Bispecific T cell engager-armed T cells targeting integrin ανß6 exhibit enhanced T cell redirection and antitumor activity in cholangiocarcinoma.

Advanced cholangiocarcinoma (CCA) presents a clinical challenge due to limited treatment options, necessitating exploration of innovative therapeutic approaches. Bispecific T cell engager (BTE)-armed T cell therapy shows promise in hematological and solid malignancies, offering potential advantages in safety over continuous BTE infusion. In this context, we developed a novel BTE, targeting CD3 on T cells and integrin αvß6, an antigen elevated in various epithelial malignancies, on cancer cells. The novel BTE was generated by fusing an integrin αvß6-binding peptide (A20) to an anti-CD3 (OKT3) single-chain variable fragment (scFv) through a G4S peptide linker (A20/αCD3 BTE). T cells were then armed with A20/αCD3 BTE (A20/αCD3-armed T cells) and assessed for antitumor activity. Our results highlight the specific binding of A20/αCD3 BTE to CD3 on T cells and integrin αvß6 on target cells, effectively redirecting T cells towards these targets. After co-culture, A20/αCD3-armed T cells exhibited significantly heightened cytotoxicity against integrin αvß6-expressing target cells compared to unarmed T cells in both KKU-213A cells and A375.ß6 cells. Moreover, in a five-day co-culture, A20/αCD3-armed T cells demonstrated superior cytotoxicity against KKU-213A spheroids compared to unarmed T cells. Importantly, A20/αCD3-armed T cells exhibited an increased proportion of the effector memory T cell (Tem) subset, upregulation of T cell activation markers, enhanced T cell proliferation, and increased cytolytic molecule/cytokine production, when compared to unarmed T cells in an integrin αvß6-dependent manner. These findings support the potential of A20/αCD3-armed T cells as a novel therapeutic approach for integrin αvß6-expressing cancers.

A preclinical platform for assessing long-term drug efficacy exploiting mechanically tunable scaffolds colonized by a three-dimensional tumor microenvironment.

BACKGROUND: Long-term drug evaluation heavily relies upon rodent models. Drug discovery methods to reduce animal models in oncology may include three-dimensional (3D) cellular systems that take into account tumor microenvironment (TME) cell types and biomechanical properties. METHODS: In this study we reconstructed a 3D tumor using an elastic polymer (acrylate-endcapped urethane-based poly(ethylene glycol) (AUPPEG)) with clinical relevant stiffness. Single cell suspensions from low-grade serous ovarian cancer (LGSOC) patient-derived early passage cultures of cancer cells and cancer-associated fibroblasts (CAF) embedded in a collagen gel were introduced to the AUPPEG scaffold. After self-organization in to a 3D tumor, this model was evaluated by a long-term (> 40 days) exposure to a drug combination of MEK and HSP90 inhibitors. The drug-response results from this long-term in vitro model are compared with drug responses in an orthotopic LGSOC xenograft mouse model. RESULTS: The in vitro 3D scaffold LGSOC model mimics the growth ratio and spatial organization of the LGSOC. The AUPPEG scaffold approach allows to test new targeted treatments and monitor long-term drug responses. The results correlate with those of the orthotopic LGSOC xenograft mouse model. CONCLUSIONS: The mechanically-tunable scaffolds colonized by a three-dimensional LGSOC allow long-term drug evaluation and can be considered as a valid alternative to reduce, replace and refine animal models in drug discovery.

Development and characterisation of suitably bioengineered microfibrillar matrix-based 3D prostate cancer model forin vitrodrug testing.

Bioengineered 3D models that can mimic patient-specific pathologiesin vitroare valuable tools for developing and validating anticancer therapeutics. In this study, microfibrillar matrices with unique structural and functional properties were fabricated as 3D spherical and disc-shaped scaffolds with highly interconnected pores and the potential of the newly developed scaffolds for developing prostate cancer model has been investigated. The newly developed scaffolds showed improved cell retention upon seeding with cancer cells compared to conventional electrospun scaffolds. They facilitated rapid growth and deposition of cancer-specific extracellular matrix through-the-thickness of the scaffold. Compared to the prostate cancer cells grown in 2D culture, the newly developed prostate cancer model showed increased resistance to the chemodrug Docetaxel regardless of the drug concentration or the treatment frequency. A significant reduction in the cell number was observed within one week after the drug treatment in the 2D culture for both PC3 and patient-derived cells. Interestingly, almost 20%-30% of the cancer cells in the newly developed 3D model survived the drug treatment, and the patient-derived cells were more resistant than the tested cell line PC3. The results from this study indicate the potential of the newly developed prostate cancer model forin vitrodrug testing.

Development of the Follow-Up Human 3D Oral Cancer Model in Cancer Treatment.

As function preservation cancer therapy, targeted radiation therapies have been developed for the quality of life of cancer patients. However, preclinical animal studies evaluating the safety and efficacy of targeted radiation therapy is challenging from the viewpoints of animal welfare and animal protection, as well as the management of animal in radiation-controlled areas under the regulations. We fabricated the human 3D oral cancer model that considers the time axis of the follow up in cancer treatment. Therefore, in this study, the 3D model with human oral cancer cells and normal oral fibroblasts was treated based on clinical protocol. After cancer treatment, the histological findings of the 3D oral cancer model indicated the clinical correlation between tumor response and surrounding normal tissue. This 3D model has potential as a tool for preclinical studies alternative to animal studies.

Anti-Cancer Effects of Artesunate in Human 3D Tumor Models of Different Complexity.

The anti-malaria drug Artesunate (ART) shows strong anti-cancer effects in vitro; however, it shows only marginal treatment results in clinical cancer studies. In this study, ART was tested in preclinical 3D cancer models of increasing complexity using clinically relevant peak plasma concentrations to obtain further information for translation into clinical use. ART reduced cell viability in HCT-116 and HT-29 derived cancer spheroids (p < 0.001). HCT-116 spheroids responded dose-dependently, while HT-29 spheroids were affected more strongly by ART than by cytostatics (p < 0.001). HCT-116 spheroids were chemo-sensitized by ART (p < 0.001). In patient-derived cancer spheroids (PDCS), ART led to inhibition of cell viability in 84.62% of the 39 samples tested, with a mean inhibitory effect of 13.87%. Viability reduction of ART was 2-fold weaker than cytostatic monotherapies (p = 0.028). Meanwhile, tumor-stimulation of up to 16.30% was observed in six (15.38%) PDCS-models. In 15 PDCS samples, ART modulated chemotherapies in combined testing, eight of which showed chemo-stimulation (maximum of 36.90%) and seven chemo-inhibition (up to 16.95%). These results demonstrate that ART's anti-cancer efficacy depends on the complexity of the tumor model used. This emphasizes that cancer treatment with ART should be evaluated before treatment of the individual patient to ensure its benefits and prevent unwanted effects.

GelMA, Click-Chemistry Gelatin and Bioprinted Polyethylene Glycol-Based Hydrogels as 3D Ex Vivo Drug Testing Platforms for Patient-Derived Breast Cancer Organoids.

3D organoid model technologies have led to the development of innovative tools for cancer precision medicine. Yet, the gold standard culture system (Matrigel®) lacks the ability for extensive biophysical manipulation needed to model various cancer microenvironments and has inherent batch-to-batch variability. Tunable hydrogel matrices provide enhanced capability for drug testing in breast cancer (BCa), by better mimicking key physicochemical characteristics of this disease's extracellular matrix. Here, we encapsulated patient-derived breast cancer cells in bioprinted polyethylene glycol-derived hydrogels (PEG), functionalized with adhesion peptides (RGD, GFOGER and DYIGSR) and gelatin-derived hydrogels (gelatin methacryloyl; GelMA and thiolated-gelatin crosslinked with PEG-4MAL; GelSH). Within ranges of BCa stiffnesses (1−6 kPa), GelMA, GelSH and PEG-based hydrogels successfully supported the growth and organoid formation of HR+,−/HER2+,− primary cancer cells for at least 2−3 weeks, with superior organoid formation within the GelSH biomaterial (up to 268% growth after 15 days). BCa organoids responded to doxorubicin, EP31670 and paclitaxel treatments with increased IC50 concentrations on organoids compared to 2D cultures, and highest IC50 for organoids in GelSH. Cell viability after doxorubicin treatment (1 µM) remained >2-fold higher in the 3D gels compared to 2D and doxorubicin/paclitaxel (both 5 µM) were ~2.75−3-fold less potent in GelSH compared to PEG hydrogels. The data demonstrate the potential of hydrogel matrices as easy-to-use and effective preclinical tools for therapy assessment in patient-derived breast cancer organoids.

Angiopoietin-2 and the Vascular Endothelial Growth Factor Promote Migration and Invasion in Hepatocellular Carcinoma- and Intrahepatic Cholangiocarcinoma-Derived Spheroids.

Aggressive hepatocellular carcinoma (HCC) overexpressing Angiopoietin-2 (ANG-2) (a protein linked with angiogenesis, proliferation, and epithelial-mesenchymal transition (EMT)), shares 95% of up-regulated genes and a similar poor prognosis with the proliferative subgroup of intrahepatic cholangiocarcinoma (iCCA). We analyzed the pro-invasive effect of ANG-2 and its regulator vascular endothelial growth factor (VEGF) on HCC and CCA spheroids to uncover posUsible common ways of response. Four cell lines were used: Hep3B and HepG2 (HCC), HuCC-T1 (iCCA), and EGI-1 (extrahepatic CCA). We treated the spheroids with recombinant human (rh) ANG-2 and/or VEGF and then observed the changes at the baseline, after 24 h, and again after 48 h. Proangiogenic stimuli increased migration and invasion capability in HCC- and iCCA-derived spheroids and were associated with a modification in EMT phenotypic markers (a decrease in E-cadherin and an increase in N-cadherin and Vimentin), especially at the migration front. Inhibitors targeting ANG-2 (Trebananib) and the VEGF (Bevacizumab) effectively blocked the migration ability of spheroids that had been stimulated with rh-ANG-2 and rh-VEGF. Overall, our findings highlight the critical role played by ANG-2 and the VEGF in enhancing the ability of HCC- and iCCA-derived spheroids to migrate and invade, which are key processes in cancer progression.

Semi-Synthetic Click-Gelatin Hydrogels as Tunable Platforms for 3D Cancer Cell Culture.

Basement membrane extracts (BME) derived from Engelbreth-Holm-Swarm (EHS) mouse sarcomas such as Matrigel® remain the gold standard extracellular matrix (ECM) for three-dimensional (3D) cell culture in cancer research. Yet, BMEs suffer from substantial batch-to-batch variation, ill-defined composition, and lack the ability for physichochemical manipulation. Here, we developed a novel 3D cell culture system based on thiolated gelatin (Gel-SH), an inexpensive and highly controlled raw material capable of forming hydrogels with a high level of biophysical control and cell-instructive bioactivity. We demonstrate the successful thiolation of gelatin raw materials to enable rapid covalent crosslinking upon mixing with a synthetic poly(ethylene glycol) (PEG)-based crosslinker. The mechanical properties of the resulting gelatin-based hydrogels were readily tuned by varying precursor material concentrations, with Young's moduli ranging from ~2.5 to 5.8 kPa. All hydrogels of varying stiffnesses supported the viability and proliferation of MDA-MB-231 and MCF-7 breast cancer cell lines for 14 and 21 days of cell culture, respectively. Additionally, the gelatin-based hydrogels supported the growth, viability, and osteogenic differentiation of patient-derived preosteoblasts over 28 days of culture. Collectively, our data demonstrate that gelatin-based biomaterials provide an inexpensive and tunable 3D cell culture platform that may overcome the limitations of traditional BMEs.

Transwell-Hypoxia Method Facilitates the Outgrowth of 3D-Printed Collagen Scaffolds Loaded with Cryopreserved Patient-Derived Melanoma Explants.

A previous study from our laboratory demonstrated the effects of in vitro three-dimensional (3D)-printed collagen scaffolds on the maintenance of cryopreserved patient-derived melanoma explants (PDMEs). However, it remains unknown whether 3D-printed collagen scaffolds (3D-PCSs) can be harmonized with any external culture conditions to increase the growth of cryopreserved PDMEs. In this study, 3D-PCSs were manufactured with a 3DX bioprinter. The 3D-printed collagen scaffold-on-frame construction was loaded with fragments of cryopreserved PDMEs (approximately 1-2 mm). 3D-PCSs loaded with patient-derived melanoma explants (3D-PCS-PDMEs) were incubated using two types of methods: (1) in transwells in the presence of a low concentration of oxygen (transwell-hypoxia method) and (2) using a traditional adherent attached to the bottom flat surface of a standard culture dish (traditional flat condition). In addition, we used six different types of media (DMEM high glucose, MEM α, DMEM/F12, RPMI1640, fibroblast basal medium (FBM), and SBM (stem cell basal medium)) for 7 days. The results reveal that the culture conditions of MEM α, DMEM/F12, and FBM using the transwell-hypoxia method show greater synergic effects on the outgrowth of the 3D-PCS-PDME compared to the traditional flat condition. In addition, the transwell-hypoxia method shows a higher expression of the MMP14 gene and the multidrug-resistant gene product 1 (MDR1) than in the typical culture method. Taken together, our findings suggest that the transwell-hypoxia method could serve as an improved, 3D alternative to animal-free testing that better mimics the skin's microenvironment using in vitro PDMEs.

3-Dimensional coculture of breast cancer cell lines with adipose tissue-Derived stem cells reveals the efficiency of oncolytic Newcastle disease virus infection via labeling technology.

Oncolytic virotherapy is one of the emerging biological therapeutics that needs a more efficient in vitro tumor model to overcome the two-dimensional (2D) monolayer tumor cell culture model's inability to maintain tissue-specific structure. This is to offer significant prognostic preclinical assessment findings. One of the best models that can mimic the in vivo model in vitro are the three-dimensional (3D) tumor-normal cell coculture systems, which can be employed in preclinical oncolytic virus therapeutics. Thus, we developed our 3D coculture system in vitro using two types of breast cancer cell lines showing different receptor statuses cocultured with adipose tissue-derived mesenchymal stem cells. The cells were cultured in a floater tissue culture plate to allow spheroids formation, and then the spheroids were collected and transferred to a scaffold spheroids dish. These 3D culture systems were used to evaluate oncolytic Newcastle disease virus AMHA1 strain infectivity and antitumor activity using a tracking system of the Newcastle disease virus (NDV) labeled with fluorescent PKH67 linker to follow the virus entry into target cells. This provides evidence that the NDV AMHA1 strain is an efficient oncolytic agent. The fluorescently detected virus particles showed high intensity in both coculture spheres. Strategies for chemically introducing fluorescent dyes into NDV particles extract quantitative information from the infected cancer models. In conclusion, the results indicate that the NDV AMHA1 strain efficiently replicates and induces an antitumor effect in cancer-normal 3D coculture systems, indicating efficient clinical outcomes.

In vitro 2D and 3D cancer models to evaluate compounds that modulate macrophage polarization.

In vitro cancer models that can identify novel immunomodulating compounds are essential. Using a 3D multicellular tumor spheroid (MCTS) model comprising cancer cells, fibroblasts, and macrophages, we tested tumor-associated macrophage (TAM)-inhibiting compounds (CCL2 Ab, CSF1R inhibitor, CSF1R Ab) and TAM-reprograming compounds (poly I:C, CD40 Ab, CD40 ligand) for their effects on monocyte infiltration and polarization in tumor spheroids. For characterization of macrophage polarization, we measured the expression of CD206, CD163, CD86, MHC II, CD40, and CD14 and measured 43 soluble factors in the 3D MCTS cultures. 2D macrophage models were evaluated for comparison. A CSF1R inhibitor prevented infiltration of monocytes into pancreatic cancer spheroids, and macrophages treated with the inhibitor showed decreased expression of M2 markers. Treatment with a CD40 ligand and poly I:C induced M1 macrophage polarization in our models. We propose that these models can be used to improve the drug screening process of anti-cancer immunotherapies targeting macrophages.

Mimicking tumor microenvironment by 3D bioprinting: 3D cancer modeling.

The tumor microenvironment (TME) typically comprises cancer cells, tumor vasculature, stromal components like fibroblasts, and host immune cells that assemble to support tumorigenesis. However, preexisting classic cancer models like 2D cell culture methods, 3D cancer spheroids, and tumor organoids seem to lack essential TME components. 3D bioprinting offers enormous advantages for developingin vitrotumor models by allowing user-controlled deposition of multiple biomaterials, cells, and biomolecules in a predefined architecture. This review highlights the recent developments in 3D cancer modeling using different bioprinting techniques to recreate the TME. 3D bioprinters enable the fabrication of high-resolution microstructures to reproduce TME intricacies. Furthermore, 3D bioprinted models can be applied as a preclinical model for versatile research applications in the tumor biology and pharmaceutical industries. These models provide an opportunity to develop high-throughput drug screening platforms and can further be developed to suit individual patient requirements hence giving a boost to the field of personalized anti-cancer therapeutics. We underlined the various ways the existing studies have tried to mimic the TME, mimic the hallmark events of cancer growth and metastasis within the 3D bioprinted models and showcase the 3D drug-tumor interaction and further utilization of such models to develop personalized medicine.

Implementation of a dynamic culture condition to the heterotypic 3D breast cancer model.

Cell culture system is used for a wide range of research and biotechnology production. Majority of in vitro cell studies are conducted as static, two dimensional (2D) dish culture system where cells grow in a monolayer. However, to better reflect the in vivo condition, three dimensional (3D) culture systems were introduced that allow investigating the cell-cell and cell-microenvironment interactions. In this work, the 3D breast cancer model was investigated. Previously, we developed a 3D breast cancer model that constituted of fibroblasts and breast cancer cells seeded on the silkworm silk scaffold. The dynamic culture condition that provides the medium flow and shear forces was implemented to the model. The dynamic conditions were compared to the static cultivation regarding its influence on the number of cells, their viability, scaffold penetration, and cells co-localization. The implication of the dynamic condition to the 3D cultures resulted in a higher number and viability of the cells compared with the static 3D cultures. In contrast to the static culture condition, during the dynamic cultivation cells penetrated entirely and evenly the inner parts of the scaffold. Moreover, in coculture, the transitions like a ratio of fibroblast to the cancer cells, fibroblast morphology, and their localization were similar in both types of culture conditions, but they proceeded much faster during the dynamic cultivation. The implementation of dynamic culture condition shortened the time needed to establish the settle 3D breast cancer model. The established dynamic cancer model can be used to study tumor biology and drug screening.

Cell-Laden Hydrogel as a Clinical-Relevant 3D Model for Analyzing Neuroblastoma Growth, Immunophenotype, and Susceptibility to Therapies.

High risk Neuroblastoma (NB) includes aggressive, metastatic solid tumors of childhood. The survival rate improved only modestly, despite the use of combination therapies including novel immunotherapies based on the antibody-mediated targeting of tumor-associated surface ligands. Treatment failures may be due to the lack of adequate in vitro models for studying, in a given patient, the efficacy of potential therapeutics, including those aimed to enhance anti-tumor immune responses. We here propose a 3D alginate-based hydrogel as extracellular microenvironment to evaluate the effects of the three-dimensionality on biological and immunological properties of NB cells. NB cell lines grown within the 3D alginate spheres presented spheroid morphology, optimal survival, and proliferation capabilities, and a reduced sensitivity to the cytotoxic effect of imatinib mesylate. 3D cultured NB cells were also evaluated for the constitutive and IFN-γ-induced expression of surface molecules capable of tuning the anti-tumor activity of NK cells including immune checkpoint ligands. In particular, IFN-γ induced de novo expression of high amounts of HLA-I molecules, which protected NB cells from the attack mediated by KIR/KIR-L matched NK cells. Moreover, in the 3D alginate spheres, the cytokine increased the expression of the immune checkpoint ligands PD-Ls and B7-H3 while virtually abrogating that of PVR, a ligand of DNAM-1 activating receptor, whose expression correlates with high susceptibility to NK-mediated killing. Our 3D model highlighted molecular features that more closely resemble the immunophenotypic variants occurring in vivo and not fully appreciated in classical 2D culture conditions. Thus, based on our results, 3D alginate-based hydrogels might represent a clinical-relevant cell culture platform where to test the efficacy of personalized therapeutic approaches aimed to optimize the current and innovative immune based therapies in a very systematic and reliable way.

Emerging In Vitro 3D Tumour Models in Nanoparticle-Based Gene and Drug Therapy.

3D models are emerging as valuable tools for personalised nanoparticle-based cancer treatments. 3D models represent in vivo cancers more realistically than 2D patterns that are grown in Petri dishes. However, creating a 3D cancer model that mimics the complexity and heterogeneity of cancers in vivo remains difficult.

Emerging Roles of Electrospun Nanofibers in Cancer Research.

This article reviews the recent progress of electrospun nanofibers in cancer research. It begins with a brief introduction to the emerging potential of electrospun nanofibers in cancer research. Next, a number of recent advances on the important features of electrospun nanofibers critical for cancer research are discussed including the incorporation of drugs, control of release kinetics, orientation and alignment of nanofibers, and the fabrication of 3D nanofiber scaffolds. This article further highlights the applications of electrospun nanofibers in several areas of cancer research including local chemotherapy, combinatorial therapy, cancer detection, cancer cell capture, regulation of cancer cell behavior, construction of in vitro 3D cancer model, and engineering of bone microenvironment for cancer metastasis. This progress report concludes with remarks on the challenges and future directions for design, fabrication, and application of electrospun nanofibers in cancer diagnostics and therapeutics.

Silk as an innovative biomaterial for cancer therapy.

Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields.