3D Cell Models Research Articles

The contradictory role of febuxostat in ABCG2 expression and potentiating hypericin-mediated photodynamic therapy in colorectal cancers.

Photodynamic Therapy (PDT) is an emerging method to treat colorectal cancers (CRC). Hypericin (HYP) is an effective mediator of PDT and the ABCG2 inhibitor, Febuxostat (FBX) could augment PDT. HT29 and HEK293 cells showed light dependant cytotoxic response to PDT in both 2D and 3D cell models. FBX co-treatment was not found to improve PDT cytotoxicity. Next, ABCG2 protein expression was observed in HT29 but not in HEK293 cells. However, ABCG2 gene expression analysis did not support protein expression results as ABCG2 gene expression results were found to be higher in HEK293 cells. Although HYP treatment was found to significantly reduce ABCG2 gene expression levels in both cell lines, FBX treatment partially restored ABCG2 gene expression. Our findings indicate that FBX co-treatment may not be suitable for augmenting HYP-mediated PDT in CRC but could potentially be useful for other applications.

Cell Membrane Fragment-Wrapped Parenteral Nanoemulsions: A New Drug Delivery Tool to Target Gliomas.

Poor prognosis in high-grade gliomas is mainly due to fatal relapse after surgical resection in the absence of efficient chemotherapy, which is severely hampered by the blood-brain barrier. However, the leaky blood-brain-tumour barrier forms upon tumour growth and vascularization, allowing targeted nanocarrier-mediated drug delivery. The homotypic targeting ability of cell-membrane fragments obtained from cancer cells means that these fragments can be exploited to this aim. In this experimental work, injectable nanoemulsions, which have a long history of safe clinic usage, have been wrapped in glioma-cell membrane fragments via co-extrusion to give targeted, homogeneously sized, sterile formulations. These systems were then loaded with three different chemotherapeutics, in the form of hydrophobic ion pairs that can be released into the target site thanks to interactions with physiological components. The numerous assays performed in two-dimensional (2D) and three-dimensional (3D) cell models demonstrate that the proposed approach is a versatile drug-delivery platform with chemo-tactic properties towards glioma cells, with adhesive interactions between the target cell and the cell membrane fragments most likely being responsible for the effect. This approach's promising translational perspectives towards personalized nanomedicine mean that further in vivo studies are foreseen for the future.

Antineoplastic Drug Synergy of Artesunate with Navitoclax in Models of High-Grade Serous Ovarian Cancer.

Artesunate belongs to a class of medications derived from the sweet wormwood plant (Artemisia annua) known as artemisinins. Artesunate has traditionally been used as a frontline treatment for severe malaria but has also demonstrated antineoplastic activity against various malignancies, including ovarian cancer. Data suggest that artesunate exacerbates cellular oxidative stress, triggering apoptosis. In the current study, we investigated the ability of navitoclax, an inhibitor of the antiapoptotic Bcl-2 protein family, to enhance artesunate efficacy in ovarian cancer cells. Artesunate and navitoclax both demonstrated antiproliferative effects on 2D and 3D ovarian cancer cell models as single agents. Upon combination of navitoclax with artesunate, antineoplastic drug synergy was also observed in each of the 2D cell lines and ovarian tumor organoid models tested. Further investigation of this drug combination using intraperitoneal CAOV3 xenograft models in BALB/scid mice showed that the artesunate/navitoclax doublet was superior to single-agent artesunate and vehicle control treatment. However, it did not outperform single-agent navitoclax. With optimization, this drug combination could provide a new therapeutic option for ovarian cancer and warrants further preclinical investigation.

Abstract 1514: A novel workflow to assess the T-cell and patient-derived organoid interaction

Abstract Immunotherapy is increasingly popular as a type of cancer treatment. These therapies include the use of Chimeric Antigen Receptor-engineered T-cells (CAR T-cells), tumor-infiltrating lymphocytes (TIL), and other genetically modified T-cells to specifically target the cancer cells. Although much success has been achieved with immunotherapy for treatment of blood cancers, its efficacy remains limited in solid tumors. One of the reasons for the low success rate is attributed to the solid tumor microenvironment (TME) where suppressive cytokines limit the tumor killing ability of T-cells. Thus, understanding the role the TME plays in T-cell responses is essential for the development of effective cancer therapies. The benefits of using 3-dimensional (3D) PDOs lie in the physical and chemical cues present within the TME that cannot be mimicked in traditional 2D monolayer cultures. Studies show that PDOs show similar responses to drugs as original tumors, suggesting the value of using PDOs to improve therapeutic outcomes. Thus, PDOs can provide more relevant physiological and pathological cancer models that recapitulate the basic features of primary tumors and is more suited for assessing the effectiveness of T-cell killing than 2D cell models. Despite the benefits associated with the use of PDOs, there are significant barriers to widespread adoption of PDOs in drug discovery. Organoid production is a costly and highly labor-intensive process. Moreover, organoid culture is a skilled manual process, and thus there can be significant variability between operators. To address the challenges associated with the use of PDOs in large scale applications, a semi-automated bioprocess has been developed for the large-scale expansion of assay ready organoids. Here, we developed a method to assess the effectiveness of T-cell invasion in solid tumors using patient derive organoids (PDOs). Using bioreactor expanded patient-derived colorectal cancer organoids (CRCs), activated PBMCs (human peripheral blood mononuclear cells) stained with CellTracker were added to CRCs (stained with MitoTracker) in a 96well microtiter plate and monitored on every 4 hours for 3 days using high content imager. To quantify T-cell invasion, we developed an image analysis method to measure the distance of each T-cell to the nearest organoid (interaction distance). We find that stimulated T-cell resulted in smaller interaction distance than non-stimulated T-cells. The results demonstrate the utility of the bioreactor-expanded organoids in large scale T-cell based screens. Citation Format: Zhisong Tong, Angeline Lim, Oksana Sirenko. A novel workflow to assess the T-cell and patient-derived organoid interaction [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 1514.

Abstract 4226: Human-derived hydrogels for 3D models of bone and osteosarcoma organoids and adipose tissue interactions

Abstract The goal of the study was to determine the metabolic and morphological changes, via high Content Imaging, in 3D models of osteosarcoma cancer cells using human derived hydrogels with different protein composition, Obagel and Obagel ECM. Furthermore, we evaluated the relevance of the adipose tissue microenvironment in Adipose Derived Stem Cells (ADSC) linage and Tumoroids development. Methods: Adipose derived Stem cells were cultured in a modified 3D human and fat and bone linage differentiation as studied via histology and Immunofluorescence. Osteosarcoma spheroids were cultured in Obatala Sciences’ human-derived hydrogels ObaGel and ObaGelECM and control media. Organoid structure and phenotypic changes were analyzed via live cell imaging on the Incucyte S3 and Nikon high content imaging device. Then, adipocyte- cancer cell crosstalk was evaluated using pooled adipocytes co-cultured with KHOS spheroids. Adipose derived Stem cells were cultured in a modified 3D human derived hydrogel dictates that promotes the differentiation potential toward fat and adipose tissue simultaneously in microenvironments within the organoid. Furthermore, the results demonstrated that ObaGel and ObaGel-ECM 3D cultures can support the growth and proliferation of KHOS tumor spheres during an extended culture period. Metabolic changes and phenotypic changes associated to ObaGel and ObaGel-ECM differentially facilitated tumoroids growth and migratory behavior. Conclusions: Human derived hydrogels are developed to support 3D culture of Bone and Osteosarcoma cells to recapitulate the phenotypic changes associated with their metastatic potential. The use of human derived 3D culture systems can accelerate the understanding of the role of the microenvironment in bone development and the pathogenesis and progression of Cancers like Osteosarcoma as well as screening for drug development. Citation Format: Cecilia G. Sanchez, Arman Raz, Haley Lassiter, Trivia Frazier. Human-derived hydrogels for 3D models of bone and osteosarcoma organoids and adipose tissue interactions [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 4226.

Abstract 3386: Changes in extracellular vesicle miRNAs from three-dimensional ovarian cancer cell models reflect physiological changes and cancer survival

Abstract Ovarian Cancer (OC) is the most common gynaecological malignancy and the eighth most diagnosed cancer in females worldwide. Currently, it is the fifth leading cause of cancer-related mortality among patients globally, largely due to delayed diagnosis, chemotherapy resistance, high metastasis rates, and subtype heterogeneity. Recent OC research highlights extracellular vesicles (EVs) as pivotal contributors to intercellular communication and disease progression, elucidating the intricacies of OC pathology. EVs, diverse membrane-derived vesicles released by most cells, carry molecular cargoes containing proteins and nucleic acids. Studies indicate that the biogenesis, packaging, and release of EVs are highly dependent and sensitive to the cellular microenvironment and depend on the in-vitro and in-vivo modelling systems. Labs use varied model systems—2D monolayers, animal models, and innovative 3D models—to investigate EVs' ovarian cancer roles. Therefore, in this study we aimed to compare the miRNA profiles associated with EVs in 3D ovarian cancer cell models, and to identify the pathophysiological relevance of the EVs isolated from these models to the patient derived EVs.In this study, two OC epithelial cell lines, SKOV-3 and OVCAR-, were initially cultured as 2D monolayers and embedded within Gelatin Methacryloyl hydrogels. Over nine days, multiple assays observed spheroid formation within the cell-laden hydrogels. EVs isolated from the cell-conditioned media were characterized per MISEV 2018 guidelines. Small RNA sequencing identified statistically significant miRNAs, subject to gene ontology and gene rank analyses. EVs from a cohort of 60 OC patients were used to identify survival-associated miRNA profiles.Our findings in the cell-laden hydrogels demonstrated OC cell growth, proliferation, and aggregation into spheroidal structures, establishing an ideal 3D model. The isolated EVs were characterized for size, concentration, morphology, and surface markers. Small RNA sequencing revealed 18 significantly different EV-associated miRNA species across 3D vs 2D models, influencing apoptosis, angiogenesis, migration, and proliferation in ovarian cancer. Notably, 3D model-derived EV-associated miRNAs mirrored patient-derived EV-associated miRNA profiles, indicating their pathophysiological relevance.This study establishes a robust 3D OC model in hydrogels, showcasing growth, proliferation, and aggregation capabilities. Differential miRNA profiles between 3D and 2D model EVs highlight the critical roles of these miRNAs in essential ovarian cancer processes. Moreover, similarities between 3D model and patient-derived EV-associated miRNA profiles emphasize the clinical relevance of our model. Citation Format: Nihar Godbole, Akhilandeshwari Ravichandran, Dominic Guanzon, Andrew Lai, Flavio Carrion, Priyakshi Kalita de Croft, Lewis Perrin, John Hooper, Laura Bray, Carlos Salomon. Changes in extracellular vesicle miRNAs from three-dimensional ovarian cancer cell models reflect physiological changes and cancer survival [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 3386.

Abstract 4258: An innovative PDX and patient-derived 3D platform for improved translatability of new medical entities

Abstract The preclinical platform landscape in oncology has radically changed in the last 15 years, mainly due to an increased ability to generate patient-derived models. Multiple 3D tumor cell cultures methods, either from established cell lines or from primary cultures, have been developed to assess the activity of therapeutic agents in vitro. These methods are strongly influenced by the experimental conditions, such as cell culture conditions, the presence of 3D supports like Matrigel, and the experimental design of the assay, such as the choice of the dose and the duration of the assay. The ability of these methods to predict drug efficacy in the patient relies not only in the optimization of the experimental settings, but also on the representativeness of the patient population, and on the prevention of model drifts due to clonal selection and/or tumor cell adaptation to culture conditions with passages. In this project, we aimed to develop an innovative 3D cell model platform that addresses all these experimental challenges. To do so, we took advantage of our low passage Tumorgraft platform, a bank of more than 1500 patient-derived xenografts (PDXs) well characterized at molecular and pharmacological level and highly representative of the patient population, to generate the Tumorgraft3D platform. We derived TumorGraft3D models from low passage PDXs, by using proprietary, indication-specific culture conditions. By keeping these models at low passage, we were able to avoid clonal drifts. Moreover, the use of indication specific media allowed the preservation of patient’s and PDX’s tumor histology, molecular traits at genetic, gene expression and proteomic level, and displayed proliferation rates that recapitulate those observed in the parental PDXs. So far, we have successfully developed over 100 models from breast, ovarian, prostate, NSCLC, colorectal and gastric indications, with success rates up to 90%. Ex-vivo testing of standard of care highlighted different response profiles in accordance with the molecular feature of these models. The tumor dissociation process and culture conditions optimized with PDXs were also used for the development of 3D cultures derived from patients’ samples. These data demonstrated that our 3D platform can integrate the complementarity of our PDX and patient-derived 3D platform, which lies in the possibility of evaluating test agent activity in the repeatable and reproducible setting provided by the patient-derived 3D models, as well as in models directly derived from patients with more heterogeneous and indication-specific establishment success rate, and throughput dependent on the amount of primary tumor material. Citation Format: Samaneh Kamali, Stefano Cairo, Fu-Ju Chou, Taylor Light, Mara Gilardi, Brandon Walling, Christine Baer, Hsiu-Wen Tsai, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. An innovative PDX and patient-derived 3D platform for improved translatability of new medical entities [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 4258.

Abstract 2047: Scale up and scale down approaches for screening of 3D patient-derived cell models

Abstract 3D cell culture is becoming more prevalent as scientists work to better model human physiology using in vitro systems. However, differences in handling 3D cell cultures compared to traditional 2D cell lines have limited adoption of 3D techniques in compound and cell therapy screening experiments. To facilitate use of patient-derived 3D tumoroid models, we developed protocols for both scale up and scale down of these models in Gibco™ OncoPro™ Tumoroid Culture Medium. OncoPro Tumoroid Culture Medium is a serum-free, conditioned medium-free culture medium designed to promote the growth of patient-derived 3D cancer models (tumoroids, cancer organoids). OncoPro medium is compatible with a suspension culture approach for easier handling compared to traditional embedded culture formats. We studied the growth of tumoroids in different flask sizes during suspension culture to optimize scale up for downstream assays. Tumoroid growth rates and morphologies were consistent across non-tissue culture treated flask sizes, and viable cell yield increased with surface area during scale up. In some cases, as many as 100 × 106 dissociated tumoroid cells can be recovered from a single Nunc™ TripleFlask™ after a week of growth. After scaling up, tumoroids were dissociated and seeded in multiwell plates to test multiple screening conditions (compound identity, compound concentration) in parallel. Dissociated cells were seeded in 96-well plates using both manual and automated liquid handling (Tecan Fluent 780). Due to the tendency of 3D cell models to aggregate and fall out of solution, initial seeding in a 96-well flat bottom plate resulted in high well-to-well variability. Optimization of cell seeding protocols led to standard deviations in tumoroid metabolic activity, number, and size that were comparable to manual seeding, with a coefficient of variation of less than 3% between wells for multiple tumoroid lines when automation was implemented. Generally, variability in cell seeding was lower when using the optimized, automated cell seeding protocol compared to manual methods. After seeding, cells were grown for three days to form tumoroids prior to the addition of compounds. At this time point, there was low well-to-well variability in tumoroid metabolic activity (CV<2%). Compounds and reagents to analyze cell health could also be added using automated liquid handling prior to analysis on plate readers or high-content imaging platforms. Citation Format: Colin Paul, Anthony Chatman, Amber Bullock, Xiaoyu Jenny Yang, Garrett Wong, Brittany Balhouse, Chris Yankaskas, Erik Willems, Matt Dallas, David Kuninger. Scale up and scale down approaches for screening of 3D patient-derived cell models [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 2047.

Polydopamine-Coated Liposomes for Methylene Blue Delivery in Anticancer Photodynamic Therapy: Effects in 2D and 3D Cellular Models.

Photodynamic therapy (PDT) is a therapeutic option for cancer, in which photosensitizer (PS) drugs, light, and molecular oxygen generate reactive oxygen species (ROS) and induce cell death. First- and second-generation PSs presented with problems that hindered their efficacy, including low solubility. Thus, second-generation PSs loaded into nanocarriers were produced to enhance their cellular uptake and therapeutic efficacy. Among other compounds investigated, the dye methylene blue (MB) showed potential as a PS, and its photodynamic activity in tumor cells was reported even in its nanocarrier-delivered form, including liposomes. Here, we prepared polydopamine (PDA)-coated liposomes and efficiently adsorbed MB onto their surface. lipoPDA@MB vesicles were first physico-chemically characterized and studies on their light stability and on the in vitro release of MB were performed. Photodynamic effects were then assessed on a panel of 2D- and 3D-cultured cancer cell lines, comparing the results with those obtained using free MB. lipoPDA@MB uptake, type of cell death induced, and ability to generate ROS were also investigated. Our results show that lipoPDA@MB possesses higher photodynamic potency compared to MB in both 2D and 3D cell models, probably thanks to its higher uptake, ROS production, and apoptotic cell death induction. Therefore, lipoPDA@MB appears as an efficient drug delivery system for MB-based PDT.

Spike structure of gold nanobranches induces hepatotoxicity in mouse hepatocyte organoid models

BackgroundGold nanoparticles (GNPs) have been extensively recognized as an active candidate for a large variety of biomedical applications. However, the clinical conversion of specific types of GNPs has been hindered due to their potential liver toxicity. The origin of their hepatotoxicity and the underlying key factors are still ambiguous. Because the size, shape, and surfactant of GNPs all affect their properties and cytotoxicity. An effective and sensitive platform that can provide deep insights into the cause of GNPs’ hepatotoxicity in vitro is therefore highly desired.MethodsHere, hepatocyte organoid models (Hep-orgs) were constructed to evaluate the shape-dependent hepatotoxicity of GNPs. Two types of GNPs with different nanomorphology, gold nanospheres (GNSs) and spiny gold nanobranches (GNBs), were synthesized as the representative samples. Their shape-dependent effects on mice Hep-orgs’ morphology, cellular cytoskeletal structure, mitochondrial structure, oxidative stress, and metabolism were carefully investigated.ResultsThe results showed that GNBs with higher spikiness and tip curvature exhibited more significant cytotoxicity compared to the rounded GNSs. The spike structure of GNBs leads to a mitochondrial damage, oxidative stress, and metabolic disorder in Hep-orgs. Meanwhile, similar trends can be observed in HepG2 cells and mice models, demonstrating the reliability of the Hep-orgs.ConclusionsHep-orgs can serve as an effective platform for exploring the interactions between GNPs and liver cells in a 3D perspective, filling the gap between 2D cell models and animal models. This work further revealed that organoids can be used as an indispensable tool to rapidly screen and explore the toxic mechanism of nanomaterials before considering their biomedical functionalities.

Ultrasmall Gold Nanoparticles Radiolabeled with Iodine-125 as Potential New Radiopharmaceutical.

The relatively high linear energy transfer of Auger electrons, which can cause clustered DNA damage and hence efficient cell death, makes Auger emitters excellent candidates for attacking metastasized tumors. Moreover, gammas or positrons are usually emitted along with the Auger electrons, providing the possibility of theragnostic applications. Despite the promising properties of Auger electrons, only a few radiopharmaceuticals employing Auger emitters have been developed so far. This is most likely explained by the short ranges of these electrons, requiring the delivery of the Auger emitters to crucial cell parts such as the cell nucleus. In this work, we combined the Auger emitter 125I and ultrasmall gold nanoparticles to prepare a novel radiopharmaceutical. The 125I labeled gold nanoparticles were shown to accumulate at the cell nucleus, leading to a high tumor-killing efficiency in both 2D and 3D tumor cell models. The results from this work indicate that ultrasmall nanoparticles, which passively accumulate at the cell nucleus, have the potential to be applied in targeted radionuclide therapy. Even better tumor-killing efficiency can be expected if tumor-targeting moieties are conjugated to the nanoparticles.

Complex in vitro Model: A Transformative Model in Drug Development and Precision Medicine.

In vitro and in vivo models play integral roles in preclinical drug research, evaluation, and precision medicine. In vitro models primarily involve research platforms based on cultured cells, typically in the form of two-dimensional (2D) cell models. However, notable disparities exist between 2D cultured cells and in vivo cells across various aspects, rendering the former inadequate for replicating the physiologically relevant functions of human or animal organs and tissues. Consequently, these models failed to accurately reflect real-life scenarios post-drug administration. Complex in vitro models (CIVMs) refer to in vitro models that integrate a multicellular environment and a three-dimensional (3D) structure using bio-polymer or tissue-derived matrices. These models seek to reconstruct the organ- or tissue-specific characteristics of the extracellular microenvironment. The utilization of CIVMs allows for enhanced physiological correlation of cultured cells, thereby better mimicking in vivo conditions without ethical concerns associated with animal experimentation. Consequently, CIVMs have gained prominence in disease research and drug development. This review aimed to comprehensively examine and analyze the various types, manufacturing techniques, and applications of CIVM in the domains of drug discovery, drug development, and precision medicine. The objective of this study was to provide a comprehensive understanding of the progress made in CIVMs and their potential future use in these fields.

Pharmaceutical Approach to Develop Novel Photosensitizer Nanoformulation: An Example of Design and Characterization Rationale of Chlorophyll α Derivative.

In this study, we described physico-chemical properties of novel nanoformulation of photosensitizer-pyropheophorbide α 17-diethylene glycol ester (XL) (chlorophyll α derivative), revealing insights into antitumor activity and maintaining quality, meeting the pharmaceutical approach of new nanoformulation design. Our formulation, based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles, increased XL solubility and selective tumor-targeted accumulation. In our research, we revealed, for the first time, that XL binding to polyvinyl alcohol (PVA) enhances XL photophysical activity, providing the rationale for PVA application as a stabilizer for nanoformulations. Results of FTIR, DSC, and XRD revealed the physical interactions between XL and excipients, including PVA, indicating that the encapsulation maintained XL binding to PVA. The encapsulated XL exhibited higher photophysical activity compared to non-encapsulated substance, which can be attributed to the influence of residual PVA. Gamma-irradiation led to degradation of XL; however, successful sterilization of the samples was achieved through the filtration. Importantly, the encapsulated and sterilized XL retained cytotoxicity against both 2D and 3D tumor cell models, demonstrating the potential of the formulated NP-XL for photodynamic therapy applications, but lacked the ability to reactivate epigenetically silenced genes. These findings provide valuable insights into the design and characterization of PLGA-based nanoparticles for the encapsulation of photosensitizers.

Advanced Cellular Models for Rare Disease Study: Exploring Neural, Muscle and Skeletal Organoids.

Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed.

Protocol for high throughput 3D drug screening of patient derived melanoma and renal cell carcinoma

High Throughput Screening (HTS) with 3D cell models is possible thanks to the recent progress and development in 3D cell culture technologies. Results from multiple studies have demonstrated different drug responses between 2D and 3D cell culture. It is now widely accepted that 3D cell models more accurately represent the physiologic conditions of tumors over 2D cell models. However, there is still a need for more accurate tests that are scalable and better imitate the complex conditions in living tissues. Here, we describe ultrahigh throughput 3D methods of drug response profiling in patient derived primary tumors including melanoma as well as renal cell carcinoma that were tested against the NCI oncologic set of FDA approved drugs. We also tested their autologous patient derived cancer associated fibroblasts, varied the in-vitro conditions using matrix vs matrix free methods and completed this in both 3D vs 2D rendered cancer cells. The result indicates a heterologous response to the drugs based on their genetic background, but not on their maintenance condition. Here, we present the methods and supporting results of the HTS efforts using these 3D of organoids derived from patients. This demonstrated the possibility of using patient derived 3D cells for HTS and expands on our screening capabilities for testing other types of cancer using clinically approved anti-cancer agents to find drugs for potential off label use.

SERS in 3D cell models: a powerful tool in cancer research.

Unraveling the cellular and molecular mechanisms underlying tumoral processes is fundamental for the diagnosis and treatment of cancer. In this regard, three-dimensional (3D) cancer cell models more realistically mimic tumors compared to conventional 2D cell cultures and are more attractive for performing such studies. Nonetheless, the analysis of such architectures is challenging because most available techniques are destructive, resulting in the loss of biochemical information. On the contrary, surface-enhanced Raman spectroscopy (SERS) is a non-invasive analytical tool that can record the structural fingerprint of molecules present in complex biological environments. The implementation of SERS in 3D cancer models can be leveraged to track therapeutics, the production of cancer-related metabolites, different signaling and communication pathways, and to image the different cellular components and structural features. In this review, we highlight recent progress in the use of SERS for the evaluation of cancer diagnosis and therapy in 3D tumoral models. We outline strategies for the delivery and design of SERS tags and shed light on the possibilities this technique offers for studying different cellular processes, through either biosensing or bioimaging modalities. Finally, we address current challenges and future directions, such as overcoming the limitations of SERS and the need for the development of user-friendly and robust data analysis methods. Continued development of SERS 3D bioimaging and biosensing systems, techniques, and analytical strategies, can provide significant contributions for early disease detection, novel cancer therapies, and the realization of patient-tailored medicine.

MMP-2 Responsive Peptide Hydrogel-Based Nanoplatform for Multimodal Tumor Therapy.

Responsive drug delivery systems hold great promise for tumor treatment as they focus on therapeutic agents directly, thus minimizing systemic toxicities and drug leakage. In this study, we covalently bound a matrix metalloproteinases-2 (MMP-2) enzyme-sensitive peptide to a tissue-penetrating peptide to rationally design a MMP-2 responsive multifunctional peptide hydrogel platform (aP/IR@FMKB) for cancer photothermal-chemo-immunotherapy. The constructed aP/IR@FMKB with bufalin (BF) loaded in trimethyl chitosan nanoparticles (TB NPs), photothermal agent IR820, and immune checkpoint inhibitor aPD-L1 by self-assembly could be dissociated in the presence of MMP-2 enzyme, triggering content release. TB NPs, IR820, and aPD-L1 were encapsulated by intermolecular self-assembly and enzyme-sensitive nanogels (aP/IR@FMKB) were constructed. The in vitro cytotoxicity of the blank gels and their ability to induce immunogenic cell death (ICD) in aP/IR@FMKB were evaluated using 4T1 cells. The promotion of deep tumor penetration and enzyme responsiveness was analyzed using a 3D cell model. The retention and antitumor activity at the tumor sites were examined using the primary tumor model. To assess the antitumor effect of aP/IR@FMKB induced by the immune response and its mechanism of action, recurrent tumor and distal tumor models were constructed. This hydrogel system demonstrated exceptional photothermal performance and displayed prolonged local retention. Furthermore, the induction of ICD through IR820 and TB NPs sensitized the PD-L1 blockade, resulting in a remarkable 3.5-fold and 5.2-fold increase in the frequency of intratumor-infiltrating CD8+ T-cells in the primary tumor and distal tumor, respectively. Additionally, this system demonstrated remarkable efficacy in suppressing primary, distal, and recurrent tumors, underscoring its potential as a highly potent therapeutic strategy. This innovative design of the responsive hydrogel can effectively modulate the tumor immune microenvironment while also demonstrating sensitivity to the PD-1/PD-L1 blockade. This significant finding highlights the promising potential of this hydrogel in the field of multimodal tumor therapy.

The cytotoxic and antiproliferative properties of ruthenium nitrosyl complexes and their modulation effect on cytochrome P450 in the HepG2 cell line.

Ruthenium nitrosyl complexes are actively investigated as antitumor agents. Evaluation of potential interactions between cytochromes P450 (CYPs) with new compounds is carried out regularly during early drug development. In this study we have investigated the cytotoxic and antiproliferative activities of ruthenium nitrosyl complexes with methyl/ethyl esters of nicotinic and isonicotinic acids and γ-picoline against 2D and 3D cultures of human hepatocellular carcinoma HepG2 and non-cancer human lung fibroblasts MRC-5, assessed their photoinduced activity at λrad = 445 nm, and also evaluated their modulating effect on CYP3A4, CYP2C9, and CYP2C19. The study of cytotoxic and antiproliferative activities against 2D and 3D cell models was performed using phenotypic-based high content screening (HCS). The expression of CYP3A4, CYP2C9, and CYP2C19 mRNAs and CYP3A4 protein was examined using target-based HCS. The results of CYP3A4 mRNA expression were confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). The ruthenium nitrosyl complexes exhibited a dose-dependent cytotoxic effect against HepG2 and MRC-5 cells. The cytotoxic activity of complexes with ethyl isonicotinate (1) and nicotinate (3, 4) was significantly lower for MRC-5 than for HepG2, for a complex with methyl isonicotinate (2) it was higher for MRC-5 than for HepG2, for a complex with γ-picoline (5) it was comparable for both lines. The antiproliferative effect of complexes 2 and 5 was one order of magnitude higher for MRC-5; for complexes 1, 3, and 4 it was comparable for both lines. The cytotoxic activity of all compounds for 3D HepG2 was lower than for 2D HepG2, with the exception of 4. Photoactivation affected the activity of complex 1 only. Its cytotoxic activity decreased, while the antiproliferative activity increased. The ruthenium nitrosyl complexes 1-4 acted as inducers of CYP3A4 and CYP2C19, while the complex with γ-picoline (5) induced of CYP3A4. Among the studied ruthenium nitrosyl complexes, the most promising potential antitumor compound is the ruthenium compound with methyl nicotinate (4).

New copper(II) and oxidovanadium(IV) complexes with a vitamin B6 Schiff base: mechanism of action and synergy studies on 2D and 3D human osteosarcoma cell models.

We report the synthesis, characterization and anticancer activity of a new Schiff base (H2L) derived from the condensation of pyridoxamine with pyridoxal and its novel copper(II) and oxidovanadium(IV) complexes: [Cu(HL)Cl] (1), [Cu(LH2)(phen)]Cl2 (2), [Cu(LH2)(amphen)]Cl2 (3), [VIVO(HL)Cl] (4), and [VIVO(LH2)(phen)]Cl2 (5), where phen is 1,10-phenanthroline and amphen is its 5-amino derivative. All compounds were characterized by analytical and spectroscopic techniques, namely FTIR, UV-vis and EPR spectroscopy. Their stability in aqueous media was evaluated, revealing that the presence of the phen co-ligand significantly increases the stability. The ternary Cu(II) complexes (2 and 3) impaired cell viability of osteosarcoma cells (MG-63) (IC50 values of 3.6 ± 0.6 and 7 ± 1.9 μM for 2 and 3), while 1 and the VIVO complexes did not show relevant anticancer activity. Complexes 2 and 3 are also more active than cisplatin (CDDP). Synergistic studies between 2 and sorafenib showed significant synergism on MG-63 cells for the following combinations: 2 (2.0 μM) + sorafenib (10.0 μM) and 2 (2.5 μM) + sorafenib (12.5 μM), whilst the combination of 2 and CDDP did not show synergy. Complex 2 interacts with DNA, inducing significant genotoxic effects on MG-63 cells from 1.0 to 2.5 μM and it increases the ROS levels 880% over basal. Moreover, 2 induces apoptosis at 1.0 and 2.0 μM, while its combination with sorafenib induces apoptosis and necrosis. Finally, compound 2 reduces the cell viability of MG-63 spheroids showing an IC50 value 7-fold lower than that of CDDP (8.5 ± 0.4 μM vs. 65 ± 6 μM). The combination of 2 and sorafenib also showed synergism on spheroids, suggesting that the combination of these drugs improves the anticancer effect against bone cancer cells.

Phosphoproteomic Analysis and Organotypic Cultures for the Study of Signaling Pathways.

Signaling pathways are involved in key cellular functions from embryonic development to pathological conditions, with a pivotal role in tissue homeostasis and transformation. Although most signaling pathways have been intensively examined, most studies have been carried out in murine models or simple cell culture. We describe the dissection of the TGF-β signaling pathway in human tissue using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) in a 3D organotypic skin model combined with quantitative proteomics and phosphoproteomics mass spectrometry. The use of human 3D organotypic cultures and genetic engineering combined with quantitative proteomics and phosphoproteomics is a powerful tool providing insight into signaling pathways in a human setting. The methods are applicable to other gene targets and 3D cell and tissue models. Key features • 3D organotypic models with genetically engineered human cells. • In-depth quantitative proteomics and phosphoproteomics in 2D cell culture. • Careful handling of cell cultures is critical for the successful formation of the organotypic cultures. • For complete details on the use of this protocol, please refer to Ye et al. 2022.