Multicellular Spheroids Research Articles

Bispecific fibrous glue synergistically boosts vascular normalization and antitumor immunity for advanced renal carcinoma therapy

Immune checkpoint blockade therapy represented by programmed cell death ligand 1 (PD-L1) inhibitor for advanced renal carcinoma with an objective response rate (ORR) in patients is less than 20%. It is attributed to abundant tumoral vasculature with abnormal structure limiting effector T cell infiltration and drug penetration. We propose a bispecific fibrous glue (BFG) to regulate tumor immune and vascular microenvironments simultaneously. The bispecific precursor glue peptide-1 (pre-GP1) can penetrate tumor tissue deeply and self-assemble into BFG in the presence of neuropilin-1 (NRP-1) and PD-L1. The resultant fibrous glue is capable of normalizing tumoral vasculature as well as restricting immune escape. The pre-GP1 retains a 6-fold higher penetration depth than that of antibody in the multicellular spheroids (MCSs) model. It also shows remarkable tumor growth inhibition (TGI) from 19% to 61% in a murine advanced large tumor model compared to the clinical combination therapy. In addition, in the orthotopic renal tumor preclinical model, the lung metastatic nodules are reduced by 64% compared to the clinically used combination. This pre-GP1 provides a promising strategy to control the progression and metastasis of advanced renal carcinoma.

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 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 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.

Abstract 3208: A nano sized system for the treatment of gastrointestinal cancers

Abstract Despite the recent progress in cancer immunotherapies, most patients do not benefit from the currently available immune checkpoint blockers, and many develop severe immune-related adverse events. Therefore, it is urgent to develop alternative, effective immunotherapeutic approaches. An example of non-responsive cancers are gastrointestinal (GI) cancers, which account for 26% of the global cancer incidence and 35% of all cancer-related deaths. In many GI cancers, the failure of immunotherapies to yield clinical response is partially attributed to the inefficient antigen presentation by DC, which results in insufficient T cell stimulation despite their expression of tumor-associated antigens (TAA). To overcome these hurdles, we designed novel polymeric-based nanoparticles (NP) to deliver a combination of GI cancers antigens of CEACAM5 protein and toll-like receptor ligands to DC. We chose to encapsulate CEACAM5 antigens because it is overexpressed in GI cancers, such as gastric cancer, colorectal cancer, and pancreatic cancer. With this approach, we aim to modulate the tumor milieu-immune cell interactions to an anti-cancer phenotype in order to control tumor growth and improve overall survival. Our NP present an average diameter below 200 nm, narrow polydispersity index, slightly negative surface charge, spherical morphology, and high antigens and immune potentiators loading. Cy5-labeled NPs were found to be extensively internalized by DC, which triggered their activation in vivo, shown by the increasing expression of DC-related co-stimulatory molecules such as CD80, CD86, and CD40. Furthermore, we assessed the immunotherapeutic effect of our NP on mouse models and on a human 3D multi-cellular spheroid ex-vivo model, composing the patient’s tumor cells and peripheral blood mononuclear cells (PBMC). We found that the NP successfully induced a potent immune-mediated anti-tumor response against CEACAM5-positive GI tumors, sensitizing the tumor microenvironment to other therapies and decreasing tumor volume, ultimately leading to prolonged survival. Taken together, the developed NP induced a strong antigen-specific immune response and overall sensitized CEACAM5-expressing GI cancers to the clinically relevant treatments in vivo, that so far have shown limited clinical outcomes in addition to a specific T cell anti-tumor activity that was shown on our patient-derived ex vivo model. Citation Format: Daniella Vaskovich, Rita Acúrcio, Ron Kleiner, Barbara Carreira, Ana Matos, Helena Florindo, Ronit Satchi-Fainaro. A nano sized system for the treatment of gastrointestinal 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 3208.

Abstract 6318: Development of an anti-PD-L1 CAR T therapeutic treating advanced gastric cancer with enhanced efficacy and lower off-tumor activities

Abstract Purpose: Driven by a relentless unmet need with unsatisfied cancer therapy, we aimed to develop chimeric antigen receptor T-cell (CAR-T) therapy for targeting advanced gastric cancer (AGC) expressing PD-L1. We constructed a 2nd generation CAR lentiviral vector expressing a unique anti-PD-L1 scFv with moderate affinity (VaxCAR001) in order to compare scFvs derived from Avelumab or Atezolizumab. This project focused on the efficacy and safety of PD-L1 CAR-Ts, which are affected by their binding affinity and dissociation with the target protein, PD-L1 during the treatment period. Methods and Results: The findings of this study revealed significant differences among PD-L1-targeting CAR-T cells with different affinities and dissociations. VaxCAR001 cells, showing intermediate affinity, demonstrated a higher CD8 ratio (up to 78% compared to 58% in Avelumab-derived and 45% in Atezolizumab-derived CAR-T), reduced expression of inhibitory markers including Tim-3, LAG-3, and TIGIT, (8%, 39% and 13% in VaxCAR001 vs 35%, 55% and 42 % in Avelumab, 19%, 51% and 34% in Atezolizumab scFv-derived CAR-T, respectively) and more sustained cytotoxicity. Notably, it maintained strong cytotoxicity even one month after production, while the Avelumab or Atezolizumab ScFv-derived CAR-T cells did not. In multicellular spheroid 3D co-culture with CAR T cells, VaxCAR001 exhibited superior infiltration into the core of spheroid and induced higher CAR-T-mediated killing for 3D tumor spheroids as indicated by LDH assay (68%, 800% increased than Avelumab or Atezolizumab scFv-derived CAR-T). Moreover, in an MSI-high AGC mouse model, treatment with VaxCAR001 cells resulted in a significant reduction in tumor size and prolonged survival until the end of the study period. On the other hand, treatment with the Avelumab or Atezolizumab scFv-derived CAR-T cells led to high mortality, with most mice succumbing within three weeks after CAR-T cell injection, suggesting an on-target off-tumor effect. Conclusion: This study demonstrates that VaxCAR001 cells bearing intermediate affinity and high dissociation rate for target PD-L1 showed the superior killing of PD-L expressing tumor cells in vitro and in vivo and manifested better safety profiles, as compared to CAR-T cells bearing scFv derived from commercial anti-PD-L1 targeting monoclonal antibodies such as avelumab and atezolizumab. VaxCAR001 revealed extended cytotoxicity, enhanced infiltration into tumor spheroids, and greater tumor size reduction in in vivo models. Importantly, it also demonstrated superior safety profiles, as evidenced by reduced expression of inhibitory markers and fewer off-tumor adverse effects. These findings would accelerate the clinical stage developments as an effective and safer treatment strategy against AGC. Citation Format: Minwoo Baek, Jae-In Yu, Eun-Ji Choi, Yeojin Lim, Jiwon Yang, Se-Ryeong Choi, Yoonjoo Choi, Mihwa Kim, Joon Haeng Rhee, Je-Jung Lee. Development of an anti-PD-L1 CAR T therapeutic treating advanced gastric cancer with enhanced efficacy and lower off-tumor activities [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 6318.

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.

Molecular engineering of a spheroid-penetrating phage nanovector for photodynamic treatment of colon cancer cells

Photodynamic therapy (PDT) represents an emerging strategy to treat various malignancies, including colorectal cancer (CC), the third most common cancer type. This work presents an engineered M13 phage retargeted towards CC cells through pentavalent display of a disulfide-constrained peptide nonamer. The M13CC nanovector was conjugated with the photosensitizer Rose Bengal (RB), and the photodynamic anticancer effects of the resulting M13CC-RB bioconjugate were investigated on CC cells. We show that upon irradiation M13CC-RB is able to impair CC cell viability, and that this effect depends on i) photosensitizer concentration and ii) targeting efficiency towards CC cell lines, proving the specificity of the vector compared to unmodified M13 phage. We also demonstrate that M13CC-RB enhances generation and intracellular accumulation of reactive oxygen species (ROS) triggering CC cell death. To further investigate the anticancer potential of M13CC-RB, we performed PDT experiments on 3D CC spheroids, proving, for the first time, the ability of engineered M13 phage conjugates to deeply penetrate multicellular spheroids. Moreover, significant photodynamic effects, including spheroid disruption and cytotoxicity, were readily triggered at picomolar concentrations of the phage vector. Taken together, our results promote engineered M13 phages as promising nanovector platform for targeted photosensitization, paving the way to novel adjuvant approaches to fight CC malignancies.

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.

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.

Engineering aggregates of julolidine-substituted aza-BODIPY nanoparticles for NIR-II photothermal therapy

Near infrared-II (NIR-II) dyes have unique advantages in biomedical applications owing to the powerful ability in penetrating biological tissues. Herein, NIR-II aza-BODIPY dye, QLD-BDP, was developed with julolidine at 1,7-sites and p-dimethylaminophenyl group at 3,5-sites. According to X-ray analysis, QLD-BDP exhibits significant distortion, and this molecule appears a bowl shaped structure. The photothermal conversion efficiency of the self-assembled QLD-BDP nanoparticles (QLD-BDP-NPs) can reach 50.5 %, with maximum emission at 998 nm by the aggregate. QLD-BDP-NPs can cause the complete destruction of 4T1 multicellular spheroids (MCSs), indicating a photothermal therapy (PTT) effect.

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.

Abstract B095: Tenascin C promotes ovarian cancer stem cells interactions with the niche

Abstract Background: Effective treatment of ovarian cancer (OC) is greatly impacted by advanced, late-stage diagnosis which usually presents with intra-peritoneal metastases, chemoresistance and high recurrence. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids disseminated in the peritoneal fluid. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components that stiffen the tumor stroma and promote the aberrant activation of oncogenic processes, including survival, invasion, and stemness. Tenascin C (TNC) is one of the molecules secreted in the ECM and correlates with migration and metastatic progression in a variety of carcinomas. However, the functional role of TNC in spheroid formation and in the maintenance of the platinum resistant ovarian cancer stem phenotype has not been investigated yet. We tested the hypothesis that the increased expression of TNC and its secretion in the ECM compartment promotes survival of OCSCs, supports OC spheroid formation and tumor progression. Methods: We compared TNC expression in OC cells grown as spheroids and monolayers and in chemoresistant vs sensitive OC cells by realtime-PCR and Western Blot (WB). TNC blockade by siRNA knockdown (KD) was evaluated in combination with carboplatin on spheroid assay, colony formation, and by qPCR of stemness related gene expression. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the TNC promoter. Results: OC cells grown as spheroids showed a significant increase in TNC expression compared to monolayers. Our results also demonstrated an increase in TNC expression levels in platinum resistant OC cell lines when compared to the platinum sensitive counterpart. A comparison of normal adjacent ovarian epithelium and primary OC tumors on a multitissue array revealed that TNC expression coincided with a pro-tumorigenic phenotype (n=88, P<0.001), correlating with poor outcome. TNC-KD in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. Mechanistically, b-catenin-KD decreased TNC expression levels and β-catenin coimmunoprecipitated with the TNC promoter, suggesting that TNC is a direct β-catenin target. Additionally, OC spheroids treated with the Wnt ligand WNT-3A showed increased TNC expression compared to control untreated cells, as demonstrated by WB. Importantly, the enhanced expression of TNC was followed by its secretion in the ECM compartment, as observed via immunofluorescence imaging, implying a direct role of TNC in the ECM-rearrangement which is responsible for CSC maintenance and spheroid formation. Conclusions: TNC is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that strategies aimed at interfering with TNC expression and/or secretion along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Amber Rogers, Maya Shebeshi, Morganna Gordon, Rula Atwani, Virginie Lazar, Salvatore Condello. Tenascin C promotes ovarian cancer stem cells interactions with the niche [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B095.

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

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.

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.

Determination of the anticancer activity of standardized extract of Centella asiatica (ECa 233) on cell growth and metastatic behavior in oral cancer cells.

The anticancer drugs used for oral cancer treatment present many disadvantages, such as low solubility, low permeability, and poor bioavailability. However, the anticancer activity of ECa 233 has not been widely studied. Therefore, the anticancer activity of ECa 233 was investigated in this study. MTT assay was carried out to determine cell viability. Characterizations of cell apoptosis were monitored using DAPI and FDA staining and Hoechst 33258 and AO staining. Confirmation of the apoptosis-induced KON cells was done using annexin V-FITC staining, and ROS generation was determined by DCFDA staining. Cell death and the cell cycle arrest activity of ECa 233 were demonstrated by a flow cytometer. The anti-migration and anti-invasion properties of ECa 233 were examined. The anti-proliferative of ECa 233 was investigated. Cellular uptake of ECa 233 was measured by TEER values. The pharmacokinetics of ECa 233 were estimated using the pkCSM web server. ECa 233 decreased the KON cell viability. Morphological analysis showed the KON cells' loss of cell stability and structure, disorganized nucleus and cytoplasm, and induced cell death. ECa 233 acted as a cell cycle arrest in the G0/G1 phase and reduced the migration and invasion ability in KON cells. TEER values significantly increased in KON cells, which decreased cell colony and multicellular spheroid formations. The pharmacokinetic profiles of the main components are of interest for future usage. ECa 233 can be used as an alternative therapy as well as a medicinal plant selected for sensitizing oral cancer cells to chemotherapy.

Tunable Self-Referenced Molecular Thermometers via Manipulation of Dual Emission in Platinum(II) Pyridinedipyrrolide Complexes.

Optical temperature sensors based on self-referenced readout schemes such as the emission ratio and the decay time are crucial for a wide range of applications, with the former often preferred due to simplicity of instrumentation. This work describes a new group of dually emitting dyes, platinum(II) pincer complexes, that can be used directly for ratiometric temperature sensing without an additional reference material. They consist of Pt(II) metal center surrounded by a pyridinedipyrrolide ligand (PDP) and a terminal ligand (benzonitrile, pyridine, 1-butylimidazol or carbon monoxide). Upon excitation with blue light, these complexes exhibit green to orange emission, with quantum yields in anoxic toluene at 25 °C ranging from 13% to 86% and decay times spanning from 8.5 to 97 μs. The emission is attributed to simultaneous thermally activated delayed fluorescence (TADF) and phosphorescence processes on the basis of photophysical investigations and DFT calculations. Rather uniquely, simple manipulations in substituents of the PDP ligand and alteration of the terminal ligand allow fine-tuning of the ratio between TADF and phosphorescence from almost 100% TADF emission (Pt(MesPDPC6F5(BN)) to over 80% of phosphorescence (Pt(PhPDPPh(BuIm)). Apart from ratiometric capabilities, the complexes also are useful as decay time-based temperature indicators with temperature coefficients exceeding 1.5% K-1 in most cases. Immobilization of the dyes into oxygen-impermeable polyacrylonitrile produces temperature sensing materials that can be read out with an ordinary RGB camera or a smartphone. In addition, Pt(PhPDPPh)Py can be incorporated into biocompatible RL100 nanoparticles suitable for cellular nanothermometry, as we demonstrate with temperature measurements in multicellular colon cancer spheroids.

3D Printing of a Vascularized Mini-Liver Based on the Size-Dependent Functional Enhancements of Cell Spheroids for Rescue of Liver Failure.

The emerging stem cell-derived hepatocyte-like cells (HLCs) are the alternative cell sources of hepatocytes for treatment of highly lethal acute liver failure (ALF). However, the hostile local environment and the immature cell differentiation may compromise their therapeutic efficacy. To this end, human adipose-derived mesenchymal stromal/stem cells (hASCs) are engineered into different-sized multicellular spheroids and co-cultured with 3D coaxially and hexagonally patterned human umbilical vein endothelial cells (HUVECs) in a liver lobule-like manner to enhance their hepatic differentiation efficiency. It is found that small-sized hASC spheroids, with a diameter of ≈50µm, show superior pro-angiogenic effects and hepatic differentiation compared to the other counterparts. The size-dependent functional enhancements are mediated by the Wnt signaling pathway. Meanwhile, co-culture of hASCs with HUVECs, at a HUVECs/hASCs seeding density ratio of 2:1, distinctly promotes hepatic differentiation and vascularization both in vitro and in vivo, especially when endothelial cells are patterned into hollow hexagons. After subcutaneous implantation, the mini-liver, consisting of HLC spheroids and 3D-printed interconnected vasculatures, can effectively improve liver regeneration in two ALF animal models through amelioration of local oxidative stress and inflammation, reduction of liver necrosis, as well as increase of cell proliferation, thereby showing great promise for clinical translation.