scholarly journals Phytogalactolipid dLGG Inhibits Mouse Melanoma Brain Metastasis through Regulating Oxylipin Activity and Re-Programming Macrophage Polarity in the Tumor Microenvironment

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4120
Author(s):  
Chung-Chih Yang ◽  
Meng-Ting Chang ◽  
Cheng-Kuei Chang ◽  
Lie-Fen Shyur
Keyword(s):  
Tumor Microenvironment ◽  
Brain Metastasis ◽  
Ex Vivo ◽  
Cytotoxic T Cells ◽  
3D Culture ◽  
Tumor Control ◽  
Cancer Therapies ◽  
Melanoma Brain Metastasis ◽  
Organ Tissue

Current conventional cancer therapies for melanoma brain metastasis (MBM) remain ineffective. In this study, we demonstrated the bioefficacy of a phyto-glyceroglycolipid, 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG) alone, or in combination with liposomal doxorubicin (Lip-DOX) or Avastin against MBM in a syngeneic B16BM4COX−2/Luc brain-seeking melanoma mouse model. Treatment with dLGG–10, dLGG–25, dLGG–10 + Avastin–5, Lipo-DOX–2, dLGG–10 + Lipo-DOX–2 or Lipo-DOX–2 + Avastin–5 suppressed, respectively, 17.9%, 59.1%, 55.7%, 16.2%, 44.5% and 72.4% of MBM in mice relative to the untreated tumor control. Metastatic PD-L1+ melanoma cells, infiltration of M2-like macrophages and CD31+ endothelial cells, and high expression levels of 15-LOX/CYP450 4A enzymes in the brain tumor microenvironment of the tumor control mice were significantly attenuated in dLGG-treated mice; conversely, M1-like resident microglia and cytotoxic T cells were increased. A lipidomics study showed that dLGG promoted B16BM4 cells to secrete oxylipins 9,10-/12,13-EpOMEs into the culture medium. Furthermore, the conditioned medium of B16BM4 cells pretreated with dLGG or 9,10-EpOMEs + 12,13-EpOMEs drove M2-like macrophages to polarize into M1-like macrophages in vitro. An ex vivo 3D-culture assay further demonstrated that dLGG, 9,10-EpOME or 9,10-EpOME + 12,13-EpOME pretreatment attenuated B16BM4 cells invading brain tissue, and prevented microglia/macrophages infiltrating into the interface of melanoma plug and brain organ/tissue. In summary, this report provides a novel therapeutic strategy and mechanistic insights into phytogalactolipid dLGG for combating MBM.

scholarly journals Advancements in 3D Cell Culture Systems for Personalizing Anti-Cancer Therapies

2021 ◽  
Vol 11 ◽  
Author(s):  
Andrew M. K. Law ◽  
Laura Rodriguez de la Fuente ◽  
Thomas J. Grundy ◽  
Guocheng Fang ◽  
Fatima Valdes-Mora ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
3D Culture ◽  
3D Cell Culture ◽  
Cancer Therapeutics ◽  
Drug Efficacy ◽  
Cancer Drug ◽  
Cancer Therapies ◽  
Anti Cancer

Over 90% of potential anti-cancer drug candidates results in translational failures in clinical trials. The main reason for this failure can be attributed to the non-accurate pre-clinical models that are being currently used for drug development and in personalised therapies. To ensure that the assessment of drug efficacy and their mechanism of action have clinical translatability, the complexity of the tumor microenvironment needs to be properly modelled. 3D culture models are emerging as a powerful research tool that recapitulates in vivo characteristics. Technological advancements in this field show promising application in improving drug discovery, pre-clinical validation, and precision medicine. In this review, we discuss the significance of the tumor microenvironment and its impact on therapy success, the current developments of 3D culture, and the opportunities that advancements that in vitro technologies can provide to improve cancer therapeutics.

scholarly journals Clinical Significance of PDCD4 in Melanoma by Subcellular Expression and in Tumor-Associated Immune Cells

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1049
Author(s):  
Thuy T. Tran ◽  
Chetan K. Rane ◽  
Christopher R. Zito ◽  
Sarah A. Weiss ◽  
Shlomit Jessel ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Brain Metastasis ◽  
Immune Cell ◽  
Tissue Microarrays ◽  
Pleckstrin Homology Domain ◽  
Neoplastic Progression ◽  
Melanoma Brain Metastasis ◽  
Pdcd4 Expression ◽  
Programmed Cell Death 4 ◽  
And Function

Little is known about the subcellular localization and function of programmed cell death 4 (PDCD4) in melanoma. Our past studies suggest PDCD4 interacts with Pleckstrin Homology Domain Containing A5 (PLEKHA5) to influence melanoma brain metastasis outcomes, as high intracranial PDCD4 expression leads to improved survival. We aimed to define the subcellular distribution of PDCD4 in melanoma and in the tumor microenvironment during neoplastic progression and its impact on clinical outcomes. We analyzed multiple tissue microarrays with well-annotated clinicopathological variables using quantitative immunofluorescence and evaluated single-cell RNA-sequencing on a brain metastasis sample to characterize PDCD4+ immune cell subsets. We demonstrate differences in PDCD4 expression during neoplastic progression, with high tumor and stromal PDCD4 levels associated with improved survival in primary melanomas and in intracranial metastases, but not in extracranial metastatic disease. While the expression of PDCD4 is well-documented on CD8+ T cells and natural killer cells, we show that it is also found on B cells and mast cells. PDCD4 expression in the tumor microenvironment is associated with increased immune cell infiltration. Further studies are needed to define the interaction of PDCD4 and PLEKHA5 and to evaluate the utility of this pathway as a therapeutic target in melanoma brain metastasis.

In Vitro and Ex Vivo Models – The Tumor Microenvironment in a Flask

2020 ◽  
pp. 431-443 ◽  
Author(s):  
Catarina Pinto ◽  
Marta F. Estrada ◽  
Catarina Brito
Keyword(s):  
Tumor Microenvironment ◽  
Ex Vivo

scholarly journals The MEMIC: An ex vivo system to model the complexity of the tumor microenvironment

2021 ◽  
Author(s):  
Libuše Janská ◽  
Libi Anandi ◽  
Nell C. Kirchberger ◽  
Zoran S. Marinkovic ◽  
Logan T. Schachtner ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Ex Vivo ◽  
Tumor Stroma ◽  
Stem Cell Differentiation ◽  
Blood Stream ◽  
Direct Access ◽  
Ex Vivo Model

There is an urgent need for accurate, scalable, and cost-efficient experimental systems to model the complexity of the tumor microenvironment. Here, we detail how to fabricate and use the Metabolic Microenvironment Chamber (MEMIC) – a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is the accessibility to the blood stream that provides key resources such as oxygen and nutrients. While some tumor cells have direct access to these resources, many others must survive under progressively more ischemic environments as they reside further from the vasculature. The MEMIC is designed to simulate the differential access to nutrients and allows co-culturing different cell types, such as tumor and immune cells. This system is optimized for live imaging and other microscopy-based approaches, and it is a powerful tool to study tumor features such as the effect of nutrient scarcity on tumor-stroma interactions. Due to its adaptable design and full experimental control, the MEMIC provide insights into the tumor microenvironment that would be difficult to obtain via other methods. As a proof of principle, we show that cells sense gradual changes in metabolite concentration resulting in multicellular spatial patterns of signal activation and cell proliferation. To illustrate the ease of studying cell-cell interactions in the MEMIC, we show that ischemic macrophages reduce epithelial features in neighboring tumor cells. We propose the MEMIC as a complement to standard in vitro and in vivo experiments, diversifying the tools available to accurately model, perturb, and monitor the tumor microenvironment, as well as to understand how extracellular metabolites affect other processes such as wound healing and stem cell differentiation.

scholarly journals ADAM22/LGI1 complex as a new actionable target for breast cancer brain metastasis

BMC Medicine ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Sara Charmsaz ◽  
Ben Doherty ◽  
Sinéad Cocchiglia ◽  
Damir Varešlija ◽  
Attilio Marino ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Metastatic Breast Cancer ◽  
Brain Metastasis ◽  
In Silico ◽  
Ex Vivo ◽  
Metastatic Breast ◽  
Peptide Mimetic ◽  
Breast Cancer Brain Metastasis

Abstract Background Metastatic breast cancer is a major cause of cancer-related deaths in woman. Brain metastasis is a common and devastating site of relapse for several breast cancer molecular subtypes, including oestrogen receptor-positive disease, with life expectancy of less than a year. While efforts have been devoted to developing therapeutics for extra-cranial metastasis, drug penetration of blood–brain barrier (BBB) remains a major clinical challenge. Defining molecular alterations in breast cancer brain metastasis enables the identification of novel actionable targets. Methods Global transcriptomic analysis of matched primary and metastatic patient tumours (n = 35 patients, 70 tumour samples) identified a putative new actionable target for advanced breast cancer which was further validated in vivo and in breast cancer patient tumour tissue (n = 843 patients). A peptide mimetic of the target’s natural ligand was designed in silico and its efficacy assessed in in vitro, ex vivo and in vivo models of breast cancer metastasis. Results Bioinformatic analysis of over-represented pathways in metastatic breast cancer identified ADAM22 as a top ranked member of the ECM-related druggable genome specific to brain metastases. ADAM22 was validated as an actionable target in in vitro, ex vivo and in patient tumour tissue (n = 843 patients). A peptide mimetic of the ADAM22 ligand LGI1, LGI1MIM, was designed in silico. The efficacy of LGI1MIM and its ability to penetrate the BBB were assessed in vitro, ex vivo and in brain metastasis BBB 3D biometric biohybrid models, respectively. Treatment with LGI1MIM in vivo inhibited disease progression, in particular the development of brain metastasis. Conclusion ADAM22 expression in advanced breast cancer supports development of breast cancer brain metastasis. Targeting ADAM22 with a peptide mimetic LGI1MIM represents a new therapeutic option to treat metastatic brain disease.

scholarly journals In vitro Models of the Small Intestine for Studying Intestinal Diseases

2022 ◽  
Vol 12 ◽  
Author(s):  
Sang-Myung Jung ◽  
Seonghun Kim
Keyword(s):  
Small Intestine ◽  
State Of The Art ◽  
Ex Vivo ◽  
3D Culture ◽  
In Vitro Models ◽  
Cellular Functions ◽  
Current State ◽  
Culture Models ◽  
Composition Structure

The small intestine is a digestive organ that has a complex and dynamic ecosystem, which is vulnerable to the risk of pathogen infections and disorders or imbalances. Many studies have focused attention on intestinal mechanisms, such as host–microbiome interactions and pathways, which are associated with its healthy and diseased conditions. This review highlights the intestine models currently used for simulating such normal and diseased states. We introduce the typical models used to simulate the intestine along with its cell composition, structure, cellular functions, and external environment and review the current state of the art for in vitro cell-based models of the small intestine system to replace animal models, including ex vivo, 2D culture, organoid, lab-on-a-chip, and 3D culture models. These models are described in terms of their structure, composition, and co-culture availability with microbiomes. Furthermore, we discuss the potential application for the aforementioned techniques to these in vitro models. The review concludes with a summary of intestine models from the viewpoint of current techniques as well as their main features, highlighting potential future developments and applications.

scholarly journals Developing a 3D B Cell Lymphoma Culture System to Model Antibody Therapy

2021 ◽  
Vol 11 ◽  
Author(s):  
Russell Foxall ◽  
Priyanka Narang ◽  
Bridget Glaysher ◽  
Elin Hub ◽  
Emma Teal ◽  
...  
Keyword(s):  
B Cell ◽  
Culture System ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
Three Dimensional ◽  
3D Culture ◽  
Antibody Therapy ◽  
B Cell Lymphoma ◽  
Large Cell

Diffuse large cell B cell lymphoma (DLBCL) accounts for approximately 30%–40% of all non-Hodgkin lymphoma (NHL) cases. Current first line DLBCL treatment results in long-term remission in more than 60% of cases. However, those patients with primary refractory disease or early relapse exhibit poor prognosis, highlighting a requirement for alternative therapies. Our aim was to develop a novel model of DLBCL that facilitates in vitro testing of current and novel therapies by replicating key components of the tumor microenvironment (TME) in a three-dimensional (3D) culture system that would enable primary DLBCL cell survival and study ex vivo. The TME is a complex ecosystem, comprising malignant and non-malignant cells, including cancer-associated fibroblasts (CAF) and tumor-associated macrophages (TAM) whose reciprocal crosstalk drives tumor initiation and growth while fostering an immunosuppressive milieu enabling its persistence. The requirement to recapitulate, at least to some degree, this complex, interactive network is exemplified by the rapid cell death of primary DLBCL cells removed from their TME and cultured alone in vitro. Building on previously described methodologies to generate lymphoid-like fibroblasts from adipocyte derived stem cells (ADSC), we confirmed lymphocytes, specifically B cells, interacted with this ADSC-derived stroma, in the presence or absence of monocyte-derived macrophages (MDM), in both two-dimensional (2D) cultures and a 3D collagen-based spheroid system. Furthermore, we demonstrated that DLBCL cells cultured in this system interact with its constituent components, resulting in their improved viability as compared to ex-vivo 2D monocultures. We then assessed the utility of this system as a platform to study therapeutics in the context of antibody-directed phagocytosis, using rituximab as a model immunotherapeutic antibody. Overall, we describe a novel 3D spheroid co-culture system comprising key components of the DLBCL TME with the potential to serve as a testbed for novel therapeutics, targeting key cellular constituents of the TME, such as CAF and/or TAM.

862 Targeting PSGL-1, a novel macrophage checkpoint, repolarizes suppressive macrophages, induces an inflammatory tumor microenvironment, and suppresses tumor growth

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A915-A915
Author(s):  
Phuong Nguyen ◽  
Ryan Phennicie ◽  
Kevin Kauffman ◽  
Dominika Nowakowska ◽  
Mohammad Zafari ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Ex Vivo ◽  
Tnf Alpha ◽  
M2 Macrophages ◽  
Humanized Mouse ◽  
Cellular Assays ◽  
Pdx Model

BackgroundMacrophages play an important role in cancer by modulating both the innate and adaptive parts of the immune system. In non-pathological conditions, multiple subsets of macrophages balance the immune response. In cancer, M2-like immune-suppressive tumor-associated macrophages (TAMs) dominate the tumor microenvironment (TME). TAMs promote tumor growth, support neo-angiogenesis and enable metastasis formation. Macrophage modulators driving macrophage repolarization from the M2-like to a pro-inflammatory M1-like phenotype are an attractive novel class of cancer immunotherapy. Here we present identification, validation, and pre-clinical data of a novel macrophage checkpoint, PSGL-1, which supports targeting this molecule for immune-oncology.MethodsTo assess the therapeutic potential of using anti-PSGL-1 antibodies to convert macrophage phenotype and the tumor microenvironment toward a more inflammatory state, we employed in vitro primary macrophage and multi-cellular assays, ex vivo patient-derived tumor cultures, and a humanized mouse PDX model.ResultsWithin the multiple subsets of macrophages, PSGL-1 is expressed at high levels on immune-suppressive TAMs and in vitro differentiated M2 macrophages. We show that targeting PSGL-1 via an antagonistic antibody repolarized M2 macrophages to a more M1-like state, both phenotypically and functionally as assessed in primary in vitro macrophage assays. Further, these repolarized M1-like macrophages enhanced the inflammatory response in complex multi-cellular assays, including SEB stimulated PBMC assays and mixed-lymphocyte reactions (MLRs).To establish a pre-clinical proof-of-concept for targeting PSGL-1, we turned to ex vivo cultures of fresh patient-derived primary tumors, where the complexity of the TME can be most preserved. RNA-seq data show that ex vivo cultures treated with anti-PD-1 antibody recapitulate TME changes in anti-PD-1 treated patients, including a strong T-cell IFN-gamma signature and a reduction in oncogenic pathway activation. Blocking PSGL-1 resulted in a robust pro-inflammatory signature driven by TNF-alpha/NF-kappa-B and chemokine-mediated signaling. The increase in TNF-alpha signaling was accompanied by reduction in oxidative phosphorylation and fatty acid metabolism. The increase in pro-inflammatory cytokine and chemokine production was confirmed by measuring secreted protein levels, further confirming the re-polarization of macrophages within a tumor setting.Lastly, we employed a humanized mouse PDX model of melanoma and show that anti-PSGL-1 treatment resulted in suppression of tumor growth favorably compared to anti-PD-1. At the cellular and molecular levels, anti-PSGL-1 treatment lead to a more enhanced inflammatory microenvironment, including a reduced M2:M1 macrophage ratio, increased antigen presentation, pro-inflammatory mediators, and effector T cell infiltration and activation.ConclusionsOur data support anti-PSGL-1 as a macrophage repolarizing agent and an effective macrophage-targeted therapy for Immuno-Oncology.

scholarly journals The adenosine 2a receptor and TIM3 directly inhibit killing of tumor cells by cytotoxic T lymphocytes through interference with cytoskeletal polarization

2021 ◽  
Author(s):  
Grace L. Edmunds ◽  
Carissa W.L. Wong ◽  
Rachel Ambler ◽  
Emily Milodowski ◽  
Hanin Alamir ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Tumor Response ◽  
Ex Vivo ◽  
Cytotoxic T Cells ◽  
Tumor Model ◽  
Cell Type ◽  
Adenosine 2A Receptor ◽  
Tumor Cell Killing

Tumors generate an immune-suppressive environment that prevents effective killing of tumor cells by CD8+ cytotoxic T cells (CTL). It remains largely unclear upon which cell type and at which stage of the anti-tumor response mediators of suppression act. We have combined an in vivo tumor model with a matching in vitro reconstruction of the tumor microenvironment based on tumor spheroids to identify suppressors of anti-tumor immunity that directly act on interaction between CTL and tumor cells and to determine mechanisms of action. An adenosine 2a receptor antagonist, as enhanced by blockade of TIM3, slowed tumor growth in vivo. Engagement of the adenosine 2a receptor and TIM3 reduced tumor cell killing in spheroids, impaired CTL cytoskeletal polarization ex vivo and in vitro and inhibited CTL infiltration into tumors and spheroids. With this focus on CTL killing, blocking A2aR and TIM3 complements therapies that enhance T cell priming, e.g. anti-PD1 and anti-CTLA-4.

scholarly journals RADI-20. Brain metastasis treatment with high energy radiotherapy and Cherenkov radiation-activated phototherapy

2021 ◽  
Vol 3 (Supplement_3) ◽  
pp. iii22-iii22
Author(s):  
Anna L Price ◽  
Russell Holden ◽  
James J H Park ◽  
Denise Dunn ◽  
Brendan Koch ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Brain Slice ◽  
Cherenkov Radiation ◽  
Uv Light ◽  
Ex Vivo ◽  
High Energy ◽  
Radiation Induced ◽  
Organotypic Brain Slice ◽  
4T1 Cells

Abstract Radiation therapy is a mainstay in the treatment of brain metastasis, yet some tumors are resistant, and elsewhere brain recurrence outside the radiation field is common. Phototherapy using UV light-activated compounds can both kill cancer cells directly and trigger an immune response to extend tumor control. Poor penetration depth of ultraviolet light, however, has limited this treatment to superficial tumors. High-energy photon beams create high energy electrons within the patient which in turn produce Cherenkov radiation in the UV spectrum while traveling through tissue. Given that this Cherenkov radiation is generated deep within the patient and has the ability to activate photosensitive compounds, we therefore developed a platform to test this phenomenon to enhance radiation therapy for brain metastasis. We first tested UV-activated psoralen derivatives in combination with UV light in vitro for activity against murine 4T1 breast cancer cells, and then irradiated an ex vivo organotypic brain slice platform using a high energy linear accelerator to generate Cherenkov radiation. We tested the survival of 4T1 cells expressing fluorescent and bioluminescent reports in the presence and absence of these psoralen compounds in this ex vivo brain metastasis model. 8-methoxypsoralen (8-MOP) and 4’-Aminomethyltrioxsalen hydrochloride (AMT) both showed 365nm UVA light-specific cell killing in vitro. We optimized AMT cell loading (1 hour) and concentrations [1μM] AMT to maximize cytotoxicity. Testing of AMT using the organotypic brain slice platform and high-energy irradiation to generate Cherenkov-UV light demonstrated similar enhanced cell death of 4T1 cells despite high baseline levels of radiation-induced tumor cell kill. Cherenkov radiation-induced photo-activation of AMT improved cell killing in an ex vivo model of breast cancer brain metastasis. This application holds promise for the re-treatment of refractory tumors with high-energy, low dose radiation, and enhanced elsewhere brain metastasis control through activation of the immune system.

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