3D Spheroid Growth Research Articles

Evaluating cell growth and hypoxic regions of 3D spheroids via a machine learning approach

Abstract This study investigated the applicability of the area of spheroids and hypoxic regions for efficient evaluation of drug efficacy using machine learning (ML). We initially developed a high-throughput detection method to obtain the area of spheroids and hypoxic regions that can handle over 10 000 images per hour with an error rate of 2%–3%. The ML models were trained using cell growth of six cell lines (i.e. HepG2, A549, Hep3B, BEAS-2B, HT-29, and HCT116) and hypoxic region variations of two cell lines (i.e. HepG2 and BEAS-2B); our model can predict the area of spheroids and hypoxic region of certain growth date with high precision. To demonstrate the applicability, HepG2 spheroids were treated with sorafenib, and the efficacy of the drug was evaluated through a comparison of differences in areas of cell size and hypoxic regions with the predicted results. Furthermore, our ML approach has been shown to be applicable to provide the model-driven evaluative criterion for toxicity and drug efficacy using spheroids.

Assessing Drug Uptake and Response Differences in 2D and 3D Cellular Environments Using Stimulated Raman Scattering Microscopy.

The architecture of cell culture, two-dimensional (2D) versus three-dimensional (3D), significantly impacts various cellular factors, including cell-cell interactions, nutrient and oxygen gradients, metabolic activity, and gene expression profiles. This can result in different cellular responses during cancer drug treatment, with 3D-cultured cells often exhibiting higher resistance to chemotherapeutic drugs. While various genetic and proteomic analyses have been employed to investigate the underlying mechanisms of this increased resistance, complementary techniques that provide experimental evidence of spatial molecular profiling data are limited. Stimulated Raman scattering (SRS) microscopy has demonstrated its capability to measure both intracellular drug uptake and growth inhibition. In this work, we applied three-band (C-D, C-H, and fingerprint regions) SRS imaging to 2D and 3D cell cultures and performed a comparative analysis of drug uptake and response with the goal of understanding whether the difference in drug uptake explains the drug resistance in 3D culture compared to 2D. Our investigations revealed that despite similar intracellular drug levels in 2D and 3D A549 cells during lapatinib treatment, the growth of 3D spheroids was less impacted, supporting an enhanced drug tolerance in the 3D microenvironment. We further elucidated drug penetration patterns and the resulting heterogeneous cellular responses across different spheroid layers. Additionally, we investigated the role of the extracellular matrix in modulating drug delivery and cell response and discovered that limited drug penetration in 3D could also contribute to lower drug response. Our study provides valuable insights into the intricate mechanisms of increased drug resistance in 3D tumor models during cancer drug treatments.

N-desmethyldauricine from Menispermum dauricum DC suppresses triple-negative breast cancer growth in 2D and 3D models by downregulating the NF-κB signaling pathway

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, for which targeted therapy regimens are lacking. The traditional Chinese medicine Menispermum dauricum DC (M. dauricum) and its compounds have been reported to have antitumor activity against various cancers; however, their anti-TNBC activity is unknown. In this work, dauricine and N-desmethyldauricine from M. dauricum were separated and identified to have anti-TNBC via a multi-component bioactivity and structure-guided method. The cell counting kit 8 assay showed that dauricine and N-desmethyldauricine inhibited the proliferation of four tested TNBC cell lines, with half maximal inhibitory concentration values ranging from 5.01 μM to 13.16 μM. Further research suggested that N-desmethyldauricine induced cell apoptosis, arrested cell cycle progression in the G0/G1 phase, and inhibited cell migration. Western blot analysis revealed that the proapoptotic protein cleaved-poly-ADP-ribose polymerase 1 was upregulated, and the G0/G1 phase-related proteins cyclin-dependent kinase 2 and cyclin D1 and the migration-related protein matrix metallopeptidase 9 were downregulated. Furthermore, N-desmethyldauricine decreased the protein expression of p65, an important subunit of nuclear factor kappa-beta (NF-κB). Moreover, an antiproliferation assay of three-dimensional (3D) tumor spheroids showed that N-desmethyldauricine diminished cell‒cell adhesion and suppressed the growth of TNBC 3D spheroids. Taken together, these findings indicate that N-desmethyldauricine inhibited the proliferation of TNBC cells and decreased the expression of p65 in the NF-κB pathway.

Next generation antimitotic β-carboline derivatives modulate microtubule dynamics and downregulate NF-κB, ERK 1/2 and phospho HSP 27

AimExploring the efficacy of β-carboline-based molecular inhibitors in targeting microtubules for the development of novel anticancer therapeutics. Materials and methodsWe synthesized a series of 1-Aryl-N-substituted-β-carboline-3-carboxamide compounds and evaluated their cytotoxicity against human lung carcinoma (A549) cells using the MTT assay. Normal lung fibroblast cells (WI-38) were used to assess compound selectivity. The mechanism of action of MJ-211 was elucidated through Western blot analysis of key pro-apoptotic and cell cycle regulatory proteins. Additionally, the inhibitory effect of MJ-211 on multicellular 3D spheroid growth of A549 cells was evaluated. Key findingsLead compound MJ-211 exhibited remarkable cytotoxicity against A549 cells with an IC50 of 4.075 μM at 24 h treatment and IC50 of 1.7 nM after 72 h of treatment, while demonstrating selectivity towards normal WI-38 cells. MJ-211 activated pro-apoptotic factors Bim and p53, and suppressed Cyclin B1, Phospho HSP 27, BubR1, Mad 2, ERK1/2, and NF-κB, indicating its potent antimitotic and pro-apoptotic effects. MJ-211 significantly suppressed the migration of cells and inhibited the growth of A549 cell-derived multicellular 3D spheroids, highlighting its efficacy in a more physiologically relevant model. SignificanceCytotoxic effect of MJ-211 against cancer cells, selectivity towards normal cells, and ability to modulate key regulatory proteins involved in apoptosis and cell cycle progression underscore its potential as a promising template for further anticancer lead optimization. Moreover, the inhibitory effect of MJ-211 on multicellular spheroid growth suggests its efficacy in combating tumor heterogeneity and resistance mechanisms, thereby offering a promising avenue for future anticancer drug development.

Abstract 7554: The histone deacetylase inhibitor romidepsin is a potential therapeutic for metastatic osteosarcoma

Abstract Osteosarcoma predominately affects adolescents, young adults, and canines. For human patients, the five-year survival rate of those with detectable lung metastases is only 30% while most canine patients with metastatic disease die within 1-2 years. Standard therapy is not effective for this patient group, so it is therefore necessary to identify novel therapies that target the progression of lung metastases. Our lab previously screened 114 FDA-approved anti-cancer drugs to identify agents that decrease the growth of 3D spheroids (sarcospheres) generated from highly metastatic osteosarcoma cell lines. The top hits included both histone deacetylase inhibitors (HDIs) that were tested. In follow-up experiments measuring potency on sarcospheres and toxicity on non-transformed cells, romidepsin was the top hit of the 114-drug panel as well of the two additional FDA-approved HDIs and seven HDIs that are in clinical oncology trials. Our goal was therefore to further evaluate romidepsin as a potential therapy for metastatic osteosarcoma. Romidepsin potently decreased viability in sarcospheres from established highly metastatic osteosarcoma cell lines (relative ED50s = 3-36nM). About 50% of the patients (both canine and human) had similar responses to romidepsin (relative ED50s = 1- 18nM) suggesting that those patients might be good candidates for romidepsin therapy. This includes patients who had been heavily pretreated with standard-of-care osteosarcoma chemotherapeutics. Mechanistically, romidepsin blocks proliferation and causes a G2/M cell cycle arrest in fast growing sarcospheres (143B and MG63.3) while in slow growing sarcospheres (LM7 and K7M2) where primarily induces cytotoxicity and there is no cell cycle arrest. Patient-derived sarcospheres are slow growing, so we predict that the primary mechanism will be cytotoxicity. Using a tail vein injection model of osteosarcoma lung metastases, romidepsin dose dependently prolonged survival and reduced the area and number of lung metastases in immunodeficient mice injected with 143B cells and a syngeneic model using K7M2 cells. Romidepsin will be evaluated in mice with metastases from patient-derived cells and if effective, will be repurposed for canine and human clinical trials to improve outcomes in metastatic osteosarcoma without the associated costs and extensive timeframe of traditional drug discovery. Citation Format: Emily E. Seiden, Spencer M. Richardson, Shrey Ramnath, Tia Whiteside, Kathryn L. Coy, Anthony L. Sinn, Maegan L. Capitano, Karen E. Pollok, Chris D. Collier, Ed M. Greenfield. The histone deacetylase inhibitor romidepsin is a potential therapeutic for metastatic osteosarcoma [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 7554.

DDDR-03. DRUG REPURPOSING VIA ULTRA-HIGH THROUGHPUT 3D SPHEROIDS SCREENING FOR HUMAN SCHWANNOMA

Abstract Schwannoma is the most common benign peripheral nerve tumor occurring sporadically or as part of neurofibromatosis syndromes. While surgical resection can be used as the primary treatment option for Schwannoma, patients could face life-long repeated surgeries due to recurrence of syndromes that decreases the quality of life. Thus, non-invasive treatments for Schwannoma are highly demanded. To discover novel therapeutics for non-invasive Schwannoma treatment, in this project, we leveraged the benefits of drug repurposing high-throughput screening (HTS) of FDA-approved drugs using 3D spheroids derived from Schwannoma patient samples. Both 2D primary cells and 3D primary spheroids models were developed from an IRB-approved tumor bank of schwannoma patient samples. The 3D spheroids were validated to recapitulate the features of patient tumors. We then conducted an ultra-HTS(uHTS) in 1536-well format of 2036 FDA-approved drugs on the growth and viability of Schwannoma patient-derived 3D spheroids. Seven FDA-approved drugs were identified from the pilot screen to inhibit the growth of Schwannoma patient-derived 3D spheroids. These drugs offer potential to serve as pharmacotherapeutics for Schwannoma non-invasive treatment. Our results have demonstrated the capability of a drug repurposing strategy for Schwannoma using uHTS and a patient-derived tumor bank. We further demonstrated that patient-derived drug sensitivity signatures could be encoded from the screening data to enable clustering of patients, which accounts for heterogeneity in Schwannoma and avoids "one-size-fits-all" solutions in future non-invasive Schwannoma treatment studies.

Cytotoxic Imidazolyl-Mesalazine Ester-Based Ru(II) Complexes Reduce Expression of Stemness Genes and Induce Differentiation of Oral Squamous Cell Carcinoma.

The aggressiveness and recurrence of cancer is linked to cancer stem cells (CSCs), but drugs targeting CSCs may not succeed in the clinic due to the lack of a distinct CSC subpopulation. Clinical Pt(II) drugs can increase stemness. We screened 15 RuII or IrIII complexes with mesalazine or 3-aminobenzoate Schiff bases of the general formulas [Ru(p-cym)L]+, [Ru(p-cym)L], and [Ir(Cp*)L]+ (L = L1-L9) and found three complexes (2, 12, and 13) that are active against oral squamous cell carcinoma (OSCC) CSCs. There is a putative oncogenic role of transcription factors (viz. NOTCH1, SOX2, c-MYC) to enhance the stemness. Our work shows that imidazolyl-mesalazine ester-based RuII complexes inhibit growth of CSC-enriched OSCC 3D spheroids at low micromolar doses (2 μM). Complexes 2, 12, and 13 reduce stemness gene expression and induce differentiation markers (Involucrin, CK10) in OSCC 3D cultures. The imidazolyl-mesalazine ester-based RuII complex 13 shows the strongest effect. Downregulating c-MYC suggests that RuII complexes may target c-MYC-driven cancers.

The Effects of Natural Epigenetic Therapies in 3D Ovarian Cancer and Patient-Derived Tumor Explants: New Avenues in Regulating the Cancer Secretome.

High mortality rates in ovarian cancer have been linked to recurrence, metastasis, and chemoresistant disease, which are known to involve not only genetic changes but also epigenetic aberrations. In ovarian cancer, adipose-derived stem cells from the omentum (O-ASCs) play a crucial role in supporting the tumor and its tumorigenic microenvironment, further propagating epigenetic abnormalities and dissemination of the disease. Epigallocatechin gallate (EGCG), a DNA methyltransferase inhibitor derived from green tea, and Indole-3-carbinol (I3C), a histone deacetylase inhibitor from cruciferous vegetables, carry promising effects in reprograming aberrant epigenetic modifications in cancer. Therefore, we demonstrate the action of these diet-derived compounds in suppressing the growth of 3D ovarian cancer spheroids or organoids as well as post-treatment cancer recovery through proliferation, migration, invasion, and colony formation assays when compared to the synthetic epigenetic compound Panobinostat with or without standard chemotherapy. Finally, given the regulatory role of the secretome in growth, metastasis, chemoresistance, and relapse of disease, we demonstrate that natural epigenetic compounds can regulate the secretion of protumorigenic growth factors, cytokines, extracellular matrix components, and immunoregulatory markers in human ovarian cancer specimens. While further studies are needed, our results suggest that these treatments could be considered in the future as adjuncts to standard chemotherapy, improving efficiency and patient outcomes.

HILPDA promotes NASH-driven HCC development by restraining intracellular fatty acid flux in hypoxia

The prevalence of non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is rising rapidly, yet its underlying mechanisms remain unclear. Herein, we aim to determine the role of hypoxia-inducible lipid droplet associated protein (HILPDA)/hypoxia-inducible gene 2 (HIG2), a selective inhibitor of intracellular lipolysis, in NASH-driven HCC. The clinical significance of HILPDA was assessed in human NASH-driven HCC specimens by immunohistochemistry and transcriptomics analyses. The oncogenic effect of HILPDA was assessed in human HCC cells and in 3D epithelial spheroids upon exposure to free fatty acids and either normoxia or hypoxia. Lipidomics profiling of wild-type and HILPDA knockout HCC cells was assessed via shotgun and targeted approaches. Wild-type (Hilpdafl/fl) and hepatocyte-specific Hilpda knockout (HilpdaΔHep) mice were fed a Western diet and high sugar in drinking water while receiving carbon tetrachloride to induce NASH-driven HCC. In patients with NASH-driven HCC, upregulated HILPDA expression is strongly associated with poor survival. In oxygen-deprived and lipid-loaded culture conditions, HILPDA promotes viability of human hepatoma cells and growth of 3D epithelial spheroids. Lack of HILPDA triggered flux of polyunsaturated fatty acids to membrane phospholipids and of saturated fatty acids to ceramide synthesis, exacerbating lipid peroxidation and apoptosis in hypoxia. The apoptosis induced by HILPDA deficiency was reversed by pharmacological inhibition of ceramide synthesis. In our experimental mouse model of NASH-driven HCC, HilpdaΔHep exhibited reduced hepatic steatosis and tumorigenesis but increased oxidative stress in the liver. Single-cell analysis supports a dual role of hepatic HILPDA in protecting HCC cells and facilitating the establishment of a pro-tumorigenic immune microenvironment in NASH. Hepatic HILPDA is a pivotal oncometabolic factor in the NASH liver microenvironment and represents a potential novel therapeutic target. Non-alcoholic steatohepatitis (NASH, chronic metabolic liver disease caused by buildup of fat, inflammation and damage in the liver) is emerging as the leading risk factor and the fastest growing cause of hepatocellular carcinoma (HCC), the most common form of liver cancer. While curative therapeutic options exist for HCC, it frequently presents at a late stage when such options are no longer effective and only systemic therapies are available. However, systemic therapies are still associated with poor efficacy and some side effects. In addition, no approved drugs are available for NASH. Therefore, understanding the underlying metabolic alterations occurring during NASH-driven HCC is key to identifying new cancer treatments that target the unique metabolic needs of cancer cells.

Assessment of Antitumor and Antiproliferative Efficacy and Detection of Protein-Protein Interactions in Cancer Cells from 3D Tumor Spheroids.

When compared to two-dimensional (2D) cell cultures, 3D spheroids have been considered suitable in vitro models for drug discovery research and other studies of drug activity. Based on different 3D cell culture procedures, we describe procedures we have used to obtain 3D tumor spheroids by both the hanging-drop and ultra-low-attachment plate methods and to analyze the antiproliferative and antitumor efficacy of different chemotherapeutic agents, including a peptidomimetic. We have applied this method to breast and lung cancer cell lines such as BT-474, MCF-7, A549, and Calu-3. We also describe a proximity ligation assay of the cells from the spheroid model to detect protein-protein interactions of EGFR and HER2. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of 3D spheroids using the hanging-drop method Basic Protocol 2: Growth of spheroids using ultra-low-attachment plates Support Protocol 1: Cell viability assay of tumor spheroids Support Protocol 2: Antiproliferative and antitumor study in 3D tumor spheroids Support Protocol 3: Proximity ligation assay on cells derived from 3D spheroids.

Generation of 3D Spheroids Using a Thiol-Acrylate Hydrogel Scaffold to Study Endocrine Response in ER+ Breast Cancer.

Culturing cancer cells in a three-dimensional (3D) environment better recapitulates in vivo conditions by mimicking cell-to-cell interactions and mass transfer limitations of metabolites, oxygen, and drugs. Recent drug studies have suggested that a high rate of preclinical and clinical failures results from mass transfer limitations associated with drug entry into solid tumors that 2D model systems cannot predict. Droplet microfluidic devices offer a promising alternative to grow 3D spheroids from a small number of cells to reduce intratumor heterogeneity, which is lacking in other approaches. Spheroids were generated by encapsulating cells in novel thiol–acrylate (TA) hydrogel scaffold droplets followed by on-chip isolation of single droplets in a 990- or 450-member trapping array. The TA hydrogel rapidly (∼35 min) polymerized on-chip to provide an initial scaffold to support spheroid development followed by a time-dependent degradation. Two trapping arrays were fabricated with 150 or 300 μm diameter traps to investigate the effect of droplet size and cell seeding density on spheroid formation and growth. Both trapping arrays were capable of ∼99% droplet trapping efficiency with ∼90% and 55% cellular encapsulation in trapping arrays containing 300 and 150 μm traps, respectively. The oil phase was replaced with media ∼1 h after droplet trapping to initiate long-term spheroid culturing. The growth and viability of MCF-7 3D spheroids were confirmed for 7 days under continuous media flow using a customized gravity-driven system to eliminate the need for syringe pumps. It was found that a minimum of 10 or more encapsulated cells are needed to generate a growing spheroid while fewer than 10 parent cells produced stagnant 3D spheroids. As a proof of concept, a drug susceptibility study was performed treating the spheroids with fulvestrant followed by interrogating the spheroids for proliferation in the presence of estrogen. Following fulvestrant exposure, the spheroids showed significantly less proliferation in the presence of estrogen, confirming drug efficacy.

Metabolic alterations and mitochondrial dysfunction underlie hepatocellular carcinoma cell death induced by a glycogen metabolic inhibitor

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths. There is an urgent need for new targets to treat HCC due to limited treatment options and drug resistance. Many cancer cells are known to have high amount of glycogen than their tissue of origin and inhibition of glycogen catabolism induces cancer cell death by apoptosis. To further understand the role of glycogen in HCC and target it for pharmacotherapy, we studied metabolic adaptations and mitochondrial function in HepG2 cells after pharmacological inhibition of glycogen phosphorylase (GP) by CP-91149 (CP). GP inhibition increased the glycogen levels in HepG2 cells without affecting overall glucose uptake. Glycolytic capacity and importantly glycolytic reserve decreased significantly. Electron microscopy revealed that CP treatment altered mitochondrial morphology leading to mitochondrial swelling with less defined cristae. A concomitant decrease in mitochondrial oxygen consumption and mitochondria-linked ATP generation was observed. Metabolomics and enzyme activity / expression studies showed a decrease in the pentose phosphate pathway. In addition, CP treatment decreased the growth of HepG2 3D tumor spheroids in a dose- and time-dependent manner. Taken together, our study provides insights into metabolic alterations and mitochondrial dysfunction accompanying apoptosis in HepG2 cells upon GP inhibition. Our study can aid in the understanding of the mechanism and development of metabolic inhibitors to treat HCC.

Small-Molecule Gankyrin Inhibition as a Therapeutic Strategy for Breast and Lung Cancer.

Gankyrin is an oncoprotein responsible for the development of numerous cancer types. It regulates the expression levels of multiple tumor suppressor proteins (TSPs) in liver cancer; however, gankyrin's regulation of these TSPs in breast and lung cancers has not been thoroughly investigated. Additionally, no small-molecule gankyrin inhibitor has been developed which demonstrates potent anti-proliferative activity against gankyrin overexpressing breast and lung cancers. Herein, we are reporting the structure-based design of gankyrin-binding small molecules which potently inhibited the proliferation of gankyrin overexpressing A549 and MDA-MB-231 cancer cells, reduced colony formation, and inhibited the growth of 3D spheroids in an in vitro tumor simulation model. Investigations demonstrated that gankyrin inhibition occurs through either stabilization or destabilization of its 3D structure. These studies shed light on the mechanism of small-molecule inhibition of gankyrin and demonstrate that gankyrin is a viable therapeutic target for the treatment of breast and lung cancer.

Epigallocatechin-3-gallate inhibits the growth of three-dimensional in vitro models of neuroblastoma cell SH-SY5Y

The aim of the study is to investigate the potential of using three-dimensional (3D) in vitro neuroblastoma models to mimic the neuroblastoma microenvironment by testing a potential therapeutic compound—the natural extract epigallocatechin-3-gallate (EGCG), and to further elucidate the roles of DYRK1A in the growth and differentiation of neuroblastoma tissue. In vitro models based on a classic neuroblastoma cell line SH-SY5Y were employed, including 3D models with extracellular matrix and co-cultured with vascular endothelial cells. Cell viability was tested using AlamarBlue and Resazurin assay. The growth and differentiation of in vitro models of SH-SY5Y were analysed based on microscopy images obtained from immunofluorescence or real-time imaging. Protein expression level was investigated using immunoblotting analysis. The two-dimensional (2D) in vitro model implies the cytotoxicity and DYRK1A inhibition effect of EGCG and shows the induction of neuronal differentiation marker TuJ1. 3D in vitro models suggest that EGCG treatment compromised the growth of SH-SY5Y multicellular 3D spheroids and the viability of SH-SY5Y cultured in 3D Matrigel matrix. In addition, co-culture of SH-SY5Y with human vascular umbilical vein endothelial cells implied the inhibitory effects by EGCG in a vascularised microenvironment. In this study, novel 3D in vitro models of neuroblastoma were established in the application of testing a potential anti-cancer candidate compound EGCG. In pursuit of the goals of the 3Rs (replacement, reduction and refinement), the usage of these 3D in vitro models has the potential to reduce and eventually replace current animal models used in neuroblastoma research. The DYRK1A inhibiting nature of EGCG, together with the facts that EGCG inhibits the growth and induces the differentiation of neuroblastoma in vitro models, suggests an oncogene role of DRYK1A.

Targeting DNA Double-Strand Break Repair Enhances Radiosensitivity of HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma to Photons and Protons.

The response of head and neck squamous cell carcinoma (HNSCC) to radiotherapy depends on human papillomavirus type 16 (HPV) status, and where improved outcome and survival is observed in HPV-positive disease. However, strategies to further radiosensitise the tumours, particularly relatively radioresistant HPV-negative HNSCC, are actively being sought. The impact of targeting the major protein kinases involved in the signaling of DNA double-strand break (DSB) repair, namely ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the catalytic subunit of DNA-dependent protein kinase (DNA-Pkcs), on the radiosensitisation of HNSCC cells was examined. The response to both conventional photon radiotherapy, but also proton beam therapy, was analysed by clonogenic assays and 3D spheroid growth. We observed that inhibition of ATM, ATR, and particularly DNA-Pkcs, caused a significant reduction in HNSCC cell survival post-irradiation with both photons and protons, with less of an impact on the most radiosensitive HPV-positive cell line. The inhibition of DNA-Pkcs and, to a lesser extent ATM, in combination with radiation was also more effective at inhibiting the growth of 3D spheroids derived from relatively radioresistant HPV-negative HNSCC. Similar effects of the inhibitors were observed comparing photon and proton irradiation, demonstrating the potential for targeting DSB repair as an effective combination treatment for HNSCC.

A Glutathione Activatable Ion Channel Induces Apoptosis in Cancer Cells by Depleting Intracellular Glutathione Levels

AbstractCancer cells use elevated glutathione (GSH) levels as an inner line of defense to evade apoptosis and develop drug resistance. In this study, we describe a novel 2,4‐nitrobenzenesulfonyl (DNS) protected 2‐hydroxyisophthalamide system that exploits GSH for its activation into free 2‐hydroxyisophthalamide forming supramolecular M+/Cl− channels. Better permeation of the DNS protected compound into MCF‐7 cells compared to the free 2‐hydroxyisophthalamide and GSH‐activatable ion transport resulted in higher cytotoxicity, which was associated with increased oxidative stress that further reduced the intracellular GSH levels and altered mitochondrial membrane permeability leading to the induction of the intrinsic apoptosis pathway. The GSH‐activatable transport‐mediated cell death was further validated in rat insulinoma cells (INS‐1E); wherein the intracellular GSH levels showed a direct correlation to the resulting cytotoxicity. Lastly, the active compound was found to restrict the growth and proliferation of 3D spheroids of MCF‐7 cells with efficiency similar to that of the anticancer drug doxorubicin.

A Glutathione Activatable Ion Channel Induces Apoptosis in Cancer Cells by Depleting Intracellular Glutathione Levels.

Cancer cells use elevated glutathione (GSH) levels as an inner line of defense to evade apoptosis and develop drug resistance. In this study, we describe a novel 2,4-nitrobenzenesulfonyl (DNS) protected 2-hydroxyisophthalamide system that exploits GSH for its activation into free 2-hydroxyisophthalamide forming supramolecular M+ /Cl- channels. Better permeation of the DNS protected compound into MCF-7 cells compared to the free 2-hydroxyisophthalamide and GSH-activatable ion transport resulted in higher cytotoxicity, which was associated with increased oxidative stress that further reduced the intracellular GSH levels and altered mitochondrial membrane permeability leading to the induction of the intrinsic apoptosis pathway. The GSH-activatable transport-mediated cell death was further validated in rat insulinoma cells (INS-1E); wherein the intracellular GSH levels showed a direct correlation to the resulting cytotoxicity. Lastly, the active compound was found to restrict the growth and proliferation of 3D spheroids of MCF-7 cells with efficiency similar to that of the anticancer drug doxorubicin.

A novel histone acetyltransferase inhibitor A485 improves sensitivity of non-small-cell lung carcinoma cells to TRAIL

Transcriptional coactivators p300 and CBP catalyze the acetylation of lysine residues in histone proteins. Upregulation of p300 and CBP has been associated with lung, colorectal and hepatocellular cancer, indicating an important role of p300 and CBP in tumorigenesis. Recently, the novel p300 and CBP-selective inhibitor A485 became available, which was shown to inhibit proliferation of 124 different cancer cell lines. Here, we found that downregulation of EP300 or CREBBP enhances apoptosis upon TRAIL stimulation in non-small-cell lung cancer (NSCLC) cells. A485 upregulates pro- and anti-apoptotic genes at the mRNA level, implying an apoptosis-modulating effect in NSCLC cells. However, A485 alone does not induce apoptosis. Interestingly, we observed that the number of apoptotic cells increases upon combined treatment with A485 and TRAIL. Therefore, A485, as a TRAIL-sensitizer, was used in combination with TRAIL in wild type of NSCLC cell lines (HCC827 and H1650) and cells with acquired erlotinib resistance (HCC827-ER and H1650-ER). Our results show that the combination of A485 and TRAIL synergistically increases cell death and inhibits long-term cell proliferation. Furthermore, this combination inhibits the growth of 3D spheroids of EGFR-TKI-resistant cells. Taken together, we demonstrate a successful combination of A485 and TRAIL in EGFR-TKI-sensitive and resistant NSCLC cells.

Molecular Entrapment in Thermophilic Ferritin for Nanoformulation in Photodynamic Therapy

AbstractNanoformulation of therapeutic and diagnostic agents is beneficial as they accumulate at tumor sites due to enhanced permeability and retention effects. However, many in vitro studies performed on 2D cultures lack the realistic dimension of in vivo systems. In this work, a photosensitizer, acridine orange (AO), used in photodynamic therapy of cancer, is nanoformulated by entrapment in thermophilic ferritin. The efficacy is tested on 2D and 3D in vitro models and complemented with studies on the cellular uptake routes. Ferritin from the archaeon Archaeoglobus fulgidus (AfFtn) exhibits the unique property of divalent metal ion or ionic strength mediated assembly, making it an interesting nanocarrier for the entrapment of small molecules. The photosensitization and toxicity studies on 2D monolayers and 3D spheroid models of colorectal cancer cells show that AO loaded in AfFtn is functional. The killing efficiency in 2D monolayers reaches 50% while the growth of 3D spheroids is arrested after the first day with a subsequent 10% reduction of tumor diameter over the next 2 days. Results show that AO loaded AfFtn has better penetration ability than free AO alone in 3D spheroid models indicating that nanocage formulation provides for an efficient delivery vehicle into the tumor tissue.

Abstract 32: Development of a three-dimensional spheroid model system to more accurately evaluate schedule dependent mechanistic differences between metronomic and conventional dosing of topotecan

Abstract Alternative treatment schedules, such as low-dose, high-frequency (metronomic; METRO) of some chemotherapeutics enhance efficacy and reduce toxicity in some cancers when compared to chemotherapy given conventionally (CONV). Previously, we demonstrated that low-dose, prolonged exposure to topotecan (30-day infusion 0.1 mg/kg/day via Alzet pump) improved survival and reduced tumor volume ~120%, compared to topotecan given CONV (4 mg/kg, every 4 days) in xenograft model of aggressive prostate cancer in athymic mice. Yet, detailed examination of the mechanisms underlying this enhanced activity and optimization of treatment schedules is limited with existing 2-dimension (2D) cell culture models that do not adequately reflect in vivo reality. Therefore, we developed and applied a 3D spheroid model that allows for the evaluation of longer drug exposures. We used PC-3-Luc-GFP, an aggressive, metastatic castration-resistant prostate cancer that expresses luciferase (Luc) and green fluorescent (GFP) that support bioluminescent and fluorescence imaging. Cell growth and cytotoxicity studies were performed using 2D or 3D models. Briefly, 3D spheroids were formed by seeding cells in a poly-HEMA coated 96-well plate with 2.5% recombinant basement membrane followed by centrifugation (5 min at 1000 g) to encourage spheroid generation. The 2D cell cultures reached confluency in 72-96 hr. While we previously demonstrated enhanced activity in vivo, the 2D model showed no benefit (improved potency) over this time frame, ie, METRO vs CONV dosing (logIC50 2.278 (177nM), 95% CI [1.781-2.776] vs logIC50 2.248 (189 nM) 95% CI [1.723-2.773]. Attempts to reduce seeding density to increase study duration resulted in poor and inconsistent cell growth and greater variability. 3D spheroids yielded reproducible round spheroids of similar size (~800 μm) that grew for up to 28 days, thus permitting evaluation of long treatment exposures. Tumor stromal cells are also known to alter tumor pathology, drug delivery and responsiveness to therapy. Therefore, 3D spheroids with PC-3-Luc-GFP and macrophages and endothelial cells in different cell proportions (1:1:1, 1:2:1, etc.,) were examined. Growth of 3D spheroids was altered based on cell type and cell proportion. Further, differences in rates of growth and cellular morphology were observed for tumor activated vs wild macrophages. Overall, we developed a high-throughput, liquid overlay, scaffold-based 3D co-culture spheroid that can be kept for 28 days. This model will permit evaluation of single agent and concomitant therapy over prolonged periods better mimicking clinical practice. Further, this model will allow for more detailed molecular analysis and ability to discern the effect of tumor microenvironment on alternate treatment schedules. Citation Format: Joshua Davis, Taraswi Mitra Ghosh, Lani Jasper, Matthew Eggert, Jeff Warner, Brian Cummings, Robert Arnold. Development of a three-dimensional spheroid model system to more accurately evaluate schedule dependent mechanistic differences between metronomic and conventional dosing of topotecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 32.