Multicellular Tumor Spheroids Research Articles

Fine particulate matter (PM2.5) promotes chemoresistance and aggressive phenotype of A549 lung cancer cells

Lung cancer is one of the most aggressive malignancies with a high mortality rate. In large cities, particulate matter (PM) is a common air pollutant. High PM levels with aerodynamic size ≤2.5 μm (PM2.5) associates with lung cancer incidence and mortality. In this work, we explored PM2.5 effects on the behavior of lung cancer cells. To this, we chronically exposed A549 cells to increasing PM2.5 concentrations collected in México City, then evaluating cell proliferation, chemoresponse, migration, invasion, spheroid formation, and P-glycoprotein and N-cadherin expression. Chronic PM2.5 exposure from 1 μg/cm2 stimulated A549 cell proliferation, migration, and chemoresistance and upregulated P-glycoprotein and N-cadherin expression. PM2.5 also induced larger multicellular tumor spheroids (MCTS) and less disintegration compared with control cells. Therefore, these results indicate lung cancer patients exposed to airborne PM2.5 as urban pollutant could develop more aggressive tumor phenotypes, with increased cell proliferation, migration, and chemoresistance.

Highly Permeable Nanoparticles Achieve Efficient Tumor Treatment through Oxygen‐Independent Photonic Thermodynamic Therapy

AbstractPhotodynamic therapy (PDT) represents a promising strategy for tumor treatment. However, the development of PDT‐based antitumor therapies faces challenges stemming from the hypoxic nature of the tumor microenvironment and the limited penetration of photosensitizers. This study presents a novel class of antitumor nanoparticles, designated as ARLN‐FP, engineered to penetrate tumor tissues deeply and inhibit tumor growth through the generation of oxygen‐independent free radicals. With the encapsulation of IR780 and dimethyl 2,2′‐azobis(2‐methylpropionate) (AIBME) in the core of ARLN‐FP, near‐infrared (NIR) laser irradiation of ARLN‐FP with induced heat generation, which subsequentially led to the decomposition of AIBME and the production of free radicals without relying on oxygen. In vitro studies indicated that ARLN‐FP penetrated more than 100 µm into multicellular tumor spheroids, demonstrating its enhanced tissue permeability. Animal studies confirmed the anti‐tumor effects of ARLN‐FP‐mediated photonic thermodynamic therapy, where the oxygen‐independent radical formation contributed to successful tumor eradication, evidenced by a significant tumor inhibition rate of 90.8% after 22 days. This research presents a feasible method to overcome the limitations of traditional oxygen‐dependent photodynamic therapies, which offers a new approach to developing nanomedicines to overcome resistance in deep and hypoxic tumor regions.

Mitochondria-targeted ruthenium complexes can be generated in vitro and in living cells to target triple-negative breast cancer cells by autophagy inhibition

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, which owned severe resistance to platinum-based anticancer agents. Herein, we report a new metal-arene complex, Ru-TPE-PPh3, which can be synthesized in vitro and in living cells with copper catalyzed the cycloaddition reaction of Ru-azide and alkynyl (CuAAC). The complex Ru-TPE-PPh3 exhibited superior inhibition of the proliferation of TNBC MDA-MB-231 cells with an IC50 value of 4.0 μM. Ru-TPE-PPh3 could induce the over production of reactive oxygen species (ROS) to initiate the oxidative stress, and further damage the mitochondria both functionally and morphologically, as loss of mitochondrial membrane potential (MMP) and cutting the supply of adenosine triphosphate (ATP), the disappearance of cristae structure. Moreover, the damaged mitochondria evoked the occurrence of mitophagy with the autophagic flux blockage and cell death. The complex Ru-TPE-PPh3 also demonstrated excellent anti-proliferative activity in 3D MDA-MB-231 multicellular tumor spheroids (MCTSs), indicating the potential to inhibit solid tumors in living cells. This study not only provided a potent agent for the TNBC treatment, but also demonstrated the universality of the bioorthogonally catalyzed lethality (BCL) strategy through CuAAC reation.

Exploration of isoxazole analogs: Synthesis, COX inhibition, anticancer screening, 3D multicellular tumor spheroids, and molecular modeling

In this study, a new series of Isoxazole-carboxamide derivatives were synthesized and characterized via HRMS, 1H-, 13CAPT-NMR, and MicroED. The findings revealed that nearly all of the synthesized derivatives exhibited potent inhibitory activities against both COX enzymes, with IC50 values ranging from 4.1 nM to 3.87 μM. Specifically, MYM1 demonstrated the highest efficacy among the compounds tested against the COX-1, displaying an IC50 value of 4.1 nM. The results showed that 5 compounds possess high COX-2 isozyme inhibitory effects with IC50 value in range 0.24–1.30 μM with COX-2 selectivity indexes (2.51–6.13), among these compounds MYM4 has the lowest IC50 value against COX-2, with selectivity index around 4. Intriguingly, this compound displayed significant antiproliferative effects against CaCo-2, Hep3B, and HeLa cancer cell lines, with IC50 values of 10.22, 4.84, and 1.57 μM, respectively, which was nearly comparable to that of doxorubicin. Compound MYM4 showed low cytotoxic activities on normal cell lines LX-2 and Hek293t with IC50 values 20.01 and 216.97 μM respectively, with safer values than doxorubicin. Furthermore, compound MYM4 was able to induce the apoptosis, suppress the colonization of both HeLa and HepG2 cells. Additionally, the induction of Reactive oxygen species (ROS) production could be the mechanism underlying the apoptotic effect and the cytotoxic activity of the compound. In the 3D multicellular tumor spheroid model, results revealed that MYM4 compound hampered the spheroid formation capacity of Hep3B and HeLa cancer cells. Moreover, the molecular docking of MYM4 compound revealed a high affinity for the COX2 enzyme, with energy scores (S) −7.45 kcal/mol, which were comparable to celecoxib (S) −8.40 kcal/mol. Collectively, these findings position MYM4 as a promising pharmacological candidate as COX inhibitor and anticancer agent.

Engineered protein cages with enhanced extracellular drug release for elevated antitumor efficacy

Human heavy chain ferritin (HFn) protein cage has been explored as a nanocarrier for targeted anticancer drug delivery. Here, we introduced a matrix metalloproteinases (MMPs)-cleavable sequence into the DE loop of HFn, creating an MMP-responsive variant, MR-HFn, for localized and extracellular drug release. The crystal structure of MR-HFn revealed that the addition of the MMPs recognition sequence did not affect the self-assembly of HFn but presented a surface-exposed loop susceptible to MMPs cleavage. Biochemical analysis indicated that this engineered protein cage is responsive to MMPs, enabling the targeted release of encapsulated drugs. To evaluate the therapeutic potential of this engineered protein cage, monosubstituted β-carboxy phthalocyanine zinc (CPZ), a type of photosensitizer, was loaded inside this protein cage. The prepared CPZ@MR-HFn showed higher uptake and stronger phototoxicity in MMPs overexpressed tumor cells, as well as enhanced penetration into multicellular tumor spheroids compared with its counterpart CPZ@HFn in vitro. In vivo, CPZ@MR-HFn displayed a higher tumor inhibitory rate than CPZ@HFn under illumination. These results indicated that MR-HFn is a promising nanocarrier for anticancer drug delivery and the MMP-responsive strategy here can also be adapted for other stimuli.

Use of non-small cell lung cancer multicellular tumor spheroids to study the impact of chemotherapy

BackgroundLung cancers represent the main cause of cancer related-death worldwide. Recently, immunotherapy alone or in combination with chemotherapy has deeply impacted the therapeutic care leading to an improved overall survival. However, relapse will finally occur, with no efficient second line treatment so far. New therapies development based on the comprehension of resistance mechanisms is necessary. However, the difficulties to obtain tumor samples before and after first line treatment hamper to clearly understand the consequence of these molecules on tumor cells and also to identify adapted second line therapies.MethodsTo overcome this difficulty, we developed multicellular tumor spheroids (MCTS) using characterized Non-Small Cell Lung Cancer (NSCLC) cell lines, monocytes from healthy donors and fibroblasts. MCTS were treated with carboplatin-paclitaxel or -gemcitabine combinations according to clinical administration schedules. The treatments impact was studied using cell viability assay, histological analyses, 3’RNA sequencing, real-time PCR, flow cytometry and confocal microscopy.ResultsWe showed that treatments induced a decrease in cell viability and strong modifications in the transcriptomic profile notably at the level of pathways involved in DNA damage repair and cell cycle. Interestingly, we also observed a modification of genes expression considered as hallmarks of response to immune check point inhibitors and immunogenicity, particularly an increase in CD274 gene expression, coding for PD-L1. This result was validated at the protein level and shown to be restricted to tumor cells on MCTS containing fibroblasts and macrophages. This increase was also observed in an additional cell line, expressing low basal CD274 level.ConclusionsThis study shows that MCTS are interesting models to study the impact of first line therapies using conditions close to clinical practice and also to identify more adapted second line or concomitant therapies for lung cancer treatment.

Benzothiazolium-based NIR AIE photosensitizers with type I and II ROS generation for efficient mitochondria-targeted photodynamic therapy.

In this work, a near-infrared emissive photosensitizer of 3,3-dimethyl-N,N-diphenyl-2-(thiophen-2-yl)-3H-indol-6-amine functionlized benzothiazolium (DPITT) was developed. DPITT exhibited aggregation-induced emission effect and potent type I and II reactive oxygen species generation capacities after white light irradiation. Taking advantage of the cationic feature, DPITT penetrated the cell membrane and selectively accumulated in the mitochondria in living cells. Upon white light irradiation, the photosensitized DPITT was able to induce mitochondrial dysfunction, leading to cell death. Photosensitized DPITT was further applied to disrupt the multicellular tumour spheroids, demonstrating its potential application in inhibiting hypoxic solid tumours.

Co-delivery of fucoxanthin and Twist siRNA using hydroxyethyl starch-cholesterol self-assembled polymer nanoparticles for triple-negative breast cancer synergistic therapy

IntroductionTriple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer with an extremely dismal prognosis and few treatment options. As a desmoplastic tumor, TNBC tumor cells are girdled by stroma composed of cancer-associated fibroblasts (CAFs) and their secreted stromal components. The rapidly proliferating tumor cells, together with the tumor stroma, exert additional solid tissue pressure on tumor vasculature and surrounding tissues, severely obstructing therapeutic agent from deep intratumoral penetration, and resulting in tumor metastasis and treatment resistance. ObjectivesFucoxanthin (FX), a xanthophyll carotenoid abundant in marine algae, has attracted widespread attention as a promising alternative candidate for tumor prevention and treatment. Twist is a pivotal regulator of epithelial to mesenchymal transition, and its depletion has proven to sensitize antitumor drugs, inhibit metastasis, reduce CAFs activation and the following interstitial deposition, and increase tumor perfusion. The nanodrug delivery system co-encapsulating FX and nucleic acid drug Twist siRNA (siTwist) was expected to form a potent anti-TNBC therapeutic cyclical feedback loop. Methods and resultsHerein, our studies constituted a novel self-assembled polymer nanomedicine (siTwist/FX@HES-CH) based on the amino-modified hydroxyethyl starch (HES-NH2) grafted with hydrophobic segment cholesterol (CH). The MTT assay, flow cytometry apoptosis analysis, transwell assay, western blot, and 3D multicellular tumor spheroids growth inhibition assay all showed that siTwist/FX@HES-CH could kill tumor cells and inhibit their metastasis in a synergistic manner. The in vivo anti-TNBC efficacy was demonstrated that siTwist/FX@HES-CH remodeled tumor microenvironment, facilitated interstitial barrier crossing, killed tumor cells synergistically, drastically reduced TNBC orthotopic tumor burden and inhibited lung metastasis. ConclusionSystematic studies revealed that this dual-functional nanomedicine that targets both tumor cells and tumor microenvironment significantly alleviates TNBC orthotopic tumor burden and inhibits lung metastasis, establishing a new paradigm for TNBC therapy.

Development of a fluorescent ligand that can illuminate nuclear G-quadruplexes and modulate oncogene expression

In this study, we developed a promising dual-function fluorescent ligand termed KS-1 by a slight structural modification on a reported carbazole-based scaffold. KS-1 was then found to mainly bind and illuminate the nuclear DNA G-quadruplexes (G4s) in a sandwich-like interacting mode, and also effectively modulate the oncogene expression through a G4-mediated manner. Furthermore, KS-1 was proved to inhibit cancer cell growth either in 2D monolayer cells or 3D multicellular tumor spheroids. To be noted, this ligand could overcome the shortcomings of other reported dual-function ligands that appeared to accumulate in the lysosomes or mitochondria, and may be used as a theranostic agent in the future.

Abstract 2018: Tumor treating fields (TTFields) disrupt cancer cell invasion by impacting cell-ECM traction forces

Abstract Introduction: Tumor Treating Fields (TTFields) are an anti-cancer treatment that use low intensity, alternating electric fields. Anti-mitotic treatment effects of TTFields have been, to date, considered the primary anti-cancer effect. Recent studies have shown additional impacts on cancer cell invasion and migration, among other effects, resulting in a complex anti-tumor response derived from a combination of factors. By deploying computational imaging tools to assess experimental conditions that apply TTFields treatment to 3D cancer multicellular spheroid cultures, the goal of our study is to elucidate important mechanisms and effect sizes on anti-tumor effects relating to the modulation of cancer cell-extracellular matrix (ECM) biophysical interactions. Methods: 3D multicellular tumor spheroids (MTS) are cultured using GFP-tagged U-87 MG glioma cells. A single 3D MTS is placed in a cell culture insert and embedded in collagen I ECM at 2.25 mg/mL. Fluorescent polystyrene microspheres are dispersed in the ECM to facilitate subsequent tracking of displacement fields induced by the MTS-ECM construct. Fluorescence microscopy imaging is acquired for all spheroids prior to treatment initiation. Then, with 3 replicates in each treatment condition, TTFields are applied for a duration of 48 hours at 200 kHz frequency with a no treatment condition as a control. At 48 hours, we acquire fluorescence microscopy images to track spheroid growth and bead-field displacement. To determine deformation fields induced by the cancer spheroid, we compare each image at 48 hours to their respective baseline image. Deformation fields are analyzed using a custom-developed analysis code that performs an initial alignment of the baseline and specific time point images using a global rigid registration. Next, we perform a B-spline based nonrigid image registration to generate an estimate of the deformation field vector at each pixel location within the image. Deformation intensity is measured within a circular region of interest (r = 900 µm) centered at the spheroid core. Results: The average magnitude of displacement for treatment and control groups is 0.52±0.2 µm and 1.07±0.1 µm, respectively. Analysis of deformation fields reveals a significant difference (p < 0.1) in the magnitude of deformation present between treatment and control groups. Conclusions: Implementation of our computational imaging analysis methods using rigid and nonrigid registration, we observe a 69.6% decrease in the magnitude of deformation in TTFields treated spheroids compared to control spheroids. Due to the decrease in deformation induced by cancer spheroids treated with TTFields, we conclude that TTFields impact cancer cell traction forces applied to the ECM to disrupt cancer cell invasion. Citation Format: Samantha M. Short, Haley J. Bowers, Jared A. Weis. Tumor treating fields (TTFields) disrupt cancer cell invasion by impacting cell-ECM traction forces [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 2018.

Abstract 5549: Multicellular spheroid structural and metabolic characterization through radial line profiling

Abstract Three-dimensional multicellular tumor spheroids have emerged as a step between in vitro and in vivo models that can offer biological insight in fields such as tumor biology. Spheroids closely resemble in vivo solid tumors regarding their heterogeneous architecture and gradients of nutrients and oxygenation. These characteristics give the spheroid model great potential for studying basic tumor biology. However, traditional characterization methods investigate changes in the discrete regions within the spheroid, limiting the amount of spatial information regarding cellular metabolism. In this study, multicellular spheroids were created through a combination of hanging drops and gentle agitation using murine RAW 264.7 macrophages and colorectal adenocarcinoma CT26 cells. Hanging drops were maintained for three days then transferred to 6-well plates and placed on an orbital shaker set to 70 RPM for seven days. Prior to metabolic imaging, spheroids were moved to a microincubator with controllable temperature and humidified gas delivery (5% CO2). A custom inverted multiphoton imaging system (Bruker) equipped with an Ultrafast Ti:Sapphire via a 60x/1.2NA water immersion objective (Olympus) and four close-proximity, high-efficiency GaAsP detectors. NADH fluorescence was captured with a 460 (± 20) nm band pass filter at 755 nm excitation and FAD fluorescence with a 525 (± 25) nm band pass filter at 855 nm excitation. NADH and FAD fluorescence were normalized by PMT gain and laser power. An integrated fluorescence lifetime imaging microscopy (FLIM) module was used to measure mitochondrial function regarding the different components contributing to NADH autofluorescence. Immunofluorescence was used to characterize macrophage populations (M1 vs M2) and cellular proliferation (Ki67) and apoptosis (CC3). CD206, a M2 marker, showed a lower normalized fluorescent intensity at the spheroid core (0.429) compared to the proliferative edge (0.570). Similar trends are also observed for CD80, a M1 marker and Ki67. CC3 showed a higher normalized fluorescent intensity at the spheroid core (0.823) compared to the proliferative edge (0.004). The optical redox ratio displays a slightly lower value at the core (0.429) compared to the proliferative edge (0.570). Overall, these results indicate that the proliferative edge shows higher numbers of immune cells and more oxidative metabolism compared to the core. Future work includes exposing this spheroid model to low levels of oxygen to investigate how environmental changes in oxygen can affect spheroid structure and metabolism. Citation Format: Shelby N. Bess, Matthew J. Igoe, Gaven K. Smart, Timothy J. Muldoon. Multicellular spheroid structural and metabolic characterization through radial line profiling [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 5549.

Abstract 3204: Antitumor activity of a coencapsulated liposomal formulation of ceramides and doxorubicin in neuroblastoma models

Abstract Background: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Targeted therapies are severely limited by the lack of specific receptors, thus chemotherapy and radiotherapy remain the main treatment choices, which expose NB patients to systemic toxicity. Recently, we have demonstrated that cell surface Nucleolin (NCL) represents a novel cellular target for NB therapy. Methods: The cytotoxic potential of pH-sensitive nanocarriers (NC), decorated with the NCL-recognizing F3 peptide and incorporating a combination of the PI3K/Akt signaling inhibitors, C6-Ceramide (C6-Cer) or C18-ceramide (C18-Cer), and the topoisomerase II inhibitor doxorubicin (DXR) (F3-NC[C6-Cer/DXR] and F3-NC[C18-Cer/DXR], respectively), was tested in vitro on a panel of NB cell lines. Cytotoxicity was evaluated in monolayer and in multicellular tumor spheroid models, by MTS proliferation and Cell Titer Glo 3D cell viability assays, respectively. Apoptotic/necrotic cell death was investigated by Real Time-Glo Annexin VApoptosis and Necrosis assay. Results: F3-NC[C6-Cer/DXR] and F3-NC[C18-Cer/DXR] resulted significantly more effective, in term of reduction of NB cell viability, compared to the corresponding “drugs”-loaded untargeted NC formulations. Both formulations triggered apoptotic cell death, even if in a different kinetic profile. In particular, NB cells treated with F3-NC[C6-Cer/DXR] underwent apoptotic cell death earlier with respect to those treated with F3-NC[C18-Cer/DXR]. Interestingly, F3-NC[C6-Cer/DXR] exerted a significant superior cell killing effect when compared both to F3-NC[C18-Cer/DXR] and to F3-NC encapsulating only DXR, suggesting the importance of the combination treatment to enhance the antitumor effect against NB cells. Conclusion: Our preliminary findings demonstrate that the co-encapsulation and delivery of C6-Ceramide and DXR by NCL-recognizing nanoparticles exert potent antitumor effects against NB models in vitro. Ongoing in vivo experiments on clinically relevant mouse NB models (i.e. recapitulating primary tumor growth, metastases, and minimal residual disease) will allow us to define the possible use of F3-NC[C6-Cer/DXR] as an innovative therapeutic strategy for relapsed/refractory NB. Funding: AIRC IG n. 24397 to PF; CInTech (PRR) C644865576-00000005 to JNM.VB is recipient of AIRC ID 24397 contract. MBC is recipient of PD/BDE/150664/2020 (FCT) fellowship. Citation Format: Veronica Bensa, Mariana Biscaia-Caleiras, Mirco Ponzoni, Joao Nuno Moreira, Chiara Brignole, Fabio Pastorino. Antitumor activity of a coencapsulated liposomal formulation of ceramides and doxorubicin in neuroblastoma 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 3204.

Abstract 6765: A patient-derived hepatocellular carcinoma multicellular spheroid platform harnessing autologous patient serum to model the tumor microenvironment and treatment response

Abstract Hepatocellular carcinoma (HCC) is the third leading and fastest rising cause of cancer-related death worldwide. The discovery and preclinical development of compounds targeting HCC is hampered by the absence of authentic tractable systems recapitulating the heterogeneity of patient HCC tumors and the tumor microenvironment (TME). Here, we established a novel, simple and robust patient-derived multicellular tumorspheroid model based on clinical HCC tumor tissues combined with the autologous patient serum. The model was robust and reproducible independently from cancer etiology and grade recapitulating key functional features of original tissue. We show that the use of autologous patient serum was essential for the TME function. The tumorspheroids comprised the main cell compartments including epithelial cancer cells as well as all major cell populations of the TME (cancer-associated fibroblasts, macrophages and T cells). Cell type proportions were variable between tumorspheroids reflecting HCC heterogeneity. Moreover, we observed differential responses to FDA HCC approved drugs (including tyrosine kinase and checkpoint inhibitors as well as VEGF-targeting agents) between donors as in patients. This patient HCC serum-tumorspheroid model provides novel opportunities for drug discovery and development as well mechanism of action studies including compounds targeting the TME. In conclusion, this HCC serum-tumorspheroid platform will contribute to improve the dismal outcome of patients with HCC. Citation Format: Emilie Crouchet, Nuno Almeida, Sarah C. Durand, Marie Parnot, Marine A. Oudot, Fabio Giannone, Cloé Gadenne, Sara Cherradi, Natascha Roehlen, Antonio Saviano, Emanuele Emanuele Felli, Patrick Pessaux, Kazuaki Chayama, Hong Tuan F. Duong, Thomas F. Baumert, Catherine Schuster. A patient-derived hepatocellular carcinoma multicellular spheroid platform harnessing autologous patient serum to model the tumor microenvironment and treatment response [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 6765.

Augmenting Cancer Therapy with a Supramolecular Immunogenic Cell Death Inducer: A Lysosome-Targeted NIR-Light-Activated Ruthenium(II) Metallacycle.

Though immunogenic cell death (ICD) has garnered significant attention in the realm of anticancer therapies, effectively stimulating strong immune responses with minimal side effects in deep-seated tumors remains challenging. Herein, we introduce a novel self-assembled near-infrared-light-activated ruthenium(II) metallacycle, Ru1105 (λem = 1105 nm), as a first example of a Ru(II) supramolecular ICD inducer. Ru1105 synergistically potentiates immunomodulatory responses and reduces adverse effects in deep-seated tumors through multiple regulated approaches, including NIR-light excitation, increased reactive oxygen species (ROS) generation, selective targeting of tumor cells, precision organelle localization, and improved tumor penetration/retention capabilities. Specifically, Ru1105 demonstrates excellent depth-activated ROS production (∼1 cm), strong resistance to diffusion, and anti-ROS quenching. Moreover, Ru1105 exhibits promising results in cellular uptake and ROS generation in cancer cells and multicellular tumor spheroids. Importantly, Ru1105 induces more efficient ICD in an ultralow dose (10 μM) compared to the conventional anticancer agent, oxaliplatin (300 μM). In vivo experiments further confirm Ru1105's potency as an ICD inducer, eliciting CD8+ T cell responses and depleting Foxp3+ T cells with minimal adverse effects. Our research lays the foundation for the design of secure and exceptionally potent metal-based ICD agents in immunotherapy.

Role of beta-(1→3)(1→6)-D-glucan derived from yeast on natural killer (NK) cells and breast cancer cell lines in 2D and 3D cultures

BackgroundBeta-(1,3)(1,6)-D-glucan is a complex polysaccharide, which is found in the cell wall of various fungi, yeasts, bacteria, algae, barley, and oats and has immunomodulatory, anticancer and antiviral effects. In the present study, we investigated the effect of beta-(1,3)(1,6)-D-glucan derived from yeast on the proliferation of primary NK cells and breast cancer cell lines in 2D and 3D models, and on the cytotoxicity of primary NK cells against breast cancer cell lines in 2D and 3D models.MethodsIn this study, we investigated the effects of different concentrations of yeast-derived beta-(1→3)(1→6)-D-glucan on the proliferation and cytotoxicity of human NK cells and breast cancer cell lines in 2D and 3D models using the XTT cell proliferation assay and the CellTiter-Glo® 2.0 assay to determine the cytotoxicity of human NK cells on breast cancer cell lines in 2D and 3D models.ResultsWe found that the co-incubation of NK cells with beta-glucan in the absence of IL2 at 48 h significantly increased the proliferation of NK cells, whereas the co-incubation of NK cells with beta-glucan in the presence of IL2 (70 U/ml) increased the proliferation of NK cells but not significantly. Moreover, beta-glucan significantly inhibited the proliferation of breast cancer cell lines in 2D model and induced a weak, non-significant growth inhibitory effect on breast cancer multicellular tumor spheroids (3D). In addition, the cytotoxicity of NK cells against breast cancer cell lines was examined in 2D and 3D models, and beta-glucan significantly increased the cytotoxicity of NK cells against MCF-7 (in 2D).ConclusionsYeast derived beta-(1,3)(1,6)-D-glucan could contribute to the treatment of cancer by enhancing NK cell immune response as well as contributing to inhibition of breast cancer cell growth.

Optical coherence tomography for multicellular tumor spheroid category recognition and drug screening classification via multi-spatial-superficial-parameter and machine learning.

Optical coherence tomography (OCT) is an ideal imaging technique for noninvasive and longitudinal monitoring of multicellular tumor spheroids (MCTS). However, the internal structure features within MCTS from OCT images are still not fully utilized. In this study, we developed cross-statistical, cross-screening, and composite-hyperparameter feature processing methods in conjunction with 12 machine learning models to assess changes within the MCTS internal structure. Our results indicated that the effective features combined with supervised learning models successfully classify OVCAR-8 MCTS culturing with 5,000 and 50,000 cell numbers, MCTS with pancreatic tumor cells (Panc02-H7) culturing with the ratio of 0%, 33%, 50%, and 67% of fibroblasts, and OVCAR-4 MCTS treated by 2-methoxyestradiol, AZD1208, and R-ketorolac with concentrations of 1, 10, and 25 µM. This approach holds promise for obtaining multi-dimensional physiological and functional evaluations for using OCT and MCTS in anticancer studies.

Dendritic nanomedicine enhances chemo-immunotherapy by disturbing metabolism of cancer-associated fibroblasts for deep penetration and activating function of immune cells

Inefficient drug penetration hurdled by the stroma in the tumor tissue leads to a diminished therapeutic effect for drugs and a reduced infiltration level of immune cells. Herein, we constructed a PEGylated dendritic epirubicin (Epi) prodrug (Epi-P4D) to regulate the metabolism of cancer-associated fibroblasts (CAFs), thus enhancing Epi penetration into both multicellular tumor spheroids (MTSs) and tumor tissues in mouse colon cancer (CT26), mouse breast cancer (4T1) and human breast cancer (MDA-MB-231) models. Enhanced cytotoxicity against CT26 MTSs and remarkable antitumor efficacy of Epi-P4D were ascribed to reduced fibronectin, α-SMA, and collagen secretion. Besides, thinning of the tumor tissue stroma and efficient eradication of tumor cells promoted the immunogenic cell death effect for dendritic cell (DC) maturation and subsequent immune activation, including elevating the CD4+ T cell population, reducing CD4+ and CD8+ T cell hyperactivation and exhaustion, and amplifying the natural killer (NK) cell proportion and effectively activating them. As a result, this dendritic nanomedicine thinned the stroma of tumor tissues to enhance drug penetration and facilitate immune cell infiltration for elevated antitumor efficacy.

Therapeutic restoration of miR-126-3p as a multi-targeted strategy to modulate the liver tumor microenvironment.

Impaired natural killer (NK) cell-mediated antitumor responses contribute to the growth of liver tumors. Expression of a disintegrin and metalloprotease 9 (ADAM9) increases shedding of membrane-bound major histocompatibility complex class I chain-related protein A and results in evasion from NK cell-mediated cytolysis. ADAM9 is also involved in angiogenesis and tumor progression and is a target of miR-126-3p, a tumor suppressor that is downregulated and alters tumor cell behavior in the liver and other cancers. We evaluated the restoration of miR-126-3p and modulation of the miR-126-3p/ADAM9 axis as a therapeutic approach to simultaneously enhance NK cell-mediated cytolysis while targeting both tumor cells and their microenvironment. Precursor miRNAs were loaded into milk-derived nanovesicles to generate therapeutic vesicles (therapeutic milk-derived nanovesicles) for the restoration of functional miR-126-3p in recipient cancer cells. Administration of therapeutic milk-derived nanovesicles increased miR-126-3p expression and reduced ADAM9 expression in target cells and was associated with an increase in membrane-bound major histocompatibility complex class I chain-related protein A. This enhanced NK cell cytolysis in adherent tumor cells and in multicellular tumor spheroids while also impairing angiogenesis and modulating macrophage chemotaxis. Moreover, IV administration of therapeutic milk-derived nanovesicles with adoptive transfer of NK cells reduced tumor burden in orthotopic hepatocellular cancer xenografts in mice. A directed RNA therapeutic approach can mitigate NK cell immune evasion, reduce angiogenesis, and alter the tumor cell phenotype through the restoration of miR-126-3p in liver tumor cells. The pleiotropic effects elicited by this multi-targeted approach to modulate the local tumor microenvironment support its use for the treatment of liver cancer.

Loss of phosphatase and tensin homolog (PTEN) increases Lysyl oxidase-like 2 (LOXL2) expression enhancing the growth of fallopian tube epithelial cells as three-dimensional spheroids

BackgroundHigh-grade serous ovarian cancer (HGSOC) accounts for 70–80% of all ovarian cancer-related deaths. Multiple studies have suggested that the fallopian tube epithelium (FTE) serves as the cell of origin of HGSOC. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and its loss is sufficient to induce numerous tumorigenic changes in FTE, including increased migration, formation of multicellular tumor spheroids (MTSs), and ovarian colonization. In murine oviductal epithelial (MOE) cells (the equivalent of human FTE) loss of PTEN results in the upregulation of transcripts associated with the extracellular matrix, with a specific focus on the elevation of lysyl oxidase-like 2 (LOXL2). Although LOXL2 is known to drive transformation and invasion in solid tumors and is associated with a poor prognosis in ovarian cancer, its specific role in the tumorigenesis of ovarian cancer originating from FTE remains unclear. Therefore, we aim to investigate whether LOXL2 mediates tumorigenesis from the fallopian tube epithelium. MethodsIn this study, we utilized clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CAS9) technology to delete LOXL2 in PTEN-deficient MOE cells to understand its role in mediating the oncogenic effects of PTEN loss. In addition, CRISPR-CAS9 was used to delete LOXL2 in OVCAR8 ovarian cancer cells. We monitored the changes in tumorigenic properties, such as migration, invasion, and growth of three-dimensional (3D) spheroids, to assess whether the loss of LOXL2 resulted in any changes. ResultsWe found that a reduction in LOXL2 expression did not significantly change the migration or invasive capabilities of PTEN-depleted MOE or human ovarian cancer cells. However, we found that a reduction in LOXL2 expression resulted in a significant reduction in 3D MTS formation and survival in both lines. ConclusionsThese results reveal for the first time that PTEN loss in FTE cells increases LOXL2 expression through downregulation of Pax2, and LOXL2 deletion blocks 3D spheroid formation.

An economical, do-it-yourself device for multicellular tumor spheroids generation brings sophisticated biochemical tools to the masses

An economical, do-it-yourself device for multicellular tumor spheroids generation brings sophisticated biochemical tools to the masses