bims-tuchim 2021-04-11 papers

bims-tuchim

Biomed News

on Tumor-on-chip models

Issue of 2021–04–11
eightteen papers selected by
Philipp Albrecht, Friedrich Schiller University

Controlling Cancer Cell Behavior by Improving the Stiffness of Gastric Tissue-Decellularized ECM Bioink With Cellulose Nanoparticles.
Endothelial struts enable the generation of large lumenized blood vessels de novo.
Stem cell-based vascularization of microphysiological systems.
Immune-regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells.
Oncolytic herpesvirus expressing PD-L1 BiTE for cancer therapy: exploiting tumor immune suppression as an opportunity for targeted immunotherapy.
Using 3D in vitro cell culture models in anti-cancer drug discovery.
Remodeling of an in vitro microvessel exposed to cyclic mechanical stretch.
Modeling human tumor-immune environments in vivo for the preclinical assessment of immunotherapies.
Single-cell RNA-seq reveals dynamic change in tumor microenvironment during pancreatic ductal adenocarcinoma malignant progression.
Cell-programmed nutrient partitioning in the tumour microenvironment.
Nonlinear elasticity of biological basement membrane revealed by rapid inflation and deflation.
Remodeling of Adhesion Network within Cancer Spheroids via Cell-Polymer Interaction.
A novel 4D cell culture mimicking stomach peristalsis altered gastric cancer spheroids growth and malignance.
Pancreatic cancer stem cells may define tumor stroma characteristics and recurrence patterns in pancreatic ductal adenocarcinoma.
Measuring Nanoparticle Penetration Through Bio-Mimetic Gels.
Role of tumor mutation burden-related signatures in the prognosis and immune microenvironment of pancreatic ductal adenocarcinoma.
Fundamentals of T Cell Metabolism and Strategies to Enhance Cancer Immunotherapy.
VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer in vivo.

Front Bioeng Biotechnol. 2021 ;9 605819

Controlling Cancer Cell Behavior by Improving the Stiffness of Gastric Tissue-Decellularized ECM Bioink With Cellulose Nanoparticles.

Jisoo Kim, Jinah Jang, Dong-Woo Cho.

  A physiologically relevant tumor microenvironment is favorable for the progression and growth of gastric cancer cells. To simulate the tumor-specific conditions of in vivo environments, several biomaterials engineering studies have investigated three-dimensional (3D) cultures. However, the implementation of such cultures remains limited because of challenges in outlining the biochemical and biophysical characteristics of the gastric cancer microenvironment. In this study, we developed a 3D cell printing-based gastric cancer model, using a combination of gastric tissue-specific bioinks and cellulose nanoparticles (CN) to provide adequate stiffness to gastric cancer cells. To create a 3D gastric tissue-specific microenvironment, we developed a decellularization process for a gastric tissue-derived decellularized extracellular matrix (g-dECM) bioink, and investigated the effect of the g-dECM bioink on promoting the aggressiveness of gastric cancer cells using histological and genetic validation methods. We found that incorporating CN in the matrix improves its mechanical properties, which supports the progression of gastric cancer. These mechanical properties are distinguishing characteristics that can facilitate the development of an in vitro gastric cancer model. Further, the CN-supplemented g-dECM bioink was used to print a variety of free-standing 3D shapes, including gastric rugae. These results indicate that the proposed model can be used to develop a physiologically relevant gastric cancer system that can be used in future preclinical trials.

Keywords:  3D cell-printing; 3D gastric cancer model; cellulose nanoparticles; gastric-derived extracelluar matrix bioink; tissue engineering

DOI:  https://doi.org/10.3389/fbioe.2021.605819
Nat Cell Biol. 2021 Apr;23(4): 322-329

Endothelial struts enable the generation of large lumenized blood vessels de novo.

Bart Weijts, Iftach Shaked, Mark Ginsberg, David Kleinfeld, Catherine Robin, David Traver.

De novo blood vessel formation occurs through coalescence of endothelial cells (ECs) into a cord-like structure, followed by lumenization either through cell-1-3 or cord-hollowing4-7. Vessels generated in this manner are restricted in diameter to one or two ECs, and these models fail to explain how vasculogenesis can form large-diameter vessels. Here, we describe a model for large vessel formation that does not require a cord-like structure or a hollowing step. In this model, ECs coalesce into a network of struts in the future lumen of the vessel, a process dependent upon bone morphogenetic protein signalling. The vessel wall forms around this network and consists initially of only a few patches of ECs. To withstand external forces and to maintain the shape of the vessel, strut formation traps erythrocytes into compartments to form a rigid structure. Struts gradually prune and ECs from struts migrate into and become part of the vessel wall. Experimental severing of struts resulted in vessel collapse, disturbed blood flow and remodelling defects, demonstrating that struts enable the patency of large vessels during their formation.

DOI: https://doi.org/10.1038/s41556-021-00664-3
Stem Cell Reports. 2021 Apr 03. pii: S2213-6711(21)00145-4. [Epub ahead of print]

Stem cell-based vascularization of microphysiological systems.

Shane Browne, Elisabeth L Gill, Paula Schultheiss, Ishan Goswami, Kevin E Healy.

Microphysiological systems (MPSs) (i.e., tissue or organ chips) exploit microfluidics and 3D cell culture to mimic tissue and organ-level physiology. The advent of human induced pluripotent stem cell (hiPSC) technology has accelerated the use of MPSs to study human disease in a range of organ systems. However, in the reduction of system complexity, the intricacies of vasculature are an often-overlooked aspect of MPS design. The growing library of pluripotent stem cell-derived endothelial cell and perivascular cell protocols have great potential to improve the physiological relevance of vasculature within MPS, specifically for in vitro disease modeling. Three strategic categories of vascular MPS are outlined: self-assembled, interface focused, and 3D biofabricated. This review discusses key features and development of the native vasculature, linking that to how hiPSC-derived vascular cells have been generated, the state of the art in vascular MPSs, and opportunities arising from interdisciplinary thinking.

DOI: https://doi.org/10.1016/j.stemcr.2021.03.015
Mol Cell. 2021 Apr 04. pii: S1097-2765(21)00214-8. [Epub ahead of print]

Immune-regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells.

Alice Clare Newman, Mattia Falcone, Alejandro Huerta Uribe, Tong Zhang, Dimitris Athineos, Matthias Pietzke, Alexei Vazquez, Karen Blyth, Oliver David Kenneth Maddocks.

  Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis.

Keywords:  IDO1; IFNγ; PDAC; cancer immunology; cancer metabolism; epacadostat; formate; immunometabolism; immunotherapy; one-carbon metabolism; pancreas; serine; stellate cells; tryptophan; tumor microenvironment

DOI:  https://doi.org/10.1016/j.molcel.2021.03.019
J Immunother Cancer. 2021 Mar;pii: e001292. [Epub ahead of print]9(4):

Oncolytic herpesvirus expressing PD-L1 BiTE for cancer therapy: exploiting tumor immune suppression as an opportunity for targeted immunotherapy.

Hena Khalique, Richard Baugh, Arthur Dyer, Eleanor M Scott, Sally Frost, Sarah Larkin, Janet Lei-Rossmann, Leonard W Seymour.

   BACKGROUND: Programmed death-ligand 1 (PD-L1) is an important immune checkpoint protein that can be regarded as a pan-cancer antigen expressed by multiple different cell types within the tumor. While antagonizing PD-L1 is well known to relieve PD-1/PD-L1-mediated T cell suppression, here we have combined this approach with an immunotherapy strategy to target T cell cytotoxicity directly toward PD-L1-expressing cells. We developed a bi-specific T cell engager (BiTE) crosslinking PD-L1 and CD3ε and demonstrated targeted cytotoxicity using a clinically relevant patient-derived ascites model. This approach represents an immunological 'volte-face' whereby a tumor immunological defense mechanism can be instantly transformed into an Achilles' heel for targeted immunotherapy.
METHODS: The PD-L1 targeting BiTE comprises an anti-PD-L1 single-chain variable fragment (scFv) or nanobody (NB) domain and an anti-CD3 scFv domain in a tandem repeat. The ability to activate T cell cytotoxicity toward PD-L1-expressing cells was established using human carcinoma cells and PD-L1-expressing human ('M2') macrophages in the presence of autologous T cells. Furthermore, we armed oncolytic herpes simplex virus-1 (oHSV-1) with PD-L1 BiTE and demonstrated successful delivery and targeted cytotoxicity in unpurified cultures of malignant ascites derived from different cancer patients.
RESULTS: PD-L1 BiTE crosslinks PD-L1-positive cells and CD3ε on T cells in a 'pseudo-synapse' and triggers T cell activation and release of proinflammatory cytokines such as interferon-gamma (IFN-γ), interferon gamma-induced protein 10 (IP-10) and tumour necrosis factor-α (TNF-α). Activation of endogenous T cells within ascites samples led to significant lysis of tumor cells and M2-like macrophages (CD11b+CD64+ and CD206+/CD163+). The survival of CD3+ T cells (which can also express PD-L1) was unaffected. Intriguingly, ascites fluid that appeared particularly immunosuppressive led to higher expression of PD-L1 on tumor cells, resulting in improved BiTE-mediated T cell activation.
CONCLUSIONS: The study reveals that PD-L1 BiTE is an effective immunotherapeutic approach to kill PD-L1-positive tumor cells and macrophages while leaving T cells unharmed. This approach activates endogenous T cells within malignant ascites, generates a proinflammatory response and eliminates cells promoting tumor progression. Using an oncolytic virus for local expression of PD-L1 BiTE also prevents 'on-target off-tumor' systemic toxicities and harnesses immunosuppressive protumor conditions to augment immunotherapy in immunologically 'cold' clinical cancers.

Keywords:  B7-H1 antigen; T-lymphocytes; immunotherapy; oncolytic virotherapy; tumor microenvironment

DOI:  https://doi.org/10.1136/jitc-2020-001292
Expert Opin Drug Discov. 2021 Apr 06.

Using 3D in vitro cell culture models in anti-cancer drug discovery.

Sigrid A Langhans.

   INTRODUCTION: The high failure rate in drug discovery remains a costly and time-consuming challenge. Improving the odds of success in the early steps of drug development requires disease models with high biological relevance for biomarker discovery and drug development. The adoption of three-dimensional (3D) cell culture systems over traditional monolayers in cell-based assays is considered a promising step towards improving the success rate in drug discovery.
AREAS COVERED: In this article, the author focuses on new technologies for 3D cell culture and their applications in cancer drug discovery. Besides the most common 3D cell culture systems for tumor cells, the article emphasizes the need for 3D cell culture technologies that can mimic the complex tumor microenvironment and cancer stem cell niche.
EXPERT OPINION: There has been a rapid increase in 3D cell culture technologies in recent years in an effort to more closely mimic in vivo physiology. Each 3D cell culture system has its own strengths and weaknesses with regard to key aspects of in vivo tumor growth and the tumor microenvironment. This requires careful consideration of which 3D cell culture system is chosen for drug discovery and needs to be based on factors like drug target and tumor origin.

Keywords:  3D culture; extracellular matrix; high-content screening, high-throughput screening; microenvironment; scaffold-based culture; spheroids

DOI:  https://doi.org/10.1080/17460441.2021.1912731
APL Bioeng. 2021 Jun;5(2): 026102

Remodeling of an in vitro microvessel exposed to cyclic mechanical stretch.

Soheila Zeinali, Emily K Thompson, Holger Gerhardt, Thomas Geiser, Olivier T Guenat.

In the lungs, vascular endothelial cells experience cyclic mechanical strain resulting from rhythmic breathing motions and intraluminal blood pressure. Mechanical stress creates evident physiological, morphological, biochemical, and gene expression changes in vascular endothelial cells. However, the exact mechanisms of the mechanical signal transduction into biological responses remain to be clarified. Besides, the level of mechanical stress is difficult to determine due to the complexity of the local distension patterns in the lungs and thus assumed to be the same as the one acting on the alveolar epithelium. Existing in vitro models used to investigate the effect of mechanical stretch on endothelial cells are usually limited to two-dimensional (2D) cell culture platforms, which poorly mimic the typical three-dimensional structure of the vessels. Therefore, the development of an advanced in vitro vasculature model that closely mimics the dynamic of the human lung vasculatures is highly needed. Here, we present the first study that investigates the interplay of the three-dimensional (3D) mechanical cyclic stretch and its magnitude with vascular endothelial growth factor (VEGF) stimulation on a 3D perfusable vasculature in vitro. We studied the effects of the cyclic strain on a perfusable 3D vasculature, made of either human lung microvascular endothelial cells or human umbilical vein endothelial cells embedded in a gel layer. The in vitro 3D vessels underwent both in vivo-like longitudinal and circumferential deformations, simultaneously. Our results showed that the responses of the human lung microvascular endothelial cells and human umbilical vein endothelial cells to cyclic stretch were in good agreement. Although our 3D model was in agreement with the 2D model in predicting a cytoskeletal remodeling in response to different magnitudes of cyclic stretch, however, we observed several phenomena in the 3D model that the 2D model was unable to predict. Angiogenic sprouting induced by VEGF decreased significantly in the presence of cyclic stretch. Similarly, while treatment with VEGF increased vascular permeability, the cyclic stretch restored vascular barrier tightness and significantly decreased vascular permeability. One of the major findings of this study was that a 3D microvasculature can be exposed to a much higher mechanical cyclic stress level than reported in the literature without any dysfunction of its barrier. For higher magnitudes of the cyclic stretch, the applied longitudinal strain level was 14% and the associated circumferential strain reached the equivalent of 63%. In sharp contrast to our findings, such strain typically leads to the disruption of the endothelial barrier in a 2D stretching assay and is considered pathological. This highlights the importance of 3D modeling to investigate mechanobiology effects rather than using a simple endothelial monolayer, which truly recapitulates the in vivo situation.

DOI: https://doi.org/10.1063/5.0010159
Cancer Immunol Immunother. 2021 Apr 08.

Modeling human tumor-immune environments in vivo for the preclinical assessment of immunotherapies.

Bethany Bareham, Nikitas Georgakopoulos, Alba Matas-Céspedes, Michelle Curran, Kourosh Saeb-Parsy.

  Despite the significant contributions of immunocompetent mouse models to the development and assessment of cancer immunotherapies, they inadequately represent the genetic and biological complexity of corresponding human cancers. Immunocompromised mice reconstituted with a human immune system (HIS) and engrafted with patient-derived tumor xenografts are a promising novel preclinical model for the study of human tumor-immune interactions. Whilst overcoming limitations of immunocompetent models, HIS-tumor models often rely on reconstitution with allogeneic immune cells, making it difficult to distinguish between anti-tumor and alloantigen responses. Models that comprise of autologous human tumor and human immune cells provide a platform that is more representative of the patient immune-tumor interaction. However, limited access to autologous tissues, short experimental windows, and poor retention of tumor microenvironment and tumor infiltrating lymphocyte components are major challenges affecting the establishment and application of autologous models. This review outlines existing preclinical murine models for the study of immuno-oncology, and highlights innovations that can be applied to improve the feasibility and efficacy of autologous models.

Keywords:  Animal models; Autologous models; Cancer; Immune-system; Immunotherapies; Preclinical Safety-assessment/risk management

DOI:  https://doi.org/10.1007/s00262-021-02897-5
EBioMedicine. 2021 Apr 02. pii: S2352-3964(21)00108-0. [Epub ahead of print]66 103315

Single-cell RNA-seq reveals dynamic change in tumor microenvironment during pancreatic ductal adenocarcinoma malignant progression.

Kai Chen, Qi Wang, Mingzhe Li, Huahu Guo, Weikang Liu, Feng Wang, Xiaodong Tian, Yinmo Yang.

   BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is most aggressive among all gastrointestinal tumors. The complex intra-tumor heterogeneity and special tumor microenvironment in PDAC bring great challenges for developing effective treatment strategies. We aimed to delineate dynamic changes of tumor microenvironment components during PDAC malignant progression utilizing single-cell RNA sequencing.
METHODS: A total of 11 samples (4 PDAC I, 4 PDAC II, 3 PDAC III) were used to construct expression matrix. After identifying distinct cell clusters, subcluster analysis for each cluster was performed. New cancer associated fibroblasts (CAFs) subset was validated by weighted gene co-expression network analysis, RNA in situ hybridization and immunofluorescence.
FINDINGS: We found that ductal cells were not dominant component while tumor infiltrating immune cells and pancreatic stellate cells gradually accumulated during tumor development. We defined several new Treg and exhausted T cell signature genes, including DUSP4, FANK1 and LAIR2. The analysis of TCGA datasets showed that patients with high expression of DUSP4 had significantly worse prognosis. In addition, we identified a new CAFs subset (complement-secreting CAFs, csCAFs), which specifically expresses complement system components, and constructed csCAFs-related module by weighted gene co-expression network analysis. The csCAFs were located in the tissue stroma adjacent to malignant ductal cells only in early PDAC.
INTERPRETATION: We systematically explored PDAC heterogeneity and identified csCAFs as a new CAFs subset special to PDAC, which may be valuable for understanding the crosstalk inside tumor.
FUNDING: This study was supported by The Natural Science Foundation of China (NO.81572339, 81672353, 81871954) and the Youth Clinical Research Project of Peking University First Hospital (2018CR28).

Keywords:  Cancer associated fibroblasts; Intra-tumor heterogeneity; Pancreatic cancer; Tumor microenvironment; scRNA-seq

DOI:  https://doi.org/10.1016/j.ebiom.2021.103315
Nature. 2021 Apr 07.

Cell-programmed nutrient partitioning in the tumour microenvironment.

Bradley I Reinfeld, Matthew Z Madden, Melissa M Wolf, Anna Chytil, Jackie E Bader, Andrew R Patterson, Ayaka Sugiura, Allison S Cohen, Ahmed Ali, Brian T Do, Alexander Muir, Caroline A Lewis, Rachel A Hongo, Kirsten L Young, Rachel E Brown, Vera M Todd, Tessa Huffstater, Abin Abraham, Richard T O'Neil, Matthew H Wilson, Fuxue Xin, M Noor Tantawy, W David Merryman, Rachelle W Johnson, Christopher S Williams, Emily F Mason, Frank M Mason, Katherine E Beckermann, Matthew G Vander Heiden, H Charles Manning, Jeffrey C Rathmell, W Kimryn Rathmell.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

DOI: https://doi.org/10.1038/s41586-021-03442-1
Proc Natl Acad Sci U S A. 2021 Mar 16. pii: e2022422118. [Epub ahead of print]118(11):

Nonlinear elasticity of biological basement membrane revealed by rapid inflation and deflation.

Hui Li, Yue Zheng, Yu Long Han, Shengqiang Cai, Ming Guo.

  Basement membrane (BM) is a thin layer of extracellular matrix that surrounds most animal tissues, serving as a physical barrier while allowing nutrient exchange. Although they have important roles in tissue structural integrity, physical properties of BMs remain largely uncharacterized, which limits our understanding of their mechanical functions. Here, we perform pressure-controlled inflation and deflation to directly measure the nonlinear mechanics of BMs in situ. We show that the BMs behave as a permeable, hyperelastic material whose mechanical properties and permeability can be measured in a model-independent manner. Furthermore, we find that BMs exhibit a remarkable nonlinear stiffening behavior, in contrast to the reconstituted Matrigel. This nonlinear stiffening behavior helps the BMs to avoid the snap-through instability (or structural softening) widely observed during the inflation of most elastomeric balloons and thus maintain sufficient confining stress to the enclosed tissues during their growth.

Keywords:  basement membrane; extracellular matrix; nonlinear mechanics; permeability; strain stiffening

DOI:  https://doi.org/10.1073/pnas.2022422118
ACS Biomater Sci Eng. 2020 10 12. 6(10): 5632-5644

Remodeling of Adhesion Network within Cancer Spheroids via Cell-Polymer Interaction.

Youngbin Cho, Seung Jung Yu, Jiwon Kim, Ung Hyun Ko, Eun Young Park, Jin Seung Choung, Goro Choi, Daehyun Kim, Eunjung Lee, Sung Gap Im, Jennifer H Shin.

  3D spheroids are considered as the improved in vitro model to mimic the distinct arrangements of the cells in vivo. To date, low-attachment surfaces have been most widely used to induce the spontaneous aggregation of cells in suspension by simply tuning the relative strength of the cell-cell adhesion over cell-substrate adhesion. However, aggregating cancer cells into 3D clusters should mean more than just adjoining the cells in the physical proximity. The tumor cell functionality is strongly affected by the adhesion networks between cancer cells and extracellular matrix (ECM). Here, we performed an in-depth analysis of how the nonmetastatic breast cancer cells (MCF7) can be transformed to gain invasive phenotypes through compact aggregation into 3D spheroids on a functional polymer film surface, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4). By comparing the adhesion networks and invasion dynamics between 3D spheroids cultured on the pV4D4 surface with those cultured on conventional ultra-low-attachment (ULA) dishes, we report that only spheroids on the pV4D4 display active and sporadic cell-surface binding activities via dynamic protrusions, which correlates strongly with an increase in integrin β1. Moreover, localized laminin expression at the core of the pV4D4-cultured spheroids confirms the prominence of the intimate integrin-laminin interactions prompted by the exposure to pV4D4. This study suggests that structurally and functionally dissimilar 3D spheroids can be generated from the same type of cells on the surfaces of different physicochemical properties without any chemical treatment or genetic manipulation.

Keywords:  3D tumor model; cancer cell invasion; cell adhesions; initiated chemical vapor deposition (iCVD); poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4)

DOI:  https://doi.org/10.1021/acsbiomaterials.0c00977
Biofabrication. 2021 Apr 09.

A novel 4D cell culture mimicking stomach peristalsis altered gastric cancer spheroids growth and malignance.

Juzhi Zhao, Ruiqi Wang, Jinyu Zhang, Yufang Zhao, Shupei Qiao, Thomas Crouzier, Hongji Yan, Weiming Tian.

  In vitro cancer models that can largely mimic the in vivo microenvironment are crucial for conducting more accurate research. Models of three-dimensional (3D) culture that can mimic some aspects of cancer microenvironment or cancer biopsies that can adequately represent tumor heterogeneity are intensely used currently. Those models still lack the dynamic stress stimuli in gastric carcinoma exposed to stomach peristalsis in vivo. This study leveraged a lab-developed four-dimensional (4D) culture model by a magnetic responsive alginate-based hydrogel to rotating magnets that can mimic stress stimuli in gastric cancer. We used the 4D model to culture human gastric cancer cell line AGS and SGC7901, cells at the primary and metastasis stage. We revealed the 4D model altered the cancer cell growth kinetics mechanistically by altering PCNA and p53 expression compared to the 3D culture that lacks stress stimuli. We found the 4D model altered the cancer spheroids stemness as evidenced by enhanced cancer stem cells (CD44) marker expression in AGS spheroids but the expression was dampened in SGC7901 cells. We examined the multi-drug resistance (MDR1) marker expression and found the 4D model dampened the MDR1 expression in SGC7901 cell spheroids, but not in spheroids of AGS cells. Such a model provides the stomach peristalsis mimic and is promising for conducting basic or translational gastric cancer-associated research, drug screening, and culturing patient gastric biopsies to tailor the therapeutic strategies in precision medicine.

Keywords:  Alginate,; gastric cancer spheroids; magnetic hydrogel; stress stimuli

DOI:  https://doi.org/10.1088/1758-5090/abf6bf
BMC Cancer. 2021 Apr 09. 21(1): 385

Pancreatic cancer stem cells may define tumor stroma characteristics and recurrence patterns in pancreatic ductal adenocarcinoma.

Gokce Askan, Ibrahim Halil Sahin, Joanne F Chou, Aslihan Yavas, Marinela Capanu, Christine A Iacobuzio-Donahue, Olca Basturk, Eileen M O'Reilly.

   BACKGROUND: Herein, we investigate the relationship between pancreatic stem cell markers (PCSC markers), CD44, and epithelial-specific antigen (ESA), tumor stroma, and the impact on recurrence outcomes in pancreatic ductal adenocarcinoma (PDAC) patients.
METHODS: PDAC patients who underwent surgical resection between 01/2012-06/2014 were identified. CD44 and ESA expression was assessed by immunohistochemistry. Stroma was classified as loose, moderate, and dense based on fibroblast content. Overall survival (OS) and relapse-free survival (RFS) were estimated using the Kaplan-Meier method and compared between subgroups by log-rank test. The association between PCSC markers and stroma type was assessed by Fisher's exact test.
RESULTS: N = 93 PDAC patients were identified. The number of PDAC patients with dense, moderate density, and loose stroma was 11 (12%), 51 (54%), and 31 (33%) respectively. PDAC with CD44+/ESA- had highest rate of loose stroma (63%) followed by PDAC CD44+/ESA+ (50%), PDAC CD44-/ESA+ (35%), CD44-/ESA- (9%) (p = 0.0033). Conversely, lack of CD44 and ESA expression was associated with the highest rate of moderate and dense stroma (91% p = 0.0033). No local recurrence was observed in patients with dense stroma and 9 had distant recurrence. The highest rate of cumulative local recurrence was observed in patients with loose stroma. No statistically significant difference in RFS and OS was observed among subgroups (P = 0.089).
CONCLUSIONS: These data indicate PCSCs may have an important role in stroma differentiation in PDAC. Our results further suggest that tumor stroma may influence the recurrence pattern in PDAC patients.

Keywords:  CD44; Cancer stem cells; Desmoplasia; ESA; Pancreatic cancer; Pancreatic ductal adenocarcinoma; Recurrence pattern; Sonic hedgehog; Tumor microenvironment; Tumor stroma

DOI:  https://doi.org/10.1186/s12885-021-08123-w
Int J Nanomedicine. 2021 ;16 2585-2595

Measuring Nanoparticle Penetration Through Bio-Mimetic Gels.

Scott C McCormick, Namid Stillman, Matthew Hockley, Adam W Perriman, Sabine Hauert.

   Background: In cancer nanomedicine, drugs are transported by nanocarriers through a biological system to produce a therapeutic effect. The efficacy of the treatment is affected by the ability of the nanocarriers to overcome biological transport barriers to reach their target. In this work, we focus on the process of nanocarrier penetration through tumour tissue after extravasation. Visualising the dynamics of nanocarriers in tissue is difficult in vivo, and in vitro assays often do not capture the spatial and physical constraints relevant to model tissue penetration.
Methods: We propose a new simple, low-cost method to observe the transport dynamics of nanoparticles through a tissue-mimetic microfluidic chip. After loading a chip with triplicate conditions of gel type and loading with microparticles, microscopic analysis allows for tracking of fluorescent nanoparticles as they move through hydrogels (Matrigel and Collagen I) with and without cell-sized microparticles. A bespoke image-processing codebase written in MATLAB allows for statistical analysis of this tracking, and time-dependent dynamics can be determined.
Results: To demonstrate the method, we show size-dependence of transport mechanics can be observed, with diffusion of fluorescein dye throughout the channel in 8 h, while 20 nm carboxylate FluoSphere diffusion was hindered through both Collagen I and Matrigel™. Statistical measurements of the results are generated through the software package and show the significance of both size and presence of microparticles on penetration depth.
Conclusion: This provides an easy-to-understand output for the end user to measure nanoparticle tissue penetration, enabling the first steps towards future automated experimentation of transport dynamics for rational nanocarrier design.

Keywords:  fast-prototyping; image processing; microfluidics; nanomedicine; tissue penetration; transport barriers

DOI:  https://doi.org/10.2147/IJN.S292131
Cancer Cell Int. 2021 Apr 07. 21(1): 196

Role of tumor mutation burden-related signatures in the prognosis and immune microenvironment of pancreatic ductal adenocarcinoma.

Rong Tang, Xiaomeng Liu, Wei Wang, Jie Hua, Jin Xu, Chen Liang, Qingcai Meng, Jiang Liu, Bo Zhang, Xianjun Yu, Si Shi.

   BACKGROUND: High tumor mutation burden (TMB) has gradually become a sensitive biomarker for predicting the response to immunotherapy in many cancers, including lung, bladder and head and neck cancers. However, whether high TMB predicts the response to immunotherapy and prognosis in pancreatic ductal adenocarcinoma (PDAC) remained obscure. Hence, it is significant to investigate the role of genes related to TMB (TRGs) in PDAC.
METHODS: The transcriptome and mutation data of PDAC was downloaded from The Cancer Genome Atlas-Pancreatic Adenocarcinoma (TCGA). Five independent external datasets of PDAC were chosen to validate parts of our results. qRT-PCR and immunohistochemical staining were also performed to promote the reliability of this study.
RESULTS: The median overall survival (OS) was significantly increased in TMB_low group compared with the counterpart with higher TMB score after tumor purity adjusted (P = 0.03). 718 differentially expressed TRGs were identified and functionally enriched in some oncogenic pathways. 67 TRGs were associated with OS in PDAC. A prognostic model for the OS was constructed and showed a high predictive accuracy (AUC = 0.849). We also found TMB score was associated with multiple immune components and signatures in tumor microenvironment. In addition, we identified a PDAC subgroup featured with TMBlowMicrosatellite instabilityhigh (MSIhigh) was associated with prolonged OS and a key molecule, ANKRD55, potentially mediating the survival benefits.
CONCLUSION: This study analyzed the biological function, prognosis value, implications for mutation landscape and potential influence on immune microenvironment of TRGs in PDAC, which contributed to get aware of the role of TMB in PDAC. Future studies are expected to investigate how these TRGs regulate the initiation, development or repression of PDAC.

Keywords:  Immune microenvironment; Microsatellite instability; Molecular oncology; Pancreatic cancer; Tumor mutation burden

DOI:  https://doi.org/10.1186/s12935-021-01900-4
Front Immunol. 2021 ;12 645242

Fundamentals of T Cell Metabolism and Strategies to Enhance Cancer Immunotherapy.

Guillermo O Rangel Rivera, Hannah M Knochelmann, Connor J Dwyer, Aubrey S Smith, Megan M Wyatt, Amalia M Rivera-Reyes, Jessica E Thaxton, Chrystal M Paulos.

  Emerging reports show that metabolic pathways can be targeted to enhance T cell-mediated immunity to tumors. Yet, tumors consume key metabolites in the host to survive, thus robbing T cells of these nutrients to function and thrive. T cells are often deprived of basic building blocks for energy in the tumor, including glucose and amino acids needed to proliferate or produce cytotoxic molecules against tumors. Immunosuppressive molecules in the host further compromise the lytic capacity of T cells. Moreover, checkpoint receptors inhibit T cell responses by impairing their bioenergetic potential within tumors. In this review, we discuss the fundamental metabolic pathways involved in T cell activation, differentiation and response against tumors. We then address ways to target metabolic pathways to improve the next generation of immunotherapies for cancer patients.

Keywords:  T cell metabolism; adoptive T cell transfer; immune checkpoint therapy; tumor infiltrating lymphocytes; tumor metabolism; tumor microenvironment

DOI:  https://doi.org/10.3389/fimmu.2021.645242
Cancer Biol Med. 2021 Apr 08. pii: j.issn.2095-3941.2020.0568. [Epub ahead of print]

VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer in vivo.

Guangbo Kang, Min Hu, He Ren, Jiewen Wang, Xin Cheng, Ruowei Li, Bo Yuan, Yasmine Balan, Zixuan Bai, He Huang.

   OBJECTIVE: We aimed to develop a novel anti-HIF-1α intrabody to decrease gemcitabine resistance in pancreatic cancer patients.
METHODS: Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1α VHH212 [a single-domain antibody (nanobody)]. Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α (HIF-1α) and VHH212. The real-time polymerase chain reaction (PCR) and Western blot analyses were performed to identify the expressions of HIF-1α and VEGF-A in pancreatic ductal adenocarcinoma cell lines. The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay. Finally, a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment. Immunohistochemistry analysis was conducted to detect the expressions of HIF-1α and VEGF-A in tumor tissues.
RESULTS: VHH212 was stably expressed in tumor cells with low cytotoxicity, high affinity, specific subcellular localization, and neutralization of HIF-1α in the cytoplasm or nucleus. The binding affinity between VHH212 and the HIF-1α PAS-B domain was 42.7 nM. Intrabody competitive inhibition of the HIF-1α heterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro. Compared with single agent gemcitabine, co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44% tumor inhibition.
CONCLUSIONS: We developed an anti-HIF-1α nanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer. The combination of VHH212 and gemcitabine significantly inhibited tumor development. These results suggested that combined use of anti-HIF-1α nanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.

Keywords:  HIF-1α inhibitor; Pancreatic cancer; chemosensitizer; gemcitabine; intracellular antibody; nanobody therapeutic

DOI:  https://doi.org/10.20892/j.issn.2095-3941.2020.0568