2022). To test our hypothesis that IFNγ signaling induces MES transition in glioma cells in vivo, we compared fractions of glioma cell subtypes in wildtype and Ifng-/- male and female hosts. In female Ifng-/- mice, MES-like GBM cell fraction was reduced while OPC-like fraction was increased, supporting our hypothesis. However, there was no difference in male hosts. Furthermore, gliomas grown in female B6 hosts showed higher infiltration of immune cells (CD45+), lymphocytes (CD3+) and fewer resident microglia cells (CD11b+CD45int), compared with Ifng-/- female hosts. In contrast, no significant difference in immune cell infiltration was observed between B6 vs Ifng-/- males. These results suggest an unanticipated and complex context-dependent role of IFNγ signaling in GBM, which include sex differences in its role in immune cell recruitment and MES transition in glioma cells.

TMIC-69. HUMAN ORGANOID TUMOR TRANSPLANTATION IDENTIFIES FUNCTIONAL GLIOBLASTOMA MICROENVIRONMENTAL COMMUNICATION MEDIATED BY PTPRZ1

Abstract Glioblastoma, the most aggressive form of primary brain cancer, is driven by both intrinsic cellular properties and extrinsic factors from the tumor microenvironment (TME). Here, we leverage our novel human organoid tumor transplantation (HOTT) system to explore how external cues modulate glioblastoma cell type specification, heterogeneity, and migration. Comparing the single-cell transcriptomes of HOTT and matched primary tumors, we found that HOTT recapitulates not only core features of major patient tumor cell types but also those of peritumor non-neoplastic cell types. This enables us to explore tumor-TME interactions and identify PTPRZ1, a receptor tyrosine phosphatase implicated in brain tumor migration, as a key mediator in intercellular communication. This finding was further confirmed by interrogation of published datasets from patients containing non-neoplastic microenvironmental cells. PTPRZ1 activation, resulting from the reactivation of developmental programs in both the tumor and its TME, highlights the relevance of the organoid system for modeling the GBM microenvironment. Moreover, HOTT uniquely allows for perturbations in either patient tumors or their microenvironments, or both, to verify PTPRZ1 function. Tumor knockdown of PTPRZ1 confirmed its role in promoting tumor migration and maintaining progenitor identity. Surprisingly, environmental PTPRZ1 knockdown in human cortical organoids before tumor transplantation inhibited tumor migration and promoted differentiation, even without direct tumor manipulation. Overall, this fundamental result demonstrates that a single treatment delivered to the tumor and its TME can exert opposite effects on the tumor. Consequently, it underscores the necessity of studying human glioblastoma within a human microenvironment context, such as HOTT, especially when evaluating therapeutic efficacy in GBM, as effective treatments must address both the tumor and its complex TME, rather than the tumor alone.

EPCO-26. LONGITUDINAL SINGLE-CELL ANALYSES IDENTIFY DRIVERS OF GENETIC, EPIGENOMIC, AND CELLULAR EVOLUTION IN IDH-MUTANT GLIOMA

Abstract Despite standard treatment, IDH-mutant gliomas inevitably recur. Previous studies suggest that therapeutic resistance may result from a combination of intratumoral cellular heterogeneity, epigenetic evolution, and acquired genetic alterations. However, how these multilayered molecular features interact to influence the evolutionary paths of IDH-mutant gliomas remains incompletely understood. To chart this evolution, 75 glioma samples were longitudinally collected from 35 patients (n = 13 oligodendroglioma, n = 22 astrocytoma) and profiled using single nucleus RNA sequencing, ATAC sequencing, and bulk DNA sequencing. Analysis of 331,016 nuclei (snRNA) revealed eight tumor microenvironment cell types and five pan-IDH-mutant malignant cellular states. The malignant states were distributed along a cellular hierarchy of stem-like populations (neural progenitor cell-like, oligodendrocyte progenitor cell-like, and undifferentiated) and more differentiated populations (mesenchymal-like and astrocyte-like) with cycling cells enriched in the stem-like populations. Joint open chromatin accessibility data (snATAC) was available for a subset of malignant cells (71,088 nuclei) and state-specific differentially accessible peaks supported that these states are epigenetically encoded. Across both subtypes, higher tumor grade was associated with reduced malignant cell differentiation and increased cycling populations. Between the two time points, we identified that there was a longitudinal increase in the cycling, undifferentiated, and mesenchymal-like populations with a corresponding decrease in the astrocyte-like population. Longitudinal genetic analysis revealed that 19 of 35 tumors acquired at least one of the following genetic alterations: treatment-associated hypermutation, cell cycle alteration, or large changes in copy number alterations. Importantly, tumors that acquired these key genetic alterations demonstrated significant shifts towards reduced differentiation and increased cycling populations while those tumors that did not had more stable malignant profiles. Collectively, our results suggest a common cellular hierarchy across IDH-mutant gliomas with a shift towards reduced differentiation, increased cycling populations during disease progression that is driven by acquired genetic alterations.

IMMU-36. MULTI-OMIC CHARACTERIZATION OF A SYNGENEIC MOUSE CANCER STEM CELL MODEL OF GBM FOR PRECLINICAL APPLICATIONS

Abstract Glioblastoma (GBM) is resistant to many therapies including immunotherapies. There is an urgent need to explore the role of the microenvironment in resistance to therapy with tailored pre-clinical models. Using multi-omic approaches we describe a syngeneic cancer stem cell mouse model of GBM, with a spontaneous amplification of Igf2. We investigate whether Igf2 influences tumour and microenvironment cells to promote immunosuppression in GBM. Three cell lines were previously established from spontaneous brain tumours in C57Bl/6 Trp53+/-/Nf1+/- mice and maintained under neural stem cell culture conditions. Whole genome sequencing (WGS) was used to determine single nucleotide, copy number, and structural variation, revealing loss of both copies of Trp53 and Nf1 in all three cell lines. However only one cell line, mBT0309, developed tumors when orthotopically allografted. Analysis of genomic alterations and transcriptomics revealed that mBT309 exhibits amplification of the Igf2 loci and overexpression was confirmed at the RNA and protein level. Spatial and single cell transcriptomics showed that Igf2 is overexpressed in mBT0309 allografted tumours and correlated with specific transcriptomic programs. A high-parameter Imaging Mass Cytometry (IMCTM) panel was used for spatial proteomic analysis, to monitor the development of tumours in a time-course experiment. In vitro growth characteristics and stem marker expression in both transcriptomic and spatial IMC data suggest that high levels of Igf2 may regulate GBM stemness features. Analysis of human GBM datasets revealed that Igf2 amplified tumours have reduced CXCL11 expression. These findings suggest that high levels of Igf2 may influence immunosuppression by reducing T-cell recruitment. This syngeneic immunocompetent GBM stem cell model harbouring Igf2 amplification adds to the suite of models to study the GBM microenvironment and its role in immunosuppression and resistance to therapy.

Re-irradiation plus pembrolizumab: Phase II study for recurrent glioblastoma patients.

Radiation therapy may enhance anti-tumor immune responses by several mechanisms including induction of immunogenic cell death. We performed a phase 2 study of pembrolizumab with re-irradiation in patients with recurrent glioblastoma. Sixty recurrent glioblastoma patients received pembrolizumab with re-irradiation alone (cohort A, bevacizumab-naïve; n=30) or with bevacizumab continuation (cohort B, n=30). Dual primary endpoints including overall response rate (ORR) and overall survival at either 12 (OS-12; cohort A) or six months (OS-6; cohort B) were assessed per cohort relative to historical benchmarks. Paired paraffin-embedded formalin-fixed tumor samples were assessed for immunologic biomarkers by immunohistochemistry using digital quantification and Co-Detection-by-InDEXing (CODEX). Study therapy was well tolerated with most toxicities being grade ≤ 3. For cohort B, the primary endpoint of OS-6 was achieved (57%), however survival was not improved for cohort A patients. The ORR was 3.3% and 6.7% for cohorts A and B, respectively. CODEX analysis of paired tumor samples from 5 patients revealed an increase of activated T cells in the tumor microenvironment after study therapy. Compared to historical controls, re-irradiation plus pembrolizumab appeared to improve survival among bevacizumab-refractory patients but not among bevacizumab-naïve patients. CODEX revealed evidence of intratumoral infiltration of activated immune effector cells. A randomized, properly controlled trial of PD-1 blockade plus re-irradiation is warranted to further evaluate this regimen for bevacizumab refractory patients, but alternative approaches are needed for bevacizumab-naïve patients.

Profiling the cancer-prone microenvironment in a zebrafish model for MPNST.

Microenvironmental contributions to soft tissue sarcoma progression are relatively undefined, particularly during sarcoma onset. Use of animal models to reveal these contributions is impeded by difficulties in discriminating between microenvironmental, precancerous, and cancer cells, and challenges in defining a precancerous microenvironment. We developed a zebrafish model that allows segregation of microenvironmental, precancerous, and cancerous cell populations by fluorescence-activated cell sorting. This model has high predilection for malignant peripheral nerve sheath tumor (MPNST), a type of soft tissue sarcoma that exhibits rapid, aggressive growth. Using RNA-seq, we profiled the transcriptomes of microenvironmental, precancerous, and cancer cells from our zebrafish MPNST model. We show broad activation of inflammation/immune-associated signaling networks, describe gene expression patterns that uniquely characterize the transition from precancerous to cancer ME, and identify macrophages as potential contributors to microenvironmental phenotypes. We identify conserved gene expression changes and candidate genes of interest by comparative genomics analysis of MPNST versus benign lesions in both humans and zebrafish. Finally, we functionally validate a candidate extracellular matrix protein, periostin (POSTN), in human MPNST. This work provides insight into how the microenvironment may regulate MPNST initiation and progression.

Emerging Technologies for the Assessment of Natural Killer Cell Activity

Understanding natural killer (NK) cell functionality is essential in developing more effective immunotherapeutic strategies that can enhance patient outcomes, especially in the context of cancer treatment. This review provides a comprehensive overview of both traditional and novel techniques for evaluating NK cell functionality, focusing on multiparameter assays and spatial methods that illuminate NK cell interactions within their microenvironment. We discuss the significance of standardized assays for assessing NK cell function across various research and clinical settings, including cancer immunotherapy, infectious diseases, and transplantation. Key factors influencing NK cell functionality include the origin of the sample, target–effector ratios, the functional state of NK cells, and the impact of pre-treatment conditions and their natural aging effect on NK cell activity. By emphasizing the importance of selecting a suitable technique for reliable measurements, especially for longitudinal monitoring, this review aims to give an overview on techniques to measure NK cell functionality in vitro and show the interaction with their microenvironment cells by spatial imaging. Ultimately, our understanding of NK cell functionality could be critical to biomarker development, drug design, and understanding of disease progression in the field of oncology or infectious disease.

Whispering Gallery Mode Micro/Nanolasers for Intracellular Probing at Single Cell Resolution.

Intracellular probing at single cell resolution is key to revealing the heterogeneity of cells, learning new cell subtypes and functions, understanding the pathophysiology of disease, and ensuring precise diagnosis and treatment. Despite the best efforts, an enormous challenge remains due to the very small size, extremely low content, and dynamic microenvironment of a single cell. Whispering gallery mode (WGM) micro/nanolasers (active WGM) offer unique advantages of small mode volume, high quality factors, bright and low threshold laser emission, and narrow line width, particularly suitable for integration within a single cell. In this review, we provide a focused overview of WGM micro/nanolasers for intracellular probing. We deliver information on WGM micro/nanolaser concepts, sensing mechanism, and biocompatibility, as well as recent progress in intracellular probing applications mainly covering cellular-level sensing, molecular-level detection, and feasibility for cellular imaging. At the end, challenges and prospects of WGM micro/nanolasers for intracellular applications are discussed.

Adipocytes reprogram the proteome of breast cancer cells in organotypic three-dimensional cell cultures.

While epidemiological evidence has long linked obesity with an increased risk of breast cancer, the intricate interactions between adipocytes and cancer cells within the tumor microenvironment remain largely uncharted territory. The use of organotypic three-dimensional (3D) cell cultures that more accurately mimic the spatial architecture of tumors represents an innovative approach to this complex issue. In the present study, we investigated the effects of adipocytes on the proteome of Hs578t breast cancer cells cultured in a 3D microenvironment. Using different treatments, we rigorously optimized the experimental conditions to induce the optimal differentiation of 3T3-L1 fibroblasts into mature adipocytes. Then, we grow the Hs578t cells in a simulated microenvironment using an on-top model for organotypic 3D cultures. Our data showed that cancer cells formed 3D stellate-like architectures when grown over an extracellular matrix proteins-enriched scaffold for 48h. Proteomic profiling using LC-MS/MS mass spectrometry of Hs578t cells grown in 3D conditions with or without the adipocyte-enriched culture discovered 916 unique proteins. Of these, 605 showed no significant changes in abundance, whereas 87 proteins were significantly upregulated and 224 downregulated after interaction with fat cells (p < 0.05, FC > 2.0). Bioinformatic analysis of upregulated proteins indicated that the most enriched GO terms and molecular functions were related to lipids transport, cell differentiation, hypoxia response, and cell junctions. In addition, several modulated proteins have been previously associated with breast cancer progression. Interestingly, lipid transport proteins, including PITPNM2, ATP2C1, ABCA12, HDLBP, and APOB, showed perturbations in their expression, which were also associated with low overall survival in breast cancer patients. Functional studies showed that the knockdown of apolipoprotein B (APOB) expression in Hs578t cells reduced the size of 3D cellular structures. Moreover, APOB-knocked cells cocultured with adipocytes for 48h exhibited a significant decrease of intracellular lipids, whereas an increase in the adipocytes was found. Our results indicate that the 3D microenvironment and the adipocytes crosstalk reprogram the proteome of breast cancer cells. These data help us understand the environmental effects in gene expression and contribute to discovering novel tumor proteins with potential intervention in breast cancer therapy.

Epigenetic Regulation of Immune Cells in Health and Disease: A Comprehensive Review

Epigenetic regulation plays a crucial role in the development, differentiation, and function of immune cells. Mechanisms such as DNA methylation, histone modifications, chromatin remodeling, and non-coding RNA interactions dynamically shape gene expression, enabling immune cells to adapt to environmental stimuli and mediate appropriate immune responses. Aberrant epigenetic regulation is implicated in a range of diseases, including autoimmune disorders, cancer, and chronic inflammatory conditions. In autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, altered DNA methylation and histone modifications contribute to immune dysregulation and tissue damage. In cancer, immune cells in the tumor microenvironment often exhibit epigenetic changes that facilitate immune evasion. Emerging therapeutic strategies targeting epigenetic pathways, including DNA methyltransferase inhibitors, histone deacetylase inhibitors, and CRISPR-based epigenome editing, offer promising approaches for treating immune-mediated diseases. This review provides a comprehensive overview of the epigenetic mechanisms governing immune cell behavior, their dysregulation in disease, and the potential of epigenetic therapies to restore immune homeostasis and improve patient outcomes. Keywords: Epigenetics, immune cells, DNA methylation, histone modifications, non-coding RNAs, autoimmune diseases, immunotherapy

FGL2172-220 peptides improve the antitumor effect of HCMV-IE1mut vaccine against glioblastoma by modulating immunosuppressive cells in the tumor microenvironment

ABSTRACT Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor characterized by poor prognosis and lack of effective treatments. In recent years, peptide vaccines that use sequences based on tumor-specific or tumor-associated antigens to activate immune responses against tumor cells have emerged as a new therapeutic strategy. In this study, we developed a novel therapeutic polypeptide vaccine targeting the tumor-associated antigen Fibrinogen-Like Protein 2 (FGL2), whose dominant epitope peptide was tandemly linked to the C-terminus of HCMV-IE1mut via a linker. We used this vaccine to compare the therapeutic efficacy of HCMV-IE1mut alone versus HCMV-IE1mut-FGL2172-220 and investigate the potential mechanism of action of HCMV-IE1mut-FGL2172-220 in glioma treatment. An in situ GBM model (GL261-IE1-luc cells) was used to determine the efficacy of the vaccine. Treatment with HCMV-IE1mut-FGL2172-220 exerted antitumor effects and extended the survival of the GL261 animal model. We observed reduced proportions of microglia, regulatory T cells (Treg), and myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME) by immunofluorescence. Flow cytometry showed that compared to HCMV-IE1mut alone, treatment with HCMV-IE1mut-FGL2172-220 increased the proportion of CD8+ T cells and tissue-resident memory T cells (TRM). ELISA analysis showed that it improved the secretion of tumor-specific IFN-γ and TNF-α by these cells and downregulated the expression of IL-6 and IL-10. Our study demonstrates that the long-peptide FGL2172-220 improves the antitumor efficacy of HCMV-IE1mut, possibly by reshaping immune cells in the glioma microenvironment. These findings lay the groundwork for the development of therapeutic antigenic peptide vaccines to improve antitumor effects for cancer.