TAMI-28. DIFFERENTIAL MIGRATION MECHANICS AND IMMUNE RESPONSES OF GLIOMA SUBTYPES

Abstract In Glioblastoma (GBM), tumor spreading is driven by tumor cells’ ability to infiltrate healthy brain parenchyma, which prevents complete surgical resection and contributes to tumor recurrence. GBM molecular subtypes, classical, proneural and mesenchymal, were shown to strongly correlate with specific genetic alterations (Mesenchymal: NF1; Classical: EGFRVIII; Proneural: PDGFRA). Here we tested the hypothesis that a key mechanistic difference between GBM molecular subtypes is that proneural cells are slow migrating and mesenchymal cells are fast migrating. Using Sleeping Beauty transposon system, immune-competent murine brain tumors were induced by SV40-LgT antigen in combination with either NRASG12V (NRAS) or PDGFB (PDGF) overexpression. Cross-species transcriptomic analysis revealed NRAS and PDGF-driven tumors correlate with human mesenchymal and proneural GBM, respectively. Similar to human GBM, CD44 expression was higher in NRAS tumors and, consistent with migration simulations of varying CD44 levels, ex vivo brain slice live imaging showed NRAS tumors cells migrate faster than PDGF tumors cells (random motility coefficient = 30µm2/hr vs. 2.5µm2/hr, p < 0.001). Consistent with CD44 function as an adhesion molecule, migration phenotype was independent of the tumor microenvironment. NRAS and human PDX/MES tumor cells were found to migrate faster and have larger cell spread area than PDGF and human PDX/PN tumors cells, respectively, in healthy mouse brain slices. Furthermore, traction force microscopy revealed NRAS tumor cells generate larger traction forces than PDGF tumors cells which further supports our theoretical mechanism driving glioma migration. Despite increased migration, NRAS cohort had better survival than PDGF which was attributed to enhanced antitumoral immune response in NRAS tumors, consistent with increased immune cell infiltration found in human mesenchymal GBM. Overall our work identified a potentially actionable difference in migration mechanics between GBM subtypes and establishes an integrated biophysical modeling and experimental approach to mechanically parameterize and simulate distinct molecular subtypes in preclinical models of cancer.

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Pediatric medulloblastoma express immune checkpoint B7-H3

Clinical & Translational Oncology ◽

10.1007/s12094-021-02762-y ◽

2022 ◽

Author(s):

S. Li ◽

G. C. Poolen ◽

L. C. van Vliet ◽

J. G. Schipper ◽

R. Broekhuizen ◽

...

Keyword(s):

Protein Expression ◽

Tumor Cells ◽

Immune Checkpoint ◽

Immune Cell ◽

Molecular Subtypes ◽

Staining Intensity ◽

Immune Checkpoints ◽

Γδt Cells ◽

Young Infants ◽

Pediatric Medulloblastoma

Abstract Purpose Medulloblastomas (MB) are highly malignant brain tumors that predominantly occur in young infants. Immunotherapy to boost the immune system is emerging as a novel promising approach, but is often hampered by inhibitory immune checkpoints. In the present study, we have studied immune checkpoint B7-H3 expression in a tissue cohort of human pediatric MB. Methods Expression of B7-H3 was detected by immunohistochemistry and classified via B7-H3 staining intensity and percentage of B7-H3 positive tumor cells. Subsequently, B7-H3 protein expression was distinguished in MB molecular subtypes and correlated to immune cell infiltrates, patient characteristics, and survival. Results B7-H3 protein expression was found in 23 out of 24 (96%) human pediatric MB cases and in 17 out of 24 (71%) MB cases > 25% of tumor cells had any level of B7-H3 expression. B7-H3 protein expression was more frequent on Group-4 MB as compared with other molecular subtypes (p = 0.02). Tumors with high B7-H3 expression showed less influx of γδT cells (p = 0.002) and CD3+ T cells (p = 0.041). Conclusion Immune checkpoint B7-H3 is differentially expressed by the large majority of pediatric MB. This further warrants the development of novel B7-H3-directed (immuno)therapeutic methods for children with incurable, metastatic, or chemo-resistant MB.

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TAMI-71. EFFECTS OF THE TREATED MICROENVIRONMENT ON GLIOMA CELL PROPERTIES IN AN ORGANOTYPIC BRAIN SLICE MODEL

Neuro-Oncology ◽

10.1093/neuonc/noab196.853 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi213-vi213

Author(s):

Vasiliki Pantazopoulou ◽

Tracy Berg ◽

Alexander Pietras

Keyword(s):

Tumor Cells ◽

Glioma Cell ◽

Radiation Treatment ◽

Ex Vivo ◽

Brain Slices ◽

Glioma Cells ◽

Cell Phenotype ◽

Slice Culture ◽

Tumor Bearing ◽

Organotypic Brain Slices

Abstract Glioblastoma is the most aggressive primary brain tumor. Despite treatment all patients invariably recur. Treatment resistance is attributed to the presence of glioma stem-like cells. Initially thought to be a distinct and static cell population, it is becoming increasingly clear that the glioma stem-like cell phenotype represents one of many cellular states and that glioma cells show plasticity between stem-like and non-stem like states. These plastic cell states are affected by the tumor microenvironment. In our lab we have shown that irradiated and hypoxic astrocytes increase the stem-like cell properties of glioma cells. In this study, we aim to evaluate how the treated microenvironment alters glioma cell properties and use ex vivo organotypic brain slices generated from tumor bearing and tumor naïve mice to assess all aspects of the microenvironment. We first characterized organotypic brain slices cultured in different oxygen tensions. We saw that tumor-bearing slices survive for at least 14 days in culture at 21%, 5% or 1% oxygen tension (O2). Tumor cells were more viable in all culture conditions and timepoints compared to non-tumor cells. Moreover, we found that astrocytes seem to be attracted to tumor areas in both 5% and 1% O2 cultures. We then used the organotypic glioma slice culture system to address how preconditioning the microenvironment using radiation or temozolomide affects the properties of glioma cells that are seeded in these pretreated, tumor naïve slices. We saw that fluorescently labelled glioma cells seeded in treated slices can be isolated after two days of culture in the slices and can be used for downstream analyses, such as temozolomide or radiation treatment and colony formation. This study will elucidate the effect of the treated microenvironment on glioma cell properties by using the medium throughput method of organotypic slice cultures.

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Gasdermin-D-dependent IL-1α release from microglia promotes protective immunity during chronic Toxoplasma gondii infection

10.1101/2020.01.22.915652 ◽

2020 ◽

Author(s):

Samantha J. Batista ◽

Katherine M. Still ◽

David Johanson ◽

Jeremy A. Thompson ◽

Carleigh A. O’Brien ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Immune Cell ◽

Ex Vivo ◽

Myeloid Cells ◽

Brain Parenchyma ◽

Parasite Control ◽

Reporter Mice ◽

Chemical Inhibition ◽

Toxoplasma Gondii Infection ◽

The Brain

AbstractMicroglia, the resident immune cells of the brain parenchyma, are thought to be first-line defenders against CNS infections. We sought to identify specific roles of microglia in the control of the eukaryotic parasite Toxoplasma gondii, an opportunistic infection that can cause severe neurological disease. In order to identify the specific function of microglia in the brain during infection, we sorted microglia and infiltrating myeloid cells from infected microglia reporter mice. Using RNA-sequencing, we find strong NF-κB and inflammatory cytokine signatures overrepresented in blood-derived macrophages versus microglia. Interestingly, we also find that IL-1α is enriched in microglia and IL-1β in macrophages, which was also evident at the protein level. We find that mice lacking IL-1R1 or IL-1α, but not IL-1β, have impaired parasite control and immune cell infiltration specifically within the brain. Further, by sorting purified populations from infected brains, we show that microglia, not peripheral myeloid cells, release IL-1α ex vivo. Finally, using knockout mice as well as chemical inhibition, we show that ex vivo IL-1α release is gasdermin-D dependent, and that gasdermin-D and caspase-1/11 deficient mice show deficits in immune infiltration into the brain and parasite control. These results demonstrate that microglia and macrophages are differently equipped to propagate inflammation, and that in chronic T. gondii infection, microglia specifically can release the alarmin IL-1α, a cytokine that promotes neuroinflammation and parasite control.

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Effect of Annexins Translocated in Extracellular Vesicles on Tumorigenesis

Current Molecular Medicine ◽

10.2174/1566524020666200825163512 ◽

2020 ◽

Vol 20 ◽

Author(s):

Qionghui Wu ◽

Haidong Wei ◽

Wenbo Meng ◽

Xiaodong Xie ◽

Zhenchang Zhang ◽

...

Keyword(s):

Tumor Cells ◽

Extracellular Vesicles ◽

Immune Cell ◽

Epithelial Mesenchymal Transition ◽

Main Role ◽

Tumor Cell Adhesion ◽

Mesenchymal Transition ◽

Calcium Dependent ◽

Tumor Neovascularization

: Annexin, a calcium-dependent phospholipid binding protein, can affect tumor cell adhesion, proliferation, apoptosis, invasion and metastasis, as well as tumor neovascularization in different ways. Recent studies have shown that annexin exists not only as an intracellular protein in tumor cells, but also in different ways to be secret outside the cell as a “crosstalk” tool for tumor cells and tumor microenvironment, thus playing an important role in the development of tumors, such as participating in epithelial-mesenchymal transition, regulating immune cell behavior, promoting neovascularization and so on. The mechanism of annexin secretion in the form of extracellular vesicles and its specific role is still unclear. This paper summarizes the main role of annexin secreted into the extracellular space in the form of extracellular vesicles in tumorigenesis and drug resistance and analyzes its possible mechanism.

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Inflamm-Aging Is Associated with Lower Plasma PTX3 Concentrations and an Impaired Capacity of PBMCs to Express hTERT following LPS Stimulation

Mediators of Inflammation ◽

10.1155/2019/2324193 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 1

Author(s):

Aaron L. Slusher ◽

Tiffany M. Zúñiga ◽

Edmund O. Acevedo

Keyword(s):

Gene Expression ◽

Young Adults ◽

Telomere Length ◽

Immune Cell ◽

Ex Vivo ◽

Middle Aged ◽

Human Telomerase Reverse Transcriptase ◽

Negatively Associated ◽

Htert Gene ◽

Age Related

Age-related elevations in proinflammatory cytokines, known as inflamm-aging, are associated with shorter immune cell telomere lengths. Purpose. This study examined the relationship of plasma PTX3 concentrations, a biomarker of appropriate immune function, with telomere length in 15 middle-aged (40-64 years) and 15 young adults (20-31 years). In addition, PBMCs were isolated from middle-aged and young adults to examine their capacity to express a key mechanistic component of telomere length maintenance, human telomerase reverse transcriptase (hTERT), following ex vivo cellular stimulation. Methods. Plasma PTX3 and inflammatory cytokines (i.e., IL-6, IL-10, TGF-β, and TNF-α), PBMC telomere lengths, and PBMC hTERT gene expression and inflammatory protein secretion following exposure to LPS, PTX3, and PTX3+LPS were measured. Results. Aging was accompanied by the accumulation of centrally located visceral adipose tissue, without changes in body weight and BMI, and alterations in the systemic inflammatory milieu (decreased plasma PTX3 and TGF-β; increased TNF-α (p≤0.050)). In addition, shorter telomere lengths in middle-aged compared to young adults (p=0.011) were negatively associated with age, body fat percentages, and plasma TNF-α (r=−0.404, p=0.027; r=−0.427, p=0.019; and r=−0.323, p=0.041, respectively). Finally, the capacity of PBMCs to increase hTERT gene expression following ex vivo stimulation was impaired in middle-aged compared to young adults (p=0.033) and negatively associated with telomere lengths (r=0.353, p=0.028). Conclusions. Proinflammation and the impaired hTERT gene expression capacity of PBMCs may contribute to age-related telomere attrition and disease.

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The Caspase-1/IL-18 Axis of the Inflammasome in Tumor Cells: A Modulator of the Th1/Tc1 Response of Tumor-Infiltrating T Lymphocytes in Colorectal Cancer

Cancers ◽

10.3390/cancers13020189 ◽

2021 ◽

Vol 13 (2) ◽

pp. 189

Author(s):

Linda Bilonda Mutala ◽

Cécile Deleine ◽

Matilde Karakachoff ◽

Delphine Dansette ◽

Kathleen Ducoin ◽

...

Keyword(s):

Colorectal Cancer ◽

T Lymphocytes ◽

Tumor Cells ◽

Ex Vivo ◽

Explant Culture ◽

T Cell Response ◽

Mrna Levels ◽

Innate And Adaptive Immunity ◽

Caspase 1 ◽

Culture Model

In colorectal cancer (CRC), a high density of T lymphocytes represents a strong prognostic marker in subtypes of CRC. Optimized immunotherapy strategies to boost this T-cell response are still needed. A good candidate is the inflammasome pathway, an emerging player in cancer immunology that bridges innate and adaptive immunity. Its effector protein caspase-1 matures IL-18 that can promote a T-helper/cytotoxic (Th1/Tc1) response. It is still unknown whether tumor cells from CRC possess a functional caspase-1/IL-18 axis that could modulate the Th1/Tc1 response. We used two independent cohorts of CRC patients to assess IL-18 and caspase-1 expression by tumor cells in relation to the density of TILs and the microsatellite status of CRC. Functional and multiparametric approaches at the protein and mRNA levels were performed on an ex vivo CRC explant culture model. We show that, in the majority of CRCs, tumor cells display an activated and functional caspase-1/IL-18 axis that contributes to drive a Th1/Tc1 response elicited by TILs expressing IL-18Rα. Furthermore, unsupervised clustering identified three clusters of CRCs according to the caspase-1/IL-18/TIL density/interferon gamma (IFNγ) axis and microsatellite status. Together, our results strongly suggest that targeting the caspase-1/IL-18 axis can improve the anti-tumor immune response in subgroups of CRC.

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602 STING agonist-based treatment promotes vascular normalization and tertiary lymphoid structure formation in the therapeutic melanoma microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0602 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A637-A637

Author(s):

Manoj Chelvanambi ◽

Ronald Fecek ◽

Jennifer Taylor ◽

Walter Storkus

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Cell ◽

Ex Vivo ◽

In Vitro Assays ◽

Type I ◽

Vascular Normalization ◽

S 100 ◽

Transcriptional Changes

BackgroundThe degree of immune infiltration in tumors, especially CD8+ T cells, greatly impacts patient disease course and response to interventional immunotherapy. Hence, enhancement of TIL prevalence is a preferred clinical endpoint, one that may be achieved via administration of agents that normalize the tumor vasculature (VN) leading to improved immune cell recruitment and/or that induce the development of local tertiary lymphoid structures (TLS) within the tumor microenvironment (TME).MethodsLow-dose STING agonist ADU S-100 (5 μg/mouse) was delivered intratumorally to established s.c. B16.F10 melanomas on days 10, 14 and 17 post-tumor inoculation under an IACUC-approved protocol. Treated and control, untreated tumors were isolated at various time points to assess transcriptional changes associated with VN and TLS formation via qPCR, with corollary immune cell composition changes determined using flow cytometry and immunofluorescence microscopy. In vitro assays were performed on CD11c+ BMDCs treated with 2.5 μg/mL ADU S-100 (vs PBS control) and associated transcriptional changes analyzed via qPCR or profiled using DNA microarrays. For TCRβ-CDR3 analyses, CDR3 was sequenced from gDNA isolated from enzymatically digested tumors and splenocytes.ResultsWe report that activation of STING within the TME leads to slowed melanoma growth in association with increased production of angiostatic factors including Tnfsf15 (Vegi), Cxcl10 and Angpt1, and TLS inducing factors including Ccl19, Ccl21, Lta, Ltb and Tnfsf14 (Light). Therapeutic responses from intratumoral STING activation were characterized by increased vascular normalization (VN), enhanced tumor infiltration by CD8+ T cells and CD11c+ DCs and local TLS neo-genesis, all of which were dependent on host expression of STING. Consistent with a central role for DC in TLS formation, ex vivo ADU S-100-activated mCD11c+ DCs also exhibited upregulated expression of TLS promoting factors including lymphotoxin-α (LTA), IL-36, inflammatory chemokines and type I interferons. TLS formation was associated with the development of a therapeutic TIL TCR repertoire enriched in T cell clonotypes uniquely detected within the tumor but not the peripheral circulation in support or local T cell cross-priming within the TME.ConclusionsOur data support the premise that i.t. delivery of STING agonist promotes a pro-inflammatory TME in support of VN and TLS formation, leading to the local expansion of unique TIL repertoire in association with superior anti-melanoma efficacy.

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Altered Spatial Composition of the Immune Cell Repertoire in Association to CD34+ Blasts in Myelodysplastic Syndromes and Secondary Acute Myeloid Leukemia

Cancers ◽

10.3390/cancers13020186 ◽

2021 ◽

Vol 13 (2) ◽

pp. 186

Author(s):

Marcus Bauer ◽

Christoforos Vaxevanis ◽

Haifa Kathrin Al-Ali ◽

Nadja Jaekel ◽

Christin Le Hoa Naumann ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Myelodysplastic Syndromes ◽

Myeloid Leukemia ◽

Multispectral Imaging ◽

Immune Cell ◽

Genetic Alterations ◽

Spatial Proximity ◽

Secondary Acute Myeloid Leukemia ◽

Acute Myeloid

Background: Myelodysplastic syndromes (MDS) are caused by a stem cell failure and often include a dysfunction of the immune system. However, the relationship between spatial immune cell distribution within the bone marrow (BM), in relation to genetic features and the course of disease has not been analyzed in detail. Methods: Histotopography of immune cell subpopulations and their spatial distribution to CD34+ hematopoietic cells was determined by multispectral imaging (MSI) in 147 BM biopsies (BMB) from patients with MDS, secondary acute myeloid leukemia (sAML), and controls. Results: In MDS and sAML samples, a high inter-tumoral immune cell heterogeneity in spatial proximity to CD34+ blasts was found that was independent of genetic alterations, but correlated to blast counts. In controls, no CD8+ and FOXP3+ T cells and only single MUM1p+ B/plasma cells were detected in an area of ≤10 μm to CD34+ HSPC. Conclusions: CD8+ and FOXP3+ T cells are regularly seen in the 10 μm area around CD34+ blasts in MDS/sAML regardless of the course of the disease but lack in the surrounding of CD34+ HSPC in control samples. In addition, the frequencies of immune cell subsets in MDS and sAML BMB differ when compared to control BMB providing novel insights in immune deregulation.

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Quantitative measurement of reactive oxygen species in ex vivo mouse brain slices

STAR Protocols ◽

10.1016/j.xpro.2021.100332 ◽

2021 ◽

Vol 2 (1) ◽

pp. 100332

Author(s):

Chirag Vasavda ◽

Solomon H. Snyder ◽

Bindu D. Paul

Keyword(s):

Reactive Oxygen Species ◽

Mouse Brain ◽

Quantitative Measurement ◽

Ex Vivo ◽

Brain Slices ◽

Reactive Oxygen ◽

Oxygen Species

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A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Function ◽

10.1093/function/zqab012 ◽

2021 ◽

Vol 2 (3) ◽

Cited By ~ 1

Author(s):

Nelly Redolfi ◽

Elisa Greotti ◽

Giulia Zanetti ◽

Tino Hochepied ◽

Cristina Fasolato ◽

...

Keyword(s):

Transgenic Mouse ◽

Fluorescent Protein ◽

Ex Vivo ◽

Brain Slices ◽

Resonance Energy ◽

Mouse Line ◽

Isolated Cells ◽

Genomic Locus ◽

Transgenic Mouse Line

AbstractMitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

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