3D Conditions Research Articles

Cellular memory function from 3D to 2D: Three-dimensional high density collagen microfiber cultures induce their resistance to reactive oxygen species

Cell properties generally change when the culture condition is changed. However, mesenchymal stem cells cultured on a hard material surface maintain their differentiation characteristics even after being cultured on a soft material surface. This phenomenon suggests the possibility of a cell culture material to memorize stem cell function even in changing cell culture conditions. However, there are no reports about cell memory function in three-dimensional (3D) culture. In this study, colon cancer cells were cultured with collagen microfibers (CMF) in 3D to evaluate their resistance to reactive oxygen species (ROS) in comparison with a monolayer (2D) culture condition and to understand the effect of 3D-culture on cell memory function. The ratio of ROS-negative cancer cells in 3D culture increased with increasing amounts of CMF and the highest amount of CMF was revealed to be 35-fold higher than that of the 2D condition. The ROS-negative cells ratio was maintained for 7 days after re-seeding in a 2D culture condition, suggesting a 3D-memory function of ROS resistance. The findings of this study will open up new opportunities for 3D culture to induce cell memory function.

Mesoscopic simulations of a fracture process in reinforced concrete beam in bending using a 2D coupled DEM/micro-CT approach

In this study, a complex fracture process in a short rectangular concrete (RC) beam reinforced by one longitudinal bar (without vertical reinforcement) and subjected to quasi-static three-point bending was numerically explored in 2D conditions. A critical diagonal shear crack in the beam caused it to fail during the experiment. The numerical simulations were conducted with a classical particle discrete element method (DEM). A three-phase concrete description (aggregate, mortar, and interfacial transitional zones (ITZs) around aggregates) accounted for the concrete heterogeneity. In mesoscopic DEM calculations based on a 2D X-ray CT scan, the actual shape and placement of aggregate particles in concrete were taken for granted. In the study, the steel bar with ribs was replicated. ITZ was also assumed between the bar and mortar. Without imposing any bond-slip law, a geometrical bar/concrete interface condition was explicitly considered. The focus was on the force–deflection diagram, fracture process, contact forces, and stresses along the bar. A good level of agreement about the evolution of the vertical force versus the deflection and failure mechanism was attained between DEM analyses and in-house laboratory tests despite simplified 2D conditions. A strong effect of concrete mesostructure on the crack pattern was found.

In Vitro and In Vivo Evaluation of the Effects of Drug 2c and Derivatives on Ovarian Cancer Cells

Background: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins. Methods: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery. Results: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers. Conclusion: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.

Modulation of Epithelial-Mesenchymal Transition Is a Possible Underlying Mechanism for Inducing Chemoresistance in MIA PaCa-2 Cells against Gemcitabine and Paclitaxel.

To elucidate the currently unknown molecular mechanisms responsible for the similarity and difference during the acquirement of resistance against gemcitabine (GEM) and paclitaxel (PTX) in patients with pancreatic carcinoma, we examined two-dimensional (2D) and three-dimensional (3D) cultures of parent MIA PaCa-2 cells (MIA PaCa-2-PA) and their GEM resistance cell line (MIA PaCa-2-GR) and PTX resistance (MIA PaCa-2-PR). Using these cells, we examined 3D spheroid configurations and cellular metabolism, including mitochondrial and glycolytic functions, with a Seahorse bio-analyzer and RNA sequencing analysis. Compared to the MIA PaCa-2-PA, (1) the formation of the 3D spheroids of MIA PaCa-2-GR or -PR was much slower, and (2) their mitochondrial and glycolytic functions were greatly modulated in MIA PaCa-2-GR or -PR, and such metabolic changes were also different between their 2D and 3D culture conditions. RNA sequencing and bioinformatic analyses of the differentially expressed genes (DEGs) using an ingenuity pathway analysis (IPA) suggested that various modulatory factors related to epithelial -mesenchymal transition (EMT) including STAT3, GLI1, ZNF367, NKX3-2, ZIC2, IFIT2, HEY1 and FBLX, may be the possible upstream regulators and/or causal network master regulators responsible for the acquirement of drug resistance in MIA PaCa-2-GR and -PR. In addition, among the prominently altered DEGs (Log2 fold changes more than 6 or less than -6), FABP5, IQSEC3, and GASK1B were identified as unique genes associated with their antisense RNA or pseudogenes, and among these, FABP5 and GASK1B are known to function as modulators of cancerous EMT. Therefore, the observations reported herein suggest that modulations of cancerous EMT may be key molecular mechanisms that are responsible for inducing chemoresistance against GEM or PTX in MIA PaCa-2 cells.

Growth and organotypic branching of lung-specific microvascular cells on 2D and in 3D lung-derived matrices

Tissue-specific endothelial cells have vital roles in maintenance and functioning of native tissues with constant reciprocal crosstalk with resident cells. Three-dimensional (3D) physio-mimetic in vitro models which incorporate lung-specific microvasculature are needed to model lung-related diseases which involve modulation of endothelial cell behavior like cancer. In this study, we investigated the growth kinetics, morphological changes and responses to biological cues of lung microvasculature on two-dimensional (2D) and in lung matrix-derived 3D hydrogels. HUVEC and HULEC-5a cells were cultured on 2D and compared for their growth, morphologies, and responses to varying growth medium formulations. Brightfield and immunofluorescence imaging was performed to assess differences in morphology. For 3D cultures, native bovine lungs were decellularized, lyophilized, solubilized, and reconstituted into hydrogel form in which endothelial cells were embedded. Cell growth and organotypic branching was monitored in 3D hydrogels in the presence of varying biological cues including lung cancer cell secretome. HUVEC and HULEC-5a cells demonstrated comparable growth and morphology on 2D. However, in 3D lung-derived ECM hydrogels, tissue-specific HULEC-5a cells exhibited much better adaptation to their microenvironment, characterized by enhanced organotypic branching and longer branches. HULEC-5a growth was responsive to lung cancer cell-conditioned medium in both 2D and 3D conditions. In 3D, the concentration of ECM ligand significantly affected cell growth in long-term culture where molecular crowding had an inhibitory role. Our data reveals that HULEC-5a cells offer a reliable alternative to frequently pursued HUVECs with comparable growth and morphology. Due to their intrinsic program for cellular crosstalk with resident cells, the use of tissue-specific endothelium constitutes a vital aspect for modeling physiological and pathological processes. Furthermore, our study is the first demonstration of the synergy between lung-specific microvasculature with lung-specific ECM within a 3D in vitro model.

Identification of a set of genes potentially responsible for resistance to ferroptosis in lung adenocarcinoma cancer stem cells

Scientific literature supports the evidence that cancer stem cells (CSCs) retain inside low reactive oxygen species (ROS) levels and are, therefore, less susceptible to cell death, including ferroptosis, a type of cell death dependent on iron-driven lipid peroxidation. A collection of lung adenocarcinoma (LUAD) primary cell lines derived from malignant pleural effusions (MPEs) of patients was used to obtain 3D spheroids enriched for stem‐like properties. We observed that the ferroptosis inducer RSL3 triggered lipid peroxidation and cell death in LUAD cells when grown in 2D conditions; however, when grown in 3D conditions, all cell lines underwent a phenotypic switch, exhibiting substantial resistance to RSL3 and, therefore, protection against ferroptotic cell death. Interestingly, this phenomenon was reversed by disrupting 3D cells and growing them back in adherence, supporting the idea of CSCs plasticity, which holds that cancer cells have the dynamic ability to transition between a CSC state and a non-CSC state. Molecular analyses showed that ferroptosis resistance in 3D spheroids correlated with an increased expression of antioxidant genes and high levels of proteins involved in iron storage and export, indicating protection against oxidative stress and low availability of iron for the initiation of ferroptosis. Moreover, transcriptomic analyses highlighted a novel subset of genes commonly modulated in 3D spheroids and potentially capable of driving ferroptosis protection in LUAD-CSCs, thus allowing to better understand the mechanisms of CSC-mediated drug resistance in tumors.

Abstract LB176: Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer spheroids

Abstract A major barrier in lung cancer pre-clinical research is the lack of simple, replicative and reproducible cell culture models. Most studies are still focused on 2D, completely ignoring cell-to-cell interactions, in vivo tissue architecture and the surrounding microenvironment. To address this void in cancer research, we aimed to develop a biomimetic 3D lung cancer spheroid model that can be used as a more reliable tool for pharmacological studies. Therefore, 3D spheroids of A549 (adenocarcinoma), H460 (large cell carcinoma) and H520 (squamous cell carcinoma) non-small cell lung cancer (NSCLC) cells were optimized and consequently submitted to AZD2014 treatment, a dual mTOR inhibitor targeting mTORC1 and mTORC2, members of the PI3K/Akt/mTOR pathway, which is commonly deregulated in lung cancer. Each cell line was seeded at 2000, 5000 or 10000 cells/spheroid in agarose micro-molds for eight days. The 3D NSCLC spheroids were further characterized for their tumor features, namely 3D dynamics and morphology, including diameter, circularity and compactness, viability properties, as well as proliferative and necrotic areas. Based on this characterization, the 2000 cells/spheroid condition was selected for further treatments and spheroid treatment time-points were selected. For each cell line, spheroids were treated with AZD2014, using concentrations based on the IC30 values (sensitization concentration) defined under 2D conditions: 10 μM, 10 μM and 0.2 μM for A549, H460 and H520 spheroids, respectively. Treated and untreated spheroids were followed during six days post-treatment, and their morphological characteristics, cell viability, necrotic core formation and proliferative areas, as well as ATP production, were assessed. Spheroids of all cell lines exhibited a significant inhibition of spheroid growth with AZD2014 treatment, which was maintained over the six days. Moreover, a reduction in spheroid intracellular ATP was observed at days three and six post-treatment. An additional treatment regimen was studied by re-treating spheroids with the same concentration of AZD2014 after 48hrs from the first treatment. Spheroids exposed to this dual treatment regimen exhibited a significant inhibition of spheroid growth, while the respective controls exhibited intensified growth. While AZD2014 did not induce significant reductions in 3D cell viability, a clear inhibition of spheroid growth was observed. Having a more intricate cellular organization, this study alleviates the increased complexity of pharmacological responses in 3D spheroids when compared to 2D models. Further studies are being carried out to define the enhanced biomimetic ability of these NSCLC spheroids and the underlying mechanisms involved when targeting the PI3K/Akt/mTOR pathway using AZD2014 as mono-therapy or in combination with two novel anti-sense oligonucleotides (ASO). Citation Format: Nathan Vella, Diogo Estêvão, Tânia Cruz, Maria José Oliveira, Anthony George Fenech, Vanessa Petroni Magri. Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB176.

Improvement of histone deacetylase inhibitor efficacy by SN38 through TWIST1 suppression in synovial sarcoma.

Synovial sarcoma (SS) is an SS18-SSX fusion gene-driven soft tissue sarcoma with mesenchymal characteristics, associated with a poor prognosis due to frequent metastasis to a distant organ, such as the lung. Histone deacetylase (HDAC) inhibitors (HDACis) are arising as potent molecular targeted drugs, as HDACi treatment disrupts the SS oncoprotein complex, which includes HDACs, in addition to general HDACi effects. To provide further molecular evidence for the advantages of HDACi treatment and its limitations due to drug resistance induced by the microenvironment in SS cells, we examined cellular responses to HDACi treatment in combination with two-dimensional (2D) and 3D culture conditions. Using several SS cell lines, biochemical and cell biological assays were performed with romidepsin, an HDAC1/2 selective inhibitor. SN38 was concomitantly used as an ameliorant drug with romidepsin treatment. Cytostasis, apoptosis induction, andMHC class I polypeptide-related sequence A/B(MICA/B) induction were monitored to evaluate the drug efficacy. In addition to the conventional 2D culture condition, spheroid culture was adopted to evaluate the influence of cell-mass microenvironment on chemoresistance. By monitoring the cellular behavior with romidepsin and/or SN38 in SS cells, we observed that responsiveness is diverse in each cell line. In the apoptotic inducible cells, co-treatment with SN38 enhanced cell death. In nonapoptotic inducible cells, cytostasis and MICA/B induction were observed, and SN38 improved MICA/B induction further. As a novel efficacy of SN38, we revealed TWIST1 suppression in SS cells. In the spheroid (3D) condition, romidepsin efficacy was severely restricted in TWIST1-positive cells. We demonstrated that TWIST1 downregulation restored romidepsin efficacy even in spheroid form, and concomitant SN38 treatment along with romidepsin reproduced the reaction. The current study demonstrated the benefits and concerns of using HDACi for SS treatment in 2D and 3D culture conditions and provided molecular evidence that concomitant treatment with SN38 can overcome drug resistance to HDACi by suppressing TWIST1 expression.

Differential Competitive Growth of Transgenic Subclones of Neuroblastoma Cells Expressing Different Levels of Cathepsin D Co-Cultured in 2D and 3D in Response to EGF: Implications in Tumor Heterogeneity and Metastasis.

Neuroblastoma (NB) is an embryonal tumor arising from the sympathetic central nervous system. The epidermal growth factor (EGF) plays a role in NB growth and metastatic behavior. Recently, we have demonstrated that cathepsin D (CD) contrasts EGF-induced NB cell growth in 2D by downregulating EGFR/MAPK signaling. Aggressive NB is highly metastatic to the bone and the brain. In the metastatic process, adherent cells detach to form clusters of suspended cells that adhere once they reach the metastatic site and form secondary colonies. Whether CD is involved in the survival of metastatic NB clones is not known. Therefore, in this study, we addressed how CD differentially affects cell growth in suspension versus the adherent condition. To mimic tumor heterogeneity, we co-cultured transgenic clones silenced for or overexpressing CD. We compared the growth kinetics of such mixed clones in 2D and 3D models in response to EGF, and we found that the Over CD clone had an advantage for growth in suspension, while the CD knocked-down clone was favored for the adherent growth in 2D. Interestingly, on switching from 3D to 2D culture conditions, the expression of E-cadherin and of N-cadherin increased in the KD-CD and Over CD clones, respectively. The fact that CD plays a dual role in cancer cell growth in 2D and 3D conditions indicates that during clonal evolution, subclones expressing different level of CD may arise, which confers survival and growth advantages depending on the metastatic step. By searching the TCGA database, we found up to 38 miRNAs capable of downregulating CD. Interestingly, these miRNAs are associated with biological processes controlling cell adhesion and cell migration. The present findings support the view that during NB growth on a substrate or when spreading as floating neurospheres, CD expression is epigenetically modulated to confer survival advantage. Thus, epigenetic targeting of CD could represent an additional strategy to prevent NB metastases.

Abstract 5848: Unraveling c-MET targeting resistance mechanisms in gastric cancer via single-cell transcriptome analysis; Novel therapeutic target discovery of ERO1A and VEGFA

Abstract Background: Gastric cancer (GC) is the fifth most prevalent malignant tumor worldwide, and the fourth most common cause of mortality. c-MET has been reported to be associated with poor prognosis in GC. Over the decades, treatment approaches targeting HGF/c-MET have been developed including small molecule tyrosine kinase inhibitors, anti-c-MET and anti-HGF monoclonal antibodies. Unfortunately, the results of previous study support the development of acquired resistance to HGF/c-Met inhibitors. They suggest a possible resistance mechanism involving poor MET-status detection, crosstalk between c-MET and compensatory signaling pathways, the occurrence of acquired a new mutation, however, knowledge regarding the mechanism of c-MET inhibitor is limited.2. Purpose: We identify new markers and mechanisms that induce drug resistance when c-MET-targeted inhibitors are treated in c-MET-amplified GC cell lines in 3D culture, and discover new therapeutic targets for GC via single cell-RNA sequencing analysis.3. Material and Methods: Single cell-RNA sequencing is performed on c-MET amplified GC cell line in the 2D and 3D culture methods according to the presence or absence of c-MET inhibitor treatment. Through DEG and clustering analysis, the candidate genes estimated to impart resistance are selected and verified by qPCR. Through over-expression, induction of resistance for c-MET inhibitor treatment with target gene is evaluated in c-MET amplified GC cell line. c-MET inhibitor selected gene is constructed as a xenograft model to reveal the mechanism of drug resistance. Retrospective analysis confirms the relationship between target gene expression level and the resistance for c-MET inhibitor or other TKI treated patients with GC. 4. Results: Treatment of c-MET-amplified GC cell lines with c-MET inhibitors under the conditions of 2D and 3D cultures confirmed that resistance was induced only in the 3D. We performed a high-precision single cell RNA-Seq analysis of 18056 single cells in total from c-MET amplified GC cell line MKN-45 in 2D/3D culture control as well as corresponding samples with volitinib treatment investigate their gene expression signatures. Fifteen selected candidate genes presumed to induce drug resistance were screened and validated by qPCR, with a total of six genes consistent with the sequencing results. After over-expression of six genes to the c-MET amplified gastric cancer cell line, c-MET inhibitors were treated and cell viability was performed to check for resistance to cell viability. Finally, ERO1A and VEGFA were over-expressed to confirm the resistant for the c-MET inhibitor treatment in vitro and in vivo.5. Discussion: We plan to build a xenograft mouse model with candidate genes such as ERO1a and VEGFA to prove whether the candidate gene overcomes drug resistance when they are on or off. Citation Format: Hajung Kim, Jinhyeon Kim, Seung-Jae Myung, Charny Park, Sun young Kim. Unraveling c-MET targeting resistance mechanisms in gastric cancer via single-cell transcriptome analysis; Novel therapeutic target discovery of ERO1A and VEGFA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5848.

Abstract 4564: Therapeutic potential of SOS1 and KRAS inhibitors in malignant peripheral nerve sheath tumors

Abstract Neurofibromatosis type 1 (NF1) is a genetic disorder that affects the nervous system. It is caused by mutations in the NF1 gene. NF1 loss of function leads to elevated RAS signaling, which plays a crucial role in cell growth and proliferation. Due to the dysregulated RAS signaling, individuals with NF1 are prone to the development of neurofibromas. These neurofibromas are typically benign and called Plexiform Neurofibromas (PNFs), in some cases, they can transform into malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are highly aggressive and invasive cancers that contribute to a significant proportion of soft tissue sarcomas. The exact mechanisms behind this transformation are not fully understood, however it involves additional genetic mutations and alterations in cellular signaling pathways. With limited effective targeted therapies available, surgical removal remains the primary treatment option. However, due to tumor size and location, surgery may not always be feasible. The MEK inihibitor, selumetinib, has received approval for the treatment of PNFs, however some patients who were treated with selumetinib continued to develop MPNSTs suggesting that MEK inhibitors may have limitations in addressing MPNSTs. To target the RAS pathway and mitigate the effects of NF1 gene alterations, we aim to explore the therapeutic potential of two novel, potent and orally bioavailable small molecule inhibitors targeting SOS1 (BI 1701963) or KRAS (KRASmulti inhibitor BI 3706674), as alternative treatment options for MPNSTs. The KRASmulti inhibitor BI 3706674 binds non-covalently to the KRAS wild type allele as well as multiple KRAS mutant alleles in the GDP-bound state and thereby disrupts KRAS signaling. Therefore, we hypothesize that both inhibitors will work in NF1 models by reducing activated KRAS levels. In this study, a series of in vitro and in vivo experiments were conducted to evaluate the efficacy of BI 1701963 and BI 3706674 inhibitors, both individually and in combination. The studies were done in comparison to the MEK inhibitor selumetinib. In vitro analysis revealed a synergistic anti-proliferative effect when combining BI 1701963 and BI 3706674 inhibitors both in 2D and 3D growth conditions, which led to the downregulation of the RAS pathway, specifically the MAPK and PI3K signaling cascade, and an increase in apoptosis. The in vivo efficacy of either BI 1701963 or BI 3706674 inhibitor alone or in combination was tested in four different in vivo MPNST models. We observed significant tumor growth stabilization as well as tumor regression in all tested models in vivo. In-depth pathway modulation and pharmaco-dynamic studies are ongoing. In conclusion, this study highlights the combined effects of SOS1 (BI 1701963) and KRAS (BI 3706674) inhibition in MPNSTs. The combination therapy of these novel therapeutics may hold promise as a potential treatment strategy for patients with MPNSTs. Citation Format: Özlem Yüce Petronczki, Laura Pisarsky, Petra Lujza Szabó, Julia Brunmair, Antonia Geyer, Norbert Kraut, Darryl McConnell, Irene Waizenegger. Therapeutic potential of SOS1 and KRAS inhibitors in malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4564.

Abstract 3335: A novel and potent IRE1α RNase inhibitor, HM100168 as a promising therapeutic strategy in solid cancers

Abstract Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network called the unfolded protein response (UPR). Inositol-requiring enzyme 1α (IRE1α) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved way of the UPR. The IRE1α-XBP1 pathway, one of the most essential branches of the UPR, is activated in various types of cancer. When IRE1α is activated, the spliced form of XBP1 (XBP1s) is generated to activate the transcription of many pro-survival genes to prevent cellular death and relieve cellular stress. IRE1α plays an instrumental pro-tumoral role in various cancers, such as glioblastoma, myeloma, lung adenocarcinoma, and breast cancers, as well as high IRE1α activity is associated with poor prognoses. Moreover, IRE1-XBP1 plays an essential role in the UPR and ER stress responses, which have been implicated in the development of drug resistance. Thereby, blockade of the IRE1α-XBP1 pathway is crucial as it is considered as a potential anti-cancer strategy. Here, we developed a potent and selective IRE1α RNase inhibitor, which have a hydroxy-aryl-aldehydes (HAA) moiety that binds to IRE1α within the RNase catalytic site. Our lead compound, HM100168, potently inhibited the RNase activity of human IRE1α at nanomolar concentrations without affecting its kinase activity. Experiments were performed on MCF7 breast cancer cells to confirm the inhibitory effect in the mRNA and protein levels of XBP1s, which was increased by tunicamycin (TM), a ER stress inducer. HM100168 strongly inhibited ​​XBP1s levels in a dose-dependent manner, displaying the potent IC50 values. We tested cell growth inhibition activity in 3D as well as 2D cell culture condition of various cancer cell lines. Interestingly, HM100168 effectively inhibited cell proliferation of diverse cancer cell lines in both 2D and 3D culture condition. In addition, we explored the promising anti-cancer effects of HM100168 and its synergistic potential when combined with anti-tumorigenic agents. As a result, HM100168 in combination with anti-tumorigenic agents significantly enhanced suppression of tumor growth in human cancer cell transplanted mice. In conclusion, we have identified a novel compound with unique properties that specifically blocked the endonuclease activity of IRE1α. HM100168 inhibited IRE1α endonuclease activity, without affecting its kinase activity after endoplasmic reticulum stress in vitro. The identification of this novel IRE1α inhibitor supports the hypothesis that the IRE1α-XBP1 axis is a promising target for anticancer therapy. Also, co-targeting of IRE1α-XBP1 in combination with anti-tumorigenic agents can be considered a novel approach to overcome cancer resistance. Further preclinical studies will be performed and reported soon after the establishment of a preclinical candidate. Citation Format: Jisook Kim, Seung Hyun Jung, Minjeong Kim, Wongi Park, Jooyun Byun, Soonki Park, Miyoung Lee, Yu-Yon Kim, Junghwa Park, Seokhyun Hong, Minhwa Kim, Young Gil Ahn. A novel and potent IRE1α RNase inhibitor, HM100168 as a promising therapeutic strategy in solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3335.

Abstract 512: Cell-cycle inhibition may influence differences in 2D vs 3D tumor sensitivity profiles

Abstract Background: Traditional cell culture methods involve growing cells on flat surfaces, which do not fully replicate the human physiological conditions. In contrast, 3D cell cultures accurately reproduce aspects of the in-vitro malignant and non-malignant cell behavior. However, differences in tumor sensitivity platforms between 2D vs. 3D models might be relevant for some, but not for other drugs. Methods: The colorectal cancer cell line HCT-116 was cultured and plated in flat bottom well plates for the 2D condition and in Ultra Low Attachment round-bottom plates for the 3D condition. After plating, cells were treated with chemotherapies and targeted therapies for four days; cellular sensitivity was assessed through a viability assay. Four-parameter logistic (4PL) dose response curves were generated and IC50s were quantified to understand the differences in responses between 2D and 3D cellular environments. Results: HCT-116 exhibited similar responses to platinum drugs, taxanes, and several other tested drugs in both platforms, with the exception of gemcitabine (2D IC50 0.04 mcM; 3D IC50 12.8 mcM), lapatinib (2D IC50 23; 3D IC50 418), tucatinib (2D IC50 23.3; 3D IC50 79.3), and palbociclib (2D IC50 7.0; 3D IC50 24.0). Interestingly, these drugs share a common mechanism of action, which is the inhibition of cell cycle progression from G1 to S phase. Hence, we hypothesize that this shared mechanism may account for the observed differences in drug efficacy between the two environments. Conclusions: Similar dose responses have been observed with various drugs on both platforms, yet drugs that inhibit cell cycle progression from G1 to S phase, revealed higher sensitivity in 2D and more resistance in the 3D platform. Confirmatory cell-cycle results analyses are in progress. Further research is needed to understand if this applies to patient samples. Citation Format: Jahanvi Kumar, Chiara Maestri, Rajeshwar Nitiyanandan, Ivan Trus, Ricardo J. Parker, Brigitte Apfel, Christian Apfel. Cell-cycle inhibition may influence differences in 2D vs 3D tumor sensitivity profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 512.

Quasi-3D: reducing convergence effort improves visual comfort of head-mounted stereoscopic displays

The diffusion of virtual reality urges to solve the problem of vergence-accommodation conflict arising when viewing stereoscopic displays, which causes visual stress. We addressed this issue with an approach based on reducing ocular convergence effort. In virtual environments, vergence can be controlled by manipulating the binocular separation of the virtual cameras. Using this technique, we implemented two quasi-3D conditions characterized by binocular image separations intermediate between 3D (stereoscopic) and 2D (monoscopic). In a first experiment, focused on perceptual aspects, ten participants performed a visuo-manual pursuit task while wearing a head-mounted display (HMD) in head-constrained (non-immersive) condition for an overall exposure time of ~ 7 min. Passing from 3D to quasi-3D and 2D conditions, progressively resulted in a decrease of vergence eye movements—both mean convergence angle (static vergence) and vergence excursion (dynamic vergence)—and an increase of hand pursuit spatial error, with the target perceived further from the observer and larger. Decreased static and dynamic vergence predicted decreases in asthenopia trial-wise. In a second experiment, focused on tolerance aspects, fourteen participants performed a detection task in near-vision while wearing an HMD in head-free (immersive) condition for an overall exposure time of ~ 20 min. Passing from 3D to quasi-3D and 2D conditions, there was a general decrease of both subjective and objective visual stress indicators (ocular convergence discomfort ratings, cyber-sickness symptoms and skin conductance level). Decreased static and dynamic vergence predicted the decrease in these indicators. Remarkably, skin conductance level predicted all subjective symptoms, both trial-wise and session-wise, suggesting that it could become an objective replacement of visual stress self-reports. We conclude that relieving convergence effort by reducing binocular image separation in virtual environments can be a simple and effective way to decrease visual stress caused by stereoscopic HMDs. The negative side-effect—worsening of spatial vision—arguably would become unnoticed or compensated over time. This initial proof-of-concept study should be extended by future large-scale studies testing additional environments, tasks, displays, users, and exposure times.

In vitro morphogenesis, antioxidant enzyme activity and secondary compound accumulation of Phyllanthus amarus under Clinostat 2D condition

In this study, 4-week-old internodes of in vitroPhyllanthus amarus with 1 cm in length were cut in a half longitudinal section and cultured under 2D clinostat and control conditions to evaluate in vitro morphogenesis, antioxidant enzyme activity and secondary compound accumulation. The results showed that 34.33% of explants induced callus and 65.67% of explants formed adventitious root under 2D clinostat compared with 100% explants induced callus under control after 4 weeks of culture. In addition, the fresh and dry weights of callus clusters (792.00 and 79.17 mg, respectively) under 2D clinostat were higher than those under control (313.33 and 30.07 mg, respectively). Superoxide dismutase (SOD), catalase (CAT), and phenolic under 2D clinostat were higher than those of control. For hypophyllanthin and phyllanthin, the concentrations in the callus under 2D clinostat condition were higher than control. The hypophyllanthin content in the callus (29.06 μg/g fresh weight) was higher than in the adventitious root (9.03 μg/g fresh weight) under 2D clinostat, while the phyllanthin only obtained in the callus that was absent in the adventitious root.

Identification of critical process parameters for expansion of clinical grade human Wharton's jelly-derived mesenchymal stromal cells in stirred-tank bioreactors.

Cell therapies based on multipotent mesenchymal stromal cells (MSCs) are traditionally produced using 2D culture systems and platelet lysate- or serum-containing media (SCM). Although cost-effective for single-dose autologous treatments, this approach is not suitable for larger scale manufacturing (e.g., multiple-dose autologous or allogeneic therapies with banked MSCs); automated, scalable and Good Manufacturing Practices (GMP)-compliant platforms are urgently needed. The feasibility of transitioning was evaluated from an established Wharton's jelly MSCs (WJ-MSCs) 2D production strategy to a new one with stirred-tank bioreactors (STRs). Experimental conditions included four GMP-compliant xeno- and serum-free media (XSFM) screened in 2D conditions and two GMP-grade microcarriers assessed in 0.25 L-STRs using SCM. From the screening, a XSFM was selected and compared against SCM using the best-performing microcarrier. It was observed that SCM outperformed the 2D-selected medium in STRs, reinforcing the importance of 2D-to-3D transition studies before translation into clinical production settings. It was also found that attachment efficiency and microcarrier colonization were essential to attain higher fold expansions, and were therefore defined as critical process parameters. Nevertheless, WJ-MSCs were readily expanded in STRs with both media, preserving critical quality attributes in terms of identity, viability and differentiation potency, and yielding up to 1.47×109 cells in a real-scale 2.4-L batch.

Direct Reprogramming of Hepatocytes Into JAK/Stat-Dependent LGR5+ Liver Cells Able to Initiate Intrahepatic Cholangiocarcinoma.

Somatic cells that have been partially reprogrammed by the factors Oct4, Sox2, Klf4, and cMyc (OSKM) have been demonstrated to be potentially tumorigenic in vitro and in vivo due to the acquisition of cancer-associated genomic alterations and the absence of OSKM clearance over time. In the present study, we obtained partially reprogrammed, SSEA1-negative cells by transducing murine hepatocytes with Δ1Δ3-deleted adenoviruses that expressed the 4 OSKM factors. We observed that, under long-term 2D and 3D culture conditions, hepatocytes could be converted into LGR5-positive cells with self-renewal capacity that was dependent on 3 cross-signaling pathways: IL6/Jak/Stat3, LGR5/R-spondin, and Wnt/β-catenin. Following engraftment in syngeneic mice, LGR5-positive cells that expressed the cancer markers CD51, CD166, and CD73 were capable of forming invasive and metastatic tumors reminiscent of intrahepatic cholangiocarcinoma (ICC): they were positive for CK19 and CK7, featured associations of cord-like structures, and contained cuboidal and atypical cells with dissimilar degrees of pleomorphism and mitosis. The LGR5+-derived tumors exhibited a highly vascularized stroma with substantial fibrosis. In addition, we identified pro-angiogenic factors and signaling pathways involved in neo-angiogenesis and vascular development, which represent potential new targets for anti-angiogenic strategies to overcome tumor resistance to current ICC treatments.

Four sides to the story: A proteomic comparison of liquid-phase and matrix-bound extracellular vesicles in 2D and 3D cell cultures.

Multipotent mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) play important roles in cellular communication and are extensively studied as promising therapeutic agents. While there is a substantial pool of studies on liquid-phase EVs, data on EVs bound to the extracellular matrix (ECM) is lacking. There is also an emerging trend of accumulating and comparing data on characteristics of EVs obtained in different culturing conditions. Aiming to reveal proteomic signatures of EVs obtained from conditioned media and ECM of MSCs cultured in 2D and 3D conditions, we performed liquid chromatography with tandem mass spectrometry. Bioinformatic analysis revealed common patterns in proteomic composition of liquid-phase EVs and matrix-bound vesicles (MBVs), namely extracellular environment organization, immune, and transport pathways enrichment. However, extracellular environmental organization pathways are more enriched in liquid-phase EVs than in MBVs, while MBVs proteins noticeably enrich enzymatic pathways. Furthermore, each type of EVs from 2D and 3D cultures has a unique differential abundance profile. We have also performed comparative functional assays, namely scratch assay to assess EVs effect on cell migration and tubulogenesis assay to evaluate EVs angiogenic potential. We found that both liquid-phase EVs and MBVs enhance cell migration, while angiogenic potential is higher in MBVs. Results of the present study suggest that while both liquid-phase EVs and MBVs have therapeutic potential, some unique features of each subgroup may determine optimal areas of their application.

DMT1-dependent endosome-mitochondria interactions regulate mitochondrial iron translocation and metastatic outgrowth.

Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains mitochondrial iron translocation via EE-mitochondria interactions in triple-negative MDA-MB-231, but not in luminal A T47D breast cancer cells. DMT1 silencing increases labile iron pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy in MDA-MB-231 cells. Mitochondrial bioenergetics and the iron-associated protein profile were altered by DMT1 silencing and rescued by DMT1 re-expression. Transcriptomic profiles upon DMT1 silencing are strikingly different between 2D and 3D culture conditions, suggesting that the environment context is crucial for the DMT1 knockout phenotype observed in MDA-MB-231 cells. Lastly, in vivo lung metastasis assay revealed that DMT1 silencing promoted the outgrowth of lung metastatic nodules in both human and murine models of triple-negative breast cancer cells. These findings reveal a DMT1-dependent pathway connecting EE-mitochondria interactions to mitochondrial iron translocation and metastatic fitness of breast cancer cells.

Core-softened colloid under extreme geometrical confinement.

Geometrical constraints offer a promising strategy for assembling colloidal crystal structures that are not typically observed in bulk or under 2D conditions. Core-softened colloids, in particular, have emerged as versatile chemical building blocks with applications across various scientific and technological areas. In this study, we investigate the behavior of a core-softened model confined between two parallel walls. Employing molecular dynamics simulations, we analyze the system's response under extreme confinement, where only one or two layers of colloids are permitted. The system comprises particles modeled by a ramp-like potential confined within slit nanoslits created by two flat, purely repulsive walls with a lateral side L separated by a distance Lz. Through a systematic analysis of the phase behavior as Lz increases, or as the system undergoes decompression, for different values of L, we identified a mono-to-bilayer transition associated with changes in the colloidal structure. In the monolayer regime, we observed solid phases at lower densities than those observed in the 2D case. Importantly, we demonstrated that confinement at specific Lz values, allowing particle arrangement into two layers, can lead to the emergence of the square phase, which was not observed under monolayer or 2D conditions. By correlating thermodynamic, translational, and orientational ordering, as well as the dynamics of this confined colloidal system, our findings offer valuable insights into the utilization of geometrical constraints to induce and manipulate structural changes.