bims-tuchim Biomed News
on Tumor-on-chip models
Issue of 2021–05–16
twenty-two papers selected by
Philipp Albrecht, Friedrich Schiller University
  1. On-chip perivascular niche supporting stemness of patient-derived glioma cells in a serum-free, flowable culture.
  2. CD8+ T cell metabolism in infection and cancer.
  3. A novel human arterial wall-on-a-chip to study endothelial inflammation and vascular smooth muscle cell migration in early atherosclerosis.
  4. The Role of Tumor Necrosis Factor in Manipulating the Immunological Response of Tumor Microenvironment.
  5. Microphysiological Systems for Studying Cellular Crosstalk During the Neutrophil Response to Infection.
  6. Extracellular Vesicle Mediated Tumor-Stromal Crosstalk Within an Engineered Lung Cancer Model.
  7. Immunotherapy for pancreatic cancer.
  8. Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments.
  9. Tumor hypoxia is associated with resistance to PD-1 blockade in squamous cell carcinoma of the head and neck.
  10. Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System.
  11. Directional Cues in the Tumor Microenvironment due to Cell Contraction against Aligned Collagen Fibers.
  12. A Simple Method for Generating, Clearing, and Imaging Pre-vascularized 3D Adipospheres Derived from Human iPS Cells.
  13. Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition.
  14. Identification, Validation, and Utilization of Immune Cells in Pancreatic Ductal Adenocarcinoma Based on Marker Genes.
  15. Mechanical plasticity of collagen directs branch elongation in human mammary gland organoids.
  16. The Dynamic Entropy of Tumor Immune Infiltrates: The Impact of Recirculation, Antigen-Specific Interactions, and Retention on T Cells in Tumors.
  17. Regulation of vascular permeability in cancer metastasis.
  18. Bispecific antibody simultaneously targeting PD1 and HER2 inhibits tumor growth via direct tumor cell killing in combination with PD1/PDL1 blockade and HER2 inhibition.
  19. Characterization of an engineered live bacterial therapeutic for the treatment of phenylketonuria in a human gut-on-a-chip.
  20. Ultraviolet light-induced collagen degradation inhibits melanoma invasion.
  21. Applications of Engineering Techniques in Microvasculature Design.
  22. Engineered red blood cells as an off-the-shelf allogeneic anti-tumor therapeutic.
  1. Lab Chip. 2021 May 10.
    On-chip perivascular niche supporting stemness of patient-derived glioma cells in a serum-free, flowable culture.
    Magda Gerigk, Harry Bulstrode, HaoTian Harvey Shi, Felix Tönisen, Camilla Cerutti, Gillian Morrison, David Rowitch, Yan Yan Shery Huang.
      Glioblastoma multiforme (GBM) is the most common and the most aggressive type of primary brain malignancy. Glioblastoma stem-like cells (GSCs) can migrate in vascular niches within or away from the tumour mass, increasing tumour resistance to treatments and contributing to relapses. To study individual GSC migration and their interactions with the perivasculature of the tumour microenvironment, there is a need to develop a human organotypic in vitro model. Herein, we demonstrated a perivascular niche-on-a-chip, in a serum-free condition with gravity-driven flow, that supported the stemness of patient-derived GSCs and foetal neural stem cells grown in a three-dimensional environment (3D). Endothelial cells from three organ origins, (i) human brain microvascular endothelial cells (hCMEC/D3), (ii) human umbilical vein endothelial cells (HUVECs) and, (iii) human lung microvascular endothelial cells (HMVEC-L) formed rounded microvessels within the extracellular-matrix integrated microfluidic chip. By optimising cell extraction protocols, systematic studies were performed to evaluate the effects of serum-free media, 3D cell cultures, and the application of gravity-driven flow on the characteristics of endothelial cells and their co-culture with GSCs. Our results showed the maintenance of adherent and tight junction markers of hCMEC/D3 in the serum-free culture and that gravity-driven flow was essential to support adequate viability of both the microvessel and the GSCs in co-culture (>80% viability at day 3). Endpoint biological assays showed upregulation of neovascularization-related genes (e.g., angiopoietins, vascular endothelial growth factor receptors) in endothelial cells co-cultured with GSCs in contrast to the neural stem cell reference that showed insignificant changes. The on-chip platform further permitted live-cell imaging of GSC - microvessel interaction, enabling quantitative analysis of GSC polarization and migration. Overall, our comparative genotypic (i.e. qPCR) and phenotypic (i.e. vessel permeability and GSC migration) studies showed that organotypic (brain cancer cells-brain endothelial microvessel) interactions differed from those within non-tissue specific vascular niches of human origin. The development and optimization of this on-chip perivascular niche, in a serum-free flowable culture, could provide the next level of complexity of an in vitro system to study the influence of glioma stem cells on brain endothelium.
    DOI:  https://doi.org/10.1039/d1lc00271f
  2. Nat Rev Immunol. 2021 May 12.
    CD8+ T cell metabolism in infection and cancer.
    Miguel Reina-Campos, Nicole E Scharping, Ananda W Goldrath.
      Cytotoxic CD8+ T cells play a key role in the elimination of intracellular infections and malignant cells and can provide long-term protective immunity. In the response to infection, CD8+ T cell metabolism is coupled to transcriptional, translational and epigenetic changes that are driven by extracellular metabolites and immunological signals. These programmes facilitate the adaptation of CD8+ T cells to the diverse and dynamic metabolic environments encountered in the circulation and in the tissues. In the setting of disease, both cell-intrinsic and cell-extrinsic metabolic cues contribute to CD8+ T cell dysfunction. In addition, changes in whole-body metabolism, whether through voluntary or disease-induced dietary alterations, can influence CD8+ T cell-mediated immunity. Defining the metabolic adaptations of CD8+ T cells in specific tissue environments informs our understanding of how these cells protect against pathogens and tumours and maintain tissue health at barrier sites. Here, we highlight recent findings revealing how metabolic networks enforce specific CD8+ T cell programmes and discuss how metabolism is integrated with CD8+ T cell differentiation and function and determined by environmental cues.
    DOI:  https://doi.org/10.1038/s41577-021-00537-8
  3. Lab Chip. 2021 May 12.
    A novel human arterial wall-on-a-chip to study endothelial inflammation and vascular smooth muscle cell migration in early atherosclerosis.
    Chengxun Su, Nishanth Venugopal Menon, Xiaohan Xu, Yu Rong Teo, Huan Cao, Rinkoo Dalan, Chor Yong Tay, Han Wei Hou.
      Mechanistic understanding of atherosclerosis is largely hampered by the lack of a suitable in vitro human arterial model that recapitulates the arterial wall structure, and the interplay between different cell types and the surrounding extracellular matrix (ECM). This work introduces a novel microfluidic endothelial cell (EC)-smooth muscle cell (SMC) 3D co-culture platform that replicates the structural and biological aspects of the human arterial wall for modeling early atherosclerosis. Using a modified surface tension-based ECM patterning method, we established a well-defined intima-media-like structure, and identified an ECM composition (collagen I and Matrigel mixture) that retains the SMCs in a quiescent and aligned state, characteristic of a healthy artery. Endothelial stimulation with cytokines (IL-1β and TNFα) and oxidized low-density lipoprotein (oxLDL) was performed on-chip to study various early atherogenic events including endothelial inflammation (ICAM-1 expression), EC/SMC oxLDL uptake, SMC migration, and monocyte-EC adhesion. As a proof-of-concept for drug screening applications, we demonstrated the atheroprotective effects of vitamin D (1,25(OH)2D3) and metformin in mitigating cytokine-induced monocyte-EC adhesion and SMC migration. Overall, the developed arterial wall model facilitates quantitative and multi-factorial studies of EC and SMC phenotype in an atherogenic environment, and can be readily used as a platform technology to reconstitute multi-layered ECM tissue biointerfaces.
    DOI:  https://doi.org/10.1039/d1lc00131k
  4. Front Immunol. 2021 ;12 656908
    The Role of Tumor Necrosis Factor in Manipulating the Immunological Response of Tumor Microenvironment.
    Dipranjan Laha, Robert Grant, Prachi Mishra, Naris Nilubol.
      The tumor microenvironment (TME) is an intricate system within solid neoplasms. In this review, we aim to provide an updated insight into the TME with a focus on the effects of tumor necrosis factor-α (TNF-α) on its various components and the use of TNF-α to improve the efficiency of drug delivery. The TME comprises the supporting structure of the tumor, such as its extracellular matrix and vasculature. In addition to cancer cells and cancer stem cells, the TME contains various other cell types, including pericytes, tumor-associated fibroblasts, smooth muscle cells, and immune cells. These cells produce signaling molecules such as growth factors, cytokines, hormones, and extracellular matrix proteins. This review summarizes the intricate balance between pro-oncogenic and tumor-suppressive functions that various non-tumor cells within the TME exert. We focused on the interaction between tumor cells and immune cells in the TME that plays an essential role in regulating the immune response, tumorigenesis, invasion, and metastasis. The multifunctional cytokine, TNF-α, plays essential roles in diverse cellular events within the TME. The uses of TNF-α in cancer treatment and to facilitate cancer drug delivery are discussed. The effects of TNF-α on tumor neovasculature and tumor interstitial fluid pressure that improve treatment efficacy are summarized.
    Keywords:  extracellular matrix; interstitial fluid pressure; transforming growth factor beta; tumor microenvironment; tumor necrosis factor-α
    DOI:  https://doi.org/10.3389/fimmu.2021.656908
  5. Front Immunol. 2021 ;12 661537
    Microphysiological Systems for Studying Cellular Crosstalk During the Neutrophil Response to Infection.
    Isaac M Richardson, Christopher J Calo, Laurel E Hind.
      Neutrophils are the primary responders to infection, rapidly migrating to sites of inflammation and clearing pathogens through a variety of antimicrobial functions. This response is controlled by a complex network of signals produced by vascular cells, tissue resident cells, other immune cells, and the pathogen itself. Despite significant efforts to understand how these signals are integrated into the neutrophil response, we still do not have a complete picture of the mechanisms regulating this process. This is in part due to the inherent disadvantages of the most-used experimental systems: in vitro systems lack the complexity of the tissue microenvironment and animal models do not accurately capture the human immune response. Advanced microfluidic devices incorporating relevant tissue architectures, cell-cell interactions, and live pathogen sources have been developed to overcome these challenges. In this review, we will discuss the in vitro models currently being used to study the neutrophil response to infection, specifically in the context of cell-cell interactions, and provide an overview of their findings. We will also provide recommendations for the future direction of the field and what important aspects of the infectious microenvironment are missing from the current models.
    Keywords:  antimicrobial functions; cell-cell interactions; in vitro models; infection; inflammation; innate immunity; microfluidics; neutrophil
    DOI:  https://doi.org/10.3389/fimmu.2021.661537
  6. Front Oncol. 2021 ;11 654922
    Extracellular Vesicle Mediated Tumor-Stromal Crosstalk Within an Engineered Lung Cancer Model.
    Kayla F Goliwas, Hannah M Ashraf, Anthony M Wood, Yong Wang, Kenneth P Hough, Sandeep Bodduluri, Mohammad Athar, Joel L Berry, Selvarangan Ponnazhagan, Victor J Thannickal, Jessy S Deshane.
      Tumor-stromal interactions within the tumor microenvironment (TME) influence lung cancer progression and response to therapeutic interventions, yet traditional in vitro studies fail to replicate the complexity of these interactions. Herein, we developed three-dimensional (3D) lung tumor models that mimic the human TME and demonstrate tumor-stromal crosstalk mediated by extracellular vesicles (EVs). EVs released by tumor cells, independent of p53 status, and fibroblasts within the TME mediate immunomodulatory effects; specifically, monocyte/macrophage polarization to a tumor-promoting M2 phenotype within this 3D-TME. Additionally, immune checkpoint inhibition in a 3D model that included T cells showed an inhibition of tumor growth and reduced hypoxia within the TME. Thus, perfused 3D tumor models incorporating diverse cell types provide novel insights into EV-mediated tumor-immune interactions and immune-modulation for existing and emerging cancer therapies.
    Keywords:  3D tissue models; extracellular vesicles; lung carcinoma; tumor-immune regulation; tumor-stromal crosstalk
    DOI:  https://doi.org/10.3389/fonc.2021.654922
  7. World J Clin Cases. 2021 May 06. 9(13): 2969-2982
    Immunotherapy for pancreatic cancer.
    Jai Hoon Yoon, Ye-Ji Jung, Sung-Hoon Moon.
      Pancreatic cancer, a highly lethal cancer, has the lowest 5-year survival rate for several reasons, including its tendency for the late diagnosis, a lack of serologic markers for screening, aggressive local invasion, its early metastatic dissemination, and its resistance to chemotherapy/radiotherapy. Pancreatic cancer evades immunologic elimination by a variety of mechanisms, including induction of an immunosuppressive microenvironment. Cancer-associated fibroblasts interact with inhibitory immune cells, such as tumor-associated macrophages and regulatory T cells, to form an inflammatory shell-like desmoplastic stroma around tumor cells. Immunotherapy has the potential to mobilize the immune system to eliminate cancer cells. Nevertheless, although immunotherapy has shown brilliant results across a wide range of malignancies, only anti-programmed cell death 1 antibodies have been approved for use in patients with pancreatic cancer who test positive for microsatellite instability or mismatch repair deficiency. Some patients treated with immunotherapy who show progression based on conventional response criteria may prove to have a durable response later. Continuation of immune-based treatment beyond disease progression can be chosen if the patient is clinically stable. Immunotherapeutic approaches for pancreatic cancer treatment deserve further exploration, given the plethora of combination trials with other immunotherapeutic agents, targeted therapy, stroma-modulating agents, chemotherapy, and multi-way combination therapies.
    Keywords:  Immune checkpoint inhibitor; Immunotherapy; Pancreatic adenocarcinoma; Pancreatic cancer
    DOI:  https://doi.org/10.12998/wjcc.v9.i13.2969
  8. Nat Commun. 2021 May 14. 12(1): 2815
    Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments.
    Erdem D Tabdanov, Nelson J Rodríguez-Merced, Alexander X Cartagena-Rivera, Vikram V Puram, Mackenzie K Callaway, Ethan A Ensminger, Emily J Pomeroy, Kenta Yamamoto, Walker S Lahr, Beau R Webber, Branden S Moriarity, Alexander S Zhovmer, Paolo P Provenzano.
      Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.
    DOI:  https://doi.org/10.1038/s41467-021-22985-5
  9. J Immunother Cancer. 2021 May;pii: e002088. [Epub ahead of print]9(5):
    Tumor hypoxia is associated with resistance to PD-1 blockade in squamous cell carcinoma of the head and neck.
    Dan P Zandberg, Ashley V Menk, Maria Velez, Daniel Normolle, Kristin DePeaux, Angen Liu, Robert L Ferris, Greg M Delgoffe.
      The majority of patients with recurrent/metastatic squamous cell carcinoma of the head and neck (HNSCC) (R/M) do not benefit from anti-PD-1 therapy. Hypoxia induced immunosuppression may be a barrier to immunotherapy. Therefore, we examined the metabolic effect of anti-PD-1 therapy in a murine MEER HNSCC model as well as intratumoral hypoxia in R/M patients. In order to characterize the tumor microenvironment in PD-1 resistance, a MEER cell line was created from the parental line that are completely resistant to anti-PD-1. These cell lines were then metabolically profiled using seahorse technology and injected into C57/BL6 mice. After tumor growth, mice were pulsed with pimonidazole and immunofluorescent imaging was performed to analyze hypoxia and T cell infiltration. To validate the preclinical results, we analyzed tissues from R/M patients (n=36) treated with anti-PD-1 mAb, via immunofluorescent imaging for number of CD8+ T cells (CD8), Tregs and the percent area (CAIX) and mean intensity (I) of carbonic anhydrase IX in tumor. We analyzed disease control rate (DCR), progression free survival (PFS), and overall survival (OS) using proportional odds and proportional hazards (Cox) regression. We found that anti-PD-1 resistant MEER has significantly higher oxidative metabolism, while there was no difference in glycolytic metabolism. Intratumoral hypoxia was significantly increased and CD8+ T cells decreased in anti-PD-1 resistant tumors compared with parental tumors in the same mouse. In R/M patients, lower tumor hypoxia by CAIX/I was significantly associated with DCR (p=0.007), PFS, and OS, and independently associated with response (p=0.028) and PFS (p=0.04) in a multivariate model including other significant immune factors. During PD-1 resistance, tumor cells developed increased oxidative metabolism leading to increased intratumoral hypoxia and a decrease in CD8+ T cells. Lower tumor hypoxia was independently associated with increased efficacy of anti-PD-1 therapy in patients with R/M HNSCC. To our knowledge this is the first analysis of the effect of hypoxia in this patient population and highlights its importance not only as a predictive biomarker but also as a potential target for therapeutic intervention.
    Keywords:  head and neck neoplasms; immunotherapy; lymphocytes; metabolic networks and pathways; tumor microenvironment; tumor-infiltrating
    DOI:  https://doi.org/10.1136/jitc-2020-002088
  10. J Vis Exp. 2021 Apr 23.
    Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System.
    Loren M Brown, Katherine L Hebert, Rakesh R Gurrala, C Ethan Byrne, Matthew Burow, Elizabeth C Martin, Frank H Lau.
      Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.
    DOI:  https://doi.org/10.3791/62009
  11. Acta Biomater. 2021 May 06. pii: S1742-7061(21)00299-3. [Epub ahead of print]
    Directional Cues in the Tumor Microenvironment due to Cell Contraction against Aligned Collagen Fibers.
    Joseph M Szulczewski, David R Inman, Maria Proestaki, Jacob Notbohm, Brian M Burkel, Suzanne M Ponik.
      It is well established that collagen alignment in the breast tumor microenvironment provides biophysical cues to drive disease progression. Numerous mechanistic studies have demonstrated that tumor cell behavior is driven by the architecture and stiffness of the collagen matrix. However, the mechanical properties within a 3D collagen microenvironment, particularly at the scale of the cell, remain poorly defined. To investigate cell-scale mechanical cues with respect to local collagen architecture, we employed a combination of intravital imaging of the mammary tumor microenvironment and a 3D collagen gel system with both acellular pNIPAAm microspheres and MDA-MB-231 breast carcinoma cells. Within the in vivo tumor microenvironment, the displacement of collagen fiber was identified in response to tumor cells migrating through the stromal matrix. To further investigate cell-scale stiffness in aligned fiber architectures and the propagation of cell-induced fiber deformations, precise control of collagen architecture was coupled with innovative methodology to measure mechanical properties of the collagen fiber network. This method revealed up to a 35-fold difference in directional cell-scale stiffness resulting from contraction against aligned fibers. Furthermore, the local anisotropy of the matrix dramatically altered the rate at which contractility-induced fiber displacements decayed over distance. Together, our results reveal mechanical properties in aligned matrices that provide dramatically different cues to the cell in perpendicular directions. These findings are supported by the mechanosensing behavior of tumor cells and have important implications for cell-cell communication within the tissue microenvironment.
    Keywords:  Collagen alignment; and mechano-signaling; cell-scale modulus; fiber displacement; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.actbio.2021.04.053
  12. Methods Mol Biol. 2021 May 13.
    A Simple Method for Generating, Clearing, and Imaging Pre-vascularized 3D Adipospheres Derived from Human iPS Cells.
    Xi Yao, Christian Dani.
      Beige/brite/brown-like adipocytes (BAs), dispersed in white adipose tissue, represent promising cell targets to counteract obesity and associated diseases. However, there are major limitations for a BA-based treatment of obesity, among which the main ones are the rareness of BAs in adult humans and the lack of a relevant cell culture condition for modeling the development of BAs. We describe in this chapter the capacity of human induced pluripotent stem cells-derived BA progenitors (hiPSC-BAPs) to self-organize in spheroids and a method for their differentiation at a high efficiency in hiPSC-derived 3D adipospheres containing UCP1-expressing cells. Enrichment of adipospheres with human dermal microvascular endothelial cells (HDMECs) allows to better mimic native adipose tissue. To observe the accumulation of lipid droplets, organization of the extracellular matrix and expression of adipogenic markers on the surface of hiPSC-adipospheres, we detail how to combine Oil Red O staining with immunostaining both imaged by fluorescence microscopy. Furthermore, to have a global view of pre-vascularized network formed by HDMECs inside of hiPSC-adipospheres, we describe a method which consists of the whole adipospheres fixation, multicolor immunostaining, clearing, and imaging.
    Keywords:  3D cell model; Clearing; EdU staining; Human iPSCs; Imaging; Multicolor immunostaining; ORO staining; Pre-vascularized adipospheres
    DOI:  https://doi.org/10.1007/7651_2021_360
  13. Cancer Res. 2021 May 12. pii: canres.2496.2020. [Epub ahead of print]
    Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition.
    George Sharbeen, Joshua A McCarroll, Anouschka Akerman, Chantal Kopecky, Janet Youkhana, John Kokkinos, Jeff Holst, Cyrille Boyer, Mert Erkan, David Goldstein, Paul Timpson, Thomas R Cox, Brooke A Pereira, Jessica L Chitty, Sigrid K Fey, Arafath K Najumudeen, Andrew D Campbell, Owen J Sansom, Rosa Mistica C Ignacio, Stephanie Naim, Jie Liu, Nelson Russia, Julia Lee, Angela Chou, Amber Johns, Anthony J Gill, Estrella Gonzales-Aloy, Val Gebski, Yi Fang Guan, Marina Pajic, Nigel Turner, Minoti V Apte, Thomas P Davis, Jennifer P Morton, Koroush S Haghighi, Jorjina Kasparian, Benjamin J McLean, Yordanos F I Setargew, Australian Pancreatic Cancer Genome Initiative Apgi, Phoebe A Phillips.
      Cancer-Associated Fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through pro-tumor signaling and the generation of fibrosis that creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11, a cystine transporter, has been identified as a potential therapeutic target in PDAC cells. However, the role of SLC7A11 in PDAC tumor stroma and its prognostic significance has not been evaluated. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poor overall survival. Orthogonal approaches showed that PDAC-derived CAFs were dependent on SLC7A11 for cystine uptake and glutathione synthesis. SLC7A11 inhibition significantly decreased CAF proliferation, reduced their resistance to oxidative stress, and inhibited their ability to remodel collagen and support PDAC cell growth. Specific ablation of SLC7A11 from the tumor compartment of transgenic mice did not affect PDAC growth, suggesting the stroma can substantially influence PDAC response to SLC7A11 inhibition. In an orthotopic PDAC mouse model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle SLC7A11-silencing drug developed by our laboratory reduced PDAC tumor growth, metastasis, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-2496
  14. Front Immunol. 2021 ;12 649061
    Identification, Validation, and Utilization of Immune Cells in Pancreatic Ductal Adenocarcinoma Based on Marker Genes.
    Willem de Koning, Diba Latifi, Yunlei Li, Casper H J van Eijck, Andrew P Stubbs, Dana A M Mustafa.
      The immune response affects tumor biological behavior and progression. The specific immune characteristics of pancreatic ductal adenocarcinoma (PDAC) can determine the metastatic abilities of cancerous cells and the survival of patients. Therefore, it is important to characterize the specific immune landscape in PDAC tissue samples, and the effect of various types of therapy on that immune composition. Previously, a set of marker genes was identified to assess the immune cell composition in different types of cancer tissue samples. However, gene expression and subtypes of immune cells may vary across different types of cancers. The aim of this study was to provide a method to identify immune cells specifically in PDAC tissue samples. The method is based on defining a specific set of marker genes expressed by various immune cells in PDAC samples. A total of 90 marker genes were selected and tested for immune cell type-specific definition in PDAC; including 43 previously used, and 47 newly selected marker genes. The immune cell-type specificity was checked mathematically by calculating the "pairwise similarity" for all candidate genes using the PDAC RNA-sequenced dataset available at The Cancer Genome Atlas. A set of 55 marker genes that identify 22 different immune cell types for PDAC was created. To validate the method and the set of marker genes, an independent mRNA expression dataset of 24 samples of PDAC patients who received various types of (neo)adjuvant treatments was used. The results showed that by applying our method we were able to identify PDAC specific marker genes to characterize immune cell infiltration in tissue samples. The method we described enabled identifying different subtypes of immune cells that were affected by various types of therapy in PDAC patients. In addition, our method can be easily adapted and applied to identify the specific immune landscape in various types of tissue samples.
    Keywords:  immune cells; immune microenvironment; mRNA expression; marker genes; pancreatic ductal adenocarcinoma
    DOI:  https://doi.org/10.3389/fimmu.2021.649061
  15. Nat Commun. 2021 May 12. 12(1): 2759
    Mechanical plasticity of collagen directs branch elongation in human mammary gland organoids.
    B Buchmann, L K Engelbrecht, P Fernandez, F P Hutterer, M K Raich, C H Scheel, A R Bausch.
      Epithelial branch elongation is a central developmental process during branching morphogenesis in diverse organs. This fundamental growth process into large arborized epithelial networks is accompanied by structural reorganization of the surrounding extracellular matrix (ECM), well beyond its mechanical linear response regime. Here, we report that epithelial ductal elongation within human mammary organoid branches relies on the non-linear and plastic mechanical response of the surrounding collagen. Specifically, we demonstrate that collective back-and-forth motion of cells within the branches generates tension that is strong enough to induce a plastic reorganization of the surrounding collagen network which results in the formation of mechanically stable collagen cages. Such matrix encasing in turn directs further tension generation, branch outgrowth and plastic deformation of the matrix. The identified mechanical tension equilibrium sets a framework to understand how mechanical cues can direct ductal branch elongation.
    DOI:  https://doi.org/10.1038/s41467-021-22988-2
  16. Front Oncol. 2021 ;11 653625
    The Dynamic Entropy of Tumor Immune Infiltrates: The Impact of Recirculation, Antigen-Specific Interactions, and Retention on T Cells in Tumors.
    Tiffany C Blair, Alejandro F Alice, Lauren Zebertavage, Marka R Crittenden, Michael J Gough.
      Analysis of tumor infiltration using conventional methods reveals a snapshot view of lymphocyte interactions with the tumor environment. However, lymphocytes have the unique capacity for continued recirculation, exploring varied tissues for the presence of cognate antigens according to inflammatory triggers and chemokine gradients. We discuss the role of the inflammatory and cellular makeup of the tumor environment, as well as antigen expressed by cancer cells or cross-presented by stromal antigen presenting cells, on recirculation kinetics of T cells. We aim to discuss how current cancer therapies may manipulate lymphocyte recirculation versus retention to impact lymphocyte exclusion in the tumor.
    Keywords:  T cell; clonality analysis; recirculation; recruitment; retention; tumor
    DOI:  https://doi.org/10.3389/fonc.2021.653625
  17. Cancer Sci. 2021 May 09.
    Regulation of vascular permeability in cancer metastasis.
    Takeshi Tomita, Masayoshi Kato, Sachie Hiratsuka.
      Enhancement of vascular permeability is indispensable for cancer metastasis. Weakened endothelial barrier function enhances vascular permeability. Circulating tumor cells moving in the microvasculature tend to invade into stromal tissue at the location where vascular permeability is enhanced. Many basic studies have identified permeability factors by using gene-modified animals and cells. These factors directly/indirectly interact with endothelial cells. Here we review vascular permeability factors and their molecular mechanisms. Interactions between tumor cells and endothelial cells are also discussed in the process of extravasation, one of the most critical steps in tumor metastasis. In some cases, primary tumors can manipulate permeability in a remote organ by secreting permeability factors. In addition, the importance of glycocalyx, which covers the endothelial cell surface, in controlling vascular permeability and tumor metastasis is also described. Furthermore, analysis of the hyperpermeable region found in mouse model study is introduced. It clearly showed that tumor-bearing mouse lungs had a hyperpermeable region due to the influence of a remote primary tumor, and fibrinogen deposition was observed in that region. Given that fibrinogen was reported to be a permeability factor and a key regulator of inflammation, eliminating fibrinogen deposition may prevent future metastasis.
    Keywords:  endothelial cell; fibrinogen; metastasis; permeability; pre-metastatic soil
    DOI:  https://doi.org/10.1111/cas.14942
  18. Acta Pharmacol Sin. 2021 May 14.
    Bispecific antibody simultaneously targeting PD1 and HER2 inhibits tumor growth via direct tumor cell killing in combination with PD1/PDL1 blockade and HER2 inhibition.
    Chang-Ling Gu, Hai-Xia Zhu, Lan Deng, Xiao-Qing Meng, Kai Li, Wei Xu, Le Zhao, Yue-Qin Liu, Zhen-Ping Zhu, Hao-Min Huang.
      Immune checkpoint blockade has shown significant clinical benefit in multiple cancer indications, but many patients are either refractory or become resistant to the treatment over time. HER2/neu oncogene overexpressed in invasive breast cancer patients associates with more aggressive diseases and poor prognosis. Anti-HER2 mAbs, such as trastuzumab, are currently the standard of care for HER2-overexpressing cancers, but the response rates are below 30% and patients generally suffer relapse within a year. In this study we developed a bispecific antibody (BsAb) simultaneously targeting both PD1 and HER2 in an attempt to combine HER2-targeted therapy with immune checkpoint blockade for treating HER2-positive solid tumors. The BsAb was constructed by fusing scFvs (anti-PD1) with the effector-functional Fc of an IgG (trastuzumab) via a flexible peptide linker. We showed that the BsAb bound to human HER2 and PD1 with high affinities (EC50 values were 0.2 and 0.14 nM, respectively), and exhibited potent antitumor activities in vitro and in vivo. Furthermore, we demonstrated that the BsAb exhibited both HER2 and PD1 blockade activities and was effective in killing HER2-positive tumor cells via antibody-dependent cellular cytotoxicity. In addition, the BsAb could crosslink HER2-positive tumor cells with T cells to form PD1 immunological synapses that directed tumor cell killing without the need of antigen presentation. Thus, the BsAb is a new promising approach for treating late-stage metastatic HER2-positive cancers.
    Keywords:  HER2; PD1 blockade; PD1 immunological synapse; Trastuzumab; antibody-dependent cellular cytotoxicity (ADCC); bispecific antibody
    DOI:  https://doi.org/10.1038/s41401-021-00683-8
  19. Nat Commun. 2021 May 14. 12(1): 2805
    Characterization of an engineered live bacterial therapeutic for the treatment of phenylketonuria in a human gut-on-a-chip.
    M Tyler Nelson, Mark R Charbonneau, Heidi G Coia, Mary J Castillo, Corey Holt, Eric S Greenwood, Peter J Robinson, Elaine A Merrill, David Lubkowicz, Camilla A Mauzy.
      Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.
    DOI:  https://doi.org/10.1038/s41467-021-23072-5
  20. Nat Commun. 2021 May 12. 12(1): 2742
    Ultraviolet light-induced collagen degradation inhibits melanoma invasion.
    Timothy Budden, Caroline Gaudy-Marqueste, Andrew Porter, Emily Kay, Shilpa Gurung, Charles H Earnshaw, Katharina Roeck, Sarah Craig, Víctor Traves, Jean Krutmann, Patricia Muller, Luisa Motta, Sara Zanivan, Angeliki Malliri, Simon J Furney, Eduardo Nagore, Amaya Virós.
      Ultraviolet radiation (UVR) damages the dermis and fibroblasts; and increases melanoma incidence. Fibroblasts and their matrix contribute to cancer, so we studied how UVR modifies dermal fibroblast function, the extracellular matrix (ECM) and melanoma invasion. We confirmed UVR-damaged fibroblasts persistently upregulate collagen-cleaving matrix metalloprotein-1 (MMP1) expression, reducing local collagen (COL1A1), and COL1A1 degradation by MMP1 decreased melanoma invasion. Conversely, inhibiting ECM degradation and MMP1 expression restored melanoma invasion. Primary cutaneous melanomas of aged humans show more cancer cells invade as single cells at the invasive front of melanomas expressing and depositing more collagen, and collagen and single melanoma cell invasion are robust predictors of poor melanoma-specific survival. Thus, primary melanomas arising over collagen-degraded skin are less invasive, and reduced invasion improves survival. However, melanoma-associated fibroblasts can restore invasion by increasing collagen synthesis. Finally, high COL1A1 gene expression is a biomarker of poor outcome across a range of primary cancers.
    DOI:  https://doi.org/10.1038/s41467-021-22953-z
  21. Front Cardiovasc Med. 2021 ;8 660958
    Applications of Engineering Techniques in Microvasculature Design.
    Aleen Al Halawani, Ziyu Wang, Linyang Liu, Miao Zhang, Anthony S Weiss.
      Achieving successful microcirculation in tissue engineered constructs in vitro and in vivo remains a challenge. Engineered tissue must be vascularized in vitro for successful inosculation post-implantation to allow instantaneous perfusion. To achieve this, most engineering techniques rely on engineering channels or pores for guiding angiogenesis and capillary tube formation. However, the chosen materials should also exhibit properties resembling the native extracellular matrix (ECM) in providing mechanical and molecular cues for endothelial cells. This review addresses techniques that can be used in conjunction with matrix-mimicking materials to further advance microvasculature design. These include electrospinning, micropatterning and bioprinting. Other techniques implemented for vascularizing organoids are also considered for their potential to expand on these approaches.
    Keywords:  bioprinting; elastin; extracellular matrix; micropatterning; microvasculature; porous scaffolds; tropoelastin; vascularized organoids
    DOI:  https://doi.org/10.3389/fcvm.2021.660958
  22. Nat Commun. 2021 May 11. 12(1): 2637
    Engineered red blood cells as an off-the-shelf allogeneic anti-tumor therapeutic.
    Xuqing Zhang, Mengyao Luo, Shamael R Dastagir, Mellissa Nixon, Annie Khamhoung, Andrea Schmidt, Albert Lee, Naren Subbiah, Douglas C McLaughlin, Christopher L Moore, Mary Gribble, Nicholas Bayhi, Viral Amin, Ryan Pepi, Sneha Pawar, Timothy J Lyford, Vikram Soman, Jennifer Mellen, Christopher L Carpenter, Laurence A Turka, Thomas J Wickham, Tiffany F Chen.
      Checkpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E711-19, 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential 'off-the-shelf' in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.
    DOI:  https://doi.org/10.1038/s41467-021-22898-3
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