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Solid tumors present particular obstacles for cell therapies, which need to be reflected in pre-clinical models for effective therapy validation. For example, tumor biology varies along with the tumor mass, and the tumor microenvironment (TME), consisting of vasculature, stromal cells and immune cells, can become corrupted and promote resistance or prevent cell therapy targeting and efficacy. We developed an in vitro vascularized human solid tumor model which recapitulates these key features of the TME in a microfluidic device, allowing for more reliable and convenient drug and cell therapy validation. The model consists of a solid tumor spheroid surrounded by a perfusable vasculature, with a permeability value similar to in vivo physiology. We used the model to identify tumor-vasculature interactions, evaluate drug response, and evaluate cell therapy infiltration and cytotoxicity. By removing the cultured tumor microtissue from the device and performing spatial transcriptomics on standard paraffin-embedded sections, we identified known and novel genes involved in creating a pro-tumorigenic microenvironment.

Adoptive cell transfer (ACT) has been highly efficient in targeting certain refractory cancers and remains potentially effective for other cancers as new targets of cancer-immune interactions are revealed. ACT involves isolating immunocompetent cells from cancer patients, expanding them ex vivo, and infusing them back into the patient. Cells used for ACT are often effector cells, typically T cells, isolated from the patient’s peripheral blood and then engineered to target cancer cells by incorporating a T cell receptor (TCR) or chimeric antigen receptor (CAR), with additional modifications to improve immune cell proliferation and persistence. While cell therapies for various lymphomas have led to dramatic tumor regressions, response in solid tumors remains varied. Solid tumors present unique challenges, such as tumor heterogeneity and the lack of tumor-specific targets, but also an immunosuppressive tumor microenvironment (TME) characterized by poor T cell infiltration and terminal T cell differentiation and exhaustion at the tumor. To overcome the hostile TME, lymphodepleting regimens combined with an infusion of large numbers of T cells is often used. However, introducing large numbers of T cells significantly increases the risk of on-target/off-target toxicity, neurotoxicity, and cytokine release syndrome. The use of engineered T cells that transiently express TCRs or CARs by mRNA gene transfer, reduces these risks. Additionally, they avoid the use of viral vectors, hence there is no risk of insertional mutagenesis and can be manufactured more easily at low cost and on a larger scale. Such transient-engineered T cells have shown significant antitumor activity in phase I clinical trials and in preclinical animal models. However, multiple infusions are often required, increasing patient discomfort and treatment cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targeting hepatocellular carcinoma, we found that short-term inhibition of G9a/GLP increased T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increased granzyme expression without terminal T cell differentiation or exhaustion and resulted in specific changes in the expression of genes and proteins involved in pro-inflammatory pathways, T cell activation, and cytotoxicity. Citation Format: Maxine S. Lam, Jose A. Reales-Calderon, Jin Rong Ow, Joey Aw, Damien Tan, Ragavi Vijayakumar, Erica Ceccarello, Tommaso Tabaglio, Yan Ting Lim, Wang Loo Chien, Fritz Lai, Anthony Tan Tanoto, Qingfeng Chen, Radoslaw M. Sobota, Giulia Adriani, Antonio Bertoletti, Ernesto Guccione, Andrea Pavesi. G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1781.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a dismal prognosis. Currently, Temozolomide (TMZ) and Bevacizumab are the main approved agents for primary and recurrent GBM, respectively. However, TMZ resistance is frequent and Bevacizumab does not improve overall survival. The heterogeneity of GBM and the presence of a highly selectively permeable blood-brain barrier (BBB) preclude the use of chemotherapeutics approved for other malignancies. Meanwhile, new therapy development is limited in part because most pre-clinical models do not recapitulate the key features of GBM that are relevant to therapy resistance and drug delivery. This is particularly important when validating immunotherapies such as engineered T cell therapy, which has shown some promise in the clinics. We therefore developed an in vitro 3D model that recapitulates the major features of the GBM tumor microenvironment (TME) that are involved in therapy resistance, including the dense tumor core and infiltrating invasive front observed in clinical tumors, surrounded by a perfusable BBB with a physiological permeability. With this model, we investigate the angiogenic nature of GBM, and the role of tumor organization and the TME in TMZ resistance and immunosuppression. We find that TMZ sensitivity decreases as complexity of tumor cell 3D organization increases, and identify pathways potentially associated with TMZ resistance and immunosuppression using proteomics and genomics approaches. Additionally, we find that tumor-associated vasculature has a higher permeability due to VEGFA from GBM tumors, which leads to the downregulation of endothelial junction proteins. Using an IL13Ra2-targetting chimeric antigen receptor (CAR) T cell as a model for engineered T cell therapy, we find that tumor-associated vasculature acts as a physical barrier to CAR-T cell extravasation, significantly impeding CAR-T cell efficacy. We then use the model to screen a CAR library to identify novel CAR combinations that can improve CAR-T cell extravasation and cytotoxicity in GBM. Citation Format: Maxine S. Lam, Joey Aw, Damien Tan, Ragavi Vijayakumar, Khloe S. Gordon, Michael E. Birnbaum, Radoslaw M. Sobota, Giulia Adriani, Andrea Pavesi. A glioblastoma and blood-brain barrier in vitro model to assess the tumor microenvironment and temozolomide resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2501.

Development of efficacious therapeutic strategies against solid tumors is limited by the lack of pre-clinical models that can reliably predict treatment outcomes in patients. This is primarily because models often do not accurately reflect the complexity of the tumor microenvironment (TME). The TME consists of vasculature, stromal cells and immune cells that can promote tumor resistance or prevent targeted drug and cell therapy. Here, we developed an in vitro vascularized liver tumor model in a microfluidic device to evaluate drug delivery and immunotherapy approaches. The model consists of a tumor spheroid surrounded by a perfusable self-organized vasculature bed with physiologically relevant permeability. Sorafenib (Nexavar) efficacy was evaluated by assessing tumor spheroid viability, quantified by the loss of fluorescence signal. The presence of vasculature showed enhanced cytotoxicity, highlighting the importance of tumor-vascular interactions for testing therapeutic efficacy. Engineered immune cells (chimeric antigen receptor (CAR) T-cell and transiently expressing T-cell receptors (TCR)) were introduced into the model to demonstrate immunotherapy validation. Our results showed that the engineered immune cells were more effective at infiltrating the tumor spheroid without a vasculature but were more cytotoxic in the presence of the vasculature. By removing the cultured microtissue from the device and performing digital spatial transcriptomics on standard paraffin-embedded sections, we identified known and novel genes involved in creating a pro-tumorigenic microenvironment. This platform can be utilized to model critical features of the in vivo TME, providing better predictability of drug responses, with potential applications towards personalized medicine. Citation Format: Jyothsna Vasudevan, Ragavi Vijayakumar, Jose Antonio Reales-Calderon, Maxine Sin Lam, Jin Rong Ow, Joey Aw, Damien Tan, Giulia Adriani, Andrea Pavesi. Microphysiological vascularized solid liver tumor model for drug and cell therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4612.

The desmoplastic nature of the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) prevents the infiltration of T cells and the penetration of chemotherapeutic drugs, posing a challenge to the validation of targeted therapies, including T cell immunotherapies. We present an in vitro 3D PDAC-TME model to observe and quantify T cell infiltration across the vasculature. In a three-channel microfluidic device, PDAC cells are cultured in a collagen matrix in the central channel surrounded, on one side, by endothelial cells (ECs) to mimic a blood vessel and, on the opposite side, by pancreatic stellate cells (PSCs) to simulate exocrine pancreas. The migration of T cells toward the tumor is quantified based on their activation state and TME composition. The presence of EC-lining drastically reduces T cell infiltration, confirming the essential role of the vasculature in controlling T cell trafficking. We show that activated T cells migrate ∼50% more than the not-activated ones toward the cancer cells. Correspondingly, in the absence of cancer cells, both activated and not-activated T cells present similar migration toward the PSCs. The proposed approach could help researchers in testing and optimizing immunotherapies for pancreatic cancer.

Emerging base editing technology exploits CRISPR RNA-guided DNA modification effects for highly specific C > T conversion, which has been used to efficiently disrupt gene expression. These tools can enhance synthetic T cell immunity by restricting specificity, addressing histocompatibility leukocyte antigen (HLA) barriers, and promoting persistence. We report lentiviral delivery of a hepatitis B-virus (HBV)-specific recombinant T cell receptor (rTCR) and a linked CRISPR single-guide RNA for simultaneous disruption of endogenous TCRs (eTCRs) when combined with transient cytosine deamination. Discriminatory depletion of eTCR and coupled expression of rTCR resulted in enrichment of HBV-specific populations from 55% (SEM, ±2.4%) to 95% (SEM, ±0.5%). Intensity of rTCR expression increased 1.8- to 2.9-fold compared to that in cells retaining their competing eTCR, and increased cytokine production and killing of HBV antigen-expressing hepatoma cells in a 3D microfluidic model were exhibited. Molecular signatures confirmed that seamless conversion of C > T (G > A) had created a premature stop codon in TCR beta constant 1/2 loci, with no notable activity at predicted off-target sites. Thus, targeted disruption of eTCR by cytosine deamination and discriminatory enrichment of antigen-specific T cells offers the prospect of enhanced, more specific T cell therapies against HBV-associated hepatocellular carcinoma (HCC) as well as other viral and tumor antigens., Graphical Abstract, The authors deploy cytosine-deamination-mediated base editing to genetically disrupt endogenous T cell receptors (eTCRs), thereby reducing competition with recombinant TCRs (rTCRs) and allowing the enrichment of engineered T cells for immunotherapy against hepatitis B-driven hepatocellular carcinoma.

Engineered T cells transiently expressing tumor-targeting receptors are an attractive form of engineered T cell therapy as they carry no risk of insertional mutagenesis or long-term adverse side-effects. However, multiple rounds of treatment are often required, increasing patient discomfort and cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targetting hepatocellular carcinoma, we found that short-term inhibition of G9a/GLP increased T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increased granzyme expression without terminal T cell differentiation or exhaustion and resulted in specific changes in expression of genes and proteins involved in pro-inflammatory pathways, T cell activation and cytotoxicity.

Protection of the central nervous system (CNS) and cerebral homeostasis depend upon the blood-brain barrier (BBB) functions and permeability. BBB restrictive permeability hinders drug delivery for the treatment of several neurodegenerative diseases and brain tumors. Several in vivo animal models and in vitro systems have been developed to understand the BBB complex mechanisms and aid in the design of improved therapeutic strategies. However, there are still many limitations that should be addressed to achieve the structural and chemical environment of a human BBB. We developed a microfluidic-based model of the neurovascular unit. A monolayer of human cerebral endothelial cells (hCMEC-D3) was grown and cocultured with human brain microvascular pericytes (hBMVPC), and human induced pluripotent stem cells differentiated into astrocytes (hiPSC-AC) and neurons (hiPSC-N). To visualize the physiological morphology of each cell type, we used fluorescent cell-specific markers and confocal microscopy. Permeation of fluorescent solutes with different molecular weights was measured to demonstrate that the developed BBB was selectively permeable as a functional barrier.

Protection of the central nervous system (CNS) and cerebral homeostasis depend upon the blood-brain barrier (BBB) functions and permeability. BBB restrictive permeability hinders drug delivery for the treatment of several neurodegenerative diseases and brain tumors. Several in vivo animal models and in vitro systems have been developed to understand the BBB complex mechanisms and aid in the design of improved therapeutic strategies. However, there are still many limitations that should be addressed to achieve the structural and chemical environment of a human BBB. We developed a microfluidic-based model of the neurovascular unit. A monolayer of human cerebral endothelial cells (hCMEC-D3) was grown and cocultured with human brain microvascular pericytes (hBMVPC), and human induced pluripotent stem cells differentiated into astrocytes (hiPSC-AC) and neurons (hiPSC-N). To visualize the physiological morphology of each cell type, we used fluorescent cell-specific markers and confocal microscopy. Permeation of fluorescent solutes with different molecular weights was measured to demonstrate that the developed BBB was selectively permeable as a functional barrier.

With the growing expansion of viticulture, the northeastern region in particular, the Submedio Vale do Sao Francisco, stands out in the cultivation of grapes. With the introduction of new cultivars in this region, there is a need to improve pruning management for the adaptation of these varieties. The objective of this work was to evaluate the influence of different bud loads on the management of the canopy for the post harvest quality of the fruits of cv. Sugrathirteen (Midnight Beauty®). The research was carried out in two years (2014 and 2015), using the DBC design in a factorial scheme, being two seasons and five treatments (6, 8, 10, 12 and 14 buds) distributed in four blocks, considering 5 plants by repetition. It can be observed that, in relation to the chemical characteristics of the fruit, the time of pruning significantly affected the content of soluble solids and total acidity. There was no influence of the yolk load on the soluble solids content, however, in the two years, they reached levels above 15 0Brix, considered adequate for this cultivar. It was also concluded that the choice of the pruning system as a function of the yolk load and the genetic characteristics of the cultivar, provided higher yields in pruning with 10 buds, without negatively affecting the quality of the grapes.

// Simone Bersini 1, * , Agnes Miermont 2, * , Andrea Pavesi 3 , Roger Dale Kamm 2, 4 , Jean Paul Thiery 3, 5 , Matteo Moretti 1, 6, 7 and Giulia Adriani 2 1 Cell and Tissue Engineering Laboratory, Istituto Ortopedico Galeazzi, Milano, Italy 2 BioSystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, Singapore 3 Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 4 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA 5 Yong Loo Lin School of Medicine, Department of Biochemistry, National University of Singapore, Singapore 6 Regenerative Medicine Technologies Laboratory, Ente Ospedaliero Cantonale, Lugano, Switzerland 7 Swiss Institute for Regenerative Medicine, Lugano, Switzerland * These authors contributed equally to this work Correspondence to: Giulia Adriani, email: giulia.adriani@smart.mit.edu Matteo Moretti, email: matteo.moretti@grupposandonato.it Keywords: microfluidics; microarrays; cancer cell extravasation; tumor microenvironment; organ-specific metastasis Received: August 01, 2018 Accepted: October 25, 2018 Published: November 16, 2018 ABSTRACT The reciprocal interaction between circulating tumor cells (CTCs) and tissue-specific cells is influential for the progression of metastases. In particular, the process of extravasation relies on the complex cross-talk between cancer cells and other cellular players such as the endothelium and the secondary tissue. However, most in vitro studies only focus on one heterotypic cell-cell interaction and often lack of physiological relevance. In this project, we investigated both CTC-endothelium and CTC-secondary site interactions during cancer cell extravasation. We first used a microarray analysis of extravasated MDA-MB-231 breast cancer cells to identify key markers involved in extravasation. Then, we developed a tri-culture microfluidic platform combining cancer cells, endothelium and a bone-mimicking (BMi) microenvironment to assess how organ tropism influences the extravasation potential of cancer cells from different tissues. Through the microarray analyses of extravasated cancer cells we found that extravasation is associated with upregulation of late-metastatic markers along with specific proteases, such as matrix metalloprotease (MMP), a-disintegrin and metalloprotease (ADAM) and a-disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family members, which are all involved in endothelium glycocalyx shedding. Through the microfluidic extravasation assay, we found that the bone-like microenvironment increased invasion and motility of breast, bladder and ovarian cancer cell (MDA-MB-231, T24 and OVCAR-3). Among the three cell types, ovarian cancer cells presented the lowest migration rate and bladder cancer cells the highest, hence recapitulating their different level of bone tropism observed in vivo . Taken together, our results shed light on the importance of intercellular communication between CTCs and other non-tumor cells essential for promoting cancer cell extravasation.

Viticulture is an activity of great economic and social importance in the Submedium region of the São Francisco River Valley, with emphasis on table grape and wine production. With the increasing expansion of the viticulture, a growing number of alternatives that do not affect fruit quality have been studied to maximize table grape yield, such as pruning and load adjustment. The aim of the present study was to evaluate the influence of different bud loads on canopy management to enable the marketable and economic production of cv. Sugrathirteen (Midnight Beauty®) in the submedium region ofthe São Francisco River Valley. This study was carried out for two years (2014/2015) in an experimental area for the introduction of new cultivars patented by Prodomo Farm in the municipality of Petrolina, Pernambuco, Brazil. The experiment was conducted in a randomized block factorial 2 × 5 design, with two seasons and 5 treatments, 6, 8, 10, 12 and 14 buds short_textwhich correspond toshort_text 17, 23, 29, 34 and 40 buds·m–2 short_textrespectivelyshort_text, distributed in 4 plots, considering five plants per replicate. Our results show that pruning seasons significantly affected sprouting percentage. However, the difference in bud load influenced this variable, with higher values in the pruning at 14 buds in both seasons. According to the results, the selection of pruning system according to bud load and to genetic features of the cultivar, and their interaction with the environment, produced higher yields in pruning with 10 buds, without negatively affecting grape quality.

Modification of specificity of T cells for the use in adoptive transfer (CAR- or TCR-redirected T cells) has revolutionized the therapy of liquid tumors and some infectious diseases. However, several obstacles are still hampering the efficacy of such potent therapy, hence concurrent modification of the function is also required to obtain successful results. Here we show the use of splice-switching antisense oligonucleotides (SSOs) as a tool to transiently modify T cell function. We demonstrate the possibility to transfect SSOs and an exogenous TCR into primary human T cells in the same electroporation reaction, without affecting viability and function of the transfected T lymphocytes. Moreover, we show that SSOs targeting T cell-specific mRNAs induce the skipping of the targeted exons, and the reduction of the protein and consequent modification of T cell function. This technical work paves the way to the use of SSOs in immune cells, not only for the knockdown of the functional isoform of the targeted proteins, but also for the protein manipulation by elimination of specific domains encoded by targeted exons.

BACKGROUND AND AIMS HBV-specific T-cell receptor (HBV-TCR) engineered T cells have the potential for treating HCC relapses after liver transplantation, but their efficacy can be hampered by the concomitant immunosuppressive treatment required to prevent graft rejection. Our aim is to molecularly engineer TCR-T cells that could retain their polyfunctionality in such patients while minimizing the associated risk of organ rejection. APPROACH AND RESULTS We first analyzed how immunosuppressive drugs can interfere with the in vivo function of TCR-T cells in liver transplanted patients with HBV-HCC recurrence receiving HBV-TCR T cells and in vitro in the presence of clinically relevant concentrations of immunosuppressive tacrolimus (TAC) and mycophenolate mofetil (MMF). Immunosuppressive Drug Resistant Armored TCR-T cells of desired specificity (HBV or Epstein-Barr virus) were then engineered by concomitantly electroporating mRNA encoding specific TCRs and mutated variants of calcineurin B (CnB) and inosine-5'-monophosphate dehydrogenase (IMPDH), and their function was assessed through intracellular cytokine staining and cytotoxicity assays in the presence of TAC and MMF. Liver transplanted HBV-HCC patients receiving different immunosuppressant drugs exhibited varying levels of activated (CD39+ Ki67+ ) peripheral blood mononuclear cells after HBV-TCR T-cell infusions that positively correlate with clinical efficacy. In vitro experiments with TAC and MMF showed a potent inhibition of TCR-T cell polyfunctionality. This inhibition can be effectively negated by the transient overexpression of mutated variants of CnB and IMPDH. Importantly, the resistance only lasted for 3-5 days, after which sensitivity was restored. CONCLUSIONS We engineered TCR-T cells of desired specificities that transiently escape the immunosuppressive effects of TAC and MMF. This finding has important clinical applications for the treatment of HBV-HCC relapses and other pathologies occurring in organ transplanted patients.

RNA methyltransferase METTL6 is implicated in tumor cell growth and in mouse energy consumption., Recently, covalent modifications of RNA, such as methylation, have emerged as key regulators of all aspects of RNA biology and have been implicated in numerous diseases, for instance, cancer. Here, we undertook a combination of in vitro and in vivo screens to test 78 potential methyltransferases for their roles in hepatocellular carcinoma (HCC) cell proliferation. We identified methyltransferase-like protein 6 (METTL6) as a crucial regulator of tumor cell growth. We show that METTL6 is a bona fide transfer RNA (tRNA) methyltransferase, catalyzing the formation of 3-methylcytidine at C32 of specific serine tRNA isoacceptors. Deletion of Mettl6 in mouse stem cells results in changes in ribosome occupancy and RNA levels, as well as impaired pluripotency. In mice, Mettl6 knockout results in reduced energy expenditure. We reveal a previously unknown pathway in the maintenance of translation efficiency with a role in maintaining stem cell self-renewal, as well as impacting tumor cell growth profoundly.

Background & Aims Virus-specific T cell dysfunction is a common feature of HBV-related hepatocellular carcinoma (HBV-HCC). Conventional T (ConT) cells can be redirected towards viral antigens in HBV-HCC when they express an HBV-specific receptor; however, their efficacy can be impaired by liver-specific physical and metabolic features. Mucosal-associated invariant T (MAIT) cells are the most abundant innate-like T cells in the liver and can elicit potent intrahepatic effector functions. Here, we engineered ConT and MAIT cells to kill HBV expressing hepatoma cells and compared their functional properties. Methods Donor-matched ConT and MAIT cells were engineered to express an HBV-specific T cell receptor (TCR). Cytotoxicity and hepatocyte homing potential were investigated using flow cytometry, real-time killing assays, and confocal microscopy in 2D and 3D HBV-HCC cell models. Major histocompatibility complex (MHC) class I-related molecule (MR1)-dependent and MR1-independent activation was evaluated in an Escherichia coli THP-1 cell model and by IL-12/IL-18 stimulation, respectively. Results HBV TCR-MAIT cells demonstrated polyfunctional properties (CD107a, interferon [IFN] γ, tumour necrosis factor [TNF], and IL-17A) with strong HBV target sensitivity and liver-homing chemokine receptor expression when compared with HBV TCR-ConT cells. TCR-mediated lysis of hepatoma cells was comparable between the cell types and augmented in the presence of inflammation. Coculturing with HBV+ target cells in a 3D microdevice mimicking aspects of the liver microenvironment demonstrated that TCR-MAIT cells migrate readily towards hepatoma targets. Expression of an ectopic TCR did not affect the ability of the MAIT cells to be activated via MR1-presented bacterial antigens or IL-12/IL-18 stimulation. Conclusions HBV TCR-MAIT cells demonstrate anti-HBV functions without losing their endogenous antimicrobial mechanisms or hepatotropic features. Our results support future exploitations of MAIT cells for liver-directed immunotherapies. Lay summary Chronic HBV infection is a leading cause of liver cancer. T cell receptor (TCR)-engineered T cells are patients’ immune cells that have been modified to recognise virus-infected and/or cancer cells. Herein, we evaluated whether mucosal-associated invariant T cells, a large population of unconventional T cells in the liver, could recognise and kill HBV infected hepatocytes when engineered with an HBV-specific TCR. We show that their effector functions may exceed those of conventional T cells currently used in the clinic, including antimicrobial properties and chemokine receptor profiles better suited for targeting liver tumours., Graphical abstract, Highlights • Mucosal-associated invariant T (MAIT) cells expanded in vitro can be engineered to kill HBV antigen-presenting hepatoma cells. • MAIT cells produce IL-17A upon encountering their HBV targets, a functional property not observed in conventional T cells currently used in immunotherapy. • HBV-specific MAIT cells migrate readily towards liver targets in a 3D microfluidics system. • Co-expression of an HBV-specific TCR does not affect the ability of MAIT cells to respond to their physiological stimuli.

Stiff fibers are used as reinforcing phases in a wide range of high-performance composite materials. Silk is one of the most widely studied bio-fibers, but alternative materials with specific advantages are also being explored. Among these, native hagfish (Eptatretus stoutii) slime thread is an attractive protein-based polymer. These threads consist of coiled-coil intermediate filaments (IFs) as nano-scale building blocks, which can be transformed into extended β-sheet-containing chains upon draw-processing, resulting in fibers with impressive mechanical performance. Here, we report artificial hagfish threads produced by recombinant protein expression, which were subsequently self-assembled into coiled-coil nanofilaments, concentrated, and processed into β-sheet-rich fibers by a "picking-up" method. These artificial fibers experienced mechanical performance enhancement during draw-processing. We exploited the lysine content to covalently cross-link the draw-processed fibers and obtained moduli values (E) in tension as high as ∼20 GPa, which is stiffer than most reported artificial proteinaceous materials.

Angiogenesis is a crucial event for tumor progression and metastasis. It is the process through which new blood vessels are formed and has become a therapeutic target in many cancer therapies. However, current anti-angiogenic drugs such as Thalidomide still have detrimental teratogenic effects. This property could be caused by the presence of chiral carbons, intrinsic to such compounds. We synthesized four different phthalimide derivatives that lack chiral carbons in their chemical structure. We hypothesized that these achiral carbon compounds would retain similar levels of anti-angiogenic activity whilst reducing teratogenic effects. We tested for their anti-angiogenic functions using an in vitro 3D microfluidic assay with human endothelial cells. All four compounds caused a drastic inhibition of angiogenesis at lower effective concentrations compared to Thalidomide. Quantification of the blood vessel sprouting in each condition allowed us to classify compounds depending on their anti-angiogenic capabilities. The most effective identified compound (C4), was tested in vivo on a zebrafish embryo model. Blood vessel development was measured using number and lengths of the stalks visible in the fli1a:EGFP transgenic line. Potential teratogenic effects of C4 were monitored over zebrafish embryonic development. The in vivo results confirmed the increased potency of C4 compared to Thalidomide demonstrated by results in embryos exposed to concentrations as low as 0.02 μM. The teratogenic analysis further validated the advantages of using C4 over Thalidomide in zebrafish embryos. This study highlights how the use of in vitro 3D model can allow rapid screening and selection of new and safer drugs.

Current adoptive T cell therapies have shown promising results in clinical trials but need further development as an effective cancer treatment. Here, we discuss how 3D microfluidic tumour models mimicking the tumour microenvironment could help in testing T cell immunotherapies by assessing engineered T cells and identifying combinatorial therapy to improve therapeutic efficacy. We propose that 3D microfluidic systems can be used to screen different patient-specific treatments, thereby reducing the burden of in vivo testing and facilitating the rapid translation of successful T cell cancer immunotherapies to the clinic., National Cancer Institute (U.S.) (Grant 1-U01CA202177-01)

Electrical stimulation (ES) of cells has been shown to induce a variety of responses, such as cytoskeleton rearrangements, migration, proliferation, and differentiation. In this study, we have investigated whether monophasic and biphasic pulsed ES could exert any effect on the proliferation and differentiation of human cardiac progenitor cells (hCPCs) isolated from human heart fragments. Cells were cultured under continuous exposure to monophasic or biphasic ES with fixed cycles for 1 or 3 days. Results indicate that neither stimulation protocol affected cell viability, while the cell shape became more elongated and reoriented more perpendicular to the electric field direction. Moreover, the biphasic ES clearly induced the upregulation of early cardiac transcription factors, MEF2D, GATA-4, and Nkx2.5, as well as the de novo expression of the late cardiac sarcomeric proteins, troponin T, cardiac alpha actinin, and SERCA 2a. Both treatments increased the expression of connexin 43 and its relocation to the cell membrane, but biphasic ES was faster and more effective. Finally, when hCPCs were exposed to both monophasic and biphasic ES, they expressed de novo the mRNA of the voltage-dependent calcium channel Cav 3.1(α1G) subunit, which is peculiar of the developing heart. Taken together, these results show that ES alone is able to set the conditions for early differentiation of adult hCPCs toward a cardiac phenotype.

Cardiac cell function is substantially influenced by the nature and intensity of the mechanical loads the cells experience. Cardiac fibroblasts (CFs) are primarily involved in myocardial tissue remodeling: at the onset of specific pathological conditions, CFs activate, proliferate, differentiate, and critically alter the amount of myocardial extra-cellular matrix with important consequences for myocardial functioning. While cyclic mechanical strain has been shown to increase matrix synthesis of CFs in vitro, the role of mechanical cues in CFs proliferation is unclear. We here developed a multi-chamber cell straining microdevice for cell cultures under uniform, uniaxial cyclic strain. After careful characterization of the strain field, we extracted human heart-derived CFs and performed cyclic strain experiments. We subjected cells to 2% or 8% cyclic strain for 24 h or 72 h, using immunofluorescence to investigate markers of cell morphology, cell proliferation (Ki67, EdU, phospho-Histone-H3) and subcellular localization of the mechanotransduction-associated transcription factor YAP. Cell morphology was affected by cyclic strain in terms of cell area, cell and nuclear shape and cellular alignment. We additionally observed a strain intensity-dependent control of cell growth: a significant proliferation increase occurred at 2% cyclic strain, while time-dependent effects took place upon 8% cyclic strain. The YAP-dependent mechano-transduction pathway was similarly activated in both strain conditions. These results demonstrate a differential effect of cyclic strain intensity on human CFs proliferation control and provide insights into the YAP-dependent mechano-sensing machinery of human CFs.

Adoptive cell therapy (ACT) is showing promising results in clinical trials but many challenges remain in understanding the key role of the tumor microenvironment. These challenges constitute a major barrier to advancing the field. Therefore, it is crucial to perform preclinical tests of the developed ACT strategies in a fast and reproducible way to assess the potential for patient therapy. Here, we describe the development of an intrahepatic tumor model in a microfluidic device for screening T cell-based immunotherapeutic strategies and the role of monocytes in these therapies. This system can be used to test also the effects of supporting cytokine administration and changes in oxygen level that are typically found in a liver tumor microenvironment. As a result, these 3D microfluidic assays provide a means to quantify T cell anti-tumor activity under different conditions to optimize existing therapeutic strategies or the design of new ones.

In the hepatitis B virus (HBV)-related hepatocellular carcinoma tumor microenvironment (TME), monocytes reportedly impede natural T cell functions via PD-L1/PD-1 signaling. However, it remains unclear if T cell receptor-redirected T cells (TCR T cells) are similarly inhibited. Hence, we developed a 3D intrahepatic TME microfluidic model to investigate the immunosuppressive potential of monocytes toward HBV-specific TCR T cells and the role of PD-L1/PD-1 signaling. Interestingly, in our 3D static microfluidic model, we observed that monocytes suppressed only retrovirally transduced (Tdx) TCR T cell cytotoxicity toward cancer cells via PD-L1/PD-1, while mRNA electroporated (EP) TCR T cell cytotoxicity was not affected by the presence of monocytes. Importantly, when co-cultured in 2D, both Tdx and EP TCR T cell cytotoxicity toward cancer cells were not suppressed by monocytes, suggesting our 3D model as a superior tool compared to standard 2D assays for predicting TCR T cell efficacy in a preclinical setting, which can thus be used to improve current immunotherapy strategies.

In the hepatitis B virus (HBV)-related hepatocellular carcinoma tumor microenvironment (TME), monocytes reportedly impede natural T cell functions via PD-L1/PD-1 signaling. However, it remains unclear if T cell receptor-redirected T cells (TCR T cells) are similarly inhibited. Hence, we developed a 3D intrahepatic TME microfluidic model to investigate the immunosuppressive potential of monocytes toward HBV-specific TCR T cells and the role of PD-L1/PD-1 signaling. Interestingly, in our 3D static microfluidic model, we observed that monocytes suppressed only retrovirally transduced (Tdx) TCR T cell cytotoxicity toward cancer cells via PD-L1/PD-1, while mRNA electroporated (EP) TCR T cell cytotoxicity was not affected by the presence of monocytes. Importantly, when co-cultured in 2D, both Tdx and EP TCR T cell cytotoxicity toward cancer cells were not suppressed by monocytes, suggesting our 3D model as a superior tool compared to standard 2D assays for predicting TCR T cell efficacy in a preclinical setting, which can thus be used to improve current immunotherapy strategies.

Adoptive cell therapy (ACT) is showing promising results in clinical trials but many challenges remain in understanding the key role of the tumor microenvironment. These challenges constitute a major barrier to advancing the field. Therefore, it is crucial to perform preclinical tests of the developed ACT strategies in a fast and reproducible way to assess the potential for patient therapy. Here, we describe the development of an intrahepatic tumor model in a microfluidic device for screening T cell-based immunotherapeutic strategies and the role of monocytes in these therapies. This system can be used to test also the effects of supporting cytokine administration and changes in oxygen level that are typically found in a liver tumor microenvironment. As a result, these 3D microfluidic assays provide a means to quantify T cell anti-tumor activity under different conditions to optimize existing therapeutic strategies or the design of new ones.

The tumor microenvironment imposes physical and functional constraints on the antitumor efficacy of adoptive T cell immunotherapy. Preclinical testing of different T cell preparations can help in the selection of efficient immune therapies, but in vivo models are expensive and cumbersome to develop, while classical in vitro 2D models cannot recapitulate the spatiotemporal dynamics experienced by T cells targeting cancer. Here, we describe an easily customizable 3D model, in which the tumor microenvironment conditions are modulated and the functionality of different T cell preparations is tested. We incorporate human cancer hepatocytes as a single cell or as tumor cell aggregates in a 3D collagen gel region of a microfluidic device. Human T cells engineered to express tumor-specific T cell receptors (TCR-T cells) are then added in adjacent channels. The TCR-T cells' ability to migrate and kill the tumor target and the profile of soluble factors were investigated under conditions of varying oxygen levels and in the presence of inflammatory cytokines. We show that only the 3D model detects the effect that oxygen levels and the inflammatory environment impose on engineered TCR-T cell function, and we also used the 3D microdevice to analyze the TCR-T cell efficacy in an immunosuppressive scenario. Hence, we show that our microdevice platform enables us to decipher the factors that can alter T cell function in 3D and can serve as a preclinical assay to tailor the most efficient immunotherapy configuration for a specific therapeutic goal.

Background & Aims Strategies to develop virus-specific T cells against hepatic viral infections have been hindered by safety concerns. We engineered nonlytic human T cells to suppress replication of hepatitis B virus (HBV) and hepatitis C virus (HCV) without overt hepatotoxicity and investigated their antiviral activity. Methods We electroporated resting T cells or T cells activated by anti-CD3 with mRNAs encoding HBV or HCV-specific T-cell receptors (TCRs) to create 2 populations of TCR-reprogrammed T cells. We tested their ability to suppress HBV or HCV replication without lysis in 2-dimensional and 3-dimensional cultures of HepG2.2.15 cells and HBV-infected HepG2-hNTCP cells. We also injected TCR-reprogrammed resting and activated T cells into HBV-infected urokinase-type plasminogen activator/severe combined immunodeficiency disease/interleukin 2γ mice with humanized livers and measured levels of intrahepatic and serological viral parameters and serum alanine aminotransferase. Livers were collected for analysis of gene expression patterns to determine effects of the TCR-reprogrammed T cells. Results TCR-reprogrammed resting T cells produced comparable levels of interferon gamma but lower levels of perforin and granzyme than activated T cells and did not lyse HCV- or HBV-infected hepatoma cells. Although T-cell secretion of interferon gamma was required to inhibit HCV replication, the HBV-specific TCR-reprogrammed resting T cells reduced HBV replication also through intracellular activation of apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3). The mechanism of APOBEC3 intracellular activation involved temporal expression of lymphotoxin-β receptor ligands on resting T cells after TCR-mediated antigen recognition and activation of lymphotoxin-β receptor in infected cells. Conclusions We developed TCR-reprogrammed nonlytic T cells capable of activating APOBEC3 in hepatoma cells and in HBV-infected human hepatocytes in mice, limiting viral infection. These cells with limited hepatotoxicity might be developed for treatment of chronic HBV infection.

In tissue engineering, several factors play key roles in providing adequate stimuli for cells differentiation, in particular biochemical and physical stimuli, which try to mimic the physiological microenvironments. Since electrical stimuli are important in the developing heart, we have developed an easy-to-use, cost-effective cell culture platform, able to provide controlled electrical stimulation aimed at investigating the influence of the electric field in the stem cell differentiation process. This bioreactor consists of an electrical stimulator and 12 independent, petri-like culture chambers and a 3-D computational model was used to characterize the distribution and the intensity of the electric field generated in the cell culture volume. We explored the effects of monophasic and biphasic square wave pulse stimulation on a mouse adipose-derived stem cell line (m17.ASC) comparing cell viability, proliferation, protein, and gene expression. Both monophasic (8 V, 2 ms, 1 Hz) and biphasic (+4 V, 1 ms and -4 V, 1 ms; 1 Hz) stimulation were compatible with cell survival and proliferation. Biphasic stimulation induced the expression of Connexin 43, which was found to localize also at the cell membrane, which is its recognized functional mediating intercellular electrical coupling. Electrically stimulated cells showed an induced transcriptional profile more closely related to that of neonatal cadiomyocytes, particularly for biphasic stimulation. The developed platform thus allowed to set-up precise conditions to drive adult stem cells toward a myocardial phenotype solely by physical stimuli, in the absence of exogenously added expensive bioactive molecules, and can thus represent a valuable tool for translational applications for heart tissue engineering and regeneration.

We developed a computational model to predict oxygen levels in microfluidic plastic devices during cell culture. This model is based on experimental evaluation of oxygen levels. Conditions are determined that provide adequate oxygen supply to two cell types, hepatocytes and endothelial cells, either by diffusion through the plastic device, or by supplying a low flow rate of medium.

The adoption of in vitro 3D cell culture models as a bridge between conventional 2D cell culture models and the complex in vivo animal models has been increasing. A carefully designed 3D model can run biological assays with animal model-like complexities but with the simplicity and affordability of traditional cell culture. For example, traditional 2D and transwell assays of immune cell infiltration efficiency are unrealistic, as cell migration is gravity-driven. AIM 3D cell culture chips offer a more realistic immune cell infiltration model by compartmentalizing immune cells and cancer spheroids in parallel channels. The chips consist of a 3D hydrogel channel and two flanking media channels. Cancer spheroids are cultured within the hydrogel channel while immune cells are seeded in one of the media channels. The seeded immune cells are required to actively invade and seek out target cancer cells in 3D hydrogel in AIM chips before they can infiltrate the cancer spheroids. This is a process similar to immune cell infiltration in vivo. By utilizing high content confocal imaging, fluorescent-labeled immune cells that migrate into the 3D hydrogel can be visualized and quantified. This assay, in combination with adoptive T cell therapy or immune checkpoint blockade, can determine the roles of tumor infiltrated lymphocytes in immunotherapy through quantifying the live: dead ratio of cancer spheroids in the chips. This is particularly useful as a quality control tool for cellular adoptive immunotherapy where the infiltration efficiency and tumoricidal activity of engineered immune cells can be accessed in vitro. In summary, AIM 3D cell culture chips create a more physiologically relevant 3D microenvironment for visualizing immune cell infiltration of cancer spheroids. Citation Format: Sei Hien Lim, Chee Mun Kuan, Marco Campisi, Valeria Chiono, Andrea Pavesi. Analyzing immune cell infiltration of cancer spheroids in a 3D cell culture platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 47.

Immunotherapy is currently a main breakthrough in cancer treatment, but therapeutic approaches for solid tumors still present limitations in the clinical scenario due to the challenges posed by the immunosuppressive tumor microenvironment (TME). Specifically, among immune cells recruited to the TME, monocytes/macrophages are especially abundant. Macrophages in vitro have been classified as classically activated M1 macrophages and alternatively activated M2 macrophages. However, macrophages are highly plastic cells and they often present mixed phenotypes in the in vivo TME. Monocytes/macrophages are involved in cancer cell proliferation, cell invasion, cell killing, vascular angiogenesis, T-cell immunosuppression, and are often correlated with a poor outcome in an extended range of cancers. However, disrupting the protumor activity of monocytes/macrophages and their interactions with the complex cellular system in the TME remains a challenge. Thus, a better understanding of the mechanisms that modulate monocyte/macrophage phenotype and their interactions with tumors may lead to make them a relevant therapeutic strategy. To study the immunosuppressive TME, we developed microfluidic-based integrated platforms with a 3-dimensional (3D) co-culture of tumor cells and immune cells to investigate how physical and molecular cues in the TME regulate the cellular interplay. Our 3D multicellular platforms offer considerable benefits and have already demonstrated in previous studies a clear advantage over classical 2D platforms to model the TME and to screen for different therapeutic approaches. Our previous studies demonstrated the crucial role of immune system interactions with cancer cells in metastasis either at a primary tumor site or at a secondary metastatic site. Further, we investigated the impact of monocytes and Programmed Death Ligand 1 (PD-L1) immune checkpoint on T-cell receptor (TCR)-engineered T-cells. We have now developed a new 3D microfluidic platform to study the effects of interstitial flow (IF), the flow of fluid through tumor stroma, which is an important component of the TME that may contribute to the polarization of macrophages toward a protumor phenotype. The microfluidic model allows the co-culture of tumor cells and monocytes/macrophages, to stimulate them with IF and to quantify cell migration in 3D. To the best of our knowledge, this study represents the first microfluidic tumor model that incorporates IF and both tumor and immune cells. Our preliminary results confirmed that the presence of tumor cells and hence tumor-cell secreted factors (TSF) increased the migration speed and directedness of macrophages toward cancer cells, potentially contributing to cancer cell dissemination. Interestingly, the presence of the IF-based mechanical cue (without tumor cells in culture) resulted in a similar increase in macrophage migration. Further, by combining both TSF and IF we observed no synergistic or additive effect on macrophage migration. A mixed population of macrophage phenotypes (M1 and M2) was observed with either the stimulus of TSF and/or IF while lower expression levels of M1 and M2 markers were observed without TSF or IF. Importantly, the understanding gained from this investigation can help in the design and functional testing of immunotherapeutic strategies to modulate macrophage polarization in the TME towards an antitumor phenotype. Citation Format: Andrea Pavesi, Siew Cheng Wong, Roger Kamm, Sharon Wei Ling Lee, Giulia Adriani. Three-dimensional microfluidic platform mimicking the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A049.

A new imaging system allows visualizing oxygen distributions in 2D. Combining sensor foils with microfluidic devices enables online monitoring of cellular oxygen consumption in whole chip areas. Furthermore, suitable device materials depending on application and cell line can be determined. Numerically simulated oxygen consumption of rat lung microvascular endothelial cells and rat hepatocytes was experimentally validated.

The limitations of current cancer therapies highlight the urgent need for a more effective therapeutic strategy. One promising approach uses an alternating electric field; however, the mechanisms involved in the disruption of the cancer cell cycle as well as the potential adverse effects on non-cancerous cells must be clarified. In this study, we present a novel microfluidic device with embedded electrodes that enables the application of an alternating electric field therapy to cancer cells in a 3D extracellular matrix. To demonstrate the potential of our system to aid in designing and testing new therapeutic approaches, cancer cells and cancer cell aggregates were cultured individually or co-cultured with endothelial cells. The metastatic potential of the cancer cells was reduced after electric field treatment. Moreover, the proliferation rate of the treated cancer cells was lower compared with that of the untreated cells, whereas the morphologies and proliferative capacities of the endothelial cells were not significantly affected. These results demonstrate that our novel system can be used to rapidly screen the effect of an alternating electric field on cancer and normal cells within an in vivo-like microenvironment with the potential to optimize treatment protocols and evaluate synergies between tumor-treating field treatment and chemotherapy.

MBNL1 proteins lacking exon 7 (−ex7) are antisurvival factors with tumor suppressive role that cancer cells tend to down-regulate in favor of MBNL +ex7 isoforms., The extent of and the oncogenic role played by alternative splicing (AS) in cancer are well documented. Nonetheless, only few studies have attempted to dissect individual gene function at an isoform level. Here, we focus on the AS of splicing factors during prostate cancer progression, as these factors are known to undergo extensive AS and have the potential to affect hundreds of downstream genes. We identified exon 7 (ex7) in the MBNL1 (Muscleblind-like 1) transcript as being the most differentially included exon in cancer, both in cell lines and in patients' samples. In contrast, MBNL1 overall expression was down-regulated, consistently with its described role as a tumor suppressor. This observation holds true in the majority of cancer types analyzed. We first identified components associated to the U2 splicing complex (SF3B1, SF3A1, and PHF5A) as required for efficient ex7 inclusion and we confirmed that this exon is fundamental for MBNL1 protein homodimerization. We next used splice-switching antisense oligonucleotides (AONs) or siRNAs to compare the effect of MBNL1 splicing isoform switching with knockdown. We report that whereas the absence of MBNL1 is tolerated in cancer cells, the expression of isoforms lacking ex7 (MBNL1 Δex7) induces DNA damage and inhibits cell viability and migration, acting as dominant negative proteins. Our data demonstrate the importance of studying gene function at the level of alternative spliced isoforms and support our conclusion that MBNL1 Δex7 proteins are antisurvival factors with a defined tumor suppressive role that cancer cells tend to down-regulate in favor of MBNL +ex7 isoforms., Graphical Abstract