Automated tracking of tumor-stroma morphology in microtissues identifies functional targets within the tumor microenvironment for therapeutic intervention

BackgroundPredictive biomarkers for response to IO therapies remain insufficient. Although multiplex immunofluorescence has the potential to provide superior biomarkers, the information garnered from these studies is frequently underleveraged. Due to the large number of markers that must be analyzed (6 - 40 +), and the complexity of the spatial information, the number of hypotheses is large and must be tested systematically and automatically. GraphITE (Graphs-based Investigation of Tissues with Embeddings) is a novel method of converting multiplex IF image analysis results into embeddings, numerical vectors which represent the phenotype of each cell as well as the immediate neighborhood. This allows for the clustering of embeddings based on similarity as well as the discovery of novel predictive biomarkers based on both the spatial and multimarker data in multiplex IF images. Here we demonstrate initial observations from deployment of GraphITE on 564 commercially-sourced NSCLC and HNSCC resections stained with a multiplex IF panel containing CD8, PDL1, PD1, CD68, Ki67, and CK.Methods4 μm FFPE tumor sections were stained with CD8, PDL1, PD1, CD68, Ki67, and CK at Akoya Biosciences using OPAL TSA-linked fluorophores and imaged on a Vectra Polaris. Images were analyzed by Computational Biology (AstraZeneca). Graphs were built by mapping each cell in the mIF image as a node, using the X, Y coordinates and connecting nodes with edges according to distance. 64-dimensional embeddings were generated using Deep Graph InfoMax (DGI).1 Embeddings are downprojected to 2 dimensions using UMAP.2. Details are available in the preprint of the GraphITE methods manuscript.3ResultsA single downprojection was developed using embeddings from 158 HNSCC and 406 NSCLC cases. 60–80 distinct clusters were observed, some of which contained embeddings from both indications and others which were exclusive to one indication. Exclusive clusters describe tissue neighborhoods observed only in one indication. Drivers of cluster exclusivity included increased cell density in HNSCC as compared to NSCLC both in PD-L1- tumor centers with few infiltrating lymphocytes as well as in PD-L1- macrophagedominated neighborhoods. HNSCC and NSCLC embeddings were more colocalized in PD-L1+ tumor centers and in tumor stroma with high CD8+ or CD68+ immune cell content and high PD-L1+ expression.ConclusionsThis study demonstrates the utility and potential of the GraphITE platform to discriminate between and describe both unique and common neighborhood-level features of the tumor microenvironment. Deploying GraphITE across multiple indications effectively leverages spatial heterogeneity and multimarker information from multiplex IF panels.References1. Veličković P, Fedus W, Hamilton WL, Liò P, Bengio Y, DevonHjelm R. Deep Graph Infomax. 2018. arxiv:1809.10341 [stat.ML].2. McInnes L, Healy J, Melville J. UMAP: Uniform manifold approximationand projection for dimension reduction. 2020; arxiv:1802.03426 [stat.ML].3. Innocenti C, Zhang Z, Selvaraj B, Gaffney I, Frangos M, Cohen-Setton J, Dillon LAL, Surace MJ, Pedrinaci C, Hipp J, Baykaner K. An unsupervised graph embeddings approach to multiplex immunofluorescence image explorationbioRxiv 2021.06.09.447654; doi: https://doi.org/10.1101/2021.06.09.447654Ethics ApprovalThe study was approved by AstraZeneca.

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

Disease Models & Mechanisms ◽

10.1242/dmm.048942 ◽

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.

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The Tumor Microenvironment as a Target for Therapeutic Intervention

Targeted Therapy in Translational Cancer Research ◽

10.1002/9781118468678.ch5 ◽

2015 ◽

pp. 47-63

Author(s):

Hua Fang ◽

Yves A. DeClerck

Keyword(s):

Tumor Microenvironment ◽

Therapeutic Intervention

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Nano-Strategies to Target Breast Cancer-Associated Fibroblasts: Rearranging the Tumor Microenvironment to Achieve Antitumor Efficacy

International Journal of Molecular Sciences ◽

10.3390/ijms20061263 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1263 ◽

Cited By ~ 15

Author(s):

Marta Truffi ◽

Serena Mazzucchelli ◽

Arianna Bonizzi ◽

Luca Sorrentino ◽

Raffaele Allevi ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Microenvironment ◽

Cytotoxic T Cells ◽

Immune Therapy ◽

Tumor Stroma ◽

Cancer Associated Fibroblasts ◽

Reactive Stroma ◽

Current State ◽

Immune Mediated ◽

And Function

Cancer-associated fibroblasts (CAF) are the most abundant cells of the tumor stroma and they critically influence cancer growth through control of the surrounding tumor microenvironment (TME). CAF-orchestrated reactive stroma, composed of pro-tumorigenic cytokines and growth factors, matrix components, neovessels, and deregulated immune cells, is associated with poor prognosis in multiple carcinomas, including breast cancer. Therefore, beyond cancer cells killing, researchers are currently focusing on TME as strategy to fight breast cancer. In recent years, nanomedicine has provided a number of smart delivery systems based on active targeting of breast CAF and immune-mediated overcome of chemoresistance. Many efforts have been made both to eradicate breast CAF and to reshape their identity and function. Nano-strategies for CAF targeting profoundly contribute to enhance chemosensitivity of breast tumors, enabling access of cytotoxic T-cells and reducing immunosuppressive signals. TME rearrangement also includes reorganization of the extracellular matrix to enhance permeability to chemotherapeutics, and nano-systems for smart coupling of chemo- and immune-therapy, by increasing immunogenicity and stimulating antitumor immunity. The present paper reviews the current state-of-the-art on nano-strategies to target breast CAF and TME. Finally, we consider and discuss future translational perspectives of proposed nano-strategies for clinical application in breast cancer.

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Role of MSC in the Tumor Microenvironment

Cancers ◽

10.3390/cancers12082107 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2107 ◽

Cited By ~ 6

Author(s):

Ralf Hass

Keyword(s):

Tumor Microenvironment ◽

Cancer Cells ◽

Tumor Development ◽

Tumor Stroma ◽

Cell Types ◽

Cellular Interactions ◽

Cell Functions ◽

Metastatic Behavior ◽

Stem Cell Niches

The tumor microenvironment represents a dynamically composed matrix in which tissue-associated cancer cells are embedded together with a variety of further cell types to form a more or less separate organ-like structure. Constantly mutual interactions between cells of the tumor microenvironment promote continuous restructuring and growth in the tumor. A distinct organization of the tumor stroma also facilitates the formation of transient cancer stem cell niches, thereby contributing to progressive and dynamic tumor development. An important but heterogeneous mixture of cells that communicates among the cancer cells and the different tumor-associated cell types is represented by mesenchymal stroma-/stem-like cells (MSC). Following recruitment to tumor sites, MSC can change their functionalities, adapt to the tumor’s metabolism, undergo differentiation and synergize with cancer cells. Vice versa, cancer cells can alter therapeutic sensitivities and change metastatic behavior depending on the type and intensity of this MSC crosstalk. Thus, close cellular interactions between MSC and cancer cells can eventually promote cell fusion by forming new cancer hybrid cells. Consequently, newly acquired cancer cell functions or new hybrid cancer populations enlarge the plasticity of the tumor and counteract successful interventional strategies. The present review article highlights some important features of MSC within the tumor stroma.

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Exosomal MicroRNAs and Organotropism in Breast Cancer Metastasis

Cancers ◽

10.3390/cancers12071827 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1827 ◽

Cited By ~ 2

Author(s):

Grace L. Wong ◽

Sara Abu Jalboush ◽

Hui-Wen Lo

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Tumor Microenvironment ◽

Cancer Metastasis ◽

Current Knowledge ◽

Metastatic Breast ◽

Tumor Stroma ◽

Breast Cancer Metastasis ◽

Exosomal Mirnas ◽

Made In

Breast cancer is the most frequent malignancy for women in which one in eight women will be diagnosed with the disease in their lifetime. Despite advances made in treating primary breast cancer, there is still no effective treatment for metastatic breast cancer. Consequently, metastatic breast cancer is responsible for 90% of breast cancer-related deaths while only accounting for approximately one third of all breast cancer cases. To help develop effective treatments for metastatic breast cancer, it is important to gain a deeper understanding of the mechanisms by which breast cancer metastasizes, particularly, those underlying organotropism towards brain, bone, and lungs. In this review, we will primarily focus on the roles that circulating exosomal microRNAs (miRNAs) play in organotropism of breast cancer metastasis. Exosomes are extracellular vesicles that play critical roles in intercellular communication. MicroRNAs can be encapsulated in exosomes; cargo-loaded exosomes can be secreted by tumor cells into the tumor microenvironment to facilitate tumor–stroma interactions or released to circulation to prime distant organs for subsequent metastasis. Here, we will summarize our current knowledge on the biogenesis of exosomes and miRNAs, mechanisms of cargo sorting into exosomes, the exosomal miRNAs implicated in breast cancer metastasis, and therapeutic exosomal miRNAs.

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The Role of Extracellular Vesicles in Cancer: Cargo, Function, and Therapeutic Implications

Cells ◽

10.3390/cells7080093 ◽

2018 ◽

Vol 7 (8) ◽

pp. 93 ◽

Cited By ~ 28

Author(s):

James Jabalee ◽

Rebecca Towle ◽

Cathie Garnis

Keyword(s):

Tumor Microenvironment ◽

Tumor Cells ◽

Extracellular Vesicles ◽

Tumor Stroma ◽

Therapeutic Implications ◽

Membrane Bound ◽

Immune Destruction ◽

And Function ◽

Cargo Molecule

Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound structures that play key roles in intercellular communication. EVs are potent regulators of tumorigenesis and function largely via the shuttling of cargo molecules (RNA, DNA, protein, etc.) among cancer cells and the cells of the tumor stroma. EV-based crosstalk can promote proliferation, shape the tumor microenvironment, enhance metastasis, and allow tumor cells to evade immune destruction. In many cases these functions have been linked to the presence of specific cargo molecules. Herein we will review various types of EV cargo molecule and their functional impacts in the context of oncology.

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Phase I/II Study of LDE225 in Combination with Gemcitabine and Nab-Paclitaxel in Patients with Metastatic Pancreatic Cancer

Cancers ◽

10.3390/cancers13194869 ◽

2021 ◽

Vol 13 (19) ◽

pp. 4869

Author(s):

Esther N. Pijnappel ◽

Nienke P. M. Wassenaar ◽

Oliver J. Gurney-Champion ◽

Remy Klaassen ◽

Koen van der Lee ◽

...

Keyword(s):

Tumor Microenvironment ◽

Phase I ◽

Phase Ii ◽

Treatment Phase ◽

Tumor Stroma ◽

Maximum Tolerated Dose ◽

Quantitative Mri ◽

Ductal Adenocarcinoma ◽

Central Feature ◽

Plasma Fraction

Background: Desmoplasia is a central feature of the tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC). LDE225 is a pharmacological Hedgehog signaling pathway inhibitor and is thought to specifically target tumor stroma. We investigated the combined use of LDE225 and chemotherapy to treat PDAC patients. Methods: This was a multi-center, phase I/II study for patients with metastatic PDAC establishing the maximum tolerated dose of LDE225 co-administered with gemcitabine and nab-paclitaxel (phase I) and evaluating the efficacy and safety of the treatment combination after prior FOLFIRINOX treatment (phase II). Tumor microenvironment assessment was performed with quantitative MRI using intra-voxel incoherent motion diffusion weighted MRI (IVIM-DWI) and dynamic contrast-enhanced (DCE) MRI. Results: The MTD of LDE225 was 200 mg once daily co-administered with gemcitabine 1000 mg/m2 and nab-paclitaxel 125 mg/m2. In phase II, six therapy-related grade 4 adverse events (AE) and three grade 5 were observed. In 24 patients, the target lesion response was evaluable. Three patients had partial response (13%), 14 patients showed stable disease (58%), and 7 patients had progressive disease (29%). Median overall survival (OS) was 6 months (IQR 3.9–8.1). Blood plasma fraction (DCE) and diffusion coefficient (IVIM-DWI) significantly increased during treatment. Baseline perfusion fraction could predict OS (>222 days) with 80% sensitivity and 85% specificity. Conclusion: LDE225 in combination with gemcitabine and nab-paclitaxel was well-tolerated in patients with metastatic PDAC and has promising efficacy after prior treatment with FOLFIRINOX. Quantitative MRI suggested that LDE225 causes increased tumor diffusion and works particularly well in patients with poor baseline tumor perfusion.

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Norrin/Frizzled4 signalling in the preneoplastic niche blocks medulloblastoma initiation

eLife ◽

10.7554/elife.16764 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 10

Author(s):

Erin A Bassett ◽

Nicholas Tokarew ◽

Ema A Allemano ◽

Chantal Mazerolle ◽

Katy Morin ◽

...

Keyword(s):

Endothelial Cells ◽

Tumor Microenvironment ◽

Mouse Model ◽

Tumor Cells ◽

Tumor Stroma ◽

Cerebellar Tumor ◽

Short Term ◽

Stromal Component ◽

Tumor Types ◽

The Relationship

The tumor microenvironment is a critical modulator of carcinogenesis; however, in many tumor types, the influence of the stroma during preneoplastic stages is unknown. Here we explored the relationship between pre-tumor cells and their surrounding stroma in malignant progression of the cerebellar tumor medulloblastoma (MB). We show that activation of the vascular regulatory signalling axis mediated by Norrin (an atypical Wnt)/Frizzled4 (Fzd4) inhibits MB initiation in the Ptch+/− mouse model. Loss of Norrin/Fzd4-mediated signalling in endothelial cells, either genetically or by short-term blockade, increases the frequency of pre-tumor lesions and creates a tumor-permissive microenvironment at the earliest, preneoplastic stages of MB. This pro-tumor stroma, characterized by angiogenic remodelling, is associated with an accelerated transition from preneoplasia to malignancy. These data expose a stromal component that regulates the earliest stages of tumorigenesis in the cerebellum, and a novel role for the Norrin/Fzd4 axis as an endogenous anti-tumor signal in the preneoplastic niche.

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Cancer-associated Fibroblasts: Origins, Heterogeneity and Functions in Tumor Microenvironment

10.20944/preprints202001.0155.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Kevin Dzobo

Keyword(s):

Tumor Microenvironment ◽

Tumor Cell ◽

Cancer Cells ◽

Stromal Cells ◽

Tumor Stroma ◽

Bioinformatic Analysis ◽

Therapy Resistance ◽

Response To Therapy ◽

The Cancer Genome Atlas ◽

Cancer Associated Fibroblasts

Current therapeutic strategies targeting cancer cells within solid tumors have displayed limited success owing to the presence of non-cancer components referred to as the tumor stroma within the tumor microenvironment (TM). These stromal cells, extracellular matrix and blood vessels influence cancer cell response to therapy and play key roles in tumor relapse and resistance. Of the stromal cells present in the TM, a lot of attention has been given to cancer-associated fibroblasts (CAFs) as they are the most abundant and are important in cancer initiation, progression and therapy resistance. In this updated review I emphasize the role of CAFs in the regulation of tumor cell behaviour and reveal how CAF-derived factors and signaling influence tumor cell heterogeneity and development of novel strategies to combat cancer. To investigate the expression of CAF markers in tumor tissues versus normal tissues, transcriptomic data from The Cancer Genome Atlas (TCGA) and the Gene Expression Profiling Interactive Analysis (GEPIA) databases was used. Bioinformatic analysis reveals differential expression of CAF markers in several cancer types, underscoring the need for further multiomics and biochemical studies on CAFs, CAF subsets and markers. Differences in CAF markers’ expression could be due to different cellular origins as well as the effect of cancer-specific tumor microenvironmental effect on CAFs. Lastly, I present recent advances in therapeutic targeting of CAFs and the success of such endeavours or its lack thereof. It is recommended that for patients’ outcomes to improve, cancer treatment be combinatorial in nature, targeting both cancer cells and stromal cells and interactions.

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