Cell Culture Models Research Articles

Abstract 6491: Epigenetic-based combinatorial therapy is synergistic in KRAS/LKB1 mutant non-small cell lung cancers

Abstract KRAS and LKB1 mutations (KL) in non-small cell lung cancer (NSCLC) patients often results in poor prognosis, a high tumor mutational burden, and resistance to conventional therapy. The metabolic changes with these mutations can create unique vulnerabilities to be targeted therapeutically. HDAC inhibition has been reported as a promising approach to overcome MEK inhibitor resistance in animal models of melanoma, pancreatic cancer, and KL mutant NSCLC. Inhibition of HDAC3 and HDAC6 have independently been shown to have therapeutic activity in KL NSCLC. We now report on the activity of two novel HDAC3/6 inhibitors in cell culture and mouse models of human KL (A549) compared to KRAS/TP53-mutant NSCLC (KP) (CALU-6) alone and individually combined with the MEK inhibitor trametinib. GB-1101 inhibits HDAC3 and HDAC6 and GB-3103 inhibits HDAC1,3,6,10. SynergyFinder multi-dose combination data analytics showed synergistic interactions for both GB-1101 or GB-3103 when combined with trametinib in KL mutant A549 cells with maximum synergy scores of δ = 9.24 and 4.21, using zero interaction potency (ZIP) for synergy scoring. GB-1101 or GB-3103 when combined with trametinib in KP mutant CALU-6 cells did not demonstrate synergistic interaction with synergy scores of δ = -1.58 and -0.53, respectively. Mice were inoculated subcutaneously with 5.0x106 A549 or CALU-6 cells and treatment with GB-1101 (75 mg/kg qd, oral), GB-3103 (5.0 mg/kg qd, subcutaneous), trametinib (1.0 mg/kg qd, oral) and each drug in combination with trametinib was initiated when tumors reached an average of 120mm3. Single agent and combination treatments were well tolerated. GB-1101 exhibited tumor growth inhibition (TGI) of 33.0% in the A549 (KL) compared to 8.5% in CALU-6 (KP). In combination with trametinib, GB-1101 showed increased activity (TGI=92.7%) compared to trametinib alone (TGI=69.5%) in KL mutant A549. In KP mutant CALU-6 tumors, combination benefit was not observed; combination of GB-1101 and trametinib yielded a TGI=93% equivalent to trametinib alone (TGI=91%). GB-3103 resulted in a TGI of 66.7% in the KL mutant A549 and TGI= 55.9% in KP mutant CALU-6. In KL mutant A549, GB-3103 in combination with trametinib, showed increased inhibition (TGI=94.3%) compared to trametinib alone (TGI=69.5%). No combination benefit was seen for GB-3103 in KP mutant CALU-6 with the combination treatment TGI of 93.1% compared to trametinib alone TGI=90.7%. These results confirm and extend the finding that for KL mutant NSCLC, HDAC3/6 inhibitors show therapeutic benefit in combination with trametinib that is not observed in KP tumors. These data reveal a promising epigenetic-based combinatorial strategy for treating lung cancers harboring concurrent KRAS/LKB1 mutations, a path for clinical translation, and the potential to improve treatments for KRAS/LKB1 mutant NSCLC patients where there are currently no robust therapeutic strategies. Citation Format: Matthew Zucker, Kendra Devore, Logan Chesney, Gregg Hirschfeld, Annissa Reyna, Betsy Linnane, Joseph Mascarenhas, Maggie Murray, Alan Miller, Paul Gonzales, Stephen Gately. Epigenetic-based combinatorial therapy is synergistic in KRAS/LKB1 mutant non-small cell lung 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 6491.

Abstract 2577: Analyzing specific and nonspecific tissue uptake of antibodies by SPECT imaging and non-linear compartmental modeling

Abstract Understanding the mechanisms driving specific target engagement versus off-target nonspecific cellular uptake of antibodies can help inform design and/or delivery strategies that maximize therapeutic indices. Receptor binding kinetics, rates of receptor-mediated internalization and nonspecific pinocytosis are often measured through in vitro cell culture models, but there have been fewer efforts to collectively measure these cellular parameters in vivo. Single photon emission computed tomography (SPECT) imaging is a powerful method of estimating these parameters by quantifying both free/bound (intact) and internalized/catabolized antibody using non-residualizing halogen labels and residualizing radiometal-chelates, respectively. Modeling longitudinal in vivo imaging data of non-residualizing antibody can inform receptor occupancy (RO), while residualizing tracer kinetics can provide additional insights into catabolism due to pinocytosis and receptor-mediated internalization in the intracellular spaces. Here, we describe SPECT imaging of tumor-targeting trastuzumab (anti-HER2) and non-targeting anti-glycoprotein D (anti-gD) antibody, each labeled with non-residualizing ([125I]SIB) and residualizing radiometal-chelate (111In-DOTA) radionuclides in HER2+ xenograft-bearing mice. A non-linear compartmental model was fitted to SPECT imaging data to characterize RO of 10 mg/kg of anti-HER2 out to 7 days post dosing as well as to estimate rate constants of pinocytosis and receptor-mediated internalization of anti-HER2 in tumor. Longitudinal SPECT imaging demonstrated statistically significant differences in tumor drug exposure between 125I- and 111In-labeled anti-HER2 relative to anti-gD (p < 0.01). Tracer kinetics of anti-HER2 and associated RO simulations in tumor showed rapid extravasation from blood to tumor with peak RO of 25% at 13 hours post administration followed by a reduction in RO, reaching ~10% by day 7. Simulations for free, bound and catabolized tracer kinetics from our fitted model showed that anti-HER2 in the tumor interstitium was primarily receptor-bound with minimal pinocytosis of unbound anti-HER2 as well as gradual receptor-mediated catabolism, reaching a peak concentration of 113 µg/mL 7 days post dosing. Rate constants of nonspecific pinocytosis and HER2-mediated receptor internalization were estimated to be 0.0145 and 0.036 hours−1, respectively. Our model allowed us to successfully capture anti-HER2 antibody receptor binding and specific and nonspecific internalization over time in vivo. These data and analyses demonstrate the power of SPECT imaging using both non-residualizing and residualizing radiolabels combined with compartmental modeling to better characterize the different biological states (free, bound, and catabolized) of antibody within interstitial and intracellular compartments. Citation Format: Nicholas Cho, Phillip Spinosa, Danielle Mandikian, Geoffrey Del Rosario, Shang-Fan Yu, Jason Ho, Greg Ferl, Charles A. Boswell. Analyzing specific and nonspecific tissue uptake of antibodies by SPECT imaging and non-linear compartmental modeling [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 2577.

Abstract 4727: Large scale drug response comparison across different neuroblastoma cell model systems

Abstract Traditionally neuroblastoma research has predominantly used 2D cell lines, which, despite their simplicity, often fall short in mirroring the physiological complexity of actual tumors. Recently, 3D models like patient-derived tumor organoids (PDO) and patient-derived xenografts (PDX) have emerged as superior alternatives, offering more realistic predictions of patient treatment responses. This study aims to discern the differences in drug response between conventional 2D cell lines and the more advanced 3D neuroblastoma models - specifically, patient-derived organoids (nPDO) and patient-derived xenograft organoids (nPDXO). Employing high-throughput drug screening of 528 cancer drugs, we evaluated the drug responses across traditional 2D cell lines, nPDOs, and nPDXOs. Additionally, focused experiments with 29 selected drugs were conducted to determine the influence of culture topology on drug efficacy. The findings were compared to a study by Hansson et al.1 that used nPDXO. Our study discovered a pronounced difference in drug sensitivity between 2D cell lines and 3D models, with 3D models showing heightened sensitivity. Interestingly, 2D cell cultures had a more heterogenous drug response compared to 3D models. The drug response correlation between nPDOs and nPDXOs was relatively high (Spearman ~0.8), indicating consistent responses across these models. Our comparison underscores the impact of model system choice on drug response studies in neuroblastoma. The study highlights important differences in drug response between neuroblastoma 2D and 3D cell culture models. However, only subtle differences were found between PDO and PDX-derived organoid lines, despite differences in treatment centers and media compositions. This suggests that while the choice of cell culture topology greatly impacts drug response, PDO and PDX derived lines can largely be used interchangeably for drug screening experiments.

Abstract 2920: In vitro assays for investigating the effects of cancer therapeutics on bone

Abstract Bone microenvironment is an important factor in regulating bone metastasis. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Stimulatory factors released from the resorbed bone matrix in turn promote proliferation of cancer cells and this process, referred as the vicious cycle, often leads to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. The vicious cycle observed in osteolytic disease occurs also in osteoblastic metastases, and in addition cancer cells produce osteoblast-stimulating factors. Cancers with bone metastases often include both osteolytic and osteoblastic lesions. Our aim was to establish in vitro cell culture models to study the effects of cancer therapeutics on osteoblast and osteoclast differentiation and activity. In the osteoclast differentiation assay, human osteoclast precursor cells (Lonza) were cultured on bovine bone slices for 7 days. Denosumab was added in the cultures at day 0, and tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) activity was measured from the culture medium collected at day 7. Osteoclast activity was studied by allowing osteoclasts resorb bone after 7 days’ maturation period for additional 3 days in the presence of odanacatib. The amount of C-terminal cross-linked telopeptides of type I collagen (CTX-I) was measured in the culture medium collected at day 10 to quantitate osteoclast activity. Denosumab and odanacatib showed strong concentration dependent inhibition of osteoclast differentiation and activity, respectively. A mouse osteoblast progenitor cell line KS483 (Pharmatest Services) was used to study differentiation and activity of osteoblasts with test compounds bone morphogenetic protein 2 (BMP-2) and estradiol (E2). In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8. In the osteoblast activity assay, the cells were cultured for 13 days. N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to indicate the effects on organic bone formation and calcium deposition was determined at the end of the study as a marker of inorganic bone formation. BMP-2 and E2 stimulated osteoblast differentiation and activity indicated by the increase in ALP activity, PINP and calcium levels. We conclude that these culture systems can be used for studying the effects of cancer therapeutics and identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. Citation Format: Jenni H. Mäki-Jouppila, Mervi Ristola, Jukka Rissanen, Katja Fagerlund. In vitro assays for investigating the effects of cancer therapeutics on bone [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 2920.

Abstract 5800: Ultrasound-mediated nano-drug delivery system to enhance cancer treatment efficacy: An in vitro study

Abstract Conventional non-targeted nano-sized drug delivery systems face limitations such as restricted drug penetration depth and treatment efficacy in solid tumors. External stimulus such as ultrasound waves can trigger targeted, on-demand drug release from the nanocarriers. We demonstrate a first-of-its-kind use of low-intensity pulsed ultrasound (LIPUS) to induce Doxorubicin release from the surface of gold nanoparticle drug carriers. Our 1-MHz LIPUS beam is capable of penetrating tissue to sufficient depths with the required power/intensity for drug release applications with a nominal intensity of around 3 W/cm2 in the region of interest (ROI). This level of intensity is necessary to elevate the tissue temperature to the mild hyperthermia range of 41 to 45 °C. Our in vitro experiments demonstrate a remarkable 22% enhancement in drug release with the addition of LIPUS. Measurements of drug release kinetics, with and without LIPUS, suggest that LIPUS shifts the drug release mechanisms from static (Fickian) to dynamic (non-Fickian) release in vitro. Thus, revealing the intricate interactions between ultrasound and drug release from gold nanoparticle drug carriers. To assess the efficacy of the delivery system, we developed an in vitro cell culture model with a customized ultrasound exposure setup. The LIPUS device was situated at the bottom base of a water tank, directing ultrasound beam upward toward a region of interest where the MDA-MB-231 breast cancer cells were located 35 mm away from the LIPUS element. We found that the LIPUS device operating at an acoustic power of around 4 W increased cell killing efficacy by approximately 15% ± 2% when compared to conventional nano-drug delivery, highlighting a significant improvement attributed to ultrasound-induced mechanical effects. At the cellular level, these results underscore the potential advantages of ultrasound-mediated nano-drug delivery over conventional chemotherapy and non-targeted nano-drug delivery. Citation Format: Farshad Moradi Kashkooli, Anshuman Jakhmola, Tyler K. Hornsby, Graham A. Ferrier, Jahangir (Jahan) Tavakkoli, Michael C. Kolios. Ultrasound-mediated nano-drug delivery system to enhance cancer treatment efficacy: An in vitro study [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 5800.

Abstract 1791: Metabolic rewiring promotes metastatic potential upon glutamine deprivation in STK11 null KRAS-driven lung adenocarcinoma

Abstract Lung adenocarcinoma (LUAD) with concurrent oncogenic KRAS and STK11 loss-of-function mutations define an aggressive subtype characterized, in part, by increased metastasis. Loss of STK11, a tumor suppressor and nutrient sensor, leads to dysregulation of many critical cellular processes, including metabolism. As such, STK11 null cancers are “glutamine addicted” to support proliferative properties and the tumor microenvironment therefore becomes glutamine depleted. We hypothesize that conditions of glutamine stress promote metastatic potential in STK11 null KRAS-driven LUAD due to metabolic rewiring. To test this hypothesis, we utilized a cell culture model of KRAS-driven LUAD cell lines and corresponding STK11 knockout cell lines (∆STK11). Parental and ∆STK11 cells underwent the Seahorse mitochondrial stress test in full and glutamine-deficient media to obtain parameters of mitochondrial respiration at baseline and under conditions reflective of the tumor microenvironment, respectively. At baseline, ∆STK11 cells had a hypermetabolic phenotype associated with enhanced glutamine utilization. Deprivation of glutamine resulted in decreased mitochondrial respiration along with a significant increase in live, detached ∆STK11 cells compared to the parental line. Upon further examination, the detached ∆STK11 cells maintained the ability to re-adhere and proliferate in full media while the detached parental cells did not. Additionally, expression of pro-survival, anti-apoptotic and EMT markers in ∆STK11 cells were increased upon glutamine deprivation. To determine the mechanism(s) underlying this pro-metastatic phenotype, we employed heavy nitrogen labeling which revealed an upregulation of the hexosamine biosynthetic pathway (HBP). The HBP is an offshoot of glycolysis that serves as a central hub to regulate many cancer fitness pathways via O-GlcNAcylation; the addition of a GlcNAc moiety to serine/threonine residues of target proteins. Further assessment of O-GlcNAcylation levels via western and far western blot analysis revealed that parental cells had decreasing HBP flux concordant with decreasing glutamine concentration while ∆STK11 cells, conversely, had enhanced HBP flux upon decreasing glutamine availability. These observations suggest ∆STK11 cells utilize the HBP as a protective shunt under decreased glutamine availability. We are currently characterizing the invasive potential and anchorage-independence of parental and ∆STK11 LUAD cells upon glutamine deprivation. Future studies aim to establish the impact of HBP perturbation, both genetically and pharmacologically, on the described measures of metastatic potential. Overall, this work reveals novel insight into the molecular mechanisms altered downstream of STK11 loss that link glutamine metabolism with metastatic properties in KRAS-driven LUAD. Citation Format: Shannon Prior, Logan Sands, Sean Lenahan, Hailey Sarausky, Melissa Scheiber, David Seward, Paula Deming. Metabolic rewiring promotes metastatic potential upon glutamine deprivation in STK11 null KRAS-driven lung adenocarcinoma [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 1791.

Abstract 3090: Development and performance of the National Cancer Institute’s HTS384 NCI60 screen: A modernized platform to support drug discovery and development by the worldwide cancer research community

Abstract The NCI60 screen was established in the 1980s and introduced in the 1990s as a screening service for the worldwide cancer research community. The screen features 60 human tumor cell lines that represent 9 cancer types including non-small cell lung, colon, central nervous system, ovarian, renal, prostate and breast, as well as leukemia and melanoma. On a weekly basis, synthetic molecules, natural products, and biologics are screened against the 60-cell line panel to evaluate their anticancer potential. On average, more than 13,000 compounds are screened annually for investigators throughout the world. Cell growth and cytotoxicity were assessed by the sulforhodamine B (SRB) assay, performed in a 96-well format, that measures cellular protein content with an absorbance readout and enabled unprecedented throughput in the 1980s. Although the SRB assay supported a robust screening platform for three decades, its sensitivity and throughput are limited. Recently the NCI’s DCTD introduced the HTS384 NCI60 as a modernized screening platform. While the same 60 cell lines are evaluated, the HTS384 NCI60 is fully automated and performed in a 384-well format to enable a higher throughput. Additionally, the 48-h test agent exposure period of the SRB assay was increased to 72 h, which may result in lower 50% growth inhibition values (GI50) for some agents. A highly sensitive CellTiter-Glo luminescence readout for cell viability, based on cellular ATP content, was selected to enable comparisons of the NCI60 monolayer data to 3D cell culture models where the same assay chemistry can be applied. The HTS384 NCI60 was evaluated by screening a library of 1,003 U.S. Food and Drug Administration-approved and investigational oncology agents at five concentrations. Assay performance of the 60 cell lines was assessed from the controls of 30 - 60 microplates per cell line. The average doubling time of the 60 cell lines during the 72-h exposure period ranged between 19 h for NCI-H460 (17 - 23 h, n = 57) to 117 h for MDA-MB-468 (43 - 272 h, n = 60). The average signal-to-background ratio of all 60 cell lines was 89 (3,413 microplates) and ranged from 6 for OVCAR-5 (3 - 13, n = 54) to 229 for SF-539 (141 - 566, n = 60). The average coefficient of variation for microplate vehicle controls (n = 14) of all 60 cell lines (n = 3,413) was 5.2% and ranged from 3.2% for SK-MEL-28 (1.4 - 7.7%, n = 60) to 11.1% for HL-60(TB) (5.2 - 20.2%, n = 59). The average Z’-factor value of all 60 cell lines (n = 3,413) was 0.82 and ranged between 0.66 for HL-60(TB) (0.39 - 0.84, n = 59) to 0.89 for MALME-3M (0.79 - 0.95, n = 59), SF-295 (0.49 - 0.97, n = 59), SK-MEL-28 (0.74 - 0.95, n = 60), and UO-31 (0.8 - 0.94, n = 54). The development and performance of the HTS384 NCI60 screen will be described. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Nathan P. Coussens, Thomas S. Dexheimer, Eric M. Jones, Thomas Silvers, John R. Britt, Ronald C. Taylor, Mark W. Kunkel, James H. Doroshow, Beverly A. Teicher. Development and performance of the National Cancer Institute’s HTS384 NCI60 screen: A modernized platform to support drug discovery and development by the worldwide cancer research community [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 3090.

Abstract 382: Pancreatic cancer-derived organoids alter muscle fiber type and increased energy consumption leading to cachexia

Abstract Cachexia, marked by continuous skeletal muscle mass loss and severe metabolic disturbances, presents a substantial challenge for pancreatic ductal adenocarcinoma (PDAC) patients. Despite its prevalence and the adverse impact on quality of life and survival rates, effective clinical treatments for cachexia remain elusive. This study aims to bridge this gap by employing innovative in vitro models to investigate the underlying mechanisms of cachexia and identify potential therapeutic targets. To enhance the relevance of cell culture models, modifications were introduced to replicate in vivo conditions, where the muscle is exposed to the ongoing kinetics of constant tumor secretion of active factors. Patient-derived PDAC cells were cultured in a dense 3D stiffness extracellular matrix (ECM) to mimic the hypoxic desmoplastic tumor microenvironment. The conditioned medium from prolonged cultured patient-derived organoids (PDOs) was then used to supplement C2C12-derived myotubes. The investigation focuses on dynamic adaptations in muscle fiber type and metabolism when exposed to tumor paracrine factors. Metabolomic investigations of PDO-conditioned medium (PDO CM) on myotubes revealed distinct metabolic differences compared to non-treatment controls. Discriminatory metabolites included those associated with glycolysis, lipid, amino acid, and fatty acid metabolism. PDO CM induced a significant shift in muscle fiber type from slow-twitch to fast-twitch phenotypes, accompanied by de novel expression of embryonic and neonatal Myosin Heavy Chain (MyHC) isoforms. Fast fiber (F59) staining also confirmed the protein expression. Concurrently, PGC-1a, a master regulator of mitochondrial biogenesis and oxidative metabolism, significantly decreased, along with reduced ATP and Mito tracker staining, indicating a metabolic switch under the influence of tumor paracrine factors. Metabolome results on C2C12 myotubes treated with PDO CM also revealed significantly changed ABC transporter-related pathways, an adaptive process required to optimize substrate use under the influence of tumor secretome. However, the genes involved in the proteasome degradation process, such as MAFbx and MuRF1, did not show altered expression. The establishment of an in vitro cachexia model, focusing on changes in energy metabolism and utilizing muscle fiber type alterations as markers, will deepen our understanding of the mechanisms of cancer-associated cachexia. The insights gained may pave the way for identifying potential therapeutic targets to mitigate muscle wasting in PDAC patients and improve overall outcomes. Citation Format: Bo Han, Shuqing Zhao, zhi Yang, Jose Trevino, Steven Grossman, Heinz-Josef Lenz, Ba Xuan Hoang. Pancreatic cancer-derived organoids alter muscle fiber type and increased energy consumption leading to cachexia [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 382.

Abstract 1705: 3D growth modulates the competition between STAT3 and STAT5 in breast cancer

Abstract Approximately 13% of women are diagnosed with invasive breast cancer. Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that is often inappropriately activated in breast cancer and is frequently associated with Triple Negative Breast Cancer (TNBC). STAT5 can also be activated and inappropriate activation of STAT5 alone or with STAT3 is generally correlated with a more favorable prognosis, a lower grade tumor, and a better response to therapies compared to inappropriate activation of STAT3 alone. Therefore, knowing the activation status of STAT5 and understanding how STAT5 activation can attenuate STAT3-driven breast cancers could help guide therapeutic choices and patient treatment. STAT3 and STAT5 can regulate a set of overlapping target genes. For example, STAT3 and STAT5 can regulate BCL6 expression, where STAT5 represses expression and STAT3 enhances it. Importantly, STAT5 outcompetes and preferentially binds in the presence of activated STAT3. This suggests that the overlapping target genes could play an important role in understanding how activation of STAT5 can lead to a more favorable breast cancer compared to activation of STAT3 alone. This previous work was conducted in a 2D cell culture model; however, 3D models can better mimic clinical settings and the use of 3D models can help bridge the gap. Therefore, understanding the relationship between STAT3 and STAT5 in 3D is important. SK-BR-3 cells (HER2+ cells with low basal STAT3 activity) and MDA-MB-231 cells (TNBC cells with constitutive STAT3 activity) are being compared between 2D cell culture and 3D media suspension. We have shown using chromatin immunoprecipitation that STAT3 DNA-binding is enhanced in 3D at certain binding sites, whereas STAT5 is not. STAT5 has reduced dominance and preferential binding over STAT3 for BCL6 in 3D compared to 2D. In other cases, STAT3 binding is decreased upon STAT5 activation in 3D but not 2D. To begin to understand these differences, we analyzed phosphorylation of these STATs in 2D and 3D. Phospho-STAT3 levels were similar between 2D and 3D in SK-BR-3 cells when activated with LIF stimulation but were higher in 3D in the MDA-MB-231 cells which contain constitutive STAT3 activity. STAT5 activation by prolactin stimulation did not directly affect STAT3 phosphorylation in 2D or 3D but does affect STAT3 binding in 3D growing cells. However, prolactin induced phospho-STAT5 seems to be reduced in 3D in both cell lines. We are currently analyzing additional binding sites to better define the differences between STAT3 and STAT5 mediated gene regulation in 2D and 3D since STAT phosphorylation does not fully address it. Overall, our data suggests that differences between 2D and 3D breast cancer models will be important for translation to patient tumors and understanding the relationship between STAT3 and STAT5 will help elucidate how STAT5 activation status can be used when treating patients with breast cancer. Citation Format: Alexandra Elizabeth Temple, Sarah R. Walker. 3D growth modulates the competition between STAT3 and STAT5 in breast cancer [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 1705.

Abstract 1566: Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer [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 1566.

In silico agent-based modeling approach to characterize multiple in vitro tuberculosis infection models.

In vitro models of Mycobacterium tuberculosis (Mtb) infection are a valuable tool for examining host-pathogen interactions and screening drugs. With the development of more complex in vitro models, there is a need for tools to help analyze and integrate data from these models. To this end, we introduce an agent-based model (ABM) representation of the interactions between immune cells and bacteria in an in vitro setting. This in silico model was used to simulate both traditional and spheroid cell culture models by changing the movement rules and initial spatial layout of the cells in accordance with the respective in vitro models. The traditional and spheroid simulations were calibrated to published experimental data in a paired manner, by using the same parameters in both simulations. Within the calibrated simulations, heterogeneous outputs are seen for bacterial count and T cell infiltration into the macrophage core of the spheroid. The simulations also predict that equivalent numbers of activated macrophages do not necessarily result in similar bacterial reductions; that host immune responses can control bacterial growth in both spheroid structure dependent and independent manners; that STAT1 activation is the limiting step in macrophage activation in spheroids; and that drug screening and macrophage activation studies could have different outcomes depending on the in vitro culture used. Future model iterations will be guided by the limitations of the current model, specifically which parts of the output space were harder to reach. This ABM can be used to represent more in vitro Mtb infection models due to its flexible structure, thereby accelerating in vitro discoveries.

Abstract 1725: Role of PRMT5 in neuroendocrine prostate cancer development

Abstract Background: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer responsible for an estimated 20-30% of castration resistant prostate cancer (CRPC) deaths. While NEPC can arise de novo, the majority of these cases emerge as treatment-induced NEPC (tNEPC). Our data establish PRMT5:MEP50 as a key epigenetic regulator in NEPC and provide a potential predictive biomarker for tNEPC. Methods: Here we generated an in vitro cell culture model and mouse model to mimic the clinical conditions in androgen signaling inhibitor (ASI) or androgen deprivation treatment (ADT) We developed an in vitro cell culture and in vivo model to mimic the clinical conditions in enzalutamide-resistant prostate cancer. Regulation and role of protein arginine methyltransferase 5 (PRMT5) and its cofactor methylosome protein 50 (MEP50) were determined by treating cells and mice tumors with doxycycline inducible-shRNAs and catalytic inhibitors followed by analysis of cell viability, neurite growth, and effects on neuroendocrine related gene transcription. Pro-NEPC development effects were analyzed by immunofluorescence, immunohistochemical analysis, and western blot. PRMT5:MEP50-dependent methyltransferase activity was further validated by observing histone deposition levels via western blot and ChIP-qPCR. For clinical correlation, PRMT5 and MEP50 expression levels were comprehensively analyzed in both CRPC and NEPC patient prostate tissue samples. Results: Our clinical and computational analyses have identified increased expression of PRMT5:MEP50 in NEPC. Overexpression of PRMT5 and MEP50 in prostate cancer cells is sufficient to induce neuroendocrine differentiation (NED) in prostate cancer cells. Gene depletion of PRMT5 and MEP50 prevented the NE phenotype induced by androgen receptor inhibition and resensitized the prostate cancer cells to ASI/ADT. PRMT5 and MEP50 facilitate chromatin modulation through the dimethylation of H4R3, which is highly correlated with the NED phenotype. Moreover, overexpression of PRMT5 and MEP50 in mouse prostate induces NEPC development, and gene downregulation can prevent NED in a xenograft mouse model. Elevated PRMT5 and MEP50 correlated with increased recurrence in prostate cancer patients receiving ADT and their recurrence probability after the treatment. Conclusion: Our data establish PRMT5:MEP50 as a key epigenetic regulator in NEPC and provide a potential predictive biomarker for tNEPC. Citation Format: Hye Seung Nam, Xuehong Deng, Chang-Deng Hu. Role of PRMT5 in neuroendocrine prostate cancer development [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 1725.

Abstract 4250: Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research

Abstract Introduction: The development of new drugs in cancer therapy comprises toxicity and efficacy tests with increasing complexity. First and foremost, in vitro experiments are performed with well-established cancer cell lines, which are subsequently validated in animal experiments as prerequisite for clinical trials. Until a few years ago, there were gaps in the complexity chain between in vitro and in vivo experiments. Ethical considerations have led to use of systems like organ on a chip or mini-organ 3D in vitro models. In cancer research, cell cultures or organoids are generated from patient tumor material. However, these cultures only reflect the tumor of one patient, which is why panels of patient tumors should be used to evaluate the effectiveness of drugs on different patients. In vivo panel screens of patient derived xenografts (PDX) mouse models need high numbers of animals and takes several months. A pre-screen with in vitro models generated from in vivo PDX tissues allows large scale and faster pre-screens complementing in vivo systems for more focused in vivo analyses. Methods: Currently we have a pool of more than 600 established PDX models of 21 tumor entities. From this pool, cancer tissues of glioblastoma, mesothelioma, gastric, head/neck, lung and breast cancer were processed in single cell suspensions and cultured under defined conditions to obtain adherent cells or spheroids. The generated PDX in vitro cultures were analyzed for cellular impurities, cancer stem cell content and perpetuation of in vivo PDX characteristics. FACS analyses for tumor specific markers, chemo sensitivity assays and growth characteristics of the PDX derived cell lines (especially for glioblastoma) were analyzed. Results: From PDX tissues used, 90% grew as adherent and/or spheroid PDX derived in vitro cultures, in which mouse cells were entirely depleted. A high percentage of these cultures showed enriched cancer stem cell features and stem cell marker expression. Tumor marker expression and standard drug sensitivity data correlate to the in vivo PDX and derived in vitro cell culture models. RNAseq data were used to predict drug sensitivities in silico for untested drugs and drug combinations on our newly established PDX derived glioblastoma cell lines. Initial screens with predicted candidates were performed. Promising conditions were successfully repeated in corresponding animal PDX models. Conclusion: The newly developed technology for establishment if in vitro cell cultures from PDX efficiently generates stably growing cell lines possessing all key features of the original PDX. These cell lines can be used for initial pre-screens to optimize and improve selection of pharmacologically active drugs or drug combinations before initiating in vivo PDX studies. Citation Format: Lars Winkler, Joshua Alcaniz, Maria Stecklum, Wolfgang Walther, Jens Hoffmann. Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research [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 4250.

Abstract 240: Mix and match - A comprehensive pipeline for matched patient-derived PDX and PD3D® modelsfor cancer research and preclinical drug development

Abstract Relevant in vivo and in vitro cancer models have been invaluable for our current understanding of cancer biology, as well as for therapy development. Using appropriate models in the drug development process significantly supports reliable and swift decision making in proceeding along the value chain. In parallel, animal experiments can be designed more specific to the scientific and regulatory needs, resulting in shorter development cycles and eventually the availability of better therapies for cancer patients. Patient-derived xenografts (PDX) are generated by implantation of cancerous tissue from a patient’s tumor either under the skin (ectopic) or into the organ of tumor origin (orthotopic) of an immunocompromised mouse and are the most used in vivo model for preclinical drug development. Patient-derived 3D cell culture models (PD3D®) are gaining increasing significance as relevant in vitro cancer models that recapitulate the original tumor tissue’s biology and are suitable for high-throughput drug screening. The combination of both platform’s increases the translational relevance of the development pipeline and offers the possibility to enlarge the respective panels of tumor models. Tackling an unmet medical need, we initially created matched mesothelioma PD3D® and PDX models. Mesothelioma is an aggressive cancer with poor prognosis and limited treatment options. Development of new therapies is hampered by shortage of available relevant tumor models. We created PD3D® models from PDX mesothelioma models and vice versa and generated drug sensitivity profiles with standard of care such as Gemcitabine, platinum derivates and a number of targeted agents to confirm their suitability as matched models for future projects. We were able to prove that models express stable histological, phenotypic features and drug sensitivity profiles across the two platforms. With this proof-of-concept study, the feasibility to combine the PD3D® biobank comprising more than 500 models from CELLphenomics with the PDX bank from CRL including 700 PDX models, has gained momentum. In consideration of the changing regulatory landscape, the combination of in vitro and in vivo platforms is a crucial step towards a drug development pipeline that incorporates the principles of 3R and increases its translational value due to fully human technologies being the corner stones of the drug development process. Citation Format: Jürgen Loskutov, Eva Oswald, Lena Wedeken, Christoph J. Reinhard, Julia Schueler, Christian R. Regenbrecht. Mix and match - A comprehensive pipeline for matched patient-derived PDX and PD3D® modelsfor cancer research and preclinical drug development [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 240.

Abstract 4235: PD3D®models as jacks-of-all-trades for cancer research and therapy response prediction

Abstract Physiologically relevant in vitro tumor models are crucial in any research setting from preclinical drug development to functional precision oncology. Patient-derived 3D cell culture models (PD3D®) are validated cancer models which recapitulate the biology of the original tumor tissue. PD3D® can be used to model intratumoral heterogeneity in medium and high throughput screens. Using a reverse clinical engineering approach, PD3D® models are already applied in personalized oncology to identify treatment for an individual patient. We successfully established a living biobank of more than 500 PD3D® models, spanning from common cancers like colorectal, breast (incl. TNBC), non-small cell lung and pancreatic carcinoma, to orphan indications including various subtypes of sarcomas. Not surprisingly, PD3D® model morphology and culture requirements differ between tumor entities. Treating PD3D® models with standard of care drugs in a semi-automated high-throughput assay, we observed heterogeneity in drug sensitivity between models of the same cancer type, recapitulating clinical response of patients. Combining drug sensitivity profiles with genomic and proteomic data of the same PD3D® models, we successfully identified a biomarker for predicting chemosensitivity towards MEK-targeting drugs. For application of PD3D® in truly personalized oncology, we developed a protocol that allows us to generate a PD3D® culture and perform a drug sensitivity assay for an individual patient within a therapy-relevant timeframe. Within 38 days we identified a systemic therapy for a young patient with a relapsed metastasized synovial sarcoma. After surgery and initiation of recommended chemotherapy, the patient is now under remission. To further expand the scope of PD3D® for therapy response prediction, we established a method for irradiation of PD3D® cultures with different dosages of photon and proton radiation. We observed significant, model specific differences in radiosensitivity between PD3D® sarcoma models which also reflect clinically heterogenous responses. Additionally, PD3D® models can be employed for a tumor organoid-on-chip platform (TumOC), delivering real-time information on physiological parameters like drug response-mediated oxygen consumption in a microfluidic system. In conclusion, PD3D® models act as jacks-of-all-trades in current and future cancer research, delivering robust and reliable data that not only is of academic value, but speeds up the drug development process, and are ready for prime time in functional precision oncology. Citation Format: Lena Wedeken, Samantha Forbrig, Katja Herrera-Glomm, Juergen Loskutov, Ulrike Pfohl, Irina Piven, Michael Poehle, Manuela J. Regenbrecht, Barbara Seller, Cynthia Yapto, Sabine Finkler, Larissa Ruhe, Quirin Graf Adelmann, Christoph Reinhard, Marie Flechner, Katja Uhlig, David Kaul, Siyer Roohani, Maya Niethard, Rica Sauer, Christian R. Regenbrecht. PD3D®models as jacks-of-all-trades for cancer research and therapy response prediction [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 4235.

Endothelial gene regulatory elements associated with cardiopharyngeal lineage differentiation

Endothelial cells (EC) differentiate from multiple sources, including the cardiopharyngeal mesoderm, which gives rise also to cardiac and branchiomeric muscles. The enhancers activated during endothelial differentiation within the cardiopharyngeal mesoderm are not completely known. Here, we use a cardiogenic mesoderm differentiation model that activates an endothelial transcription program to identify endothelial regulatory elements activated in early cardiogenic mesoderm. Integrating chromatin remodeling and gene expression data with available single-cell RNA-seq data from mouse embryos, we identify 101 putative regulatory elements of EC genes. We then apply a machine-learning strategy, trained on validated enhancers, to predict enhancers. Using this computational assay, we determine that 50% of these sequences are likely enhancers, some of which are already reported. We also identify a smaller set of regulatory elements of well-known EC genes and validate them using genetic and epigenetic perturbation. Finally, we integrate multiple data sources and computational tools to search for transcriptional factor binding motifs. In conclusion, we show EC regulatory sequences with a high likelihood to be enhancers, and we validate a subset of them using computational and cell culture models. Motif analyses show that the core EC transcription factors GATA/ETS/FOS is a likely driver of EC regulation in cardiopharyngeal mesoderm.

Lipid-Restricted Culture Media Reveal Unexpected Cancer Cell Sensitivities.

Cancer cell culture models frequently rely on fetal bovine serum as a source of protein and lipid factors that support cell survival and proliferation; however, serum-containing media imperfectly mimic the in vivo cancer environment. Recent studies suggest that typical serum-containing cell culture conditions can mask cancer dependencies, for example, on cholesterol biosynthesis enzymes, that exist in vivo and emerge when cells are cultured in media that provide more realistic levels of lipids. Here, we describe a high-throughput screen that identified fenretinide and ivermectin as small molecules whose cytotoxicity is greatly enhanced in lipid-restricted media formulations. The mechanism of action studies indicates that ivermectin-induced cell death involves oxidative stress, while fenretinide likely targets delta 4-desaturase, sphingolipid 1, a lipid desaturase necessary for ceramide synthesis, to induce cell death. Notably, both fenretinide and ivermectin have previously demonstrated in vivo anticancer efficacy despite their low cytotoxicity under typical cell culture conditions. These studies suggest ceramide synthesis as a targetable vulnerability of cancer cells cultured under lipid-restricted conditions and reveal a general screening strategy for identifying additional cancer dependencies masked by the superabundance of medium lipids.

A partial Drp1 knockout improves autophagy flux independent of mitochondrial function

BackgroundDynamin-related protein 1 (Drp1) plays a critical role in mitochondrial dynamics. Partial inhibition of this protein is protective in experimental models of neurological disorders such as Parkinson’s disease and Alzheimer’s disease. The protective mechanism has been attributed primarily to improved mitochondrial function. However, the observations that Drp1 inhibition reduces protein aggregation in such neurological disorders suggest the involvement of autophagy. To investigate this potential novel protective mechanism of Drp1 inhibition, a model with impaired autophagy without mitochondrial involvement is needed.MethodsWe characterized the effects of manganese (Mn), which causes parkinsonian-like symptoms in humans, on autophagy and mitochondria by performing dose-response studies in two cell culture models (stable autophagy HeLa reporter cells and N27 rat immortalized dopamine neuronal cells). Mitochondrial function was assessed using the Seahorse Flux Analyzer. Autophagy flux was monitored by quantifying the number of autophagosomes and autolysosomes, as well as the levels of other autophagy proteins. To strengthen the in vitro data, multiple mouse models (autophagy reporter mice and mutant Drp1+/− mice and their wild-type littermates) were orally treated with a low chronic Mn regimen that was previously reported to increase α-synuclein aggregation and transmission via exosomes. RNAseq, laser captured microdissection, immunofluorescence, immunoblotting, stereological cell counting, and behavioural studies were used.Results in vitrodata demonstrate that at low non-toxic concentrations, Mn impaired autophagy flux but not mitochondrial function and morphology. In the mouse midbrain, RNAseq data further confirmed autophagy pathways were dysregulated but not mitochondrial related genes. Additionally, Mn selectively impaired autophagy in the nigral dopamine neurons but not the nearby nigral GABA neurons. In cells with a partial Drp1-knockdown and Drp1+/− mice, Mn induced autophagic impairment was significantly prevented. Consistent with these observations, Mn increased the levels of proteinase-K resistant α-synuclein and Drp1-knockdown protected against this pathology.ConclusionsThis study demonstrates that improved autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of its role in mitochondrial fission. Given that impaired autophagy and mitochondrial dysfunction are two prominent features of neurodegenerative diseases, the combined protective mechanisms targeting these two pathways conferred by Drp1 inhibition make this protein an attractive therapeutic target.Graphical

Design and characterization of BSA-mycophenolic acid nanocomplexes: Antiviral activity exploration

The interactions between bovine serum albumin (BSA) and mycophenolic acid (MPA) were investigated in silico through molecular docking and in vitro, using fluorescence spectroscopy. Dynamic light scattering and scanning electron microscopy were used to figure out the structure of MPA-Complex (MPA-C). The binding affinity between MPA and BSA was determined, yielding a Kd value of (12.0 ± 0.7) μM, and establishing a distance of 17 Å between the BSA and MPA molecules. The presence of MPA prompted protein aggregation, leading to the formation of MPA-C. The cytotoxicity of MPA-C and its ability to fight Junín virus (JUNV) were tested in A549 and Vero cell lines. It was found that treating infected cells with MPA-C decreased the JUNV yield and was more effective than free MPA in both cell line models for prolonged time treatments. Our results represent the first report of the antiviral activity of this type of BSA-MPA complex against JUNV, as assessed in cell culture model systems. MPA-C shows promise as a candidate for drug formulation against human pathogenic arenaviruses.

Endogenous BAX and BAK form mosaic rings of variable size and composition on apoptotic mitochondria

One hallmark of apoptosis is the oligomerization of BAX and BAK to form a pore in the mitochondrial outer membrane, which mediates the release of pro-apoptotic intermembrane space proteins into the cytosol. Cells overexpressing BAX or BAK fusion proteins are a powerful model system to study the dynamics and localization of these proteins in cells. However, it is unclear whether overexpressed BAX and BAK form the same ultrastructural assemblies following the same spatiotemporal hierarchy as endogenously expressed proteins. Combining live- and fixed-cell STED super-resolution microscopy, we show that overexpression of BAK results in novel BAK structures, which are virtually absent in non-overexpressing apoptotic cells. We further demonstrate that in wild type cells, BAK is recruited to apoptotic pores before BAX. Both proteins together form unordered, mosaic rings on apoptotic mitochondria in immortalized cell culture models as well as in human primary cells. In BAX- or BAK- single-knockout cells, the remaining protein is able to form rings independently. The heterogeneous nature of these rings in both wild type as well as single-knockout cells corroborates the toroidal apoptotic pore model.