A precision medicine tool “Knowledge Frames” to explain poor results of STING agonist trials.

e15632 Background: STING activating cyclic-di-nucleotides result in the production of interferons, activating immune cells. We show that “Precision Medicine Knowledge Frames” (PMKF) are analyses inherently involving 2 processes: Frame 1 – Bench Science - biochemical functions of the experimental product, and Frame 2 - Human Organism Level - actions from product biochemical effects to tumor cell killing. Methods: STING agonists meet this PMK frame1 and activate STING (step 1) effectively. Activated STING binds TBK1 (step 2), then phosphorylates IRF3 (step 3), enters the nucleus (step 4), dimerizes (step 5) and binds specific regions of the DNA (step 6) for interferon transcription (step 7), which are secreted (step 8). Frame 2, the Human Organism Level - final common pathway requires many additional actions: Interferon must interact with immune cells (step 9) [within tumor or peripheral locations]. Very warm or hot tumors [contain functional immune cells] actively allow trafficking (homing) to the tumor (step 10) and infiltrating the tumor microenvironment (step 11). An activated immune cell can kill the tumor cell(s) (step 12). Cold tumors may not allow homing (step 10) to the tumor and/or infiltration (step 11) of the microenvironment. Results: PMKF modeling was applied to STING administration public data. Lack of efficacy was predicted if one of the critical pathway steps to tumor cell death are dysfunctional. Early steps (Frame 1) to interferon production and excretion into the environment occur within normal cells (antigen presenting cells) predictably. However, in the setting of malignancy, Frame 2 steps are often aberrant at the Organism Level. Cold tumors do not allow immune cell homing (step 10) and inhibit tumor infiltration (Step 11), tumor cell localization and killing of the tumor cell (step 12). Conclusions: STING Agonist are cutting edge therapies that perform PMKF Frame 1 well and increase interferon production. Application of the PMKF - Frame 2 demonstrate poor clinical activity may be explained by poor immune cell homing, infiltration and functional killing within the cold tumor microenvironment (dysfunctional steps 10, 11 and 12). Population selection may improve these results.

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619 Evaluating the effectiveness of targeted ADC therapy in a patient-derived ex vivo tumoroid model, 3D-EX, for quantitative tumor cell killing

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0619 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A655-A655

Author(s):

Jenny Kreahling ◽

Jared Ehrhart ◽

Mibel Pabon ◽

Stephen Iwanowycz ◽

Tina Pastoor ◽

...

Keyword(s):

Cell Death ◽

Tumor Microenvironment ◽

Tumor Cell ◽

Immune Cells ◽

Immune Cell ◽

Confocal Imaging ◽

Tumor Cell Death ◽

Drug Conjugates ◽

Tumor Cell Killing ◽

Antibody Drug

BackgroundAntibody drug conjugates (ADCs) are an effective tool for site directed delivery of cytotoxic agents to cancer cells. Tailoring of ADC-specificity to the uniqueness of a patient‘s tumor can aid in direct-targeting of tumor cells and potentially improve drug responsiveness. Here we evaluate the potential of using an ADC therapy for targeted tumor cell death and immune cell activation in combination with checkpoint inhibitors in 3D tumoroids.MethodsAll human tumor samples were obtained with proper patient consent and IRB approval. Fresh patient tumor tissue of various histologic types including CRC and NSCLC were processed to generate uniform sized live 3D tumoroids measuring 150 µm in size. Treatment groups included a conjugated ADC therapeutic antibody alone or in combination with PD-1/PD-L1 inhibitors. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells in combination with high-content confocal imaging to determine extent of tumor cell death in the intact tumor extracellular matrix.ResultsUsing fresh patient-derived tumoroids, we observed ADC-mediated cell death and activation of immune cells within the tumor microenvironment. Production of pro-inflammatory cytokines correlated with increased activation of tumor infiltrating immune cell populations. The improved immune response led to increased tumor cell killing within the 3D tumor microenvironment observed by high-content confocal imaging.ConclusionsIn this study we demonstrate that our physiologically relevant 3D tumoroid model is an effective system to assess novel antibody drug conjugates and to develop rational drug combinations with other immuno-oncology agents. Furthermore, implementation of 3D-EX platform, in the clinical setting, may also allow for determination of the most effective combinatorial immuno-oncology treatment strategies for individualized patient care.Ethics ApprovalThe study was approved by Chesapeake IRB Pro00014313.

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557 Determining the efficacy of ADCC by the 3D-EX ex vivo platform utilizing tumoroids of fresh patient tumor samples with intact tumor microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0557 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A591-A591

Author(s):

Jenny Kreahling ◽

Jared Ehrhart ◽

Stephen Iwanowycz ◽

Mibel Pabon ◽

Tina Pastoor ◽

...

Keyword(s):

Cell Death ◽

Tumor Microenvironment ◽

Tumor Cell ◽

Immune Cells ◽

Immune Cell ◽

Ex Vivo ◽

Cell Killing ◽

Confocal Imaging ◽

Tumor Cell Death ◽

Tumor Cell Killing

BackgroundAntibody-dependent cell-mediated cytotoxicity (ADCC) is an effective tool where antibody-coated cells are targeted and killed by effector immune cells. The application of ADCC therapies has been expanded for both solid tumors as well as hematologic malignancies. However, the immunosuppressive mechanisms present in the immune tumor microenvironment (TME) pose a formidable challenge to immune cell efficacy in addition to hinderance of immune cell infiltration by tumor stromal elements. Hence, it is important to develop clinically relevant platforms to assess the efficacy of antibodies for ADCC. Here we utilized our 3D-EX platform using tumoroids of fresh patient tumor samples to assess ADCC-mediated tumor cell killing.MethodsAll human tumor samples were obtained with proper patient consent and IRB approval. Fresh patient tumor tissue of various histologic types including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) was processed to generate uniform sized live 3D tumoroids measuring 150 µm in size. Treatment groups included cetuximab alone or in combination with nivolumab and/or ipilimumab. Culture supernatants were collected for multiplex analysis of cytokine release in media. Multiplex flow cytometry was used to assess the activation profile of tumor resident immune cells in combination with high-content confocal imaging to determine extent of ADCC-mediated tumor cell death in the intact tumor extracellular matrix.ResultsUsing fresh patient-derived tumor organoids, we observed ADCC-dependent death of EGFR expressing tumor cells. Flow cytometric analysis of immune cell populations demonstrated treatment mediated activation of resident immune cells, which coincided with cytokine profiles determined by Luminex multiplex cytokine analysis. Additionally, tumor cell killing observed through high-content confocal imaging and quantitative image analysis showed tumor cell death with the 3D tumoroids.ConclusionsIn this comprehensive study we demonstrate that the 3D-EX ex vivo model is a robust system to assess the efficacy of ADCC and to develop novel therapeutic combinations with other immuno-oncology therapies. Furthermore, implementation of this platform in clinical studies may also allow for determination of the most effective combinatorial immuno-oncology therapy strategies for specialized individual patient care.Ethics ApprovalThe study was approved by Chesapeake IRB Pro00014313.

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Autophagic Markers in Chordomas: Immunohistochemical Analysis and Comparison with the Immune Microenvironment of Chordoma Tissues

Cancers ◽

10.3390/cancers13092169 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2169

Author(s):

Georgia Karpathiou ◽

Maroa Dridi ◽

Lila Krebs-Drouot ◽

François Vassal ◽

Emmanuel Jouanneau ◽

...

Keyword(s):

Tumor Cell ◽

Immune Cells ◽

Immune Cell ◽

Positive Association ◽

Immunohistochemical Analysis ◽

Vascular Density ◽

Immune Microenvironment ◽

Significant Positive Association ◽

Sequestosome 1 ◽

Cell Expression

Chordomas are notably resistant to chemotherapy. One of the cytoprotective mechanisms implicated in chemoresistance is autophagy. There are indirect data that autophagy could be implicated in chordomas, but its presence has not been studied in chordoma tissues. Sixty-one (61) chordomas were immunohistochemically studied for autophagic markers and their expression was compared with the expression in notochords, clinicopathological data, as well as the tumor immune microenvironment. All chordomas strongly and diffusely expressed cytoplasmic p62 (sequestosome 1, SQSTM1/p62), whereas 16 (26.2%) tumors also showed nuclear p62 expression. LC3B (Microtubule-associated protein 1A/1B-light chain 3B) tumor cell expression was found in 44 (72.1%) tumors. Autophagy-related 16‑like 1 (ATG16L1) was also expressed by most tumors. All tumors expressed mannose-6-phosphate/insulin-like growth factor 2 receptor (M6PR/IGF2R). LC3B tumor cell expression was negatively associated with tumor size, while no other parameters, such as age, sex, localization, or survival, were associated with the immunohistochemical factors studied. LC3B immune cell expression showed a significant positive association with programmed death-ligand 1 (PD-L1)+ immune cells and with a higher vascular density. ATG16L1 expression was also positively associated with higher vascular density. Notochords (n = 5) showed different immunostaining with a very weak LC3B and M6PR expression, and no p62 expression. In contrast to normal notochords, autophagic factors such as LC3B and ATG16L1 are often present in chordomas, associated with a strong and diffuse expression of p62, suggesting a blocked autophagic flow. Furthermore, PD-L1+ immune cells also express LC3B, suggesting the need for further investigations between autophagy and the immune microenvironment.

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492 Integration of high dimensional datasets in an immunocompetent mammary mouse model reveals pathways of tolerance and resistance to immune checkpoint blockade

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0492 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A528-A528

Author(s):

Lin Ma ◽

Jian-Hua Mao ◽

Mary Helen Barcellos-Hoff ◽

Jade Moore

Keyword(s):

Tumor Microenvironment ◽

Immune Cells ◽

Transforming Growth Factor ◽

Immune Cell ◽

Systemic Disease ◽

Ct Imaging ◽

High Dimensional ◽

Cytokine Signaling ◽

Immune Infiltrate ◽

Pet Ct

BackgroundCheckpoint inhibitors can induce robust and durable responses in a subset of patients. Extending this benefit to more patients could be facilitated by better understanding of how interacts with immune cells with the tumor microenvironment, which is a critical barrier to control both local and systemic disease. The composition and pattern of the immune infiltrate associates with the likelihood of response to immunotherapy. Inflamed tumors that exhibit a brisk immune cell infiltrate are responsive, while those in which immune cells are completely or partially excluded are not. Transforming growth factor β (TGFβ) is immunosuppressive and associated with the immune excluded phenotype.MethodsUsing an immune competent mammary tumor derived transplant (mTDT) model recently developed in our lab, exhibits inflamed, excluded or deserts immune infiltrate phenotypes based on localization of CD8 lymphocytes. Using whole transcriptome deep sequencing, cytof, and PET-CT imaging, we evaluated the tumor, microenvironment, and immune pathway activation among immune infiltrate phenotypes.ResultsThree distinct inflamed tumors phenotypes were identified: ‘classically’ inflamed characterized by pathway evidence of increased CD8+ T cells and decreased PD-L1 expression, inflamed tumors with pathways indicative of neovascularization and STAT3 signaling and reduced T cell mobilization, and an inflamed tumor with increased immunosuppressive myeloid phenotypes. Excluded tumors were characterized by TGFβ gene expression and pro-inflammatory cytokine signaling (e.g. TNFα, IL1β), associated with decreased leukocytes homing and increased immune cell death of cells. We visualized and quantified TGFβ activity using PET-CT imaging of 89Zr-fresolimumab, a TGFβ neutralizing antibody. TGFβ activity was significantly increased in excluded tumors compared to inflamed or desert tumors, which was supported by quantitative pathology (Perkin Elmer) of its canonical signaling target, phosphorylated SMAD2 (pSMAD2). pSMAD2 was positively correlated with PD-L1 expression in the stroma of excluded tumors. In contrast, in inflamed tumors, TGFβ activity positively correlated with increased F4/80 positive macrophages and negatively correlated with expression of PD-L1. CyTOF analysis of tumor and spleen immune phenotypes revealed increased trafficking of myeloid cells in mice bearing inflamed tumors compared to excluded and deserts.ConclusionsThe immunocompetent mTDT provides a model that bridges the gap between the immune landscape and tumor microenvironment. Integration of these high-dimensional data with further studies of response to immunotherapies will help to identify tumor features that favor response to treatment or the means to convert those that are unresponsive.

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When Cells Suffocate: Autophagy in Cancer and Immune Cells under Low Oxygen

International Journal of Cell Biology ◽

10.1155/2011/470597 ◽

2011 ◽

Vol 2011 ◽

pp. 1-13 ◽

Cited By ~ 16

Author(s):

Katrin Schlie ◽

Jaeline E. Spowart ◽

Luke R. K. Hughson ◽

Katelin N. Townsend ◽

Julian J. Lum

Keyword(s):

Tumor Microenvironment ◽

Cancer Cells ◽

Immune Cells ◽

Immune Cell ◽

Treatment Strategies ◽

Patient Treatment ◽

Low Oxygen ◽

Tumor Environment ◽

And Function ◽

The Impact

Hypoxia is a signature feature of growing tumors. This cellular state creates an inhospitable condition that impedes the growth and function of all cells within the immediate and surrounding tumor microenvironment. To adapt to hypoxia, cells activate autophagy and undergo a metabolic shift increasing the cellular dependency on anaerobic metabolism. Autophagy upregulation in cancer cells liberates nutrients, decreases the buildup of reactive oxygen species, and aids in the clearance of misfolded proteins. Together, these features impart a survival advantage for cancer cells in the tumor microenvironment. This observation has led to intense research efforts focused on developing autophagy-modulating drugs for cancer patient treatment. However, other cells that infiltrate the tumor environment such as immune cells also encounter hypoxia likely resulting in hypoxia-induced autophagy. In light of the fact that autophagy is crucial for immune cell proliferation as well as their effector functions such as antigen presentation and T cell-mediated killing of tumor cells, anticancer treatment strategies based on autophagy modulation will need to consider the impact of autophagy on the immune system.

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In Vitro Evaluation of CD276-CAR NK-92 Functionality, Migration and Invasion Potential in the Presence of Immune Inhibitory Factors of the Tumor Microenvironment

Cells ◽

10.3390/cells10051020 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1020

Author(s):

Stefan Grote ◽

Guillermo Ureña-Bailén ◽

Kenneth Chun-Ho Chan ◽

Caroline Baden ◽

Markus Mezger ◽

...

Keyword(s):

Tumor Microenvironment ◽

Solid Tumors ◽

Immune Cells ◽

Immune Cell ◽

Nk Cell ◽

Migration And Invasion ◽

Cellular Immunotherapy ◽

Knock Out ◽

Immunosuppressive Tumor Microenvironment

Background: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME). Methods: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity. Results: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity. Conclusions: The strong cytotoxic effect of a CD276-specific CAR in combination with an “off-the-shelf” NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.

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Identification of Immune-Related Prognostic Biomarkers in Pancreatic Cancer

Nanoscience and Nanotechnology Letters ◽

10.1166/nnl.2020.3251 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1355-1367

Author(s):

Xiaoyan Lin ◽

Jiakang Ma ◽

Kaikai Ren ◽

Mingyu Hou ◽

Bo Zhou ◽

...

Keyword(s):

Pancreatic Cancer ◽

Tumor Microenvironment ◽

Risk Score ◽

Immune Cells ◽

Immune Cell ◽

Cox Regression ◽

Individual Treatment ◽

Survival Prediction ◽

Lasso Regression ◽

Immune Related Genes

Immunotherapy for pancreatic cancer (PC) faces significant challenges. It is urgent to find immunerelated genes for targeted therapy. We aimed to identify immune-related messenger ribonucleic acids (mRNAs) with multiple methods of comprehensive immunoenrichment analysis in predicting survival of PC. PC genomics and clinical data were downloaded from TCGA. We analyzed relative enrichment of 29 immune cells using ssGSEA and classified PC samples into three immuneinfiltrating subgroups. Immune cell infiltration level and pathways were evaluated by ESTIMATE data and KEGG. Independent risk factors were derived from the combined analysis of WGCNA, LASSO regression and Cox regression analyses. Immune risk score was calculated according to four mRNAs to identify its value in predicting survival. PPI analysis was used to analyze the connections and potential pathways among genes. Finally, PC samples were classified into three immuneinfiltrating subgroups. Immunity high subgroup had higher immune score, soakage of immune cells, HLA/PD-L1 expression level, immune-related pathways enrichment and better survivability. Four potential prognostic immune-related genes (ITGB7, RAC2, DNASE1L3, and TRAF1) were identified. Immune risk score could be a potential survival prediction indictor with high sensitivity and specificity (AUC values = 0.708, HR = 1.445). A PPI network with seven nodes and five potential targeted pathways were generated. In conclusion, we estimated the state of immune infiltration in the PC tumor microenvironment by calculating stromal and immune cells enrichment with ssGSEA algorithms, and identified four prognostic immune-related genes that affect the proportion and distribution of immune cells infiltration in the tumor microenvironment. They lay a theoretical foundation to be important immunity targets of individual treatment in PC.

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Applications of Single-Cell Omics to Dissect Tumor Microenvironment

Frontiers in Genetics ◽

10.3389/fgene.2020.548719 ◽

2020 ◽

Vol 11 ◽

Author(s):

Tingting Guo ◽

Weimin Li ◽

Xuyu Cai

Keyword(s):

Tumor Microenvironment ◽

Single Cell ◽

Immune Cells ◽

Immune Cell ◽

Cell Types ◽

Immune Checkpoint Blockade ◽

Lineage Tracing ◽

Great Success ◽

Novel Technologies ◽

Single Cell Sequencing

The recent technical and computational advances in single-cell sequencing technologies have significantly broaden our toolkit to study tumor microenvironment (TME) directly from human specimens. The TME is the complex and dynamic ecosystem composed of multiple cell types, including tumor cells, immune cells, stromal cells, endothelial cells, and other non-cellular components such as the extracellular matrix and secreted signaling molecules. The great success on immune checkpoint blockade therapy has highlighted the importance of TME on anti-tumor immunity and has made it a prime target for further immunotherapy strategies. Applications of single-cell transcriptomics on studying TME has yielded unprecedented resolution of the cellular and molecular complexity of the TME, accelerating our understanding of the heterogeneity, plasticity, and complex cross-interaction between different cell types within the TME. In this review, we discuss the recent advances by single-cell sequencing on understanding the diversity of TME and its functional impact on tumor progression and immunotherapy response driven by single-cell sequencing. We primarily focus on the major immune cell types infiltrated in the human TME, including T cells, dendritic cells, and macrophages. We further discuss the limitations of the existing methodologies and the prospects on future studies utilizing single-cell multi-omics technologies. Since immune cells undergo continuous activation and differentiation within the TME in response to various environmental cues, we highlight the importance of integrating multimodal datasets to enable retrospective lineage tracing and epigenetic profiling of the tumor infiltrating immune cells. These novel technologies enable better characterization of the developmental lineages and differentiation states that are critical for the understanding of the underlying mechanisms driving the functional diversity of immune cells within the TME. We envision that with the continued accumulation of single-cell omics datasets, single-cell sequencing will become an indispensable aspect of the immune-oncology experimental toolkit. It will continue to drive the scientific innovations in precision immunotherapy and will be ultimately adopted by routine clinical practice in the foreseeable future.

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The Immune Endocannabinoid System of the Tumor Microenvironment

International Journal of Molecular Sciences ◽

10.3390/ijms21238929 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8929

Author(s):

Melanie Kienzl ◽

Julia Kargl ◽

Rudolf Schicho

Keyword(s):

Tumor Microenvironment ◽

Tumor Progression ◽

Immune Cells ◽

Cannabinoid Receptors ◽

Immune Cell ◽

Tumor Development ◽

Endocannabinoid System ◽

Cell Functions ◽

In Vitro Effects

Leukocytes are part of the tumor microenvironment (TME) and are critical determinants of tumor progression. Because of the immunoregulatory properties of cannabinoids, the endocannabinoid system (ECS) may have an important role in shaping the TME. Members of the ECS, an entity that consists of cannabinoid receptors, endocannabinoids and their synthesizing/degrading enzymes, have been associated with both tumor growth and rejection. Immune cells express cannabinoid receptors and produce endocannabinoids, thereby forming an “immune endocannabinoid system”. Although in vitro effects of exogenous cannabinoids on immune cells are well described, the role of the ECS in the TME, and hence in tumor development and immunotherapy, is still elusive. This review/opinion discusses the possibility that the “immune endocannabinoid system” can fundamentally influence tumor progression. The widespread influence of cannabinoids on immune cell functions makes the members of the ECS an interesting target that could support immunotherapy.

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Myeloid Immune Cells CARriying a New Weapon Against Cancer

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.784421 ◽

2021 ◽

Vol 9 ◽

Author(s):

Rodrigo Nalio Ramos ◽

Samuel Campanelli Freitas Couto ◽

Theo Gremen M. Oliveira ◽

Paulo Klinger ◽

Tarcio Teodoro Braga ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

Tumor Microenvironment ◽

Solid Tumors ◽

Immune Cells ◽

Scientific Community ◽

Immune Cell ◽

Clinical Results ◽

Mononuclear Phagocytes ◽

Soluble Factors

Chimeric antigen receptor (CAR) engineering for T cells and natural killer cells (NK) are now under clinical evaluation for the treatment of hematologic cancers. Although encouraging clinical results have been reported for hematologic diseases, pre-clinical studies in solid tumors have failed to prove the same effectiveness. Thus, there is a growing interest of the scientific community to find other immune cell candidate to express CAR for the treatment of solid tumors and other diseases. Mononuclear phagocytes may be the most adapted group of cells with potential to overcome the dense barrier imposed by solid tumors. In addition, intrinsic features of these cells, such as migration, phagocytic capability, release of soluble factors and adaptive immunity activation, could be further explored along with gene therapy approaches. Here, we discuss the elements that constitute the tumor microenvironment, the features and advantages of these cell subtypes and the latest studies using CAR-myeloid immune cells in solid tumor models.

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