bims-tuchim 2021-03-21 papers

bims-tuchim

Biomed News

on Tumor-on-chip models

Issue of 2021–03–21
twenty-one papers selected by
Philipp Albrecht, Friedrich Schiller University

Matrix Degradation Assay to Measure the Ability of Tumor Cells to Degrade Extracellular Matrix.
TGF-β1-activated cancer-associated fibroblasts promote breast cancer invasion, metastasis and epithelial-mesenchymal transition by autophagy or overexpression of FAP-α.
Immunogenicity risk assessment for biotherapeutics through in vitro detection of CD134 and CD137 on T helper cells.
Engineering Tissue Barrier Models on Hydrogel Microfluidic Platforms.
Chimeric antigen receptor engineered NK cellular immunotherapy overcomes the selection of T-cell escape variant cancer cells.
In Vitro 3D Models of Tunable Stiffness.
Combining IL-10 and Oncolytic Adenovirus Demonstrates Enhanced Antitumor Efficacy Through CD8+ T Cells.
Engineering the early bone metastatic niche through human vascularized immuno bone minitissues.
Heparin-modified alginate microspheres enhance neovessel formation in hiPSC-derived endothelial cells and heterocellular in vitro models by controlled release of vascular endothelial growth factor.
Metabolism of Dendritic Cells in Tumor Microenvironment: For Immunotherapy.
Modelling human blastocysts by reprogramming fibroblasts into iBlastoids.
Imaging of Human Cancer Cells in 3D Collagen Matrices.
Targeting loss of heterozygosity for cancer-specific immunotherapy.
Probing Intravascular Adhesion and Extravasation of Tumor Cells with Microfluidics.
Extracellular Matrix-Based Hydrogels to Tailoring Tumor Organoids.
Beyond PD-1: The Next Frontier for Immunotherapy in Melanoma.
Engineered models of tumor metastasis with immune cell contributions.
Circulating mucosal-associated invariant T cells identify patients responding to anti-PD-1 therapy.
Anti-CD19 CAR T cells potently redirected to kill solid tumor cells.
Tissue factor potentiates adherence of breast cancer cells to human umbilical vein endothelial cells under static and flow conditions.
Label-free deep profiling of the tumor microenvironment.

Methods Mol Biol. 2021 ;2294 151-163

Matrix Degradation Assay to Measure the Ability of Tumor Cells to Degrade Extracellular Matrix.

Rosa Fontana, Jing Yang.

  During the metastatic process, carcinoma cells form invadopodia, F-actin enriched protrusive structures, to degrade the extracellular matrix (ECM) in order to invade the surrounding stroma and intravasate into the circulatory system. In this chapter, we describe the 2D-fluorescent matrix degradation assay, a highly sensitive and reproducible in vitro method used to measure invadopodia-mediated ECM degradation. We provide a detailed protocol on how to prepare the glass coverslips with fluorescent gelatin matrix and a standardized method to quantify gelatin degradation and invadopodia formation in order to evaluate cell invasion.

Keywords:  Cancer metastasis; ECM; Fluorescent gelatin degradation assay; ImageJ; Invadopodia; Invadosome; Invasion; Podosome; Rosettes

DOI:  https://doi.org/10.1007/978-1-0716-1350-4_11
Biochem Pharmacol. 2021 Mar 16. pii: S0006-2952(21)00123-4. [Epub ahead of print] 114527

TGF-β1-activated cancer-associated fibroblasts promote breast cancer invasion, metastasis and epithelial-mesenchymal transition by autophagy or overexpression of FAP-α.

Minli Huang, Mengru Fu, Jia Wang, Chunhua Xia, Hong Zhang, Yuqing Xiong, Jiake He, Jianming Liu, Bingchen Liu, Siyi Pan, Fanglan Liu.

  Cancer-associated fibroblasts (CAFs) play an important role in the initiation, metastasis, and invasion of breast cancer. However, whether autophagy acts as a tumor promotion mechanism by inducing epithelial-mesenchymal transition (EMT) is still controversial and remains undefined at the mechanistic levels. In this study, we investigated whether autophagy or FAP-α is required for the invasion, pulmonary metastasis and EMT of breast cancer cells and underlying mechanism. We employed an in vitro model of NIH3T3 fibroblasts treated with H2O2 and confirmed that TGF-β1 could convert fibroblasts into CAFs through autophagy under oxidative stress in the tumor microenvironment. Modulation of autophagy by rapamycin, 3-methyladenine or ATG-5 knockdown regulated the expression of CAFs markers, suggesting a role of autophagy in the tumor promotion mechanism of TGF-β1-induced CAFs activation. Furthermore, we established an indirect co-culture model and a mixed xenograft as a corresponding in vivo model. We demonstrated that TGF-β1-activated CAFs promote tumor invasion, pulmonary metastasis and EMT, which act through autophagy and overexpression of FAP-α in both models, while autophagy inhibitor 3-methyladenine blocked these effects induced by TGF-β1-activated CAFs. Moreover, the co-localization of LC3β and EMT marker vimentin in mixed xenograft also revealed that TGF-β1-activated CAFs promote tumor growth, pulmonary metastasis, and EMT program partly through autophagy. In addition, knockdown of FAP-α resulted in reversed EMT and abolished tumor invasion and pulmonary metastasis induced by TGF-β1-activated CAFs. Taken together, we conclude that both autophagy and FAP-α are required for breast cancer cell invasion and metastasis. Targeting autophagy or FAP-α rather than both can serve as a potential approach to improve the prognosis for human breast cancer.

Keywords:  Autophagy; Cancer-associated fibroblasts; Epithelial-mesenchymal transition; FAP-α; TGF-β1

DOI:  https://doi.org/10.1016/j.bcp.2021.114527
MAbs. 2021 Jan-Dec;13(1):13(1): 1898831

Immunogenicity risk assessment for biotherapeutics through in vitro detection of CD134 and CD137 on T helper cells.

Sivan Cohen, Srividya Myneni, Anna Batt, Joyce Guerrero, Jochen Brumm, Shan Chung.

  Biotherapeutics, which are biologic medications that are natural or bioengineered products of living cells, have revolutionized the treatment of many diseases. However, unwanted immune responses still present a major challenge to their widespread adoption. Many patients treated with biotherapeutics develop antigen-specific anti-drug antibodies (ADAs) that may reduce the efficacy of the therapy or cross-react with the endogenous counterpart of a protein therapeutic, or both. Here, we describe an in vitro method for assessing the immunogenic risk of a biotherapeutic. We found a correlation between clinical immunogenicity and the frequency with which a biotherapeutic stimulated an increase in CD134, CD137, or both cell surface markers on CD4+ T cells. Using high-throughput flow cytometry, we examined the effects of 14 biotherapeutics with diverse rates of clinical immunogenicity on peripheral blood mononuclear cells from 120 donors with diverse human leukocyte antigen class II-encoding alleles. Biotherapeutics with high rates of ADA development in the clinic had higher proportions of CD4+ T cells positive for CD134 or CD137 than biotherapeutics with low clinical immunogenicity. This method provides a rapid and simple preclinical test of the immunogenic potential of a new candidate biotherapeutic or biosimilar. Implementation of this approach during biotherapeutic research and development enables rapid elimination of candidates that are likely to cause ADA-related adverse events and detrimental consequences.

Keywords:  CD134; CD137; HLA II type; Immunogenicity; PMBC; T cell activation; T cell epitopes; anti-drug antibodies; biotherapeutics

DOI:  https://doi.org/10.1080/19420862.2021.1898831
ACS Appl Mater Interfaces. 2021 Mar 19.

Engineering Tissue Barrier Models on Hydrogel Microfluidic Platforms.

Daniel Vera, María García-Díaz, Núria Torras, Mar Álvarez, Rosa Villa, Elena Martinez.

  Tissue barriers play a crucial role in human physiology by establishing tissue compartmentalization and regulating organ homeostasis. At the interface between the extracellular matrix (ECM) and flowing fluids, epithelial and endothelial barriers are responsible for solute and gas exchange. In the past decade, microfluidic technologies and organ-on-chip devices became popular as in vitro models able to recapitulate these biological barriers. However, in conventional microfluidic devices, cell barriers are primarily grown on hard polymeric membranes within polydimethylsiloxane (PDMS) channels that do not mimic the cell-ECM interactions nor allow the incorporation of other cellular compartments such as stromal tissue or vascular structures. To develop models that accurately account for the different cellular and acellular compartments of tissue barriers, researchers have integrated hydrogels into microfluidic setups for tissue barrier-on-chips, either as cell substrates inside the chip, or as self-contained devices. These biomaterials provide the soft mechanical properties of tissue barriers and allow the embedding of stromal cells. Combining hydrogels with microfluidics technology provides unique opportunities to better recreate in vitro the tissue barrier models including the cellular components and the functionality of the in vivo tissues. Such platforms have the potential of greatly improving the predictive capacities of the in vitro systems in applications such as drug development, or disease modeling. Nevertheless, their development is not without challenges in their microfabrication. In this review, we will discuss the recent advances driving the fabrication of hydrogel microfluidic platforms and their applications in multiple tissue barrier models.

Keywords:  hydrogel; microfabrication; microfluidics; organ-on-chip; tissue barrier

DOI:  https://doi.org/10.1021/acsami.0c21573
J Immunother Cancer. 2021 Mar;pii: e002128. [Epub ahead of print]9(3):

Chimeric antigen receptor engineered NK cellular immunotherapy overcomes the selection of T-cell escape variant cancer cells.

Maxwell Y Lee, Yvette Robbins, Cem Sievers, Jay Friedman, Houssein Abdul Sater, Paul E Clavijo, Nancy Judd, Edward Tsong, Chris Silvin, Patrick Soon-Shiong, Michelle R Padget, Jeffrey Schlom, James Hodge, Christian Hinrichs, Clint Allen.

   BACKGROUND: As heterogeneous tumors develop in the face of intact immunity, tumor cells harboring genomic or expression defects that favor evasion from T-cell detection or elimination are selected. For patients with such tumors, T cell-based immunotherapy alone infrequently results in durable tumor control.
METHODS: Here, we developed experimental models to study mechanisms of T-cell escape and demonstrated that resistance to T-cell killing can be overcome by the addition of natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR) targeting programmed death ligand-1 (PD-L1).
RESULTS: In engineered models of tumor heterogeneity, PD-L1 CAR-engineered NK cells (PD-L1 t-haNKs) prevented the clonal selection of T cell-resistant tumor cells observed with T-cell treatment alone in multiple models. Treatment of heterogenous cancer cell populations with T cells resulted in interferon gamma (IFN-γ) release and subsequent upregulation of PD-L1 on tumor cells that escaped T-cell killing through defects in antigen processing and presentation, priming escape cell populations for PD-L1 dependent killing by PD-L1 t-haNKs in vitro and in vivo.
CONCLUSIONS: These results describe the underlying mechanisms governing synergistic antitumor activity between T cell-based immunotherapy that results in IFN-γ production, upregulation of PD-L1 on T-cell escape cells, and the use of PD-L1 CAR-engineered NK cells to target and eliminate resistant tumor cell populations.

Keywords:  adoptive; combined modality therapy; head and neck neoplasms; immunotherapy

DOI:  https://doi.org/10.1136/jitc-2020-002128
Methods Mol Biol. 2021 ;2294 27-42

In Vitro 3D Models of Tunable Stiffness.

Elysse C Filipe, Amelia L Parker, Antonia L Cadell, Gretel Major, David R Croucher, Thomas R Cox.

  Three-dimensional models of spheroid formation have been routinely used in the cancer field to test the colony forming capacity of malignant cells in an in vitro setting. Use of such a model provides a robust surrogate for in vivo testing, enabling large-scale interrogation into the effect of certain treatment conditions. This adapted protocol describes a high throughput and readily accessible composite alginate hydrogel system for spheroid formation, within a biomechanically tunable three-dimensional environment. This model therefore allows users to examine the effect of certain treatment conditions while cells are embedded within a hydrogel of defined stiffness. This is particularly important in the context of cancer where cells experience a wide range of mechanical properties within their microenvironment, driven by widespread changes in the extracellular matrix composition and architecture.This protocol describes a high-throughput method which results in homogeneous interpenetrating polymer networks of collagen and alginate. We show that this network readily supports single-cell spheroid formation in numerous malignant cell lines (breast cancer, lung cancer, and melanoma) and that these can be robustly analyzed for colony formation measures such as spheroid size, spheroid number, and overall cell viability; therefore, allowing users to undertake high-throughput, in vitro screening against a controlled biomechanical background.

Keywords:  Alginate; Colony formation; Extracellular matrix; High throughput; Spheroid; Stiffness

DOI:  https://doi.org/10.1007/978-1-0716-1350-4_3
Front Immunol. 2021 ;12 615089

Combining IL-10 and Oncolytic Adenovirus Demonstrates Enhanced Antitumor Efficacy Through CD8+ T Cells.

Duo Chen, Luyu Huang, Haiyu Zhou, Yuhui Zhang.

  Oncolytic viruses are of growing importance in cancer therapeutics since they combine direct oncolytic effect and the stimulation of antitumor immunity. Emerging evidences showed that the function of oncolytic viruses is dependent on immune response in tumor microenvironment, and the modulation of immunity could influence their efficacy. Here we combined the interleukin 10 (IL-10) and oncolytic adenovirus Ad-hTERT to treat lung cancer and explored the underlying mechanism under combination therapy. Lewis lung carcinoma (LLC) and B16F10 tumor-bearing immunocompetent C57BL/6 mice that received Ad-hTERT or IL-10 alone showed mild antitumor effect, while the combination therapy shrink tumor bulks and prolonged survival remarkably. In addition, IL-10 didn't show direct influence on tumor cell viability or Ad-hTERT mediated tumor cell lysis in vitro. To further explore the influence of combination therapy mediated antitumor capacity, we eliminated CD8+ T, CD4+ T or natural killer (NK) cells in LLC and B16F10-bearing C57BL/6 mice, and found that CD8+ T cells were critical mediator in the combination therapy. The combination therapy induced intensive infiltration of CD8+ T cells in tumors, increased tumor-specific IFN-γ secretion by CD8+ T cells. The long-term tumor-specific immune memory induced by the combination therapy rejected rechallenge by respective tumor cell lines. This study demonstrated that the therapy combining IL-10 and Ad-hTERT augmented antitumor efficacy which was CD8+ T cells dependent. Our findings paved the way to combine cytokines and oncolytic viruses to enhance antitumor immunotherapy in treating cancer.

Keywords:  Ad-hTERT; CD8+ T cells; IL-10; cancer; combination therapy; oncolytic adenovirus

DOI:  https://doi.org/10.3389/fimmu.2021.615089
Biofabrication. 2021 Mar 18.

Engineering the early bone metastatic niche through human vascularized immuno bone minitissues.

Maria Vittoria Colombo, Simone Bersini, Chiara Arrigoni, Mara Gilardi, Veronica Sansoni, Enrico Ragni, Gabriele Candiani, Giovanni Lombardi, Matteo Moretti.

  Bone metastases occur in 65-80% advanced breast cancer patients. Although significant progresses have been made in understanding the biological mechanisms driving the bone metastatic cascade, traditional 2D in vitro models and animal studies are not effectively reproducing breast cancer cells (CCs) interactions with the bone microenvironment and suffer from species-specific differences, respectively. Moreover, simplified in vitro models cannot realistically estimate drug anti-tumoral properties and side effects, hence leading to pre-clinical testing frequent failures. To solve this issue, a 3D metastatic bone minitissue is designed with embedded human osteoblasts, osteoclasts, bone-resident macrophages, endothelial cells and breast CCs. This minitissue recapitulates key features of the bone metastatic niche, including the alteration of macrophage polarization and microvascular architecture, along with the induction of CC micrometastases and osteomimicry. The minitissue reflects breast CC organ-specific metastatization to bone compared to a muscle minitissue. Finally, two FDA approved drugs, doxorubicin and rapamycin, have been tested showing that the dose required to impair CC growth is significantly higher in the metastatic bone minitissue compared to a simpler CC monoculture minitissue. The metastatic bone minitissue allows the investigation of metastasis key biological features and represents a reliable tool to better predict drug effects on the metastatic bone microenvironment.

Keywords:  3D in vitro models; bone metastases; bone-tumor interactions; breast cancer; drug efficacy

DOI:  https://doi.org/10.1088/1758-5090/abefea
J Biomed Mater Res A. 2021 Mar 17.

Heparin-modified alginate microspheres enhance neovessel formation in hiPSC-derived endothelial cells and heterocellular in vitro models by controlled release of vascular endothelial growth factor.

Fabiola Munarin, Carly Kabelac, Kareen L K Coulombe.

  A formidable challenge in regenerative medicine is the development of stable microvascular networks to restore adequate blood flow or to sustain graft viability and long-term function in implanted or ischemic tissues. In this work, we develop a biomimetic approach to increase the binding affinity of the extracellular matrix for the class of heparin-binding growth factors to localize and control the release of proangiogenic cues while maintaining their bioactivity. Sulfate and heparin moieties are covalently coupled to alginate, and alginate microspheres are produced and used as local delivery depots for vascular endothelial growth factor (VEGF). Release of VEGF from sulfate-alginate and heparin-alginate bulk hydrogels and microspheres was sustained over 14 days. In vitro evaluation with human induced pluripotent stem cell (hiPSC)-derived endothelial cells and aortic ring assay in a chemically defined hydrogel demonstrates development of primitive three-dimensional vessel-like networks in the presence of VEGF released from the chemically modified alginate microspheres. Furthermore, our results suggest that the sulfate groups available on the chemically modified alginate microspheres promote some new vessel formation even in VEGF-free samples. Based on this evidence, we conclude that sulfate- and heparin-alginate hydrogels are adaptive and bioactive delivery systems for revascularization therapy and translational vascular tissue engineering.

Keywords:  VEGF delivery; alginate; angiogenesis; heparin; microspheres

DOI:  https://doi.org/10.1002/jbm.a.37168
Front Immunol. 2021 ;12 613492

Metabolism of Dendritic Cells in Tumor Microenvironment: For Immunotherapy.

Xin Peng, Youe He, Jun Huang, Yongguang Tao, Shuang Liu.

  Dendritic cells (DCs) are a type of an antigen-presenting cell which undertake a job on capturing antigens coming from pathogens or tumors and presenting to T cells for immune response. The metabolism of DCs controls its development, polarization, and maturation processes and provides energy support for its functions. However, the immune activity of DCs in tumor microenvironment (TME) is inhibited generally. Abnormal metabolism of tumor cells causes metabolic changes in TME, such as hyperglycolysis, lactate and lipid accumulation, acidification, tryptophan deprivation, which limit the function of DCs and lead to the occurrence of tumor immune escape. Combined metabolic regulation with immunotherapy can strengthen the ability of antigen-presentation and T cell activation of DCs, improve the existing anti-tumor therapy, and overcome the defects of DC-related therapies in the current stage, which has great potential in oncology therapy. Therefore, we reviewed the glucose, lipid, and amino acid metabolism of DCs, as well as the metabolic changes after being affected by TME. Together with the potential metabolic targets of DCs, possible anti-tumor therapeutic pathways were summarized.

Keywords:  amino acid; dendritic cells; glucose; lipid; metabolism; therapy; tumor microenvironment

DOI:  https://doi.org/10.3389/fimmu.2021.613492
Nature. 2021 Mar 17.

Modelling human blastocysts by reprogramming fibroblasts into iBlastoids.

Xiaodong Liu, Jia Ping Tan, Jan Schröder, Asma Aberkane, John F Ouyang, Monika Mohenska, Sue Mei Lim, Yu B Y Sun, Joseph Chen, Guizhi Sun, Yichen Zhou, Daniel Poppe, Ryan Lister, Amander T Clark, Owen J L Rackham, Jennifer Zenker, Jose M Polo.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development1-4. However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization.

DOI: https://doi.org/10.1038/s41586-021-03372-y
Bio Protoc. 2021 Jan 20. 11(2): e3889

Imaging of Human Cancer Cells in 3D Collagen Matrices.

Karin Pfisterer, Brooke Lumicisi, Maddy Parsons.

  Research on cell migration and interactions with the extracellular matrix (ECM) was mostly focused on 2D surfaces in the past. Many recent studies have highlighted differences in migratory behaviour of cells on 2D surfaces compared to complex cell migration modes in 3D environments. When embedded in 3D matrices, cells constantly sense the physicochemical, topological and mechanical properties of the ECM and adjust their behaviour accordingly. Changes in the stiffness of the ECM can have effects on cell morphology, differentiation and behaviour and cells can follow stiffness gradients in a process called durotaxis. Here we introduce a detailed protocol for the assembly of 3D matrices consisting of collagen I/fibronectin and embedding cells for live cell imaging. Further, we will show how the matrix can be stiffened via non-enzymatic glycation and how collagen staining with fluorescent dyes allows simultaneous imaging of both matrix and cells. This approach can be used to image cell migration in 3D microenvironments with varying stiffness, define cell-matrix interactions and the cellular response to changing ECM, and visualize matrix deformation by the cells.

Keywords:  3D collagen matrix; 3D imaging; Cancer cell migration; Cell-matrix interaction; Filopodia; Live cell imaging

DOI:  https://doi.org/10.21769/BioProtoc.3889
Proc Natl Acad Sci U S A. 2021 Mar 23. pii: e2022410118. [Epub ahead of print]118(12):

Targeting loss of heterozygosity for cancer-specific immunotherapy.

Michael S Hwang, Brian J Mog, Jacqueline Douglass, Alexander H Pearlman, Emily Han-Chung Hsiue, Suman Paul, Sarah R DiNapoli, Maximilian F Konig, Drew M Pardoll, Sandra B Gabelli, Chetan Bettegowda, Nickolas Papadopoulos, Bert Vogelstein, Shibin Zhou, Kenneth W Kinzler.

  Developing therapeutic agents with potent antitumor activity that spare normal tissues remains a significant challenge. Clonal loss of heterozygosity (LOH) is a widespread and irreversible genetic alteration that is exquisitely specific to cancer cells. We hypothesized that LOH events can be therapeutically targeted by "inverting" the loss of an allele in cancer cells into an activating signal. Here we describe a proof-of-concept approach utilizing engineered T cells approximating NOT-gate Boolean logic to target counterexpressed antigens resulting from LOH events in cancer. The NOT gate comprises a chimeric antigen receptor (CAR) targeting the allele of human leukocyte antigen (HLA) that is retained in the cancer cells and an inhibitory CAR (iCAR) targeting the HLA allele that is lost in the cancer cells. We demonstrate that engineered T cells incorporating such NOT-gate logic can be activated in a genetically predictable manner in vitro and in mice to kill relevant cancer cells. This therapeutic approach, termed NASCAR (Neoplasm-targeting Allele-Sensing CAR), could, in theory, be extended to LOH of other polymorphic genes that result in altered cell surface antigens in cancers.

Keywords:  cancer immunotherapy; cell engineering; chimeric antigen receptor; human leukocyte antigen; loss of heterozygosity

DOI:  https://doi.org/10.1073/pnas.2022410118
Methods Mol Biol. 2021 ;2294 111-132

Probing Intravascular Adhesion and Extravasation of Tumor Cells with Microfluidics.

Naël Osmani, Gautier Follain, Valentin Gensbittel, María Jesús García-León, Sébastien Harlepp, Jacky G Goetz.

  Cancer metastasis is a multistep process during which tumor cells leave the primary tumor mass and form distant secondary colonies that are lethal. Circulating tumor cells (CTCs) are transported by body fluids to reach distant organs, where they will extravasate and either remain dormant or form new tumor foci. Development of methods to study the behavior of CTCs at the late stages of the intravascular journey is thus required to dissect the molecular mechanisms at play. Using recently developed microfluidics approaches, we have demonstrated that CTCs arrest intravascularly, through a two-step process: (a) CTCs stop using low energy and rapidly activated adhesion receptors to form transient metastable adhesions and (b) CTCs stabilize their adhesions to the endothelial layer with high energy and slowly activated adhesion receptors. In this methods chapter, we describe these easy-to-implement quantitative methods using commercially available microfluidic channels. We detail the use of fast live imaging combined to fine-tuned perfusion to measure the adhesion potential of CTC depending on flow velocities. We document how rapidly engaged early metastable adhesion can be discriminated from slower activated stable adhesion using microfluidics. Finally, CTC extravasation potential can be assessed within this setup using long-term cell culture under flow. Altogether, this experimental pipeline can be adapted to probe the adhesion (to the endothelial layer) and extravasation potential of any circulating cell.

Keywords:  Adhesion; Circulating tumor cells (CTCs); Extravasation; Live imaging; Metastasis; Microfluidics

DOI:  https://doi.org/10.1007/978-1-0716-1350-4_8
ACS Biomater Sci Eng. 2021 Mar 16.

Extracellular Matrix-Based Hydrogels to Tailoring Tumor Organoids.

Hyun Jin Lee, Siwon Mun, Duc M Pham, Pilnam Kim.

  When embedded into a three-dimensional (3D) matrix, cancer stem cells (or cancer-initiating cells) can grow into self-organizing organotypic structures called tumor organoids. During organoid formation, the matrix not only provides structural support but also delivers biochemical signals. Although increasing evidence indicates that the extracellular matrix (ECM) is an essential component of the tumor microenvironment during tumor development and progression, the influence of the ECM on organoid formation has been largely ignored; the ECM has only recently been recognized to play a role in the regulation of cancer cell phenotypes. We reviewed ECM-based hydrogels to tailoring tumor organoids and highlight the potential role of the ECM in the development of recapitulating malignant/invasive tumor organoids with enhanced capacity for in vitro representation of ECM-regulated tumor progression.

Keywords:  3D culture; extracellular matrix (ECM); tumor microenvironment; tumor organoids

DOI:  https://doi.org/10.1021/acsbiomaterials.0c01801
Front Oncol. 2021 ;11 640314

Beyond PD-1: The Next Frontier for Immunotherapy in Melanoma.

Anjali Rohatgi, John M Kirkwood.

  The advent of first and second-generation immune checkpoint blockade (ICI) has resulted in improved survival of patients with metastatic melanoma over the past decade. However, the majority of patients ultimately progress despite these treatments, which has served as an impetus to consider a range of subsequent therapies. Many of the next generation of immunotherapeutic agents focus on modifying the immune system to overcome resistance to checkpoint blockade. ICI resistance can be understood as primary, or acquired-where the latter is the most common scenario. While there are several postulated mechanisms by which resistance, particularly acquired resistance, occurs, the predominant escape mechanisms include T cell exhaustion, upregulation of alternative inhibitory checkpoint receptors, and alteration of the tumor microenvironment (TME) into a more suppressive, anti-inflammatory state. Therapeutic agents in development are designed to work by combating one or more of these resistance mechanisms. These strategies face the added challenge of minimizing immune-related toxicities, while improving antitumor efficacy. This review focuses upon the following categories of novel therapeutics: 1) alternative inhibitory receptor pathways; 2) damage- or pathogen-associated molecular patterns (DAMPs/PAMPs); and 3) immune cell signaling mediators. We present the current state of these therapies, including preclinical and clinical data available for these targets under development.

Keywords:  TLR (Toll-like receptors); checkpoint inhibition/blockade; cytokines; melanoma; pathogen recognition receptor (PRR)

DOI:  https://doi.org/10.3389/fonc.2021.640314
iScience. 2021 Mar 19. 24(3): 102179

Engineered models of tumor metastasis with immune cell contributions.

Pamela L Graney, Daniel Naveed Tavakol, Alan Chramiec, Kacey Ronaldson-Bouchard, Gordana Vunjak-Novakovic.

  Most cancer deaths are due to tumor metastasis rather than the primary tumor. Metastasis is a highly complex and dynamic process that requires orchestration of signaling between the tumor, its local environment, distant tissue sites, and immune system. Animal models of cancer metastasis provide the necessary systemic environment but lack control over factors that regulate cancer progression and often do not recapitulate the properties of human cancers. Bioengineered "organs-on-a-chip" that incorporate the primary tumor, metastatic tissue targets, and microfluidic perfusion are now emerging as quantitative human models of tumor metastasis. The ability of these systems to model tumor metastasis in individualized, patient-specific settings makes them uniquely suitable for studies of cancer biology and developmental testing of new treatments. In this review, we focus on human multi-organ platforms that incorporate circulating and tissue-resident immune cells in studies of tumor metastasis.

Keywords:  biochemical assay; bioengineering; cancer; components of the immune system

DOI:  https://doi.org/10.1016/j.isci.2021.102179
Nat Commun. 2021 03 15. 12(1): 1669

Circulating mucosal-associated invariant T cells identify patients responding to anti-PD-1 therapy.

Sara De Biasi, Lara Gibellini, Domenico Lo Tartaro, Simone Puccio, Claudio Rabacchi, Emilia M C Mazza, Jolanda Brummelman, Brandon Williams, Kelly Kaihara, Mattia Forcato, Silvio Bicciato, Marcello Pinti, Roberta Depenni, Roberto Sabbatini, Caterina Longo, Massimo Dominici, Giovanni Pellacani, Enrico Lugli, Andrea Cossarizza.

Immune checkpoint inhibitors are used for treating patients with metastatic melanoma. Since the response to treatment is variable, biomarkers are urgently needed to identify patients who may benefit from such therapy. Here, we combine single-cell RNA-sequencing and multiparameter flow cytometry to assess changes in circulating CD8+ T cells in 28 patients with metastatic melanoma starting anti-PD-1 therapy, followed for 6 months: 17 responded to therapy, whilst 11 did not. Proportions of activated and proliferating CD8+ T cells and of mucosal-associated invariant T (MAIT) cells are significantly higher in responders, prior to and throughout therapy duration. MAIT cells from responders express higher level of CXCR4 and produce more granzyme B. In silico analysis support MAIT presence in the tumor microenvironment. Finally, patients with >1.7% of MAIT among peripheral CD8+ population show a better response to treatment. Our results thus suggest that MAIT cells may be considered a biomarker for patients responding to anti-PD-1 therapy.

DOI: https://doi.org/10.1038/s41467-021-21928-4
PLoS One. 2021 ;16(3): e0247701

Anti-CD19 CAR T cells potently redirected to kill solid tumor cells.

Christine Ambrose, Lihe Su, Lan Wu, Fay J Dufort, Thomas Sanford, Alyssa Birt, Benjamin J Hackel, Andreas Hombach, Hinrich Abken, Roy R Lobb, Paul D Rennert.

Successful CAR T cell therapy for the treatment of solid tumors requires exemplary CAR T cell expansion, persistence and fitness, and the ability to target tumor antigens safely. Here we address this constellation of critical attributes for successful cellular therapy by using integrated technologies that simplify development and derisk clinical translation. We have developed a CAR-CD19 T cell that secretes a CD19-anti-Her2 bridging protein. This cell therapy strategy exploits the ability of CD19-targeting CAR T cells to interact with CD19 on normal B cells to drive expansion, persistence and fitness. The secreted bridging protein potently binds to Her2-positive tumor cells, mediating CAR-CD19 T cell cytotoxicity in vitro and in vivo. Because of its short half-life, the secreted bridging protein will selectively accumulate at the site of highest antigen expression, ie. at the tumor. Bridging proteins that bind to multiple different tumor antigens have been created. Therefore, antigen-bridging CAR-CD19 T cells incorporate critical attributes for successful solid tumor cell therapy. This platform can be exploited to attack tumor antigens on any cancer.

DOI: https://doi.org/10.1371/journal.pone.0247701
Cell Adh Migr. 2021 Dec;15(1): 74-83

Tissue factor potentiates adherence of breast cancer cells to human umbilical vein endothelial cells under static and flow conditions.

Yanling Jin, Wei Liu, Fengxia Wang, Min Wang, Kai Xu, Aijun Yang, Chenyu Wang, Lihan Zhang, Fangfang Zhang, Min Li.

  Tissue factor (TF) has been extensively studied for tumor metastasis, but its role in mediating cancer cell adhesion to vasculature remains unknown. This study aimed to measure the ability of TF to mediate the adhesion of breast cancer cells to human umbilical vein endothelial cells (HUVECs). MDA-MB-231 cells expressed the highest TF level and adhered more to HUVECs under static and flow conditions, a neutralizing TF antibody abolished the enhanced adhesion of MDA-MB-231 cells to HUVECs. Recombinant human soluble TF (rTF) bonded β1integrin on HUVECs surfaces, β1 or α3integrin antibody combined with TF antibody abolished more cell-cell adhesion. These data suggested that TF mediated adhesion of breast cancer cells to endothelial cells may rely on β1integrin on HUVECs surfaces.

Keywords:  Tissue factor; adhesion; breast cancer; endothelium; β1 integrin

DOI:  https://doi.org/10.1080/19336918.2021.1898709
Cancer Res. 2021 Mar 19. pii: canres.3124.2020. [Epub ahead of print]

Label-free deep profiling of the tumor microenvironment.

Sixian You, Eric J Chaney, Haohua Tu, Yi Sun, Saurabh Sinha, Stephen A Boppart.

Label-free nonlinear microscopy enables non-perturbative visualization of structural and metabolic contrast within living cells in their native tissue microenvironment. Here a computational pipeline was developed to provide a quantitative view of the microenvironmental architecture within the cancerous tissue from label-free nonlinear microscopy images. To enable single-cell and single-extracellular vesicle (EV) analysis, individual cells, including tumor cells and various types of stromal cells, and EVs were segmented by a multiclass pixelwise segmentation neural network and subsequently analyzed for their metabolic status and molecular structure in the context of the local cellular neighborhood. By comparing cancer tissue with normal tissue, extensive tissue re-organization and formation of a patterned cell-EV neighborhood was observed in the tumor microenvironment. The proposed analytical pipeline is expected to be useful in a wide range of biomedical tasks that benefits from single-cell, single-EV, and cell-to-EV analysis.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-3124